b y t

V khoa hc v o to








Kim nghim dc phm
(Sch dng o to dc s i hc)
M s: .20.Z.08













Nh xut bn Y hc
H ni - 2005






Ch bin
PGS.TS. Trn T An
Tham gia bin son
PGS.TS. Trn T An
CN. Trn Tch
DS. Nguyn Vn Tuyn
TS. Chu Th Lc
ThS.Nguyn Th Kiu Anh
Tham gia t chc bn tho
ThS. Ph Vn Thm









Bn quyn thuc B Y t (V Khoa hc v o to)

2

Li ni u


Thc hin Ngh nh 43/2000/N-CP ngy 30/8/2000 ca Chnh ph quy
nh chi tit v hng dn trin khai Lut Gio dc, B Gio dc & o to v
B Y t ph duyt, ban hnh chng trnh khung cho o to Dc s i
hc. B Y t t chc thm nh sch v ti liu dy hc cc mn c s v
chuyn mn theo chng trnh mi nhm tng bc xy dng b sch chun
trong cng tc o to dc s i hc ca Ngnh Y t.
Kim nghim l mt khu quan trng trong h thng qun l cht lng
ton din ca dc phm. N c mt trong cc cng on sn xut (kim
nghim nguyn liu, bn thnh phm v kim nghim sn phm cui cng),
trong tn tr, lu thng v s dng thuc. Cc k thut c s dng trong
kim nghim rt phong ph v a dng thuc cc lnh vc vt l, ho hc v
sinh hc. Chnh v vy cc kin thc c bn cu 3 b mn ny - c bit l cc
mn hc chuyn ngnh nh ho phn tch, ho l, vi sinh - thc s l c s cho
mn hc kim nghim dc phm cn phi c nghin cu trc. Tuy nhin,
vic la chn mt k thut no cho nh gi cht lng mt nguyn liu
hoc ch phm thuc khng ch ph thuc vo c im ca k thut phn
tch m cn ph thuc vo c im ca i tng phn tch l dc phm, cho
nn cc kin thc v ho dc, dc liu, bo ch l rt cn thit cho kim
nghim dc phm.
Trong thc hnh kim nghim dc phm ngi ta s dng hu ht cc
k thut phn tch, cc php o vt l nh o im chy, im si, ch s khc
x, p sut thm thu,... cng l nhng k thut thng gp. Tuy nhin, trong
gio trnh vt l nm th nht, cc ni dung ny c trnh by chi tit c
l thuyt v thc hnh. V vy cc ni dung trn khng a vo chng trnh
Kim nghim dc phm. Cc phng php ha hc v ha l c s dng
rt ph bin: t loi thc hnh n gin nh chun th tch, so mu, n
nhng loi i hi cc thit b hin i nh sc k, quang ph, khi ph ...
Nhng vi thi lng l 2 n v hc trnh l thuyt cho sinh vin nm th 4
nn mc tiu mn hc c hn ch 2 vn :
1. Gii thch c nguyn l ca mt s phng php ho hc, ho l v
vi sinh thng dng trong kim nghim.
2. Trnh by c h thng m bo cht lng thuc v v tr ca cng
tc kim nghim trong h thng ny.
Cun sch ny c chia lm 6 chng. Sau chng 1 gii thiu s lc v
h thng m bo cht lng thuc v vai tr ca kim nghim, trong 3 chng
tip theo trnh by 3 nhm phng php ho hc, ho l v sinh hc. C s l
3
thuyt ca cc phng php kim nghim ny c nghin cu trong cc
mn c s nh: phn tch, ho l, vi sinh. y ch yu gii thiu nguyn tc
ng dng ca chng nh: hiu chun thit b, k thut xc nh nng , gim
thiu sai s... trong kim nghim. Do hn ch ca thi lng mn hc nn ch
cp n mt s phng php thng dng nh: chun mi trng khan,
quang ph phn t, HPLC. Chng 5 trnh by s lc ni dung kim nghim
cc dng bo ch: thuc vin, thuc tim, thuc dng ngoi ...
Nhm mc ch cung cp cho sinh vin mt s kin thc c bn v tnh
bn vng ca thuc, lm c s gii thch nhng bt thng c th gp trong
qu trnh kim nghim nh gi cht lng, chng ti gii thiu thm rt s
lc chng cui cng: n nh v tui th ca thuc.
Kt qu phn tch nh gi cht lng l sn phm ca thc hnh Kim
nghim dc phm. V vy sn phm ny cng nh bao loi sn phm khc lu
hnh trong x hi phi c cht lng tha mn nhu cu ngi tiu dng.
m bo cht lng ca kt qu, cn thc hin nhiu bin php ng b. l:
Trang thit b phng th nghim,
K nng ca kim nghim vin,
Qui trnh k thut kim nghim.
Thc hnh tt phng th nghim (GLP) l c s thc hin cc bin
php nu trn. Nhng ni dung ny s c gii thiu trong chng trnh cao
hc thuc chuyn ngnh Kim nghim dc phm v c cht.
V cch tip cn ni dung, trong cun sch khng nhc li qui trnh k
thut c trong Dc in m cung cp nhng kin thc cn thit sinh
vin c th gii thch c qui trnh v thc hnh kim nghim cho kt qu
tin cy.
Sch Kim nghim dc phm c cc ging vin B mn Ho Phn tch
Trng i hc Dc H Ni bin son. Sch c Hi ng chuyn mn thm
nh sch giao khoa v ti liu dy hc chuyn ngnh Dc ca B Y t thm
nh v c B Y t ban hnh lm ti liu dy hc chnh thc ca Ngnh Y t
trong giai on hin nay.
V Khoa hc v o to xin chn thnh cm n cc ging vin B mn Ho
Phn tch Trng i hc Dc tham gia bin son cun sch ny. V l ln u
xut bn nn chc chn cn nhiu thiu st, chng ti mong nhn c kin
ng gp ca ng nghip v sinh vin cun sch ngy cng hon thin hn.

V khoa hc v o to
B Y t
4
Mc lc
Li ni u 3
Chng 1
AI CONG
Trn Tch
1.1. Cht lng thuc v m bo cht lng 9
1.1.1. Thuc v yu cu cht lng 9
1.1.2. Kim tra cht lng thuc 12
1.1.3. H thng t chc kim tra cht lng thuc 14
1.2. Cng tc tiu chun ho 16
1.2.1. Khi nim 16
1.2.2. Cng tc xy dng tiu chun 18
1.2.3. Cng tc p dng tiu chun trong thc t 22
1.2.4. Gii thiu Dc in Vit Nam 23
1.3. Kim nghim thuc theo tiu chun 26
1.3.1. Ly mu kim tra 26
1.3.2. Tin hnh kim nghim 31
1.3.3. Ni dung chnh ca thc hnh tt phng kim nghim (GLP) 34
Ti liu tham kho 36
Cu hi t lng gi 36
Chng 2
KIM NGHIM THUC BNG CC PHONG PHP HO HC


*
Trn Tch, Trn T An
2.1. Cc phn ng nh tnh 37
2.2. Th gii hn cc tp cht trong thuc 48
2.2.1. Mc ch 48

*Phn 2.1 & 2.2. Trn Tch, phn 2.3. & 2.4.: Trn T An
5
2.2.2. Phng php xc nh gii hn tp cht trong thuc 48
2.2.3. Mt s thuc th trong cc phn ng ho hc xc nh
gii hn tp cht
50
2.3. Chun acid - base trong mi trng khan 51
2.3.1. Vai tr ca dung mi 51
2.3.2. Khi nim pH 52
2.3.3. Xc nh im tng ng 53

2.3.4. ng dng kim nghim thuc
54
2.4. Xc nh hm lng nc bng thuc thi Karl fischer 58
2.4.1. Nguyn tc 58
2.4.2. Pha ch v xc nh chun 58
2.4.3. Xc nh im tng ng 59

2.4.4. ng dng
59
2.5. nh lng mt s cht hu c a chc bng thuc th
periodat
60
2.6.
ng dng cp ion trong kim nghim thuc
61
Ti liu tham kho
Cu hi t lng gi
65
66
Chng 3
CC PHONG PHP HO L TRONG KIM NGHIM THUC
Nguyn Vn Tuyn

3.1. Phng php quang ph phn t 68
3.1.1. Quang ph hp th UV VIS 68
3.1.2. Quang ph hng ngoi (IR) 79
3.1.3. Quang ph hunh quang 82
3.2. Phng php sc k lng hiu nng cao (HPLC) 84
3.2.1. Cc thng s c trng ca qu trnh sc k 84
3.2.2. My HPLC 86
3.2.3. Cc k thut HPLC 88
3.2.4. Hng dn chn k thut HPLC 94
3.2.5. Chun ho ct HPLC 100
6
3.2.6. nh lng bng phng php HPLC 102
3.2.7. Cc phng php nh lng 104
Ti liu tham kho
Cu hi t lng gi
111
111
Chng 4
KIM NGHIM THUC BNG PHONG PHP SINH HC
Chu Th Lc

4.1. M u 115
4.1.1. Nguyn tc 115
4.1.2. Cht chun 116
4.1.3. nh gi kt qu 116
4.2. Kim nghim thuc bng phng php th trn ng vt 116
4.3. Kim nghim thuc bng phng php th vi sinh vt 117
4.3.1. i cng v vi sinh vt 117
4.3.2. Mi trng nui cy vi sinh vt 121
4.3.3. Th v trng 124
4.3.4. Th gii hn vi sinh vt 128
4.3.5. Xc nh hot lc khng sinh bng phng php th vi sinh vt 131
Ti liu tham kho
Cu hi t lng gi
137
138
Chng 5
KIM NGHIM CC DANG BAO CH
Nguyn Th Kiu Anh

5.1. Kim nghim thuc bt 140
5.2. Kim nghim thuc vin nang 145
5.3. Kim nghim thuc vin nn 147
5.4. Kim nghim thuc tim, thuc tim truyn 149
5.5. Kim nghim thuc nh mt 153
5.6. Kim nghim thuc ung dng lng 154
5.7. Kim nghim thuc m 157
7
5.8. Kim nghim thuc n, thuc trng 159
5.9. Th ho tan ca vin nn v vin nang 160
5.10 Th r ca vin nn v vin nang 166
Ti liu tham kho
Cu hi t lng gi
169
169

Chng 6
N |NH VA TUI TH CA THUC
Trn T An
6.1. Qu trnh pht trin nghin cu n nh ca thuc 171
6.2. i cng v n nh ca thuc 172
6.2.1. nh ngha 172
6.2.2. Mt s thut ng lin quan 173
6.2.3. Mc tiu nh gi n nh 174
6.2.4. Tiu chun nh gi n nh 175
6.2.5. Phn vng kh hu 176
6.3. ng ho hc dung dch 177
6.3.1. Bc ca phn ng 177

6.3.2. nh hng ca nhit
181
6.4. Xc nh n nh ca thuc 182
6.4.1. Ly mu 182
6.4.2. Phng php th cp tc 183
6.4.3. Phng php th di hn 185
6.4.4. Phng php phn tch nh gi kt qu 185
6.5. Cc dc cht km bn vng 186
Ti liu tham kho
Cu hi t lng gi
188
189



8
Chng 1
i cng
Mc tiu hc tp
1. Trnh by c h thng m bo cht lng thuc v v tr ca cng
tc kim nghim trong h thng ny.
2. Trnh by c cc nhim v ch yu ca cng tc kim nghim.
1.1. Cht lng thuc v m bo cht lng
1.1.1. Thuc v yu cu cht lng
Khi nim v thuc:
Theo T chc y t th gii, thuc l mt cht hay mt hn hp cc cht
c sn xut em bn, cung cp bn hay gii thiu s dng nhm mc
ch: iu tr, lm gim, phng hay chn on bnh tt, tnh trng c th bt
thng hoc triu chng bnh; khi phc, hiu chnh, thay i chc nng hu
c ca c th ngi (hay ng vt - thuc th y).
Vn dng vo Vit Nam, trong "iu l thuc phng bnh, cha bnh" ban
hnh 24/1/1991 qui nh: Thuc l nhng sn phm c ngun gc t ng vt,
thc vt, khong vt, hay sinh hc c sn xut dng cho ngi nhm:
Phng bnh, cha bnh
Phc hi, iu chnh chc nng c th
Lm gim triu chng bnh
Chn on bnh
Phc hi hoc nng cao sc kho
Lm mt cm gic mt b phn hay ton thn
Lm nh hng qu trnh sinh sn
Lm thay i hnh dng c th.
Vt liu dng trong khoa rng, bng bng, ch khu y t, cng c
coi l thuc.
Thuc lu hnh trn th trng a phn l cc tn dc v thuc y hc
c truyn (l cc thuc c sn xut theo phng php y hc c truyn).
Trong c nhiu thuc di dng bit dc (bit dc l nhng thuc mang
tn ring cn gi l tn thng mi ring ca mt c s sn xut hay mt
9
hng sn xut ln u t cho n v c php a ra th trng ang
c bo h quyn s hu cng nghip).
Cht lng thuc v yu cu cht lng:
Cht lng ca mt thuc l tng hp cc tnh cht c trng ca thuc
(th d; c cha ng cc thnh phn theo t l qui nh, c tinh khit
theo yu cu, ng gi c nhn qui nh) c th hin mt mc ph
hp vi nhng yu cu k thut nh trc tu theo iu kin xc nh v
kinh t, k thut, x hi nhm m bo cho thuc t cc mc tiu sau:
C hiu lc phng bnh v cha bnh
Khng c hoc t c tc dng c hi
n nh v cht lng trong thi hn xc nh
Tin dng v d bo qun.
Thuc l sn phm hng ho c bit, c quan h trc tip n sc kho
cng ng, n cht lng v hiu qu ca vic phng bnh, cha bnh. V th
thuc phi c bo m cht lng trong ton b qu trnh sn xut t
nguyn liu cho n l thnh phm, trong qu trnh bo qun, lu thng phn
phi n ngi s dng.
Mc tiu ca m bo cht lng trn ch c coi l t khi no thuc
p ng c cc yu cu c bn sau:
Thuc c cha ng cc thnh phn theo t l qui nh ca cng thc
c ng k v c cp php (nh tnh, nh lng).
Thuc c php sn xut v sn xut theo ng cc qui trnh ng
k v c php.
C tinh khit t yu cu qui nh.
Thuc c ng gi trong cc ng v bao gi vi nhn thch
hp v ng qui cch ng k.
Thuc c bo qun, phn phi, qun l theo qui nh cht lng
ca thuc c duy tr trong sut tui th ng k hay thi hn
bo hnh.
t cc mc tiu trn, cn phi c nhiu yu t, trong 3 yu t c
bn phi c l:
Thc hnh tt sn xut thuc (GMP)
Thc hnh tt phng kim nghim thuc (GLP)
Thc hnh tt bo qun thuc (GSP)
1.1.1.1. Thc hnh tt sn xut thuc (Good Manufacture Practice)
Bao gm nhng qui nh cht ch v chi tit v mi mt ca qu trnh sn
xut nh: T chc, nhn s, c s v tin nghi, my mc, trang thit b, kim
10
tra nguyn ph liu, bao b, ng gi, kim tra bn thnh phm, thnh phm
cui cng, nhm sn xut ra c thuc theo d kin v t tiu chun k
thut t ra. Mun vy, phi thc hin c cc yu cu c bn sau:
Tt c cc quy trnh sn xut phi c qui nh r rng v chc chn
c kh nng t mc ch ra.
Cc cn b, nhn vin phi c o to t yu cu; c s v din tch
ph hp, cc trang thit b v phng tin phc v, nguyn liu, ph
liu, bao b, ng gi, nhn, ng quy cch v theo yu cu ra.
Trong sut qu trnh sn xut phi c s kim tra, theo di, ghi chp
y chng minh rng tt c cc giai on ca qui trnh sn xut
u c thc hin nghim chnh, c cht lng v s lng sn phm
ph hp vi qui nh.
C cc h s sn xut v phn phi d dng xem xt lch s ca mt
l thuc no .
C mt h thng t chc cn thit khi cn c th thu hi bt k l
thuc no cp pht hoc bn ra.
1.1.1.2. Thc hnh tt phng kim nghim thuc (Good Laboratory Practice)
Bao gm nhng quy nh cht ch v chi tit cc yu t tham gia vo qu
trnh kim tra cht lng thuc, nhm m bo kt qu c chnh xc, ng,
khch quan.
thc hnh tt qu trnh kim tra cht lng thuc, cn phi p ng
cc yu cu c bn sau y:
Cn b c o to chu o, c t chc tt, c trnh cao, c thc
trch nhim.
C s vt cht, trang thit b tt, p ng yu cu k thut.
Ho cht, thuc th, cht chun tt.
iu kin v sinh tt
Ly mu, lu mu tt
Kt qu th nghim tt, ghi chp x l s liu tt.
H s lu tr tt.
Vn ny s c cp k hn phn nghip v ca cng tc kim nghim.
1.1.1.3. Thc hnh tt bo qun thuc (Good storage practice)
Bao gm nhng quy nh cht ch v nghim ngt cho vic tn tr, iu
kin bo qun, xut nhp nguyn ph liu, bao b, ch phm, nhn thuc...
Tt c nhm gim st kim tra cht ch thuc m bo cht lng n
11
ngi s dng. Mun thc hnh tn tr thuc tt cn lu lm tt cc yu
cu sau:
Cn b phi c o to ph hp, c trch nhim
Nh ca v phng tin bo qun tt
iu kin v sinh tt
Thc hin tt qui trnh bo qun: nguyn liu, bn sn phm, sn
phm, bao b, ng gi
Quy trnh bo qun, vn chuyn phi c tun th nghim chnh.
1.1.2. Kim tra cht lng thuc (Drug quality control)
1.1.2.1. Khi nim
Kim tra cht lng thuc hay kim nghim thuc l vic s dng cc
phng php phn tch: l hc, ho hc, ho l, sinh vt, vi sinh vt, qui
nh xc nhn mt thuc hay mt nguyn liu lm thuc c t hay khng
t tiu chun qui nh. Ni mt cch c th l kim tra cht lng thuc
nhm tr li cc cu hi:
y c phi l thuc cn kim tra khng?
C m bo hot lc hay hm lng ng k v c duyt ?
C t tinh khit theo yu cu hay khng?
C b phn hu hay bin cht hay khng?
bao gi, nhn c ng qui cch khng?
Nh vy, mc tiu c bn ca cng tc kim tra cht lng thuc l:
ngi s dng dng c thuc m bo cht lng, t hiu qu s
dng cao.
Pht hin thuc khng t tiu chun, thuc gi, thuc km phm
cht x l v khng cho php lu hnh trn th trng.
Trong ngnh y t quy nh: Tt c cc thuc v nguyn liu lm
thuc u phi c kim nghim v xc nh cht lng, nu t tiu chun
qui nh mi c a vo s dng. Bi vy thuc phi c kim tra cht
lng mt cch nghim ngt, cht ch, bo m cho thuc t cht lng
trong mi hot ng sn xut, lu thng phn phi, xut nhp khu, qun l
v s dng thuc.
1.1.2.2. Cc yu t nh hng n cht lng thuc
C nhiu yu t, trong c th l:
Nhng ri ro hoc nhm ln trong sn xut, do khng chp hnh
nhng qui nh ca GMP.
12
Nhng sn phm do v tnh hay c trong sn xut to ra sn
phm c hm lng thp hn qui nh.
Do la chn cng thc bo ch cng nh k thut bo ch cha ng
nn thuc b phn hu bin cht.
Qu trnh bo qun cha tt.
Do li ch c nhn bt chnh lm hng gi la ngi tiu dng gy
ra tc hi nghim trng
1.1.2.3. Khi nim v thuc t v khng t tiu chun, thuc gi mo, thuc
km phm cht




Thuc t tiu chun (thuc bo m cht lng):
L thuc p ng y cc yu cu k thut tiu chun ra (hay
thuc p ng y cc mc cht lng trong tiu chun cht lng
ng k).
Thuc khng t tiu chun:
L thuc khng p ng y cc yu cu k thut tiu chun
ra. Mc khng t c th l mt hay mt s ch tiu. Thuc khng t tiu
chun thuc loi thuc km cht lng.
Thuc gi:
Theo qui nh ca T chc y t th gii, thuc gi l ch phm c sn
xut khng ng vi nhn kha cnh nhn dng hay ngun gc thuc, vi s
c v mang tnh cht la o ca nh sn xut. Sn xut sai thnh phn
cng thc ng k, khng c hay khng hm lng hot cht, hoc c
ng gi trong cc bao b gi mo. Nh vy, c th ni thuc gi l nhng sn
xut thuc ca ngi sn xut mang la o, gian ln c th da vo mt
s biu hin pht hin:
Thuc khng c hoc c t dc cht
Thuc c cha dc cht khc vi tn dc cht ghi trn nhn
Nhn, bao gi ging hay gn ging vi nhn, bao gi ca mt thuc khc
Thuc km phm cht:
L thuc khng t tiu chun k thut m trc n t. Mc
khng t tiu chun c nhiu hnh thi khc nhau. Tt c cc nguyn nhn
gy ra c th xc minh c bng cc phng php khoa hc, k thut cho
php. Cc nguyn nhn c th l:
Do k thut sn xut, bo qun khng ng, do thuc t bin cht.
Do bao gi khng t tiu chun, nn a tp cht vo thuc.
13
Do tui th (hn dng) ht
Do nguyn ph liu khng t tiu chun
Do tc ng ca mi trng: nhit , nh sng, m
1.1.3. H thng t chc qun l, kim tra cht lng thuc
Nh nc giao cho B Y t chu trch nhim qun l ton din cht lng
thuc. V vy, h thng t chc, qun l, kim tra cht lng thuc ca nghnh
y t c chia lm 3 phn: H thng qun l cht lng thuc; H thng kim
tra cht lng thuc; H thng thanh tra Dc.
1.1.3.1. H thng qun l cht lng thuc


Cc qun l Dc Vit Nam:
L c quan c B Y t u quyn thc hin cc nhim v trong lnh vc
qun l nh nc v cht lng thuc:
Xy dng quy hoch, k hoch v qun l cht lng thuc v t chc
k hoch c ph duyt.
Xy dng cc vn bn php quy v tiu chun v cht lng thuc
B ban hnh, hng dn kim tra vic thc hin cc vn bn trn.
Qun l vic ng k tiu chun c s. Cung cp thng tin khoa hc k
thut lin quan n m bo cht lng thuc.
Ch o, gim st h thng kim tra cht lng thuc trn ton quc.
Kim tra v cp giy chng nhn; c s sn xut t tiu chun Thc
hnh tt sn xut thuc v phng kim nghim t tiu chun Thc
hnh tt kim nghim thuc.
T chc hng dn nghip v cho cn b qun l cht lng thuc ca
ngnh y t, t chc o to v bi dng nghip v v tiu chun - o
lng - cht lng thuc.
Phi hp vi thanh tra B Y t thc hin chc nng kim tra, thanh tra
nh nc v cht lng thuc v x l vi phm php lut v cht lng
thuc theo thm quyn.
C quan qun l nh nc v cht lng thuc a phng:
S Y t ch o qun l ton din v cht lng ca thuc a phng
(thng u quyn cho phng nghip v Dc) c nhim v:
Ph bin, hng dn v t chc thc hin cc vn bn php lut v
qun l cht lng thuc ti a phng.
Thc hin chc nng kim tra, thanh tra nh nc v cht lng thuc
v x l vi phm v cht lng thuc trong phm vi a phng.
14
1.1.3.2. H thng kim tra cht lng thuc
C quan kim tra cht lng thuc ca nh nc v thuc

Trung ng l Vin Kim nghim thuc Quc gia ( H Ni) v Phn
vin Kim nghim ( TP. H Ch Minh).
Gip B Y t qun l ch o cng tc kim tra cht lng thuc trong
ton quc v mt k thut. C nhim v:
+ Nghin cu xy dng tiu chun Vit Nam v thuc.
+ Kim tra xc nh cht lng thuc lu hnh trn th trng.
+ Thm tra k thut, gip B xt duyt cc tiu chun kim nghim
xt cp ng k sn xut v lu hnh thuc Vit Nam.
+ Pht hnh cc cht chun v cht i chiu dng trong kim nghim.
+ Lm trng ti v cht lng khi c tranh chp khiu ni v cht
lng thuc.
+ Tham gia o to cn b lm cng tc kim nghim .
+ T vn v chnh sch cht lng thuc quc gia.
+ Xy dng tiu chun phng kim nghim thuc t tiu chun v gip
, kim tra cng nhn cc phng kim nghim thuc trong c nc.
+ Kim tra vic kim nghim thuc theo tiu chun trong phm vi ton quc.
tnh, thnh ph trc thuc Trung ng l Trung tm kim nghim
dc phm, m phm.
L c quan trc thuc S y t, c nhim v nh Vin Kim nghim nhng
gii hn trong phm vi tnh, thnh ph.
H thng t kim tra cht lng cc c s (phng kim nghim):
Cng tc kim tra cht lng thuc c thc hin tt c cc c s c
lin quan ti thuc. Tu theo qui m ca c s sn xut (x nghip, cng ty
trung ng hoc a phng, cc c s nh), kinh doanh (cng ty, ca
hng) bnh vin (trung ng, a phng) m lp phng kim nghim hay t
kim nghim t kim tra cht lng thuc.Vi cc c s sn xut, bt buc
phi c b phn t kim tra cht lng. B phn ny phi c kh nng kim
nghim xc nh cht lng thuc c sn xut c s mnh theo tiu chun
duyt. Phi kim nghim, theo di c cht lng ca thuc trong sut
qu trnh sn xut t nguyn liu, bn thnh phm, thnh phm. Phi lp h
s theo di tng l sn phm. C s sn xut phi chu trch nhim v thuc
do c s sn xut ra.
Cc c s bo qun, phn phi thuc phi c b phn t kim nghim (vi
cc cng ty ln) hoc kim tra, kim sot qun l, nh gi cht lng
thuc, theo di cht lng trong qu trnh bo qun, cung cp h s cht lng
cho n v s dng thuc.
15
Cc bnh vin, ty theo quy m ln, nh cng phi c b phn kim
nghim cc thuc t pha ch, kim tra, kim sot cht lng thuc trc khi
phn phi n ngi s dng.
1.1.3.3. H thng thanh tra dc
Cng cc c quan qun l nh nc v cht lng thuc, thc hin chc
nng kim tra, thanh tra nh nc v cht lng thuc c t chc t trung
ng n a phng.
1.2. Cng tc tiu chun ho
1.2.1. Khi nim
1.2.1.1. Mt s nh ngha
Tiu chun ho l mt lnh vc hot ng bao gm hai ni dung: Xy
dng ra cc tiu chun v p dng cc tiu chun trong thc t nhm a
cc hot ng ca x hi (c bit trong lnh vc sn xut v kinh doanh) i
vo n np t c hiu qu chung c li nht cho mi ngi v cho x hi.
Tiu chun l nhng qui nh thng nht v hp l c trnh by di
dng mt vn bn hoc mt th thc nht nh do mt c quan c thm
quyn ban hnh bt buc p dng cho nhng ni c lin quan. Ni mt
cch khc tiu chun l mt vn bn mang tnh php ch trong ra
nhng qui nh thng nht v hp l bt buc p dng nhm m bo cht
lng cho mt sn phm no .
Cht lng sn phm l mt tp hp cc ch tiu c trng th hin tnh
nng s dng nhm tho mn yu cu c xc nh trc cho mt sn
phm trong nhng iu kin xc nh ph hp hon cnh kinh t, khoa hc k
thut, x hi.
i vi thuc, tiu chun l mt vn bn khoa hc k thut mang tnh
php ch trong qui nh: qui cch, ch tiu yu cu k thut, phng php
th, ng gi, ghi nhn, bo qun v cc vn khc lin quan n vic nh
gi cht lng ca mt thuc. y l c s cc cc c quan kim nghim
(hoc ngi kim nghim) tin hnh thc nghim, nh gi kt qu, cng b
kt qu (bng phiu kim nghim) nh gi cht lng thuc l t hay khng
t v c c php lu hnh (hoc s dng) hay khng.
V mt lch s, cng tc tiu chun ho gn lin vi lch s sn xut ca
loi ngi, ca cc phng thc sn xut ca tng ch x hi khc nhau.
Nhng hnh thc s khai ca tiu chun ho c t thi c i, nhng pht
trin c tnh t chc rng ri trn phm vi quc t th ch c t u th k 20.
1.2.1.2. i tng ca cng tc tiu chun ho
Bao gm rt rng, hu nh trn tt c mi lnh vc, v d:
16
Cc my mc, dng c, trang thit b cng ngh
Nguyn, nhin vt liu
Nng lm hi sn, hng tiu dng
Cc nguyn tc, phng php, th tc, cc vn t chc, qun l
Thut ng, k hiu, o lng
Sn phm v bn sn phm.
Trong ngnh Dc, mi hot ng sn xut, kinh doanh, s dng thuc,
mi vn c lin quan n cc i tng nu trn u phi tiu chun ho.
1.2.1.3. Ni dung chnh ca mt tiu chun v thuc
Tiu chun v thuc l mt tiu chun ton din, bao gm rt nhiu ni
dung th hin c mc cht lng ca thuc c to ra v duy tr cht
lng cho ti khi s dng hoc ht tui th (hn dng) ca thuc.
Ni dung chnh ca mt tiu chun v thuc (nguyn liu hay thnh
phm) thng gm cc mc sau:
Tiu : Trong nu r tn nguyn liu hoc thnh phm, tn n v
ban hnh tiu chun, loi tiu chun, hiu lc thi hnh.
Yu cu k thut: L loi tiu chun rt quan trng v n ra mc
cht lng hp l ca sn phm (mi yu cu k thut gi l mt ch
tiu hay tiu ch, l yu t cu thnh ca yu cu k thut) v l c
s cho vic lp k hoch sn xut (v s lng, cht lng), vic thc
hin k hoch, vic qun l (kinh t, k thut, gi c, k kt hp ng,
khiu ni).
Phng php th: Tiu chun ny bao gi cng phi qui nh km theo
cc ch tiu yu cu k thut. y l phn khng th thiu v n chnh
l qui trnh th nghim hay qui trnh phn tch m t chi tit ton b
qu trnh thc hnh thc hin xem mt ch tiu no ca yu cu
k thut c t hay khng t yu cu t ra.
ng gi, ghi nhn, bo qun: iu ny phi c qui nh r v tt c
cc thuc lu hnh trn th trng phi c nhn thuc n n v ng
gi nh nht. Nhn thuc phi: c ni dung cn thit gip nhn
bit c thuc, cch s dng, trnh nhm ln, tn c s sn xut, c s
ng k, s l sn xut, s kim sot, hn dng, iu kin bo qun.
1.2.1.4. Cc cp tiu chun v thuc
Trc y c 3 cp tiu chun:
Tiu chun Vit Nam (tiu chun quc gia, Dc in Vit Nam) TCVN.
Tiu chun ngnh y t Vit Nam.
17
Hai cp ny c hiu lc v phm vi p dng trong c nc.
Tiu chun c s (TC hoc TCZ): do cc c s sn xut bin son, c
hiu lc trong phm vi qui nh ca cc cp qun l.
Hin nay, ch cn 2 cp tiu chun l: Tiu chun Dc in Vit Nam v
tiu chun c c (theo quyt nh s 2412/1998/Q-BYT ngy 15/09/1998).
Tiu chun c s l tiu chun do c s sn xut bin son, p dng i
vi cc sn phm do c s sn xut ra.
C hai loi tiu chun c s:
Tiu chun c s ca nhng sn phm lu hnh th trng: phi ng
k vi c quan c thm quyn. Cc mc tiu chun cht lng khng
c thp hn cc mc quy nh trong tiu chun Dc in Vit Nam.
Tiu chun c s ca cc thuc pha ch trong n v (khng lu hnh
trn th trng): do th trng n v xt duyt v ban hnh.
1.2.2. Cng tc xy dng tiu chun
Tiu chun Vit Nam v thuc c nh nc y quyn cho B Y t t
chc bin son, xt duyt v ban hnh sau khi ng k ti cc tiu chun o
lng cht lng nh nc Vit Nam, cc tiu chun ny c tp hp trong
mt b sch gi l Dc in Vit Nam (Hi ng Dc in c B Y t giao
trch nhim ny).
Mt s thuc cha c TCVN s s dng TC do cc c s sn xut bin
son v c cp c thm quyn duyt.
1.2.2.1. Th tc xy dng tiu chun
Thng c tin hnh qua cc giai on sau:
Chun b ti liu, kho st thc t, lp cng.
Xy dng d tho tiu chun ln th nht gm cc vic: Tham kho ti
liu, xut cc ch tiu v phng php th, kho st thc t v sn
xut v s dng sn phm. Sn xut th, lm thc nghim phn tch
cc tiu chun d kin, vit d tho tiu chun v thuyt minh, lp d
tho bin php p dng (th d sa i hoc xut cc qui nh k
thut, qui ch th tc trong tin hnh sn xut theo GMP). Thng
qua d tho c s.
Hon thnh d tho tiu chun sau khi tin hnh cc vic: Ly kin
gp (cn th t chc hi tho). Thu thp gp , sa cha, hon chnh
h s gi ti c quan qun l v xt duyt.
Xt duyt v ban hnh tiu chun: C quan qun l cp tiu chun no
th xt duyt v ban hnh tiu chun . Trc khi xt duyt, tiu
chun phi c gi qua c quan thm tra k thut (Vin kim nghim
18
v phn vin kim nghim nu thuc trung ng; Trung tm kim
nghim tnh, thnh ph nu thuc a phng) v cc c quan php ch
(Cc qun l Dc trung ng v Phng nghip v dc a
phng). Th trng B Y t hoc gim c S Y t k duyt sau khi
tham kho cc c quan thm tra.
Vic xem xt v sa i tiu chun cng c tin hnh nh th tc xy
dng tiu chun.
H s tiu chun: Bao gm tt c cc vn bn khoa hc k thut, kho
st nghin cu, cng vn trnh duyt, quyt nh xt duyt.
1.2.2.2. Phng php xy dng tiu chun v yu cu k thut
Khi xy dng tiu chun v yu cu k thut cn ch ti vic kt hp
gia cc loi ch tiu phn nh ht c cht lng ca thuc ph hp vi
iu kin k thut, kinh t v thc t: Cc ch tiu phn nh v cng dng ca
thuc (th hin tinh khit v hm lng). Ch tiu phn nh v mc
tin cy v an ton ( c, bn, hn dng). Tiu chun v tm sinh l
(dng thuc - qua cc ng vo c th). Ch tiu v thm m (hnh thc ng
gi, trnh by p, bo qun).


Cc ni dung chnh ca yu cu k thut:
Thng thng cc ni dung chnh ca yu cu k thut ca mt tiu
chun v thuc bao gm:
Cng thc pha ch: Phi trnh by cng thc pha ch ca thuc, ghi r
tn nguyn liu, ph liu vi s lng ghi bng s v bng ch, cng
tiu chun ca chng (v d: t dc t tiu chun Dc dng).
Cht lng thnh phm: Phi nu r v hnh thc cng nh tnh cht
cm quan, mc tinh khit nh: Hnh dng, th cht, mu sc, mi
v, cc c im c bit, bn c hc (vi thuc vin), tan r, sai s
khi lng, sai s th tch.
Yu cu v nh tnh, th tinh khit
Yu cu v hm lng.
Phng php xy dng cc ch tiu:
Da trn cc ni dung chnh ca yu cu k thut, cn c vo thc t,
iu kin k thut, la chn ra cc ch tiu thch hp, khng qu nhiu hoc
qu t nhng ni ln c trng cht lng thuc, ph hp vi iu kin c
th. Sau phi xy dng c mc cho cc ch tiu trn. Mc ch tiu l gi
tr c th hay l khong gi tr m thuc phi t c. xy dng mc ch
tiu c tt, trc ht nn xem xt:
Nu loi ch tiu ny c sn trong cc qui nh th ch vic kim tra,
xem xt li, p dng hoc xut (th d: nu l thuc vin, khi lng
trung bnh vin qui nh sai x%; nu l thuc nc, th tch sai y%).
19
Nu loi ch tiu ny cha c trong qui nh th phi lm thc nghim,
t a ra s liu cho ph hp.
Khi xy dng mc phi c tin hnh lm th vi mu t nht ly t 3
l sn xut th hay 3 l sn phm c qu trnh sn xut n nh; tt
nht nn s dng phng php thng k (xt sai s, chnh xc,
khong tin cy).
Gii hn tin cy tm c qua th nghim , cn cn c thm vo cc
iu kin kinh t, thc t (kh nng sn xut, trnh k thut, tiu
chun dung sai chung) m qui nh mc ch tiu a vo tiu chun.
Nu sn phm ly nhiu l hoc c sn xut nhiu c s th nn
chn ch tiu trung bnh tin tin ) ( tt x
x =
x + x
max
2
(tt)

Trong :
x: ch tiu trung bnh ca nhiu c s t c
x
max
: ch tiu cao nht mt c s t c.
1.2.2.3. Phng php xy dng tiu chun v phng php th:
Cc loi qui trnh v phng php th:
Mc ch chnh ca vic tiu chun ho mt phng php th l chn cho
c mt qui trnh th hay qui trnh phn tch sao cho khi p dng s cho mt
kt qu gn vi gi tr thc nht. Trong kim nghim thuc, cc loi qui trnh
th thng c chia thnh 3 loi:
Cc php th nh tnh: l cc php th nhm chng minh rng cht
cn phn tch c mt trong mu em th. Php th c thc hin
bng cch tin hnh cc phn ng nh tnh, o cc thng s c
trng cc cht cn th (tnh cht ph UV-VIS, IR, sc k) hoc so
snh vi cht chun i chiu.
Cc php th v tinh khit: L cc php th nhm chng minh rng
mu em th t (hay khng t) v mc tinh khit. Php th c
thc hin bng cch s dng mt s tnh cht c hiu (mu sc,
c) so snh nh lng tp cht c trong mu th v mu chun
t xc nh c gii hn ca tp cht c trong mu em phn tch.
Cc php th nh lng: L nhng php th nhm xc nh hm lng
ca mu th (hoc thnh phn chnh ca mu) hay hm lng ca cc
hot cht c trong mu em th. Cc php th ny c tin hnh bng
cc phng php nh lng ho hc, ho l, vt l, sinh hc
20
Cc yu cu cht lng i vi mt phng php th:
c th hin mt s im chnh sau:
* C tnh tin tin: Th hin ng, chnh xc, tnh chn lc, c hiu.

+ ng: Mt phng php c coi l c ng cao ngha l khi p
dng s cho kt qu gn vi gi tr thc nht tc l sai s
100%

cng nh cng tt (trong x : gi tr trung bnh xc

nh c; : gi tr thc ).
Mun c gi tr thc so snh, phi c mu chun bit trc
hoc mt mu in hnh c sn xut theo mt phng php m
bo ng nht v i din cho sn phm.
ng ca phng php cng cn c biu th bng t l thu hi
100%

(: l hm lng cht chun cho vo;

x
: l hm lng xc
nh c). T l thu hi cng cao (nhng khng c vt qu 100%),
ng cng tt.
+ chnh xc: C chnh xc p ng c yu cu. chnh xc cho
bit s ph hp gia cc kt qu (hay dao ng) ca cc php xc
nh song song, thng c biu th bng lch chun S hoc
lch chun tng i RSD :
( )
1 n
x
i
x
S

=
hoc
% 100 %
X
S
RSD =

(n: s th nghim; x
i
: kt qu xc nh ln th i;
x
: kt qu trung bnh
ca n th nghim).
+ Tnh chn lc - c hiu: Phng php cho php xc nh ng cht
cn phn tch v t chu nh hng bi s c mt ca cc cht khc
trong mu th.
+ C tnh cht tuyn tnh: i vi trng hp khi xc nh kt qu phn
tch da vo ng chun. ng chun l ng biu din y = f(x).
Trong x l nng dung dch, y l i lng tn hiu o (mt
quang, din tch pic sc k, chiu cao sng cc ph) ph thuc vo x.
Tnh cht tuyn tnh c biu th bng cch tnh h s hi qui theo
phng php bnh phng ti thiu. Nu h s ny cng gn 1 th
hm y cng tuyn tnh.
21
* C tnh thc t: Phng php th a ra phi ph hp vi iu kin
thc t, c tnh kh thi cao (ph hp trang thit b, my, k thut, ho cht,
thuc th, trnh con ngi ).
* C tnh kinh t: Phng php th a ra t tn km m vn p ng
cc yu cu nu trn.
* C tnh an ton cao: An ton lao ng v bo v sc khe (t dng ho
cht c hi, trnh c cc thao tc k thut phc tp nguy him ).
Kinh nghim tin hnh xy dng phng php th:

Phi da trn yu cu k thut chn xy dng phng php th cho
thch hp.
Tham kho cc ti liu, cc tiu chun tng t c.
Kt hp vi t nghin cu, la chn.
Phi ly mu v tin hnh lm thc nghim.
Vit chng trnh d tho c km bn thuyt minh, a ra cc bng kt
qu thc nghim, x l kt qu, ti liu tham kho.
1.2.3. Cng tc p dng tiu chun trong thc t
1.2.3.1. Mc ch ngha
p dng tiu chun l mt mt cng tc ca ton b cng tc tiu chun
ho. Qua p dng :
Kim chng li cc kt qu nghin cu khi xy dng tiu chun (mc
ch tiu c ph hp khng? Phng php th ng hay sai? ).
Xc nh hiu qu kinh t ca tiu chun (tiu chun c kch thch cho
sn xut, kch thch tiu th). Trn c s tip tc sa i, hon thin
nng cao cht lng ca tiu chun.
Ngn chn vic a cc thuc khng t tiu chun ra lu hnh, s
dng. Pht hin thuc gi, thuc km cht lng, pht hin nguyn
nhn vi phm v tm cc bin php khc phc.
1.2.3.2. Cc hnh thc p dng tiu chun trong thc t
p dng tiu chun trong sn xut:
Ngha l phi thc hin ng cc tiu chun (k thut, th tc, nguyn
tc v cc qui nh c lin quan) sn xut ra thuc t tiu chun. Th d:
Phi kim tra x l nguyn vt liu t tiu chun mi a vo sn xut;
trong qu trnh sn xut phi kim tra; tt c cc cng on, 100% l sn xut
ti c s; kim tra thu nhn sn phm
22





p dng tiu chun trong kim tra cht lng:
Ngha l tin hnh thc nghim tt c cc ch tiu yu cu k thut theo
ng phng php th nu nh gi xem thuc c t tiu chun hay khng.
1.2.3.3 Cc cng vic phi thc hin
Ph bin, gii thch, cung cp tiu chun tu theo cp qun l:
Cc c quan chc nng: B Y t, S Y t chu trch nhim cung cp tiu
chun sau khi duyt.
Cc c quan k thut: Vin Kim nghim, Trung tm Kim nghim tnh,
thnh ph hng dn gii thch v thi hnh vic p dng tiu chun.
Lp k hoch cc bin php p dng tiu chun:
V t chc: Qui nh trch nhim, quyn hn ca cc cp qun l, cc
b phn thc hin. Qui nh v con ngi (phi c o to c th
p ng c vic thc hin tiu chun ).
V k thut: Sot xt li trang thit b, ho cht, cht chun, thuc
th, nh xng, phng th nghim p ng y theo yu cu
ca tiu chun ra.
Kim tra p dng tiu chun:
Cng vic ny c tin hnh thng xuyn cc c s hoc h thng
kim tra nh nc: Vin Kim nghim, Trung tm Kim nghim, thanh tra
Dc Ni dung kim tra bao gm:
Kim tra c s vt cht ca cng tc kim nghim: Ti liu, trang thit
b, phng th nghim, ho cht, thuc th
Kim tra nguyn liu, bn thnh phm, thnh phm v tt c cc
thuc ang lu hnh trn th trng.
1.2.3.4. Sa i, sot xt li tiu chun
c tin hnh khi thy tiu chun khng cn ph hp.
1.2.4. Gii thiu Dc in Vit Nam
1.2.4.1. Mt s nt chung v Dc in Vit Nam
Dc in Vit Nam l mt ti liu bao gm:
Tp hp cc tiu chun nh nc (TCVN) v thuc (ho dc v cc ch
phm, huyt thanh v vaccin, dc liu, ch phm ng dc). Mi tiu
chun cn c gi l mt chuyn lun.
23
Nhng qui nh chung trong cng tc kim nghim, gii thiu cc
phng php kim nghim chung, cc ho cht, thuc th, thuc
chun, ch th dng phn tch v nh gi cht lng thuc.
Mt s ph lc, cc bng tra cu
Dc in Vit Nam c gi tn theo ln xut bn (ging cc nc khc),
tun theo qui tc ln sau ph nhn ln trc :

Dc in Vit Nam I: gm 638 chuyn lun tn dc v 284 chuyn
lun ng dc (tp 1 in 1970, tp 1 b sung in 1977, tp 2 ng dc
in ln 1 vo 1983 ).
Dc in Vit nam II: gm 357 chuyn lun tn dc, 64 chuyn lun
ng dc, v 32 chuyn lun v vaccin (tp 1 in 1990, tp 2 in 1991 v
tp 3 in 1994).
Dc in Vit nam III: gm 342 chuyn lun ho dc, 276 chuyn
lun v dc liu, 37 chuyn lun v ch phm ng dc, 47 chuyn
lun ch phm sinh hc v 500 chuyn lun v ho cht thuc th.
(Qu trnh bin son chun b t 1995-2000, d tho in ly kin 2000,
in chnh thc 2002).
1.2.4.2. Mt s qui nh chung khi s dng Dc in Vit Nam (trong cng tc
kim nghim thuc)
C nhiu qui nh, di y nu tm tt ni dung chnh ca 10 qui nh:
1. Tn cc chuyn lun: ly tn chnh l tn Vit Nam, sau tn Vit
Nam l tn Latin v tn thng dng khc nu c.
2. n v o lng: s dng theo ng yu cu ca c quan o lng nh
nc Vit Nam.
3. Khi nim cn chnh xc ngha l cn trn cn phn tch c nhy
n 0,1 mg (0,0001 g). Khi nim ly khong c ngha l ly mt
lng vi chnh khng qu 10% so vi yu cu. Khi nim n
khi lng khng i ngha l x l ch phm n khi no sai gia 2
ln k tip nhau < 0,5 mg.
4. Nng phn trm khng c ch dn g coi l cch biu din theo %
KL/TT. Cn cc trng hp khc s ghi c th.
5. Khi nim alcol khng c ch dn g c ngha l alcol cha khong
96% (TT/TT) ethanol (C
2
H
6
O). Ethanol khng c ch dn g khc
ngha l ethanol tuyt i.
6. tan : Qui c:
Mt cht l d tan : Khi ho 1g cht di 1ml dung mi
24
Rt tan : -nt- 1 - 10
ml
-nt-
Tan : -nt- > 10-30
ml
-nt-
Hi tan : -nt- > 30-100
ml
-nt-
Kh tan : -nt- > 100-1000
ml

-nt-
Rt kh tan : -nt- >1000-10.000
ml
-nt-
Thc t khng tan : -nt- >10.000
ml
-nt-
7. V nhit : Dng thang bch phn k hiu
o
C. Khi khng ghi c
th, qui c :
Nhit chun: 20
o
C
Nhit thng: 20-30
o
C
Nc m: 40-50
o
C
Nc nng: 70-80
o
C
Nc cch thu: 98-100
o
C
Nhit trong th v mt khi lng do lm kh cho php hiu
l: 100
o
C

2
o
C.
Nhit ni bo qun :
Rt lnh: <-10
o
C
Lnh: 2-10
o
C
Mt: 10-20
o
C
Nhit phng : 20-35
o
C
Nhit phng c iu nhit : 20-25
o
C
Nng: 35-40
o
C
Rt nng: > 40
o
C
Nung : 400
o
C
thm: 600
o
C
trng: 900
o
C
8. V phng php: C th dng phng php hay phng tin khc vi
qui nh trong Dc in hoc TC nhng vi iu kin cc kt qu c
chnh xc tng ng nhau. Nu cc kt qu khc nhau th coi
phng php hoc phng tin qui nh trong Dc in hay TC l
chnh thc.
9. V hm lng: Nu trong chuyn lun khng ghi gii hn trn th c
ngha l khng c qu 101,0%.
25
10. Khi th tinh khit, nu pht hin thy tp cht l khng ghi trong
chuyn lun th vn phi ghi vo kt qu th.
1.3. Kim nghim thuc theo tiu chun
Nh trn nu, kim nghim thuc l vic tin hnh phn tch mt mu
thuc i din cho l thuc bng cc phng php ho hc, l hc, ho l,
sinh hc c qui nh xem thuc t hay khng t tiu chun t
quyt nh xem c c php lu hnh hoc s dng hay khng. s nh
gi ny chnh xc, i hi phi lm tt 3 vic sau: ly mu kim nghim, thc
hnh phn tch, nh gi kt qu v vit phiu tr li (phiu kim nghim,
phiu phn tch).
1.3.1. Ly mu kim nghim
1.3.1.1. Mt s khi nim
L thuc: L mt lng thuc xc nh ca cng mt loi sn phm
c sn xut trong mt chu k nht nh p ng yu cu GMP,
c coi l ng nht v c ghi bng s l ca c s sn xut trn
nhn cc bao b.
Tng th: L ton b cc n v sn phm c xt. Tu theo tng
trng hp tng th c th l mt l, mt s l hay mt qu trnh
sn xut.
n v bao gi: L dng bao gi sn phm lp li trong l (thng,
hm, hp).
n v ng gi: L dng c ng gi trc tip sn phm (chai ng
thuc vin, v thuc ).
n v sn phm: L i tng qui c hoc c th ca mt lng sn
phm nht nh (vin thuc, ng thuc, 1g, 1kg ).
Mu: L mt s n v sn phm ly t tng th th v c dng
lm c s c nhng thng tin quyt nh v tng th . S n v
sn phm c trong l gi l c l, s n v c trong mu gi l c mu.
Mu ban u: L mt lng sn phm ca l thuc c ly trong
mt ln mt hay nhiu n v bao gi. Mi bao gi ly mt ln.
Mu ring: L mt lng sn phm c ly t nhng mu ban u
c gp li v trn u ca mt bao gi.
Mu chung: L mt lng sn phm c ly t nhng mu ring ca
tng n v bao gi gp li v trn u.
Mu trung bnh th nghim: L mt lng sn phm c ly ra t
mu chung dng tin hnh cc php th qui nh (k c lm li).
26
Mu lu: c ly t mu trung bnh th nghim hay t mu ban u
tng ng vi lng mu th. Mu lu dng lu li khi cn
thit hoc lm cc th nghim trng ti.
1.3.1.2. Qui nh v ly mu
Ly mu l mt tp hp cc thao tc nhm ly ra mt lng mu thuc
i din kim tra cht lng. Do vy kt lun v mu thuc mang tnh
php l, cn phi tun th mt cch nghim ngt cc qui nh v th tc ly
mu nh sau:
i tng ly mu:


Vi h thng t kim tra: L cc nguyn liu dng lm thuc, bao b
ng gi, sn phm trung gian, sn phm cha ng gi, thnh phm.
Vi h thng qun l nh nc: Thuc v cc nguyn liu lm thuc
ang trong qu trnh lu thng hoc tn tr trong kho.
Cc trng hp ly mu:
Trng hp t kim tra cht lng: Phi ly mu kim tra ton b cc
l thuc ti cc c s sn xut, lu tr, phn phi. Vi cc c s sn
xut thuc, yu cu 100% s l phi c kim tra. Vic ly mu do cn
b chuyn mn ca phng kim tra cht lng sn phm (KCS) tin
hnh, c s chng kin ca cn b n v ly mu. Th trng n v
cn c vo qui nh chung c th c nhng qui nh c th hn cho ph
hp vi tnh hnh ca c s.
Trng hp kim tra gim st cht lng hoc thanh tra: u tin ly
mu kim tra v gim st l cc thuc cha bnh, c gi tr kinh t cao,
c cht lng khng n nh v c bit l c nghi ng v hm lng
hoc hiu lc tc dng.
Ly mu kim tra gim st cht lng ca cc c s sn xut (ly 10%
s l sn xut trong nm) hoc ly theo qui nh ca B Y t, S Y t.
Ly mu thanh tra t xut trong nhng trng hp c thng tin v
cht lng thuc xu, thuc khng an ton, t hiu lc v c bit l thuc gi
hay thuc km phm cht.
Vic ly mu c thc hin bi cc thanh tra vin hoc cc cn b c giy
u nhim ca c quan kim tra, thanh tra v c s chng kin ca cn b c s.
Cc iu kin cn lu khi ly mu:
Ni ly mu: Ti ni cha sn phm, mi trng xung quanh khng
c gy nhim bn hoc tc ng lm thay i tnh cht ca mu v
ngc li khng mu tc ng xu n mi trng.
Ngi ly mu: Phi l ngi c chuyn mn nht nh v p ng
c yu cu ca qu trnh ly mu.
27
Phi quan st kim tra s b l hng (phn loi nu cn), nhn xt v
phi ghi vo bin bn ly mu.
Dng c ly mu: Sch, kh, p ng yu cu cn ly mu.
ng mu: p ng yu cu ly mu (sch, khng lm hng mu,
kh, c nhn ghi chp y ).
Thao tc ly mu: Phi thn trng, t m, quan st cn thn
Phng thc ly mu: Ngi ly mu phi t tay ly mu, ghi nhn,
lm bin bn, ng gi nim phong bo m v bo qun mu. c bit
lu phi ly ch k xc nhn ca n v c ly mu.
1.3.1.3. Tin hnh ly mu
S ly mu:
Vic ly mu phi bo m cho c tnh khch quan, i din cho c
cht lng ca thuc cn ly kim tra. V vy, phi ly theo hng dn ca qui
trnh (hnh 1.1).
L thuc
Mu ring
Mu chung
Mu lu ti b
phn kim nghim
Mu lm
kim nghim
Mu trung bnh
th nghim
Mu gi
(hoc ly) kim nghim
Mu lu ti
c s
Mu lu
thanh tra


Hnh 1.1. S qui trnh ly mu, lu mu
28
- T l sn xut ly ra cc n v bao gi mt cch ngu nhin, c mu
ban u ly theo ch dn phn sau.
- Trn u cc mu ban u v gp thnh nhng mu ring ca tng n
v bao gi.
- Trn u cc mu ring thnh mu chung.
- T mu chung ly ra mt lng mu trung bnh th nghim.
- T mu trung bnh th nghim ly ra thnh cc mu lu v mu th
kim nghim
Sau khi ly mu xong, ngi ly mu t tay dn nhn nim phong, bao gi
(phi c ch k xc nhn ca ngi ly mu v c s c ly mu) v bin
bn ly mu (cng phi c ch k xc nhn).
Ly mu c th:
Cn c vo l thuc phi ly mu, xem xt phn loi tin hnh ly nh sau:
Ly mu thuc c phn liu (l sn phm thuc dng thuc c phn liu).
T l sn phm ly ra cc n v bao gi mt cch ngu nhin bt k: Cc
gi c ly ra phi c lp vi d kin ca ngi ly mu (khng nn la
chn theo cm quan ly mu xu hay mu tt). L thuc phi ng nht, hp
l v s lng hay khi lng (Th d khng qu 500.000 vin vi thuc vin,
khng qu 50.000 ng vi dng ng ).
S bao gi trong l ly ra to mu ban u tnh theo cng thc :
N n = 4 , 0
Trong :
n: s bao gi ly ra
N: s n v bao gi cui cng trong l (th d vi thuc tim: cc ng
ng trong hp giy, cc hp giy ng trong hm th n v bao gi cui cng
l hm).
Ch :
+ Khi tnh theo cng thc trn, nu phn thp phn nh hn 0,5 b qua,
nu ln hn 0,5 th tng thm mt n v.
+ Khi N > 100 lu n
max
30
+ Khi N < 100 c th dng bng:
N n
1 -10
11 - 40
41 - 80
81 - 100
1
2
3
4
29
Ly mu sn phm l cht rn (ht, bt, vin):
1. Trng hp mt bao gi:

+ Trc khi ly, xem xt sn phm c ng nht khng, nu khng ng
nht phi chn ring ra tng loi v ly theo cc loi .
+ Trng hp sn phm l ht, cc, tr trng hp phi xc nh c ht,
cn tt c phi c nghin nh thnh bt (khng c lm nh
hng ti tnh cht ca sn phm).
+ Ly mu ban u 3 v tr khc nhau: trn, gia, di sau trn
thnh mu chung.
+ Dn u lng mu chung thnh lp phng hnh vung dy khng
qu 2 cm, chia mu thnh 2 ng cho, b 2 phn i din, trn u
2 phn cn li v chia tip cho n khi lng mu cn li tng ng
vi 2-4 ln mu th cn ly, l mu trung bnh th nghim.
+ Chia mu trung bnh th nghim thnh cc mu lu v mu kim
nghim.



B
B
2. i vi thuc vin ch ng trong mt bao gi: cng ly mu ba v tr
khc nhau trong bao gi, chai, l sau trn thnh mu chung, mu trung
bnh th nghim nh trn.
3. Trng hp sn phm ng thnh nhiu bao gi: ly mu ban u theo
cng thc: N n = 4 , 0 nu trn.
Ly mu l sn phm lng:
1. Trng hp mt bao gi: Nu sn phm l ng nht th ly mu bt
k v tr no cng c. Nu khng ng nht, trc khi ly mu phi khuy
u, sau mi ly mu.
2. Trng hp nhiu bao gi : ly theo cng thc : N n = 4 , 0
Nu l nhng chai l nh th c th ly ht th tch.
Ly mu l cc sn phm thuc m, bt nho:
Tin hnh ly mu nh cc sn phm lng, rn nhng ch khuy k,
trn u c hn hp ng nht, sau mi ly mu.

Ly mu thnh phm cha ng gi l:
Thng cc thnh phm ny cha trong cc bao gi ln chuyn ch
n c s ng gi l. Nu l sn phm ch c 1 - 2 bao gi th m c 2 bao gi;
30
nu l sn phm c t 3 bao gi tr ln th m 3 bao gi. Ly t nht 3 mu
ban u 3 v tr khc nhau ca mi bao gi. Sau trn cc mu ban u li
vi nhau to mu th nghim.
Bao gi v dn nhn:
Sau khi ly mu v cho vo ng, ngi ly mu bao gi, dn nhn v
nim phong mu, lm bin bn ly mu. Lu phi c ch k xc nhn ca c
s c ly mu nhn nim phong v bin bn ly mu.
1.3.2. Tin hnh kim nghim
1.3.2.1. Nhn mu
B phn nhn mu ca c quan kim nghim phi kim tra xem mu c
p ng cc yu cu sau khng:
Mu phi c ly theo ng cc th tc qui nh trn.
Mu phi c ng gi nim phong v c nhn ghi cc thng tin
cn thit (nhn gc, tn thuc, s l sn xut, tn tiu chun yu cu
kim tra ).
Cc mu do thanh tra ly v phi c km bin bn ly mu.
Cc mu gi phi km cng vn hoc giy gii thiu.
Nu mu xin php sn xut phi km cc ti liu theo qui nh thuc
xin ng k sn xut.
Nu mu nhn qua ng bu in, phi kim tra k nim phong sau
bo li cho ni gi mu, ch sau khi nhn c kin tr li ca ni
gi mu mi tin hnh kim nghim.
1.3.2.2. Kim nghim, x l kt qu
Cng vic ny do b phn k thut thc hin. Thng thng gm cc ni
dung sau :
Chun b ti liu: theo TCVN hoc TC
Chun b dng c, ho cht, my p ng yu cu m tiu chun
qui nh. B tr th nghim mt cch hp l c mu lm v khng
lm nhim bn hoc bin cht mu cn th.
Tin hnh cc th nghim phn tch theo tiu chun.
Ngi lm kim nghim phi c cun s ghi chp y cc s liu khi
tin hnh th nghim, s ny gi l S tay kim nghim vin. S tay
kim nghim vin c coi l chng t gc ca cc s liu sau ny cng
b trn phiu tr li kt qu kim nghim (gi l phiu kim nghim).
X l cc s liu thc nghim quyt nh xem cc ch tiu th
theo tiu chun t hay khng t yu cu.
31
1.3.2.3. Vit phiu tr li kt qu
Bng phiu kim nghim hay phiu phn tch.
Phiu kim nghim l vn bn php l ca cc t chc kim tra cht
lng thuc, xc nhn kt qu kim nghim theo ti liu k thut hp php
ca mt mu thuc.
Phiu phn tch l vn bn php l xc nhn kt qu phn tch ca mt
hay nhiu tiu ch trong tiu chun k thut ca mt mu thuc.
Do vy, sau khi hon thnh cc th nghim v x l s liu nh gi kt
qu, kim nghim vin phi vit vo phiu tr li ni b (cha phi phiu
chnh thc), k tn chu trch nhim v a cho cn b ph trch phng duyt
li, trc khi a phng chc nng trnh lnh o duyt ln cui, sau tr
li chnh thc bng phiu ca c quan kim nghim (gi l phiu kim nghim
hay phiu phn tch). Phiu kim nghim ch cn c ch k v con du ca
gim c c quan kim nghim hoc n v.
Cu ch vit trong phiu kim nghim phi r rng, chnh xc, gn, y
v thng nht. Ni dung chnh ca mt phiu kim nghim phi c: Phn
tiu (bao gm tn c quan kim nghim, s phiu kim nghim, tn mu
kim nghim, l lch mu kim nghim), cc ch tiu th v kt qu, kt
lun cui cng v mu thuc kim nghim Di y l mu thng dng ca
mt phiu kim nghim (xem trang sau).
1.3.2.4. Lu mu kim nghim ti c quan kim nghim
Mu lu phi c nh s cng vi s ng k mu th cng loi,
nhng c nhn ring vi ch mu lu v bo qun trong iu kin theo qui
nh chung, mu ny c s dng n trong trng hp c tranh chp v kt
qu cng b ( phiu kim nghim). Thng thng, mu lu ly t mt
phn mu ly th, do vy s lng ly phi ging nh mu ly th.










32
B Y t
Tn c quan
kim nghim thuc

a ch c quan
Cng ho x hi ch ngha Vit Nam
c lp - T do - Hnh phc

Phiu kim nghim
Mu kim nghim: Vin nang Cephalexin 500mg
Ni sn xut: Cng ty Dc phm X
S l, hn dng:. S ng k:.
Ngi v ni gi mu:
Yu cu kim nghim (ghi r ni dung, s, ngy, thng, nm ca cng
vn hay giy t km theo):
Ngy thng, nm nhn mu: S ng k KN:
Ngi nhn mu:.
Th theo; Dc in Vit Nam III
Tnh trng mu khi nhn v m nim phong kim nghim:
........................................................................................................................
Yu cu Kt qu
1. Tnh cht: Nang nhn bng, khng mo m, bt thuc bn trong
ng nht.
2. nh tnh: Ch phm phi c phn ng c trng ca cephalexin
3. Nc: Khng c qa 10%
4. ng u khi lng: Khi lng trung bnh vin 7,5%
5. ho tan: Khng t hn 80% cephalexin (C
16
H
17
O
4
S) ghi trn
nhn c ho tan trong 45 pht.
6. nh lng: Hm lng cephalexin khan (C
16
H
17
O
4
S) t 92,5110%
so vi lng ghi trn nhn.
t

ng
t (6,75%)
t
t (86,5%)

t (98,6%)
Kt lun: Mu th ny t yu cu kim tra cht lng theo tiu chun
Dc in Vit Nam III.
H Ni, ngy thng . nm ..
Th trng c quan
(K v ng du)
33
Cc mu lu phi c gi li theo ng thi gian qui nh. Cc mu c
hn dng phi lu tip 3 thng k t khi ht hn dng. Khi ht thi gian lu,
c quan lp bin bn x l theo qui ch.
S sch, phiu kim nghim phi lu gi t nht l 3 nm. Khi ht hn
lu, mun hu phi c gim c c quan duyt.
1.3.3. Ni dung chnh ca thc hnh tt phng kim nghim (GLP)
Mc ch c bn ca GLP l nhm xy dng c mt n v lm cng
tc kim nghim p ng y mi yu cu ca cng tc kim tra cht lng
thuc ra, m bo rng kt qu cc php phn tch thu c l c tnh
chn lc cao, chnh xc v ng n, c tnh php l. ng thi gip cho vic
tra cu v tm c nhanh chng ngun gc ca cc sai st xy ra khi gp
phi. V vy, GLP l nhng qui nh nhm m bo thuc c cht lng tt
trong qu trnh sn xut, tn tr ti kho v lu thng phn phi n tay ngi
s dng. C th l nhng qui nh cht ch v c chun ho cho mt c s
kim nghim phi tun th v mi mt: nhn s, t chc, c s vt cht, trang
thit b, ho cht thuc th, qui trnh th nghim, cc iu khon kim tra,
bo co kt qu, lu gi s liu
1.3.3.1. V t chc v nhn s
Mt phng kim nghim thuc phi c cc b phn chuyn mn, hu
cn, ng k v lu tr mu ... c chc nng nhim v r rng, c
xy dng v c ngi c thm quyn ban hnh.
Cc nhn vin phi c o to v hun luyn v chuyn mn c th
thc hin c cc nhim v c giao c tinh thn trch nhim cao.
Cc nhn vin phi mc trang phc theo qui nh, phi c kim tra
sc kho, khng lm nhim bn vo cc php th v cc dng c, h
thng th nghim...
1.3.3.2. V c s vt cht chung
Phi c din tch lm vic ph hp vi yu cu t ra: C din tch,
c qui m,kch thc xy dng thch hp cho php sp xp trt t, hp
l cc dng c, trang thit b. C mi trng tt v: chiu sng, thng
gi, nhit , m
Phi m bo tnh bit lp: c s th nghim phi khng b nhim
bn do mi trng hoc cc b phn ln cn khc, bo m v sinh
theo qui nh.
Bo m cng tc an ton, cc phng tin bo h lao ng, s dng v
bo qun cht c hi, x l cht thi ...
34
1.3.3.3. Trang thit b
Phi c trang b y cc trang thit b p ng thch hp cc
yu cu th nghim. Cc trang thit b ny cn c la chn, chun
ho, c cht lng.
Thit b phi t v tr thch hp tin cho s dng, vn hnh,kim tra,
v sinh, bo dng.
nh k phi c kim tra, chun ho, bo dng.
Phi c ni qui vn hnh, hng dn s dng, c c nhn chu trch nhim.
1.3.3.4. C s vt cht cho cc php th
Tt c cc php th phi c qui trnh c vit chi tit v c chun
ho.Mi ho cht, thuc th, cht chun phi p ng ng cc yu
cu qui nh.
Thuc th, ho cht, cc dung dch b bin cht hay qu hn
khng c php s dng.
1.3.3.5. Qui nh v nguyn tc, c s cho cc php th v nh tnh, th
tinh khit, nh lng v hm lng, hay hot lc ca thuc, bn vng ca
thuc, qui nh v mu th, thuc th v cht i chiu
1.3.3.6. Qui nh v qui trnh v hng dn th nghim
Qui trnh v hng dn th nghim phi r rng, chnh xc, c
chun ho v c lnh o duyt thng qua.
Kim nghim vin tin hnh th nghim phi bo co mt cch chi tit
nhng hin tng gp phi trong qu trnh th nghim vi ph trch.
1.3.3.7. Qui trnh v bo co kt qu
Phi bo co y cho thy r : mc ch ca php th nghim, ni
th nghim, tn mu th, ngy th nghim, phng php p dng th,
ngi th nghim, cc phng php thng k p dng phn tch s liu, kt
qu th nghim v bn lun, kt lun, k xc nhn, ni lu tr mu lu, h
s, ti liu
35
Ti liu tham kho
1. B Y t (2002). Dc in Vit Nam III. NXB Y hc. H Ni.
2. B Y t (2002). Cc vn bn qun l nh nc trong lnh vc Dc.
NXB Y hc. H Ni.
3. ng Vn Ho (2001). Gio trnh kim nghim thuc. i hc Y Dc
Thnh ph H Ch Minh.
4. Lin hip cc x nghip Dc Vit Nam (1990). Hng dn thc hnh
sn xut thuc tt (sch dch). NXB Ngoi Vn. H Ni.
Cu hi t lng gi
1.1. Mc tiu ca cng tc kim tra cht lng thuc? Cc yu cu c
bn t mc tiu trn ?
1.2. Ni dung chnh ca cng tc kim tra cht lng thuc? iu kin
thuc c a vo lu thng, phn phi, s dng? Cc yu t
nh hng n cht lng thuc ?
1.3. Th no l thuc t v khng t tiu chun? Thuc gi mo?
Thuc km phm cht?
1.4. Trnh by h thng t chc kim tra cht lng thuc ca Vit Nam?
1.5. Ni dung chnh ca cng tc tiu chun ho? Ni dung chnh ca
mt tiu chun v thuc?
1.6. Trnh by phng php xy dng tiu chun v yu cu k thut?
v phng php th? Yu cu cht lng i vi mt phng
php th ?
1.7. Ni dung chnh ca cng tc p dng tiu chun trong thc t?
1.8. Trnh by mt s qui nh chung khi s dng Dc in Vit Nam
(dng trong cng tc kim nghim thuc)?
1.9. Cch tin hnh ly mu kim nghim?
1.10. Trnh by cc nhim v ch yu khi tin hnh kim nghim?
1.11. Ni dung chnh ca thc hnh tt phng kim nghim thuc?


36
Chng 2
kim nghim thuc bng cc phng php ha hc
Mc tiu hc tp
3. Trnh by c cch nh tnh v xc nh gii hn tp cht trong thuc.
4. Gii thch c k thut nh lng cc acid, base v cc loi mui
trong mi trng khan.
5. Trnh by c cch s dng thuc th Karl Fischer xc nh hm
lng nc trong cc mu phn tch rn, trong dung mi hu c.
6. Vit c phng trnh phn ng nh lng mt s cht hu c
(polyol, v amino alcol) bng thuc th periodat.
7. Phn tch c ng dng ca cp ion trong kim nghim thuc.
2.1. Cc phn ng nh tnh
2.1.1. Acetat
Phn ng vi cc acid (mnh hn acid acetic) gii phng acid acetic c
mi chua:




CH
3
COO

+ H
+
= CH
3
COOH
Vi dung dch FeCl
3
long cho phc mu [Fe
3
(OH)
2
(CH
3
COO)
6
]
+
. Khi
pha long v un si s cho kt ta mu :
[Fe
3
(OH)
2
(CH
3
COO)
6
]
+
+ 4H
2
O = 3Fe(OH)
2
CH
3
COO + 3CH
3
COOH + H
+
Vi H
2
SO
4
c v C
2
H
5
OH to ra este etyl acetat c mi thm:
O
2
H
5
H
2
COOC
3
CH
dc SO H
5
H
2
OHC COOH
3
CH
4 2
+ +
2.1.2. Amoni (mui)
B phn hu khi un nng vi dung dch NaOH, gii phng kh NH
3
:
NH
4
+
OH
-
= NH
3
H
2
O + +

Nhn bit NH
3
bng cc du hiu: Mi khai, lm xanh giy qu tm
t, lm hng giy tm phenolphtalein.
37
Thuc th Nessler (dung dch kim ca mui Kaliiodomercurat K
2
[HgI
4
]
phn ng vi NH
3
cho ta mu (lng nh cho dung dch mu vng):

NH
4
OH
-
= NH
3
H
2
O +
+
+


[ ] = + + 3KOH
4
HgI
2
2K
3
NH I

+ 7KI + 2H
2
O
NH O

2.1.3. Arseniat






Phn ng vi acid hypophosphor hoc dung dch hypophosphit (thuc th
Bugo hay Tile): to ra kt ta As nguyn t c mu nu:
+
+ + 6H PO 5H 4AsO
2 3
3
4
4As nu + 5H
3
PO
4
+ 3H
2
O
Phn ng vi AgNO
3
: to kt ta nu Ag
3
AsO
4
, ta ny khng tan trong
CH
3
COOH, tan trong HNO
3
, dung dch amoniac.
Phn ng vi hn hp magnesi (MgCl
2
+ NH
4
OH + NH
4
Cl ): Cho kt ta
tinh th trng magnesi amonarseniat :
AsO
4
3
+ Mg
2+
+ NH
4
+
= MgNH
4
AsO
4

trng
2.1.4. Arsenit
Phn ng Tile:
4AsO
3
3
+ 3H
3
PO
2
+ 12H
+
= 4As

nu
+ 3H
3
PO
4
+ 6H
2
O
Phn ng vi AgNO
3
: to ra kt ta trng hi vng Ag
3
AsO
3
, ta ny tan
trong HNO
3
, trong dung dch amoniac.
Phn ng kh bng hydro mi sinh (do Zn trong mi trng acid to ra) :
cc hp cht ca Arsen (c AsO
3
3
v AsO
4
3
) u thnh AsH
3
dng kh:
AsO
3
3
+ 3Zn + 9H
+
= AsH
3
+ 3Zn
2+
+ 3H
2
O
C th nhn ra AsH
3
bng:
Cho tc dng vi AgNO
3
:
AsH
3
+ 6AgNO
3
3HNO
3
+ AsAg
3
.3AgNO
3
(mu vng)
Hg
2
Hg
38
Sn phm mu vng ny d b thu phn to ra Ag en:
AsAg
3
.3AsNO
3
+ H
2
O H
3
AsO
3
+ 3HNO
3
+ 6Ag en
Cho tc dng vi HgCl
2
: to thnh hp cht c mu t vng sang nu:
AsH
2
(HgCl); AsH(HgCl)
2
; As(HgCl)
3
; As
2
Hg
3
Phn ng vi CuSO
4
: cho ta ng hydroarsenit mu xanh lc:
H
3
AsO
3
+ CuSO
4
= CuHAsO
3

xanh lc
+ H
2
SO
4
Nu thm NaOH v un nng s c kt ta mu ca Cu
2
O:
2CuHAsO
3
+ 6NaOH = Na
3
AsO
4
+ Cu
2
O

+ 4H
2
O + NaAsO
3
(Phn ng ny dng phn bit gia AsO
4
3-
v AsO
3
3-
).




2.1.5. Bc (mui)
Phn ng vi HCl cho ta trng AgCl, ta khng tan trong HNO
3
nhng
tan trong dung dch amoniac:
Ag
+
+ Cl

AgCl trng
AgCl + 2NH
3
Ag(NH
3
)
2
+
+ Cl

Phn ng vi formol trong mi trng kim b kh thnh Ag c mu en

(phn ng trng gng):
Ag
+
+ NH
4
OH Ag
2
O Ag(NH
3
)
2
+
+ Ag + HCOOH
HCHO
2.1.6. Barbiturat
Khi un nng vi kim c, vng ureid b m gii phng ra cc sn phm
khc nhau:
O C
NH
NH
CO
CO
C
R
1
R
2
NaOH
H
2
O +
O C
NH
2
NH
2
C
COONa
R
2
COONa
R
1
+

O C
NH
2
NH
2
t
o
+ H
2
O
NH
3
2 CO
2
+

To phn ng phc c mu vi cc ion kim loi nh Cu
2+
, Co
2+

39
2.1.7. Bari (mui)
Phn ng t cho mu ngn la xanh lc hi vng.


Phn ng vi H
2
SO
4
cho kt ta BaSO
4
mu trng, khng tan trong cc acid
v c.
2.1.8. Bismuth (mui)
Phn ng thu phn:
Bi
3+
Cl
-
H
2
O BiOCl trng H
+
2 + + +

Tc dng vi dung dch kim: cho ta Bi(OH)
3
mu trng, khi un nng
vi nc ng mu vng:

OH
-
Bi
3+
H
2
O + + = Bi(OH)
3
trng (BiO)OH vng

Phn ng vi S
2-
: cho ta nu en Bi
2
S
3



Phn ng vi thioure: trong mi trng acid cho mu vng da cam hay
vng xanh l cy (nhiu cho kt ta):
[Bi (H
2
N CS NH
2
)
3
](NO
3
)
2
: vng da cam
[Bi (H
2
N CS NH
2
)
3
]Cl
3
: vng xanh l cy
2.1.9. Borat
Hn hp mui borat vi ethanol (hoc methanol) v H
2
SO
4
c s to ra
ester trietyl borat, em t chy cho ngn la mu lc:

Na
2
B
4
O
7
. H
2
O H
+
Na
+
H
3
BO
3
H
2
O 6 +2 2 + 4 +5

H
3
BO
3
C
2
H
5
OH B
OC
2
H
5
OC
2
H
5
OC
2
H
5
H
2
O
+ + 2
H
+
3

Trong mi trng acid, borat chuyn thnh acid boric, acid boric phn ng
vi giy ngh (hoc cn ngh) cho mu nu , sau tm t bng dung
dch kim long (amoniac hoc natrihydroxyd) mu nu chuyn thnh
mu lam hay lc (do s to phc ca cucumin trong ngh vi acid boric).

40
2.1.10. Bromid
Phn ng vi AgNO
3
: cho ta vng nht AgBr, ta ny kh tan trong dung
dch amoniac 10M.



Phn ng oxy ho Br
-
thnh Br
2
bng: PbO
2
+ CH
3
COOH hoc KMnO
4
+
H
2
SO
4
. Nhn bit Br
2
bng cch chit vo cloroform c mu vng hoc
nu:
2Br

+ PbO
2
+ 4H
+
= Br
2
+ Pb
2+
+ 2H
2
O
10Br

+ 2MnO
4

+ 16H
+
= 5Br
2
+ 2Mn
2+
+ 8H
2
O
2.1.11. Calci (mui)
Phn ng vi amoni oxalat: trong mi trng trung tnh hoc CH
3
COOH
long cho kt ta mu trng, kt ta ny d tan trong cc acid v c :
Ca (NH
4
)
2
C
2
O
4
= CaC
2
O
4
trng NH
4
+
2+
+ 2
+

Phn ng vi Kali ferocyanid: trong mi trng NH
4
Cl cho ta mu trng:
Ca K
4
[(Fe(CN))
6
] NH
4

2+
2
+
+
+ = Ca(NH
4
)
2
[Fe(CN)
6
] trng
4K
+
+


2.1.12. Ch (mui)
Phn ng vi dung dch KI: cho kt ta mu vng, tan trong KI tha:
Pb
2+
+ 2I
-
= PbI vng
2

+ 2I
-
= PbI
2
PbI
4
2-

Ta PbI
2
tan trong nc nng, khi ngui kt ta tr li.
Phn ng vi dung dch K
2
CrO
4
cho ta mu vng, ta d tan trong HCl
v NaOH:


Pb
2+
+ K
2
CrO
4
=

PbCrO
4
vng + 2K
+
2.1.13 Citrat (C
6
H
5
O
7
3
)
Cho ta vi Ca
++
khi un nng. Ta ny d tan trong dung dch CH
3
COOH6M.
41
Phn ng to thnh acid acetondicarboxylic: khi un nng vi H
2
SO
4
c
(hay dung dch KMnO
4
), acid citric s b oxi ho thnh acid
acetondicarbonic CO(CH
2
COOH)
2
. Acid ny to ta vi mui Hg
2+
.




2.1.14. Clorat
Khng kt ta vi dung dch AgNO
3
.
un nng vi dung dch HCl, H
2
SO
4
long s b phn hu thnh Cl
2
bay ra:
3HClO
3
= HClO
4
+ Cl
2
+ 2O
2
+ H
2
O
Tc dng vi NaNO
2
: kh thnh Cl

:
ClO
3

+ 3NO
2

= Cl

+ 3NO
3

2.1.15. Clorid
Phn ng vi AgNO
3
: cho kt ta AgCl mu trng, ta ny tan trong dung
dch amoniac v kt ta tr li trong HNO
3

+ AgCl NH
3
2
Ag(NH
3
)
2
Cl
=
+
AgCl
HNO
3

Phn ng vi KMnO
4
trong mi trng acid: mt mu KMnO
4
:



2MnO
4

+ 10Cl

+ 17H
+
2Mn
2+
+ 5Cl
2
+ 8H
2
O
Nhn bit Cl
2
do c mi c bit hoc lm xanh giy tm h tinh bt c KI:
Cl
2
+ 2I

2Cl

+ I
2
(I
2
lm xanh h tinh bt)
2.1.16. ng (mui)
Phn ng vi K
4
[Fe(CN)
6
] Cho ta mu nu khng tan trong acid acetic:
Cu
2+
+ K
4
[Fe(CN)
6
] = CuK
2
[Fe(CN)
6
]
nu
+ 2K
+
Phn ng vi dung dch amoniac: cho ta mui base mu xanh Cu
2
(OH)
2
2+
,
mui ny tan trong amoniac d thnh phc mu xanh Cu(NH
3
)
4
2+

2CuSO
4
+ 2NH
4
OH = (NH
4
)
2
SO
4
+ Cu
2
(OH)
2
SO
4

Cu
2
(OH)
2
SO
4
+ (NH
4
)
2
SO
4
+ 6NH
3
= Cu(NH
3
)
4
2+

+ 2SO
4
2
+ 2H
2
O
2.1.17. Ethanol
Tc dng vi acid acetic (mi trng H
2
SO
4
) to ra ethyl acetat c mi thm:
42
C
2
H
5
OH + CH
3
COOH C
2
H
5
COOCH
3
+ H
2
O
Tc dng vi dung dch I
2
trong mi trng kim to ta mu vng
iodoform (CHI
3
) c mi c bit:
CH
3
CH
2
OH + 4I
2
+ 6NaOH = CHI
3
+ 5NaI + HCOONa + 5H
2
O





2.1.18. Iodid
Phn ng vi AgNO
3
cho ta mu vng AgI, ta ny khng tan trong amoniac.
Phn ng vi Fe
3+
:
2I

+ 2Fe
3+
= I
2
+ 2Fe
2
+
I
2
gii phng ra c th chit vo lp cloroform c mu tm .
2.1.19. Kali (mui )
Phn ng mu ngn la: mui Kali t cho ngn la mu tm (vch quang
ph c = 768 nm v 404 nm ).
Phn ng vi natri hexanitrocobantat : Cho kt ta mu vng (trong mi
trng CH
3
COOH long):
K
+

2
Na
3
[Co(NO
2
)
6
] + =
K
2
Na[Co(NO
2
)
6
] vng Na
+
+

2.1.20. Km ( mui )
Tc dng vi dung dch NaOH cho ta trng Zn(OH)
2
, ta ny tan trong
kim d thnh mui Zincat, khi thm Na
2
S s cho kt ta trng ZnS:
Zn
2+
+ 2OH

= Zn(OH)
2
trng
Zn(OH)
2
+ 2OH = ZnO
2
2
+ 2H
2
O
ZnO
2
2
+ Na
2
S + 2H
2
O = ZnS trng + 2NaOH + 2H
2
O

2.1.21. Magnesi (mui)

Phn ng vi dinatrihydrophosphat trong mi trng (NH
4
Cl + NH
4
OH)
cho kt ta mu trng Magnesi amoniphosphat, soi trn knh hin vi c hnh
l dng x:
Mg
2+
HPO
4
2-
+ NH
4
+
+ + OH
-
H
2
O
5 + = MgNH
4
PO
4
. H
2
O trng 6

43
2.1.22. Natri (mui)
Phn ng mu ngn la: Mui Natri t cho ngn la mu vng (vch
quang ph c = 589 nm).

Phn ng vi Kalidihydro antimonat: cho kt ta mu trng (trong mi
trng trung tnh hoc acid nh).
Na
+
KH
2
SbO
4
= NaH
2
SbO
4
trng K
+
+
+

Phn ng vi km uranyl acetat : cho kt ta mu vng natri km uranyl
acetat (trong mi trng CH
3
COOH long) :
(UO
2
)(CH
3
COO)
2
. H
2
O
2 3 +
Zn(CH
3
COO)
2
CH
3
COOH Na
+
+ +
=
H
+
NaZn(UO
2
)
3
(CH
3
COO)
9
. H
2
O vng
+ 6

(C th dng mui magnesi cng c).

2.1.23. Nhm (mui)
Vi thuc th alizarin S to hp cht ni phc c mu .
2.1.24. Nitrat
Phn ng vi FeSO
4
+ H
2
SO
4
c: to ra NO, Fe
2+
d s kt hp vi NO to
thnh st (II) nitrososulfat c mu nu:
FeSO
4
NO
3
H
2
SO
4
H
+
= Fe
2
(SO
4
)
3
H
2
O [FeNO]SO
4
2 3 2 3 4 2 8
+ + + + +
-


Phn ng vi nitrobenzen: trong mi trng H
2
SO
4
c to ra m-
dinitrobenzen, hp cht ny phn ng vi aceton trong mi trng kim
to thnh phc c mu tm (Phn ng Janovsky):
NO
2
+
NO
2
NO
2
NO
3
-
H
2
SO
4


NO
2
NO
2
+
NO
2
NO
2
CH
2
COCH
3
H
CH
3
COCH
3
NaOH
Na
+

44
2.1.25. Oxalat
Phn ng vi CaCl
2
cho kt ta trng CaC
2
O
4
, ta ny khng tan trong cc
acid v c, khng tan trong acid acetic long.

Lm mt mu dung dch KMnO
4
trong mi trng acid :
2MnO
4

+ 5C
2
O
4

2
+ 8H
+
= 2Mn
2+
+ 10CO
2
+ 4H
2
O
2.1.26. Peroxyd
Phn ng vi K
2
Cr
2
O
7
trong mi trng acid:
Cr
2
O
7
2
+ 4H
2
O
2
+ 2H
+
= 2H
2
Cr
2
O
6
+ 3H
2
O
(acid pecromic)
Acid pecromic c mu xanh c chit bng ether (acid ny d b phn
hu trong mi trng nc).
Phn ng vi KI gii phng I
2
c mu :

H
2
O
2
+ 2I

+ 2H
+
= I
2
+ 2H
2
O
2.1.27. Phosphat
Phn ng vi (NH
4
)
2
MoO
4
trong mi trng HNO
3
: cho ta mu vng
(lng t cho dung dch mu vng) amoniphosphomolipdat
(NH
4
)
3
[PMo
12
O
40
]:
H
3
PO
4
+ 12(NH
4
)
2
MoO
4
+ 21HNO
3
[NH
4
]3P[Mo
12
O
40
] + 21NH
4
NO
3
+ 12H
2
O
Phn ng vi AgNO
3
: cho kt ta bc phosphat mu vng, ta ny d tan
trong acid v c v dung dch amoniac:
+ 3
4
PO + 3Ag
+
= AgPO
4
vng
2.1.28. Salicylat (C
6
H
4
OHCOO

)
Phn ng vi dung dch FeCl
3
long cho phc mu tm Fe(OH)
2
C
7
H
5
O
3

Phn ng vi HCl long cho ta acid salicylic C
6
H
4
OHCOOH (c nung
chy 156
o
C - 161
o
C).
2.1.29. St (II)
Phn ng vi dung dch kalifericyanid to thnh ta c mu xanh lam
(nng nh cho dung dch keo mu xanh l).
45
Khng tan trong dung dch HCl 2M:
3Fe
2+
+ 2K
3
[Fe(CN)
6
] = Fe
3
[Fe(CN)
6
]
2
xanh + 6K
+
2.1.30. St (III)
Phn ng vi KCNS to phc mu Fe(CNS)
x
(3x)+
; (x t 1-6). Phc ny
chit c trong ether, alcol (hoc mt mu khi thm Hg
2+
)
Fe
3+
+ x(CNS

) = Fe(CNS)
x
(3x)+
()
Fe(CNS)
4

+ Hg
2+
= Hg(CNS)
4

2
+ Fe
3+




Phn ng vi kaliferocyanid to thnh ta xanh lam khng tan trong dung
dch HCl 2M :
4Fe
3+
+ 3K
4
[Fe(CN)
6
] = Fe
4
[Fe(CN)
6
]
3
xanh + 12K
+
2.1.31. Silicat
Hp cht ca Silic khi un vi H
2
SO
4
c v NaF (hay CaF
2
) trong chn
bch kim hay ch s to ra tetraflorid silic SiF
4
dng hi (hoc
hexaflorosilicat SiF
6
), d b thu phn to ra ta l acid metasilisic H
2
SiO
3

(hoc acid octosilisic H
4
SiO
4
).
Phn ng vi amonimolipdat trong mi trng HNO
3
cho ta mu vng :
+ H
2
SiO
3
(NH
4
)
2
MoO
4
HNO
3
= (NH
4
)
3
[SiMo
12
O
40
] NH
4
NO
3
H
2
O
12 + +20 11

+ 20
(amonisilicomolipdat)

2.1.32. Stibi (mui)
Cho phn ng vi natrisulfid (Na
2
S) to thnh ta Sb
2
S
3
hoc Sb
2
S
5
c
mu vng cam. Cc ta ny tan trong dung dch sulfid d hoc polysulfd
kim hay amoni cho cc mui SbS
5

hoc SbS
4

.
Ghi ch:
Thng ho tan cc dng ca mui Stibi bng dung dch natrikalitartrat,
khi Stibi di dng mui ni phc tartrat kp K v Sb d tan: KOOC-
CHOH - CHOH-COOSbO. Sau cho phn ng to Sb
2
S
3
hoc Sb
2
S
5
.
2.1.33. Sulfat
Cho phn ng kt ta vi dung dch BaCl
2
(to ta BaSO
4
) mu trng,
ta ny khng phn ng vi I
2
, SnCl
2
).
46
2.1.34. Sulfid (S
2
)
Tc dng vi HCl long gii phng kh H
2
S c mi thi








Phn ng vi Pb
2+
to kt ta mu en PbS.
2.1.35. Bisulfit v sulfit (HSO
3

v SO
3
2
)
Tc dng vi dung dch HCl gii phng kh SO
2
c mi c bit.
Lm mt mu I
2
:
SO
3
2
+ I
2
+ H
2
O = H
2
SO
4
+ 2I

2.1.36. Tartrat (C
4
H
4
O
6
2
v HC
4
H
4
O
6

)
Khi phn ng vi H
2
SO
4
c + resorcin: acid tartric gii phng ra v
phn hu thnh aldehyd glycolic OHH
2
CCHO, cht ny s to mu tm m
vi resorcin.
2.1.37. Thiosulfat
Phn ng lm mt mu dung dch I
2
;
S
2
O
3
2
+ I
2
= S
4
O
6
2
+ 2I

Tc dng vi HCl cho kt ta S v gii phng kh SO

2
:
S
2
O
3
2
+ 2H
+
= S + SO
2
+ H
2
O
Tc dng vi AgNO
3
cho kt ta vng chuyn dn sang en:
S
2
O
3
2
+ 2Ag
+
= Ag
2
S
2
O
3
vng Ag
2
Sen
2.1.38. Thu ngn (I v II)
Phn ng hn hng vi ng:
Hg
2+
+ Cu = Hg + Cu
2+
Khi lm, trn ming ng s thy vt hn hng sng bng, t nng vt
bng s mt (v Hg bay i).
Vi KI:
Hg
2+
I
-
+ 2 = HgI
2

+ I
-
2
HgI
4
-
(khng mu)

I
-
+ 2 = Hg
2
I
2
vng Hg
2
2+
+ I
-
2
HgI
4
2-
Hg en + lc

47
2.2. Th gii hn cc tp cht trong thuc
2.2.1. Mc ch
Xc nh gii hn tp cht trong thuc thc cht l th tinh khit ca
thuc nhm xc nh phm cht ca thuc. Nu thuc cng tinh khit th hiu
qu tc dng cng cao.
Cc tp cht trong thuc mc d rt nh nhng n c th:
Gy tc hi cho sc kho (th d tp cht bari tan, arsen, ch ).
Gy hin tng tng k ho hc, nh hng n phm cht hay bn
vng cu thuc.
Mt s tp cht c th khng c tc dng c hi nhng li l nhng
cht xc tc y nhanh qu trnh phn hu thuc (th d: cc vt kim
loi, m ).
Mt s tp cht khng gy hi, khng gy tng k ho hc, khng lm
phn hu thuc, khng gy phn ng ho hc nhng n biu th cho
mc sch (hay mc tinh ch cha ) ca thuc.
Khi bit mc tinh khit ca thuc (c bit trong trng hp khng
t yu cu) cho php xem xt cc ngun gc gy ra cc tp cht ny v
tm bin php khc phc. Cc nguyn nhn c th l:
+ Nguyn liu, ph liu hoc bn thnh phm dng sn xut thuc
cha tinh khit.
+ Qui trnh sn xut qui nh khng c thc hin nghim chnh.
+ nh hng ca cc dng c s dng.
+ Phng php sn xut cha tt.
+ Trong qu trnh bo qun, cc phn ng ph do nhiu yu t nh: mi
trng, vn v sinh, cht bo qun lm pht sinh cc tp cht.
+ Do dng gian ln ca ngi sn xut
Bi vy, TCVN (Dc in) v TC thng qui nh cho php mi thuc ch
c c nhng lng rt nh cc tp cht nht nh bo m cho thuc c
sch nht nh tc l thuc c cht lng, t hiu qu tc dng cao nht.
2.2.2. Phng php xc nh gii hn tp cht trong thuc
2.2.2.1. Phng php xc nh
Xc nh gii hn tp cht trong thuc tc l xc nh xem cc tp cht
c vt qu gii hn cho php hay khng, cc phn ng th tp cht c tnh
cht bn nh lng v c thc hin bng phng php so snh:
Ly hai bnh (thng l 2 ng nghim) thc hin phn ng.
48
Bnh 1: Ly mt th tch dung dch thuc em th.
Bnh 2: Ly mt th tch dung dch mu. (Dung dch mu l dung dch c
cha tp cht cn th vi s lng cho php).
Sau tin hnh song song phn ng th tp cht vi cng mt thuc
th. So snh kt qu phn ng hai bnh (thng l so mu hoc so c) t
xc nh c gii hn tp cht cn th c trong mu thuc em th.
Trong qu trnh thc hnh cn phi theo cc qui nh sau:
Nc v nhng ho cht, thuc th s dng khng c c tp cht
ang cn th.
Khi pha dung dch mu phi s dng cn phn tch v dng c th tch
chnh xc.
Hai bnh phn ng so snh phi ging nhau: bng thu tinh khng
mu, c ng knh bng nhau, dy nh nhau
Khi so snh, quan st c th nhn t trn xung, quan st mu th
nhn ngang trn nn trng.
Phi cho cc thuc th vo hai bnh phn ng ging nhau v: thi gian,
s lng v th tch cui.
Khi phn tch, nu pht hin c mt tp cht l th phi ghi li v
bo co.
2.2.2.2. Pha cc dung dch mu
pha dung dch mu ca mt tp no , ch cn cn lng chnh xc
cht tinh khit ca tp (cht gc) pha vo mt th tch xc nh theo tnh
ton ta s c mu tp chun c nng xc nh (thng biu th theo
mg/ml; % hoc phn triu).
Th d: Pha dung dch mu Cl
-
nh sau :
Dung dch A: Cn chnh xc 0,8238g NaCl tinh khit ho tan trong
nc v thm nc cho 1 lt (dng bnh nh mc). c dung dch
c cha:
/1ml Cl g 0,0005
1000 58,5
0,8238 35,5

=

Dung dch B (dung dch mu chun em th): Ly chnh xc 1
ml
00
dung dch A pha long bng nc cho 100,0 ml. Dung dch ny c
cha ml Cl g 000005 0
100
0005 0
/ ,
,

= (tng ng 0,005 mg Cl

/ ml hay
dung dch 0,0005% hoc 5 phn triu).

49
2.2.2.3. Pha dung dch th
pha, gi thit mu em kim tra c cha mt lng tp cht cho php
ti a, t tnh h s pha long thch hp, sau tin hnh pha theo tnh
ton ny.
Th d: Pha dung dch th tp Cl

trong paracetamol (theo tiu chun

Cl

khng c qu 0,01% ):
V dung dch mu Cl

khi em th l dung dch c cha 0,0005% (hay 5

phn triu). Do h s pha long dung dch th s l 20
0005 0
01 0
=
,
,
Ta c
cch pha nh sau: Cn 1,000 g paracetamol ho tan trong nc cho 20,00
ml, lc. Ly 10,00 ml dch lc em th v so snh vi 10,

00 ml dung dch mu
chun Cl

0,0005%.
(Thng trong Dc in hoc TC c ghi r cch pha dung dch th l
da trn c s tnh ny).
2.2.3. Mt s thuc th trong cc phn ng ho hc xc nh gii hn tp cht
Ion cn th
(tp cht)
Thuc th Sn phm Hin tng quan st
Cl
-
AgNO
3
AgCl Ta trng
SO
4
2-
BaCl
2
BaSO
4

Ta trng
NH
4
+

Nessler

I
Mu vng (nu nhiu c
nu )
Ca
2+
(NH
4
)
2
C
2
O
4
CaC
2
O
4
Ta trng
Arsen Zn + HCl AsH
3

Giy tm HgCl
2
chuyn
t vng sang nu
Kim loi nng
- Na
2
S (H
2
S)
- thioacetamid
PbS en hoc nu
St
- acid mercapto-
acetic
- acid sunfosalicylic
- Feri mercaptoacetat
Ferisulfisalicylat
Mu hng
nu hay vng
Hg
2
Hg
O NH
50
Nhm
8-hydroxyquinolin
( pH 6)
N
Al/
3
O

(oxyquinolat nhm)
Mu vng rm
(tan trong CHCl
3
)
Magnesi
8-hydroxyquinolin
( pH 10)
Oxyquinolat Mg
Mu vng
(tan trong CHCl
3
)
Phosphat Sulphomolybdic (NH
4
)
3
H
4
[P(Mo
2
O
7
)
6
] Mu vng
Km K
4
[Fe(CN)
6
] K
2
Zn
3
[Fe(CN)
6
]
2
Ta trng
2.3. Chun acid - base trong mi trng khan
Chun trong mi trng khan da trn phn ng trung ho gia
acid v base. Cho n nay c th thng k 4 thuyt chnh pht trin khi
nim acid - base:
Thuyt in ly acid - base trong mi trng nc ca Arrhenius - Ostwald
Thuyt proton ca Bronsted-Lowry,
Thuyt in t ca Lewis,
Thuyt acid - base tng qut ca Usanovich.
Vi mc ch gii thch ng dng cc phn ng acid - base trong kim
nghim thuc, chng ta x dng thuyt proton ca Bronsted - Lowry. Theo
thuyt ny, acid v base to ra nhng cp acid - base lin hp, chng khc
nhau mt proton. Phn ng acid - base l phn ng gia 1 acid v 1 base
thuc hai cp acid - base lin hp: acid 1/ base1 v acid 2/ base 2.
Acid 1 + base 2 Acid 2 + base 1
Cp th hai c th l phn t cht tan hoc phn t dung mi. Phn ng
acid - base l phn ng cho nhn proton.
2.3.1. Vai tr ca dung mi
Trong phn ng acid - base, dung mi c th tc ng theo 2 hng.
Solvat ha cht tan:
Nu dung mi c tnh acid, n lm tng tnh base ca cht tan B

51
V d:
B + HS BH
+
+ S
-

+
+
+ COO
3
CH
3
H N R COOH
3
CH
2
NH R
Ngc li nu dung mi c tnh base, n s tng tnh acid ca cht tan HA
HA + S A

+ S
+
H
Trong cc dung mi tr (dung mi khng cho hoc khng nhn proton),
qu trnh solvat ha c thc hin do c ch khc (lin kt hydro, phc , lc
Van der Waals).
Tc ng ln qu trnh in ly ca cp ion:
Trong dung mi c hng s in mi ln (nc, formamid) hu ht cp
ion to ra do qu trnh solvat ha cht tan u phn ly thnh cc ion t do.
Ngc li, trong dung mi c hng s in mi b, cc ion ch yu tn ti
di dng cp ion.
Qu trnh in ly ca cp ion do hng s quyt nh. C th phn chia
s b nh sau:
> 50 nh nc, formamid, dimetylsulfoxid: acid v base tn ti ch
yu di dng ion t do .
< 30 nh ethanol, aceton: tn ti nhiu cp ion
< 10 nh benzen, cloroform, acid acetic: tn ti cp ion l ch yu.
Cn lu l hng s ch tc ng ln qu trnh in ly ca cp ion. Nu
trong phn ng acid - base khng to ra cc ion c in tch ngc du, do
khng to cp ion th hng s t tc ng ln qu trnh in ly.
Phng trnh tng qut m t qa trnh ion ha (th hin hng s K
i
)
v qu trnh in ly (th hin hng s K
d
) ca cht tan HA trong dung mi S
c th hin nh sau:
k
i
k
d
HA + S HS
+
A

HS
+
+ A

solvat ha in ly
2.3.2. Khi nim pH
Trong dung dch nc, ngi ta nh ngha pH = - lga
H
+
Trong dung mi khan, ngi ta xc nh pH biu kin.
52
Thang ny lin quan n hng s t in ly K
S
ca dung mi.
HS + HS H
2
S+ + S

H
2
S
+
l ion lionium, S
-
l anionlyat, pH biu kin l pH = - lg a y
+
S H
2
a
+
S H
2

. a
S

= K
S
V d:
2 CH
3
COOH CH
3
COOH
2
+
+ CH
3
COO
-
K
S
= [CH
3
COOH
2
+
] [CH
3
COO
-
]
2C
2
H
5
OH C
2
H
5
OH
2
+
+ C
2
H
5
O
-
K
S
= [C
2
H
5
OH
2
+
] [C
2
H
5
O
-
]
di ca thang pH biu kin tu thuc vo hng s t in ly ca dung
mi. V d:
Methanol: pK
S=
= 17
Ethanol: pK
S
= 20
Acid acetic: pK
S
= 6,35
2.3.3. Xc nh im tng ng
nh lng trong mi trng khan thng c thc hin bng cch
chun . pht hin im tng ng thng dng 2 phng php:


Ch th mu pH:
Theo di s i mu ca ch th. Thng dng tm tinh th, tm metyl.
Ngoi ra cn dng cc loi ch th mu hn hp.
Ch th o th:
y in cc so snh c th l in cc calomel hoc bc clorid, in
cc ch th thng l in cc thu tinh (nu c th, dng dung dch trong bu
thu tinh c cng dung mi vi mi trng chun ).
c kt qu tin cy khi chun trong mi trng khan cn lu :
Khi xc nh im tng ng theo di s thay i ca in th,
khng phi ca pH,
Cn trung ho dung mi trc khi chun ,
Cn x l in cc ch th ph hp.
53
2.3.4. ng dng kim nghim thuc
Chun trong mi trng khan c p dng khi:
Cht phn tch khng ho tan trong nc. Trong kim nghim thuc,
thng gp cc acid v base c khi lng phn t ln t tan trong nc.
Sc acid, base qu yu trong nc nn kh pht hin im tng ng.
Cc acid, base a chc c cc hng s in ly trong nc t khc bit nhau.
Sau y s gii thiu cc loi dung mi, dung dch chun trong nh
lng cc acid v base.
2.3.4.1. nh lng acid
Cc cht hu c c tnh acid yu thng c chun bng base trong
mi trng khan nh:
Cc acid carboxylic,
Dn xut enol, imid, sulfonamid,
Dn xut th phenol nh polyclorophenol, polynitrophenol.
Hn hp cc cht c tnh acid hoc acid a chc .


Dung mi
Thng chn dung mi c tnh base tng tnh acid ca cht phn tch
nh: pyridin, dimetylformamid (DMFA). Ngoi ra tert - butanol thng c
dng lm dung mi cho chun acid carboxylic, dn xut ca phenol.
Dung dch chun
Thng dng cc dung dch chun base nh:
KOH trong alcol (thng dng trong methanol),
Metylat kim loi kim nh natri, kali,
Tetraalkyl amonium hydroxyd: thng dng tert - Bu
4
NOH trong hn
hp dung mi benzen - methanol (95:5).
Khi dng cc dung dch chun ny cn lu :
Dung dch chun kim loi kim gy sai s base cho in cc thu tinh
khi chun o th.
Dung dch chun R
4
NOH l base mnh, mnh hn dung dch hydroxyd
kim nh KOH, cho nn c th chun cc acid rt yu. Tuy nhin
cc dung dch ny c 2 nhc im:
+ c do c benzen
+ Pha ch mt nhiu thi gian, kh bo qun (d phn ng vi CO
2
ca
khng kh).
54
Cht chun
Cc cht chun acid thng dng l:




Acid benzoic: C
6
H
5
COOH, E = M = 122,12
Acid succinic (CH
2
COOH)
2
, E = M/2 = 59,05
Acid sulfamic NH
2
SO
3
H, E = M = 97,09
Kalihydrophtalat, E = M = 204,22.
Phn ng chun
Ly dung mi in hnh l pyridin
HA + C
5
H
5
N C
5
H
5
N + HA
chun cc enol, thiol, ngi ta thng thm AgNO
3
vo mi trng
RSH + C
5
H
5
N + AgNO
3
RSAg + C
5
H
5
N
+
HNO
3
-
2.3.4.2. nh lng cc base hu c
Cc alcaloid v base nit tng hp thng c chun bng cc acid
chun trong dung mi acid.
Dung mi
tng tnh base ca cht phn tch ngi ta dng cc dung mi acid
nh acid acetic khan.
Acid acetic khan thng c x dng ph bin nht v t c, r tin.
Dng anhydrid acetic cng hay dng v c th nh lng cc base rt yu.
Hn na bc nhy th im tng ng trong anhydrid acetic thng ln
hn trong acid acetic. Tuy nhin anhydrid acetic c nhc im l d acetyl
ha cc amin bc nht v bc hai nht l khi un nng.
(CH
3
CO)
2
O + RNH
2
CH
3
CONHR + CH
3
COOH
Trong trng hp nh lng amin khng dng dung mi ny. Ngoi acid
acetic, acetonitril c dng nh lng cc base vi dung dch chun acid
percloric trong 1,4 dioxan.
Dung dch chun
Dung dch acid percloric trong acid acetic khan thng c dng nhiu
nht. Dung dch ny pha ch t acid percloric thng mi 72% (kl/kl). V vy
khi pha ch phi thm anhydrid acetic loi nc v 48 gi trc khi dng.
Ngoi acid acetic khan, cn dng 1,4 dioxan pha dung dch chun acid
percloric. Dung dch ny km n nh, d chuyn thnh mu nu trong qu
trnh bo qun. V vy ch pha v s dng khi cn thit.
55
Cht chun
Thng dng kali hydrophtalat.




Phn ng chun
Thng c vit thnh 2 giai on
Solvat ha
RNH
2
+ CH
3
COOH RNH
3
+
CH
3
COO
-
Trung ho
RNH
3
+
CH
3
COO
-
+ HClO
4
RN
+
H
3
Cl
-
O
4
+ CH
3
COOH
Nu cht cn nh lng t tan, phi un nng. Nhng cn thn trng v
c th acetyl ha chc amin I hoc amin II. i khi ngi ta thm acid formic
vo dung mi acid acetic khan thay cho un nng lm tng tan (do hng
s ca dung mi tng ln).
2.3.4.3. nh lng cc mui
Nhiu dc cht l mui ca cc base hu c. Chn phng php nh
lng chng trong acid acetic tu thuc vo anion to mui vi base.
Mui ca cc acid yu hn acid acetic BH
+
Y
-

Y
-
l anion propionat, maleat, benzoat, salicylat,... Trong trng hp ny
mui BH
+
Y
-
c chun trc tip nh mt base bng HClO
4
:
Anion Y
-
c trung ho bng HClO
4
. V vy phn ng khng c hiu.
Mui ca acid mnh hn acid acetic BH
+
X
-

X
-
l cc halogenid, anion sulfat. Ngi ta phn ra hai trng hp:
Mui halogenid (thng hydroclorid, hydrobromid)
Cch n gin nht l nh lng X
-
nh Y
-
trong trng hp trn.
Nhng nu X

l base rt yu s khng phn ng ton lng vi HClO

4
.
gii quyt kh khn ny, ngi ta dng 2 phng php:
+ Phng php Pifer - Wollish
Thm vo mi trng mt lng d Hg(II) acetat gii phng ion
acetat. Trong mi trng acid acetic, n l base mnh c chun bng HClO
4
2BH
+
X
-
+ Hg(CH
3
COO)
2
2CH
3
COO
-
BH
+
+ HgX
2
2CH
3
COO
-
BH
+
+ 2HClO
4
2CH
3
COOH + 2BH
+
ClO
4
-
y nh lng mui BH
+
X
-
thng qua anion CH
3
COO
-
(thay cho anion
X
-
) tng t Y
-
trong trng hp trn.
56
+ Phng php Billon
Ho tan mui trong ethanol, BH
+
trong dung mi ny l acid tng i
mnh, c th c trung ho bng NaOH 0,1N. Phn ng nh lng tri qua
2 giai on
u tin thm mt lng d HCl chuyn ht base B (nu c trong
BH
+
X
-
) sang dng mui
B + HCl BH
+
+ Cl
-
Hn hp BH
+
X
-
v HCl d c trung ho bng NaOH
HCl + NaOH NaCl + H
2
O
BH
+
X
-
+ NaOH NaX + B + H
2
O
Pht hin im tng ng bng chun o th: c 2 bc nhy th
ng vi trung ho HCl d v BH
+
X
-
trong mu phn tch.
Nhc im chnh ca phng php ny l s chnh lch sc acid ca
HCl v BH
+
X
-
phi ln c 2 bc nhy. Mt khc B l base khng qu
yu trong ethanol.
Mui sulfat
H
2
SO
4
c th to nhiu dng mui vi base hu c n chc (stricnin,
atropin) hoc a chc (quinin).
+ Mui dng BH
+
HSO
4
-
y anion HSO
4
-
l base kh mnh c th nh
lng bng HClO
4
trong acid acetic.
+ Nu base hu c to mui trung tnh: dng (RN)
2
H
2
SO
4
hoc
(RNH
+
)
2
SO
4
2-
nh strycnin sulfat, atropin sulfat
(RNH
+
)
2
SO
4
2-
+ HClO
4
RNH
+
ClO
4
-
+ RNH
+
HSO
4
-
Vi quinin bisulfat cng xy ra tng t
R (NH
+
)
2
SO
4
2-
+ HClO
4
(NH
+
HSO
4
-
)R (NH
+
. ClO
4
-
)
1 mol HClO
4
ng vi 1 mol mui sulfat
+ Mui khng trung tnh nh quinin sulfat basic
R(NH
+
N)
2
SO
4
2-
+ 3 HClO
4
(N
+
H

ClO
4
-
)R(NH
+
HSO
4
) + R(NH
+
ClO
4
-
)
2
1 mol mui cn 3 mol HClO
4
trung ho.
Mui tetraalkyl amonium bc bn
Cation R
4
N
+
c th lin kt vi hydroxyd, halogenid (X
-
), phosphat. Cc
anion tham gia vo phn ng trung ho nh mt base.
57
+
Vi hydroxyd: trung ho trc tip bng HClO
4
+ Vi halogenid: thm Hg (II) acetat nh trong phng php Pifer -
Wollish.
2.4. Xc nh hm lng nc bng thuc th karl fischer
C nhiu k thut xc nh hm lng nc trong cht rn, trong dung
mi hu c. y gii thiu phng php dng thuc th Karl Fischer da
vo tnh cht oxy ha ca iod.
2.4.1. Nguyn tc
Thuc th ny gm c iod, SO
2
, methanol v pyridin. Hn hp phn ng
vi nc theo hai phng trnh sau:
C
5
H
5
N.I
2
+ C
5
H
5
N.SO
2
+ C
5
H
5
N + H
2
O 2C
5
H
5
N.HI + C
5
H
5
N.SO
3
C
5
H
5
N. SO
3
+ CH
3
OH C
5
H
5
N(H)SO
4
CH
3
Hn hp c lng ln pyridin nn cc cht tham gia phn ng v sn
phm u tn ti di dng phc. Phn ng u tiu th 1 phn t nc.
Phn ng th 2 xy ra khi c d CH
3
OH l cn thit, quyt nh s thnh
cng ca chun v phc SO
3
cng phn ng vi nc
C
5
H
5
N. SO
3
+ H
2
O C
5
H
5
N(H)SO
4
H
y l phn ng ph. loi b phn ng ny, ngi ta dng methanol
d trong hn hp.
2.4.2. Pha ch v xc nh chun
Pha ch:
C ch phn ng ch r mt phn t iod oxy ho mt phn t SO
2
tiu
th mt phn t nc trong mi trng c d pyridin v methanol. Khi pha
thuc th ngi ta dng mt lng d SO
2
v pyridin. V vy phn ng ca
thuc th vi nc do lng I
2
quyt nh. chun ca thuc th thng 2
n 5 mg H
2
O/ ml. Lng SO
2
d gp 2 ln, pyridin gp 3 - 4 ln.
chun ca thuc th gim dn trong qu trnh bo qun. V vy
thng ch pha ch trc 1 - 2 ngy. C trng hp pha thnh hai dung dch:
Dung dch A: SO
2
v pyridin trong methanol,
Dung dch B: I
2
trong methanol khan.
Khi dng trn 1 th tch A vi 1 th tch B.
58
Xc nh chun ca thuc th: c 2 cch.
1. Xc nh hm lng nc di 1%, ngi ta chn mt ha cht c hm
lng nc kt tinh xc nh, sy kh loi m. Cho thuc th tc dng
vi ha cht ri tnh ra ng lng. Mui natri tartrat dihydrat (C
4
H
4
Na
2
.
2H
2
O) hay c la chn.
2. Xc nh hm lng nc trn 1%, xc nh hm lng nc theo dung
dch chun nc/ methanol.
2.4.3. Xc nh im tng ng
Hai cch ph bin xc nh im tng ng ca phn ng nh lng:
1. Theo lng tha ca iod khi nc phn ng ht. S i mu do
tha thuc th.
2. Chun amper vi 2 in cc platin (chun n im dng). Mt s
nh sn xut cho ra i cc dng c chun t ng dng thuc th Karl
Fischer.
2.4.4. ng dng
Thuc th Karl Fischer c dng xc nh hm lng nc trong
nhiu dng mu khc nhau. Da vo c im ca mu ngi ta xy dng qui
trnh phn tch cho ph hp.
Nu mu d tan trong methanol, ngi ta dng chun trc tip
nc trong cc cht hu c nh acid, alcol, ester, anhydrid, k c cc
mui ngm nc.
Nu mu t tan trong thuc th, ngi ta dng chun tha tr: cho
mt lng tha thuc th. Sau thi gian phn ng thch hp, xc
nh lng d bng dung dch chun nc trong methanol.
Mt ci tin khc l chit hi lu nc trong mu bng methanol
khan. Xc nh lng nc chit xut c bng chun trc tip.
K thut ny rt thch hp cho nh lng nc hp th, nc kt
tinh.
Cn lu l mt s phn ng ha hc cn tr phng php Karl
Fischer do to thnh nc sau phn ng:
Cc hp cht carbonyl tc dng vi methanol:
R CHO + 2CH
3
OH

R CH (OCH
3
)
2
+ H
2
O
Oxyd kim loi phn ng vi HI
MO + 2 HI MI
2
+ H
2
O
59
Ngoi ra cc cht oxy ha kh cng thng cn tr phng php Karl
Fischer v cht oxy ha s phn ng vi iodid l sn phm ca thuc th, cn
cht kh phn ng vi iod ca thuc th.
2.5. nh lng mt s cht hu c a chc bng thuc th periodat
2.5.1. Nguyn tc
y l phng php nh lng da vo tnh cht oxy ha ca cp I
7+
/ I
5+
.
Tu theo mc hydrat ha ca I
2
O
7
ta c 2 dng acid:

I
2
O
7
+ H
2
O 2HIO
4
: acid metaperiodic
I
2
O
7
+ 5H
2
O 2H
5
IO
6
: acid paraperiodic
Trong dung dch nc cc acid v ion periodic nm trng thi cn bng,
H
4
IO
6
-
, IO
4
-
, H
3
IO
6
2-
.
Cp oxy ha kh periodic
H
5
IO
6
+ H
+
+ 2e IO
3
-
+ 3H
2
O
Th chun khong 1,6 V. Periodat l cht oxy ha mnh
Phn ng oxy ha ca periodat vi cc cht hu c c d bo theo 4
nguyn tc sau:


B gy lin kt C C c mang 2 nhm chc cnh nhau nh: chc
C = O, - NH
2
, - OH.
Nguyn t C c nhm chc OH b oxy ha thnh aldehyd hoc ceton .
Nguyn t C c nhm chc ceton c chuyn thnh acid COOH.
Nguyn t C c nhm - NH
2
s chuyn thnh aldehyd ng thi to ra NH
3
.
Ba phn ng sau minh ho 4 nguyn tc trn
H
3
C CO CO CH
3
+ HIO
4
+ H
2
O HIO
3
+ 2 CH
3
COOH
2H
3
C CO CH(OH) CH
3
+ H
2
O + HIO
4
HIO
3
+ CH
3
COOH + CH
3
CHO
H
2
C CH
2
+ HIO
4
2HCHO + NH
3
+ HIO
3
HO NH
2
Trong cc phn ng trn, ngi ta thng nh lng bng cch cho tha
thuc th HIO
4
vo mu phn tch. Sau khi phn ng kt thc cho lng
d As
2
O
3
v dung dch KI. Iod gii phng c chun bng dung dch
Na
2
S
2
O
3
. Song song lm mt mu trng. l nguyn tc ca phng
php Fleury. C trng hp ngi ta dng phng php iod xc nh
nng periodat ch vi lng d KI, khng cn dng As
2
O
3
.
60



Dung dch chun
Nu phn ng oxy ha thc hin trong mi trng acid nh dng dung
dch H
5
IO
6
. Nu trong mi trng acid mnh dng dung dch NaIO
4
hoc
Na
3
H
2
IO
6
(thng pha trong dung dch H
2
SO
4
).
nh lng thng tin hnh theo cch gin tip, c hiu chnh vi mu
trng.
2.5.2. ng dng
Ngi ta t quan tm n ng dng ca chun periodat cho nh lng
cc cht kh v c bi c nhiu thuc th oxy ha d thc hin, khng t
tin nh Cr
2
O
7
2-
, MnO
4
-
, I
2
. Ngc li ngi ta ch n phn ng ca
periodat vi cc cht hu c a chc do tnh cht oxy ha chn lc ca n.
Trc y cc phn ng ny c dng trong phn tch cu trc. Hin nay,
tnh oxy ha ca periodat c s dng trong phn tch nh lng.
C th dng thuc th periodat nh lng nhiu cht hu c nh:
Cc .diol: etylenglycol, propradiol 1,2
Polyol : phn ng tng qut
H
2
COH (CHOH)
n
CH
2
OH + (n+1) HIO
4
2HCHO + (n) HCOOH + (n+1)HIO
3
+ H
2
O
Cn lu l :
+ S phn t formadehyd to thnh bng s chc alcol bc nht.
+ S phn t acid formic to thnh bng s chc alcol bc 2.
Vi cc aldose ta c th vit:
CH
2
OH(CHOH)
n
CHO+(n+1)HIO
4
(n+1)HIO
3
+HCHO + (n+1)HCOOH
Cc diamin nh etylen diamin, amino - alcol nh ephedrin.
C th minh ho ng dng ca phng php periodat thng qua nh
lng glycerin ca Dc in Php X
+ Dng HIO
4
c d oxy ha ht glycerin
+ Thm propylenglycol loi ht HIO
4
d
+ Dng dung dch chun NaOH trung ha ht acid to thnh sau phn
ng (HIO
3
v HCOOH)
+ Song song lm mt mu trng. Tnh kt qu da vo lng NaOH
dng cho mu phn tch v mu th.
2.6. ng dng cp ion trong kim nghim thuc
K thut to cp ion c trin khai u tin trong phng php chit
lng - lng vi mc tiu tng hiu sut chit t dung dch nc ca mt s ion.
61
Sau , k thut ny c ng dng nhanh chng nh lng nhiu dc
cht mang tnh acid hoc base di dng phn tch th tch hoc o quang.
Hin nay, k thut to cp ion xm nhp vo lnh vc sc k khng ch
nh gi cht lng thuc m c trong nghin cu dc ng hc v phn tch
ha sinh. Trong phm vi gio trnh kim nghim thuc, chng ti ch trnh
by s lc khi nim v cp ion v nguyn tc ng dng ca n.
2.6.1. nh ngha cp ion
Cp ion c hnh thnh do tng tc tnh in gia 2 ion c in tch
tri du: 1 ion dng v 1 ion m. Theo thuyt lin hp Bjerrum (nm 1926)
th hai ion solvat ha c in tch tri du khi chuyn ng nhit trong dung
dch s tin li gn nhau do lc ht Couloms. n mt khong cch no
gia 2 ion c gi l khong cch c trng q, chng to thnh mt tiu phn
ng hc chuyn ng t do nh cc ion. Tiu phn ng hc l cp ion (ion
pairs). Vi cc cht in ly i xng (1 -1, 2 2, ... ) cp ion to thnh khng
mang in tch. Vi cc cht in ly khng i xng (1 - 2, 1 3, 2 3, ... ) cp
ion c in tch nh hn in tch ca cc ion ban u. Trong c hai trng
hp dn in ca dung dch s gim xung.
Khong cch c trng q ni trn c xc nh bi t l gia nng lng
to cp ion v nng lng chuyn ng nhit trung bnh tch ring cc ion.
Gi tr q c tnh theo phng trnh sau:
2
k
e
T 2
z z
q
k a
=
Trong :
z
a
v z
k
l in tch ca anion v cation,
e l in tch ca in t
k l hng s Boltzmann
l hng s in mi ca dung dch,
T l nhit tuyt i (
0
K)
Khong cch q ph thuc vo in tch z ca ion v hng s : nu in
tch cng ln v cng nh th q cng tng, cp ion to ra cng nhiu.
Vi cht in ly 1 -1 trong dung dch nc 25
0
C th q = 3,6 A
0
(1A
0
= 10
-8

cm). iu c ngha l trong dung dch nc, cht in ly 1-1 ho tan, v d
mui KCl, khi 2 ion K
+
v Cl
-
khong cch q < 3,6 A
0
s to thnh cp ion.
Chng chuyn ng t do trong dung dch nh cc ion K
+
, Cl
-
v phn t khng
in ly. Cp ion trung ho in tch li do va chm lm gim bt lp v hydrat
ha trong dung dch nc nn d chuyn sang ho tan trong dung mi hu c.
62
2.6.2. Cc yu t nh hng n s to thnh cp ion
Ngoi yu t in tch z v hng s , s to thnh cp ion cn chu nh
hng ca 2 yu t sau:


c im ca ion
Nu ion cng ln v cng s nc, cng t b hydrat ha trong dung dch
nc th cng d b solvat ha bi cc phn t dung mi hu c.
pH ca dung dch nc
c th to cp ion t cc cht mang tnh acid, base th pH dung dch
phi bo m tn ti c cation (t base) v anion (t acid).
V d:
Cc alcaloid v base nit tng hp c th mang in tch dng khi
cng proton:
R NH
2
+ H
+

+
3
NH R
Chng c th to cp ion pH acid.
Vi cc cht c tnh acid, chng to ra anion cng ty theo pH:
R- COOH RCOO
-
+ H
+
to ra cp ion ' cn c pH thch hp.
3
OCR O H N R
+
2.6.3. ng dng
K thut to cp ion c ng dng nhiu trong phn tch th tch, o
quang v sc k.
2.6.3.1. Phn tch th tch
Thng dng cch chun hai pha. V d: chun metoclopramid
hydroclorid (MetH
+
Cl
-
) bng dung dch chun natri laurylsulfat (Na
+
LS
-
) trong
h dung mi nc metylen clorid (CH
2
Cl
2
) vi ch th metyl (MIn).
Khi anion LS
-
phn ng vi cation MetH
+
(to cp ion MetH
+
LS
-
), anion
ny s kt hp vi ch th to cp ion MInH
+
LS
-
tan trong dung mi CH
2
Cl
2
.
Pha ny chuyn t mu vng (do c phn t ch th MIn ha tan) sang mu
(mu ca cp ion MInH
+
LS
-
).


63
Met H
+
Cl
-
Cl
-
+ Met

H
+
H
2
O MIn + H
+
MInH
+
LS
-
+ Na
+
NaLS


CH
2
Cl
2
Met H
+
LS
-
MInH
+
LS
-

Nhiu alcaloid v base tng hp c chun theo nguyn tc ny.
2.6.3.2. Chit o quang
y l phng php thng c dng nh lng bng o hp th
ca cc cht c tnh acid hoc base.


Chit cc base dng ch th mu acid:
Cc ch th ny l i ion to cp vi base mang in tch dng (khi cng
hp proton) pH thch hp. Cc ch th thng dng thuc nhm azoic (metyl
da cam, tropeolin 00, ...), bromophenol, bromothymol, ...
Chit cp ion bng cloroform v o quang. Cc alcaloid v base tng hp
c nh lng theo nguyn tc ny.
V d:
Alcaloid: morphin, codein, cocain, atropin, quinin, ...
Base tng hp: novocain, quinolon, ...
Mui amonium bc bn: tetraetyl amonium clorid (Et
4
NCl), tetrabutyl
amonium iodid (Bu
4
NI), ...
Chit cc acid hu c
Cc acid ny khi phn ly thnh anion s to cp ion vi cc cation l cc
ch th mu base (lc malachit, tm tinh th, rodamin v dn xut ...).
Cc dn xut phenol v acid hu c c chit di dng cp ion v o
quang nh:
Dn xut phenol: 2,4 dinitrophenol, acid picric.
Dn xut ca acid benzoic nh: acid 3,5 dinitrobenzoic, acid 2 nitrobenzoic.
Dn xut ca acid salicylic nh: acid 4 - nitro salicylic, acid 3,5
dinitrosalicylic.
Mui alkyl sulfat: lauryl sulfat, dodecyl sulfat.
Mui acid hu c mch thng nh: oleat, stearat.
64
2.6.3.3. Sc k lng hiu nng cao (HPLC)
Khi xy dng chng trnh HPLC phn tch cc dc cht, thng thng
ngi ta s dng k thut to cp ion. V d, nh lng cc cht hu c
mang in tch dng, ngi ta thng thm vo pha ng cc cht c th to
ra anion to cp nh natri lauryl sulfat, natri heptansulfonat, natri
octansulfonat. Vn ny s c cp c th hn mn hc cc phng
php sc k trong chng trnh cao hc.
Ti liu tham kho
1. B Y t (2002). Dc in Vit Nam III. NXB Y hc, H Ni.
2. H Nh Ph (1971). Kim nghim thuc. NXB Y hc, H Ni.
3. Phm Hi Tng, Phm Gia Hu (1987). Ho hc phn tch. NXB Y
hc, H Ni.
4. Nguyn B Hip (1988). Kim nghim Dc Phm. NXB KHKT, H Ni.
5. Block (1959). Phn tch nh tnh (phn II phn ng cation) Bn dch
ting Vit. NXB Gio dc, H Ni.
6. British pharmacopeia BP 2001
The United States of Pharmacopeia USP XXIV (2000).
7. Conors K. A (1982). A Textbook of Pharmaceutical Analysis, third
edition, John Wiley & Sons, Inc, pp 3 - 92.
8. Delvordre - Steinmetz A. C., Prognon P. (1992). Protomtrie en milieu
aqueux et non aqueux dans Analyse pratique du mdicament.
Coordonateur D. Pradeau, Editions Mdicales internationales, Paris,
pp 288 - 344.
9. Le Hoang M. D, Prognon (1992). Oxydo - reductimtrie, dans
Analyse pratique du mdicament. Coordonateur D. Pradeau,
Edition Mediales internationales, Paris, pp. 352 - 385.
10. Skoog D. A, West D. M, Holler F. J (1988). Fundamentals of
Analytical chemistry. Sounders College Publishing, pp 233 - 344.
11. The Merck Index (1996). 12
th
edition.
12. The United States of Pharmacopeia USP XXIV (2000).


65
cu hi t lng gi
2.1. Gii thch phn ng th nh tnh ca:
Cc ion: Amoni; Nitrat; Phosphat; Arseniat v arsenit; Thu ngn; St
(II v III)
Phn t: Ethanol
2.2. Gii thch mc ch ca vic th gii hn tp cht trong thuc.
2.3. Trnh by phng php xc nh gii hn tp cht trong thuc
2.4. Cc cch pha dung dch mu v dung dch th xc nh gii hn tp
cht trong thuc.
2.5. Trong dung dch nc, amin l mt base yu, nhng trong dung mi acid
acetic, n l mt base mnh. Ti sao ?
2.6. Pha dung dch chun acid percloric bng cch ly 17,0 ml acid acid
percloric c 72% (kl/kl) pha va thnh 1000 ml dung dch acid acetic
khan. Tnh s ml anhydrid acetic cn thit phn ng ht vi lng nc
c trong 17 ml acid percloric c dng. Bit khi lng ring ca acid
percloric l 1,60 g/ ml v ca anhydrid acetic l 1,02 g/ ml. (42,3 ml)
2.7. Vit phng trnh phn ng gii thch qu trnh nh lng acid
barbituric trong pyridin vi s c mt ca AgNO
3
. Dung dch chun l
KOH/ methanol.
Nu cht cn nh lng l mui Na barbiturat, qu trnh phn ng c g
khc khng ?
2.8. Ly 20 vin phenobarbital c khi lng 6,025g, nghin mn, cn 2,000 g
ho tan trong DMFA, chun bng dung dch Lithi metylat 0,1000N
ht 8,50ml. Tnh khi lng trung bnh ca phenobarbital trong vin,
bit M = 232,2 (29,8 mg/ vin).
2.9. Cn 0,6120 g diphenhydramin hydroclorid (M = 291,8) ho tan trong acid
acetic bng. Thm 15 ml thu ngn acetat 3,2% v chun bng dung
dch chun HClO
4
0,1145N ht 17,12 ml. Tnh t l % ca dc cht trong
mu (93,45%).
2.10. So snh nh gi 2 phng php Pifer - Wollish v Billon trong nh
lng mui halogenid.
2.11. Ha tan 0,5404g mui vo methanol v nh mc thnh 50,00ml (dung
dch A). Ly 5,00ml dung dch A thm 20,0ml methanol v chun vi
thuc th Karl Fischer ht 9,00ml. Khi xc nh nc trong methanol
trn dng ht 0,80ml thuc th Karl Fischer cho 25,0ml. Tnh
chun ca thuc th Karl Fischer (6,21 mg H
2
O/ml).
66
2.12. Cn 0,2310 g mu phn tch ha tan vo 10,00 ml methanol ( bi tp
2.11) v chun bng thuc th Karl Fischer ht 2,40 ml. Tnh t l %
nc trong mu phn tch trn (5,59%).
2.13. Vit phn ng xc nh nng dung dch chun H
5
IO
6
theo phng
php iod.
2.14. Ch r s mol ca mi sn phm to thnh khi 1 mol cc cht sau phn
ng vi HIO
4
. Vit phn ng
CH
2
OH(CHOH)
4
CH
2
OH
CH
3
CH CHO CH
3
CH CO CH
3
NH
2
NH
2
CH
3
CH CHO C
6
H
5
CH CH
2
CH
2
OH
OH OH
2.15. V s gii thch nh lng HIO
4
d theo phng php Fleury.
2.16. C g khc nhau gia phn t trung ha in v cp ion (xt v cu to
v tnh cht).
2.17. Ti sao nhiu cp ion c th chit c (t dung dch nc) bng dung
mi hu c t phn cc?
2.18. Gii thch ti sao pH dung dch l yu t quan trng nht n s hnh
thnh cp ion?
2.19. Cetyl pyridin clorid l mt base nit bc bn c th to cp ion vi ch th
vng metyl. Cp ion ny d tan trong cloroform. Anh (ch) xut
nguyn tc chit o quang nh lng base ny.
2.20. Mt phng php dc in nh lng cetyl pyridin clorid di dng
chun hai pha bng dung dch chun natri lauryl sulfat 0,004M vi
ch th vng metyl (bi 2.19). Hy v s gii thch chun ny.
67
Chng 3
cc phng php ho l trong kim nghim thuc
Mc tiu hc tp
Gii thch c cch hiu chun my quang ph t ngoi - kh kin.
Trnh by c cc phng php nh lng bng quang ph t ngoi
kh kin.
Gii thch v vn dng c k thut sc k pha o v pha o cp
ion trong cng tc kim nghim thuc.
Trnh by c cc phng php nh lng bng sc k lng hiu
nng cao: phng php chun ngoi, chun ni.
3.1. Phng php quang ph phn t
Phng php quang ph phn t: Ph UV-VIS, ph hng ngoi, ph
hunh quang ng vai tr quan trng trong kim nghim thuc. Hu ht cc
dc in p dng phng php ny trong nh tnh, nh lng v th
tinh khit cc thuc v ch phm.
3.1.1. Quang ph hp th UV.Vis
3.1.1.1. hp th
Khi cho bc x n sc i qua mt mi trng c cha cht hp th th
hp th ca bc x t l vi nng ca cht hp th v chiu dy ca mi
trng hp th (dung dch cht hp th). Mi quan h ny tun theo nh
lut Lambert Beer v c biu din bng phng trnh sau:


Trong :
T: truyn qua
I
0
: cng nh sng n sc ti
I: cng nh sng n sc sau khi truyn qua dung dch.
K: l h s hp th ph thuc , thay i theo cch biu th nng
L: l chiu dy ca lp dung dch
C: nng cht tan trong dung dch

L C K
I T
A
I
. . lg
1
lg
0
= = =
(3.1)
68
Trng hp C tnh bng mol/l, L tnh bng cm
A = . L . C (3.2)
Khi C = 1 mol/l v L = 1 cm th A = (h s hp th phn t )
c trng cho bn cht ca cht tan trong dung dch, n ch ph thuc
vo bc sng. nh lut Lambert-Beer ch ng khi dng bc x n sc.
3.3
C L
A
E C L E A
cm cm
.
. .
% 1
1
% 1
1
= =
Trng hp nng C tnh theo %(Kl/TT) v bng 1%, L = 1cm th:
E (1%, 1cm ) l h s hp th ring, n c trng cho mi cht.
Trong phn tch kim nghim hay dng E (1%, 1cm).
3.1.1.2. iu kin p dng nh lut Lambert-Beer
nh sng phi n sc,
Khong nng phi thch hp: nh lut Lambert-Beer ch ng trong
mt gii hn nht nh ca nng .
Dung dch phi trong sut.
Cht th phi bn trong dung dch v bn di tc dng ca nh sng
UV-VIS.
3.1.1.3. My quang ph
My quang ph thch hp cho vic o ph vng t ngoi v kh kin
bao gm mt h quang hc c kh nng to nh sng n sc trong vng t
200 n 800nm v mt thit b thch hp o hp th.
Ngun sng cho vng t ngoi: n deuteri hoc hydro,
Ngun sng cho vng kh kin: n tungsten.
Hai cc o (cuvet) dng cha dung dch th v dung dch so snh phi c
c tnh quang hc nh nhau.
my loi 2 chm tia, cuvet dung mi c t bn c chm tia so
snh (reference beam) i qua.
Cuvet thch anh (quartz): dng o vng t ngoi v kh kin.
Cuvet thu tinh: ch o vng kh kin.
3.1.1.4. Hiu chun my quang ph (Calibration)
Trong nh tnh v nh lng cc cht bng phng php quang ph
UV-VIS, vic chun ho my ng vai tr quan trng, c bit l trong k
thut nh lng bng o ph trc tip.
69
Hu ht dc in cc nc (Vit Nam, Anh, n , Nht Bn, Chu
u,...) u yu cu kim tra 6 ch tiu k thut ca my quang ph t ngoi
kh kin nh sau:
Kim tra thang di sng: Bng cch s dng cc i hp th ca
dung dch holmium perchlorate.
Vch ph ca n ngun hydro, n deuteri hoc cc vch ph ca n
hi thu ngn c ch ra di y vi dung sai cho php vng t ngoi l +
1nm, vng kh kin l + 3nm:
241,15 nm (H
0
) 404,66 nm (Hg)
253,70 nm (Hg) 435,83 nm (Hg)
287,15 nm (H
0
) 486,00 nm (D)
302,25 nm (Hg) 486,10 nm (H)
313,16 nm (Hg) 536,30 nm (H
0
)
334,15 (Hg) 546,07 nm (Hg)
361,50 (H
0
) 576,96 nm (H
0
)
365,48 (Hg) 579,07 nm (Hg)
y l phng php kim tra ca DVN, D Anh, D n 1996, D
Chu u 1998.
D Nht XII s dng bc x ca n Hg p sut thp bc sng
253,65 nm, 365,02 nm, 435,84 nm v 546,07 nm hoc bc x ca n
deuteri 486,00 v 656,1 nm.
D Trung Quc 1997: s dng cc vch ph sau y ca n thu ngn:
237,83nm ; 253,65nm ; 275,28nm ; 296,73nm
313,16nm ; 334,15nm ; 365,02nm ; 404,66nm
435,83nm ; 546,97nm ; 576,96nm ;
Cc cc i hp th ca dung dch holmium perchlorat khi dng n
hydro: 379,79; 456,13 v 656,28 nm.
n deuteri: 486,02 v 656,10nm.
Dng knh holmium: c cc cc i hp th : 279,4; 287,5; 333,7; 360,9;
460,0; 484,5; 536,2 v 637,5nm.
Kim tra hp th
kim tra hp th, a s cc dc in dng dung dch kali
dicromat trong acid sulfuric

(VN, Anh, Chu u, Nht, Trung quc...). M hay
dng dung dch kali cromat trong kali hydroxyd.
70
Di y l mt s phng php chun ha hp th ca mt s dc in:
o hp th ca dung dch kali dicromat cc di sng ch nh
bng 3.1. Trong bng mi di sng c mt gi tr chnh xc ca hp th
ring E(1%, 1cm) v cc gi hn cho php.
Bng 3.1. hp th ring ca dung dch kali dicromat
di sng nm E(1%, 1cm) Gii hn cho php
235 124,5 122,9 126,2
257 144,0 142,4 145,7
313 48,6 47,6 50,3
350 106,6 104,9 109,2
Pha ch dung dch kali dicromat:
Dc in VN, D Anh, D Chu u, Nht XII, Trung Quc qui nh
nh sau: Ho tan 57,0 n 63,0 mg kali dicromat sy kh n khi lng
khng i 130
0
C trong acid sulfuric 0,005 M/l vi th tch va 1000ml.
Dc in VN, Chu u cn cho thm bng hp th ca dung dch
kali dicromat cha ng 60,06mg K
2
CrO
7
trong va 1000ml acid sulfuric
0,005M/l, dng cuvet dy 1cm (bng 3.2.).
Bng 3.2. hp th ca dung dch kali dicromat
di sng (nm) hp th
235 0,748
257 0,864
313 0,292
350 0,640
Gii hn nh sng lc (stray light)
nh sng lc l nh sng khng i vo mu th m i thng vo detector.
nh sng lc c th c pht hin di sng cho vi knh lc hoc
cc dung dch thch hp.
D VN, Anh, Chu u, n qui nh : kim tra nh sng lc nh sau:
o hp th ca dung dch kali clorid (TT) 1,2% trong cuvet 1cm
= 200nm, so snh vi nc ct, phi ln hn 2,0.
D Trung Quc (1997): kim tra nh sng lc vi dung dch natri iodid
1,00% v dung dch natri nitrit 5,00% (KL/TT).
71
truyn qua ca cc dung dch ny c o so snh vi nc ct trong
cuvet thch anh 1cm phi nh hn 0,8% bc sng 220nm v 380nm.



phn gii (cho phn tch nh tnh)
Khi chuyn lun yu cu, tin hnh o phn gii ca my nh sau:
ghi ph ca dung dch toluen (TT) 0,02% trong hexan (TT) (v/v).
Gi tr ti thiu ca t s gia cc i hp th 269 nm v cc tiu hp
th 266 nm c qui nh trong chuyn lun ring.
rng gii ph ngun (cho phn tch nh lng)
trnh sai s gy ra do rng gii ph ngun, khi s dng my c
rng gii ph thay i di sng chn th rng ca gii ph phi nh
so vi na rng ca bng hp th. Song rng ny phi ln thu
c gi tr cao ca cng nh sng I
o
v vic thu hp rng gii ph phi
khng lm cho hp th tng ln.
Cuvet
Dung sai ca chiu dy lp dung dch ca cc cuvet l + 0,005cm. Khi
c np y vi cng mt dung mi, cc cuvet c nh cha dung dch
th v dung dch so snh phi c truyn qua nh nhau. Nu iu ny
khng c p ng th phi c s hiu chnh thch hp.
Cc cuvet phi c lm sch v chm sc cn thn trnh b xc. C
th bo qun chng trong cn tuyt i trnh mc.
3.1.1.5. ng dng ph UV-Vis trong kim nghim thuc
nh tnh v th tinh khit
Cc cc i hp th l c trng nh tnh ca cc cht.
V d:

Da vo cc i hp th:
Trong dung dch nc
+ Vitamin B
12
c 3 cc i hp th cc bc sng:
278 nm + 1 nm
361 nm + 1 nm
548 nm + 2 nm
+ Vitamin B
2
c 4 cc i hp th cc bc sng:
223nm 375 nm
267nm 444nm
+ Cloramphenicol c 1 cc i hp th bc sng: 278 nm;
72
+ Phenoxymethyl penicillin c 2 cc i hp th 268 nm v 274nm.
T s gia cc cc i hp th cng l i lng c trng cho nh
tnh cc cht.
V d:
+ Vitamin B
12
(USP XXII) : C t s
A
361
/ A
278
= 1,79 - 1,90
+ Phenoxymethyl penicillin (BP.80):
A
268
/ A
274
= 1,20 - 1,25
H s match: h s ny nh gi s tng ng gia 2 ph, mt ph
chun v mt ph th. H s match (Mat) c tnh theo biu thc sau:



Trong :
x v y l hp th ca ph th nht v ph th hai cng mt bc sng.
n l s im chn 2 ph.
Cch nh gi Mat:
[ ]
( ) ( )

n
y
y
n
x
x
y x y x
Mat
2 2
2
2
. ( .
3
10
) 4 . 3 (
+ < 900: hai ph khc nhau.
+ 900 - 990: hai ph c nhng im tng ng cn cn nhc khi kt lun.
+ > 990: hai ph tng t nhau.
+ Xp x 1000: hai ph ging nhau hon ton.
nh lng
Chn iu kin xy dng qui trnh nh lng
xy dng qui trnh nh lng, chng ta cn kho st chn cc
iu kin thch hp .
Chn bc sng hoc knh lc :
+ Chn bc sng ng vi cc cc i hp th, khi ng chun c
dc ln nht, tc l vi cng mt sai s ca mt quang th sai s
ca nng C v bc sng l nh nht.
+ i vi my dng knh lc (quang k): Mu ca knh lc v mu
dung dch phi ph nhau. Nhng cng c trng hp c th chn
knh lc khc.
73
Chn khong nng thch hp :
Khong nng c quan h tuyn tnh vi hp th.
Chn cc iu kin khc :
+ Loi tr nh hng ca cht l: chit cht cn nh lng ra khi hn
hp phc tp (cc dng bo ch). Dng mu trng c cc thnh phn
nh dung dch th nhng khng c cht cn nh lng.
+ Chn pH v dung mi thch hp
+ Thc hin phn ng mu: s hp th ca cc cht l, thuc th cng
c th nh hng ti kt qu nh lng. V vy trong qu trnh lm
phn ng to mu phi ch ti iu kin v cc thnh phn tham
gia phn ng.
Cc phng php nh lng
Phng php o ph trc tip
o hp th A ca dung dch, tnh nng C ca n da vo gi tr
hp th ring (c trong cc bng tra cu).


p dng phng php ny, cn phi chun ho my quang ph c v
bc sng ln hp th.
1%
1cm
C 1cm L
E
A
C L E A
cm
= = = . .
% 1
1
) 5 . 3 (
Phng php gin tip
Cc phng php gin tip nh: phng php ng chun, so snh v
thm chun tng t nh cc phng php ho l khc.
c im ca phng php gin tip:
+ Phi c cht chun so snh,
+ C th khng cn phi chun my.
Hu ht dc in cc nc u s dng c 2 phng php trc tip v
gin tip. Ring dc in M (USP XXI, XXII, XXIII, XXIV) qui nh ch
dng phng php so snh vi chun.
Phng php so snh
Theo nh lut Lambert-Beer, sau khi o hp th ca dung dch
chun so snh (S) v dung dch mu th (X) ta c :
A
S
= K . L . C
S

A
X
= K . L . C
X
y:
+ A
S
: l hp th ca dung dch chun c nng C
S
,
74
+ A
X
: l hp th ca dung dch mu th nng C
X
.
V h s hp th K v b dy L ca cuvet l nh nhau nn ta c:

S
X
S X
S
X
S
X
A
A
C C
C
C
A
A
= = ) 6 . 3 (

Ch : Nng ca dung dch th C
X
v dung dch chun C
S
khng
c chnh lch nhau qu nhiu. Cc nng ny cng gn nhau, kt qu
cng chnh xc.
Phng php thm chun so snh.
Trong phng php quang ph, loi tr cc yu t nh hng gy sai
s cho qu trnh nh lng: X l mu (chit xut), sai lch do thit b v ho
cht thuc th..., ngi ta p dng phng php thm.
* Nguyn tc tin hnh:
Ly hai lng ging nhau ca mt mu th.
Thm mt lng cht chun bit vo mt mu. Tin hnh x l c 2
mu trong cng iu kin (chit xut, pha long...), thu c 2 dung dch:
Dung dch th v dung dch th thm chun.
o hp th ca c 2 dung dch. Thu c:
A
X
: hp th ca dung dch th.
A
X
: hp th ca dung dch th thm chun.
Vi C
S
l nng ca dung dch chun, ta tnh c nng C
X
theo
phng trnh:



X X
X
S X
X S
X
X
X
A A
A
C C
C C
C
A
A

=
+
=
' '
) 7 . 3 (
Phng php ng chun
y l phng php cng hay dng trong phn tch quang ph.
Chun b mt dy chun khong 5 dung dch c cc nng cht chun
C
S
khc nhau.
o hp th A
S
ca dy chun v lp th ca A theo C
o hp th A
X
ca dung dch mu th v da vo ng chun ta xc
nh c nng mu th C
X
.
Ch :
Khi xy dng ng chun nn kho st khong tuyn tnh ca nng
vi hp th.
75
Trng hp dy chun khng tun theo nh lut Lambert-Beer (ng
chun cong) th cn lm thm mt s im chun na vi cc nng gn
nhau hn (khc nhau khng qu 10%).
V th i qua cc v tr gn nht vi cc im thc nghim hoc da
vo bng chun xy dng phng trnh hi qui tuyn tnh hay phi tuyn v
tnh h s tng quan r. Nu r > 0,995 l tt.








A

A
X



0 C
X
C
Hnh 3.1. th ca phng php ng chun A = f (C)
Phng php thm ng chun
Thm nhng th tch ging nhau ca dung dch th vo dy chun cha
nhng lng khc nhau v chnh xc ca cht chun
o hp th ca c dy r v ng chun quan h gia mt quang
vi lng cht chun thm vo.
Giao im ca ng chun vi trc honh (nng ) cho ta nng ca
cht cn nh lng(hnh 3.2.)








A
0,3

0,2
dd th
0,1 thm chun
A th
C dd th
C chun thm vo
Hnh 3.2. th ca phng php thm ng chun
76
K thut o quang vi sai theo bc sng
Trong kim nghim cc dng thuc bo ch, trc tin phi qua cng
on chit hot cht ra khi t dc. Dch chit kh trnh khi mang theo tp
cht. Tp cht ny c th gy sai s cho qu trnh nh lng bng phng
php o quang.
loi tr sai s ny, ngi ta thng s dng k thut o quang vi sai:
Trn ph ca cht nghin cu, chn 2 bc sng
1
v
2
, hiu s
hp th A l ln nht .
A
max
= A

1
A

2
(3.8)
Chun b mt dy dung dch chun vi cc nng khc nhau v o
hp th A 2 bc sng
1
v
2
.
Kho st khong nng tun theo nh lut Lambert-Beer ca A, v
th quan h A - C.
o hp th A ca cht th 2 bc sng trn.
Da vo th A - C suy ra nng cht th trong mu.
Phng php nh lng hn hp
Do hp th c tnh cng tnh nn nu c mt dung dch hn hp gm n
cht th hp th ca hn hp bng tng hp th ca n cht ring r c
cng nng nh trong hn hp. Ti mt bc sng xc nh ta c:
A
hh
= A
1
+ A
2
+ . . . + A
n
= E
1
C
1
+ E
2
C
2
+ . . . + E
n
C
n
(3.9)
thc hin phng php ny, cn phi bit h s hp th ring ca tng
cht cc bc sng khc nhau. (E
j
i
= hp th ring ca cht th i
j
)
Khi s bc sng bng s cht ta c h n phng trnh vi n n s.
(3.10)
( )
( )
( ) n C E C E C E A
C E C E C E A
C E C E C E A
n
n
n
n n n
hh
n n hh
n n hh



+ + + =
+ + + =
+ + + =
...
...... .......... .......... .......... .......... ..........
2 ...
1 ...
2 2 1 1
2
2
2
2 1
2
1
2
1
2
1
2 1
1
1
1
Gii h phng trnh ny s tm c nng ca cc thnh phn trong
hn hp: C
1
; C
2
; . . . ; C
n
Hin nay c nhiu chng trnh my tnh gii h phng trnh nhiu n
s xc nh nng cc thnh phn trong dung dch.
77
Trng hp n gin nht l hn hp gm hai thnh phn, ta c:
A
hh
= A
1
+ A
2
Chn 2 bc sng
1
v
2
v o hp th ca hn hp 2 bc sng .
2
2
2 1
2
1
2
2
1
2 1
1
1
1
C E C E A
C E C E A
hh
hh


+ =
+ =

Gii h phng trnh trn ta tm c:
1
2
2
1
2
2
1
1
2
1
1 1
1
2
2
1
2
2 2
2
1
1



E E E E
E A E A
C
E A E A
C
hh hh
hh hh
=

=

Phng php ph o hm
Ph o hm l mt k thut bao gm s chuyn i ph hp th thnh
ph o hm bc nht, bc hai hoc bc cao hn.
* C s ca phng php ph o hm:
Theo nh lut Lambert-Beer ta c :
hp th A = E.C.L ( C: %,L: cm )
C th ly o hm bc 1, bc 2, hoc bc cao hn ca A i vi bc sng .
A = f( ) (3.11)
+ Ph o hm bc nht l th ca tc bin thin ca hp th
(dA/d) i vi bc sng .
dA/d = dE/d .C.L (3.12)
+ Ph o hm bc hai l th ca cong ca ph hp th (d
2
A/d
2
)
i vi bc sng.
d
2
A/d
2
= d
2
E/d
2
.C.L (3.13)
Do E l hng s v L khng i nn gi tr ca o hm ca A ch cn
ph thuc tuyn tnh vi nng C ca dung dch.
* Ph o hm ca hn hp nhiu cht :
78
Ging nh ph hp th, ph o hm cng c tnh cht cng tnh
A
hh
= A
1
+ A
2
+ ... + A
n
= A
i
( i = 1 .... n ) (3.14)
dA
hh
/d = dA
1
/d + dA
2
/d + ... + dA
n
/d
d
2
A
hh
/d
2
= d
2
A
1
/d
2
+ d
2
A
2
/d
2
+

... + d
2
A
n
/d
2
(3.15)
Theo ngha ton hc ca o hm th :
Ti cc im cc tr, o hm bc 1 bng 0.
Ti cc im un, o hm bc 2 bng 0.
Ti gi tr 0 ca ph o hm ca cht ny c th gp gi tr khc 0 ca
ph o hm ca cht kia.
Nu hp th tun theo nh lut Lambert-Beer th o hm bc hai
bc sng bt k no cng c lin h vi nng bi phng trnh sau :
L C
d
E d
d
A d
.
2
2
2
2

= (3.16)
Ti mt bc sng xc nh, E l mt hng s .
y:
A: hp th bc sng
E: hp th ring bc sng
C: nng %(kl/tt ) ca cht hp th
L: b dy ca lp dung dch cht hp th (cm)
u im ca phng php ph o hm:
Ph o hm c th gip nh lng ring bit c cc cht c cc cc
i hp th gn nhau trong hn hp, trong khi phng php o ph
thng thng khng th gii quyt c.
Trong mt s trng hp, ph o hm gip xc nh c hm lng
ca cht th trong s c mt ca cc tp cht gy cn tr.
Hin nay phng php ph o hm c mt s nc a vo dc
in: Anh, n 1996, Vit Nam.
3.1.2. Quang ph hng ngoi
3.1.2.1. M u
Ph hng ngoi l phng php o s hp th bc x hng ngoi (IR) khi
n i qua mt lp cht cn th, cc s sng khc nhau.
79
Vng bc x hng ngoi s dng trong cc my quang ph IR thng
thng l 600-4000cm
-1
.
Cc my hin nay c th m rng vng bc x (100-10.000cm
-1
).
Trong phn t khi c nhm nguyn t no hp th nng lng v
thay i trng thi dao ng th to nn mt di hp th trn ph IR.
C mi tng quan gia nhm nguyn t v di hp th nn c th da
vo s c mt ca di hp th nhn bit mt nhm chc no .
Nhiu nhm chc c cc di ph hp th c trng. y l c s ca vic
phn tch cu trc bng IR (bng 3.3.).
Vic xc nh c s c mt ca cc nhm chc trong phn t gip
chng ta c th s dng ph IR nh tnh mt cht.
Trong kim nghim thuc hu ht cc dc in ch yu ch s dng IR
vo vic nh tnh, t c dng trong nh lng.
Bng 3.3. Mt s pic hp th hng ngoi c trng
Pic hp th
Nhm chc
S sng (cm
-1
) Bc sng m
O-H Mch thng, vng thm 3600-3000 2,8-3,3
NH
2
Bc hai v bc ba 3600-3100 2,8-3,2
C-H Thm 3150-3000 3,2-3,3
C-H Mch thng 3000-2850 3,3-3,5
CN Nitril 2400-2200 4,2-4,6
CC- Alkyn 2260-2100 4,4-4,8
COOR Ester 1750-1700 5,7-5,9
COOH Acid carboxylic 1740-1670 5,7-6,0
C=O Aldehyd v ceton 1740-1660 5,7-6,0
CONH
2
Amid 1720-1640 5,8-6,1
C=C- Alken 1670-1610 6,0-6,2
Ar-O-R Thm 1300-1180 7,7-8,5
R-O-R Mch thng 1160-1060 8,6-9,4
3.1.2.2. My
My quang ph ghi ph trong vng hng ngoi bao gm mt h quang
hc c kh nng cung cp nh sng n sc trong gii ph t 4000cm
-1
n 670
cm
-1
(2,5m-15m) hoc trong mt vi trng hp ti 200cm
-1
(50m) v mt
phng tin o t s gia cng nh sng truyn qua v nh sng ti.
80
3.1.2.3. ng dng ph hng ngoi trong nh tnh
Phng php quang ph hng ngoi ch yu c ng dng trong nh
tnh cc cht hu c. Vic nh tnh ny da trn 2 nguyn tc:
So snh s ph hp gia ph cht th vi ph chun cho sn trong sch
tra cu (atlas) hoc trong th vin ph lu gi trong my tnh.
So snh s ph hp gia ph cht th vi ph ca ho cht chun c
ghi trong cng iu kin.
Phng php nh tnh bng ph hng ngoi ca hu ht cc dc in
trn th gii u da trn hai nguyn tc ny.


nh tnh s dng cht chun so snh
Chun b mu th v mu cht chun so snh ri o ph ca chng t
4000cm
-1
n 670cm
1
(2,5m - 15m) trong cng iu kin .
Cc i hp th ph ca cht th phi tng ng vi ph ca cht
chun v v tr v tr s.
nh tnh s dng ph chun trong atlas hoc th vin ph .
c th so snh ph ca cht th vi ph chun tra cu trong atlas
hoc th vin ph ca my tnh, trc ht chng ta cn phi chun ho my
quang ph.
Tt c cc dc in u chun ho 2 thng s c bn ca my: phn
gii v thang s sng. Tm tt cch lm nh sau:
Chun ho phn gii:
Ghi ph ca phim polystyren dy 0,05mm
+ Hiu s gia phn trm truyn qua cc tiu hp th A 2870cm
-1
(3,48m) v cc i hp th B 2849cm
-1
(3,51m) phi ln hn 18.
+ Hiu s gia phn trm truyn qua cc tiu hp th C
1589,5cm
-1
(6,29m) v cc i hp th D 1583cm
-1
(6,32m) phi
ln hn 12.
Chun ho thang s sng
Cng c th chun ho thang s sng bng cch s dng phim polystyren.
Phim ny c cc tiu truyn qua (cc i hp th) cc s sng (cm
-1
) (bng 3.4.).
81
Bng 3.4. Cc i hp th ca phim polystyren (cm
-1
)
3027,1 (+ 0,3 ) 1583,1 (+ 0,3 )
2924,0 (+ 2 ) 1181,4 (+ 0,3 )
2850,7 (+ 0,3 ) 1154,3 (+ 0,3 )
1944,0 (+ 1 ) 1069,1 (+ 0,3 )
1871,0 (+ 0,3 ) 1028,0 (+ 0,3 )
1801,6 (+ 0,3 ) 906,7 (+ 0,3 )
1601,4 (+ 0,3 ) 698,9 (+ 0,5 )
3.1.3. Quang ph hunh quang (Fluorometry)
3.1.3.1. M u
Ph hunh quang l phng php ph pht x phn t. Sau khi hp th
nng lng ca bc x t ngoi, kh kin hoc cc bc x in t khc (bc x
kch thch), phn t b kch thch s tr li trng thi c bn v gii phng
nng lng di dng bc x, c gi l bc x hunh quang.
Cng hunh quang F ca mt dung dch long t l thun vi nng
C (mol/l) trong iu kin xc nh khi cng v bc sng ca bc x
kch thch l hng s.
F = K . I
0
. . . C . L (3.17)
y :
K : hng s
I
0
: cng ca bc x kch thch
: hiu sut hunh quang
S photon pht x
= ------------------------- ( 0 < < 1 ) (3.18)
S photon hp th
: H s hp th mol ca cht bc sng kch thch.
Rt gn li ta c: F = K

. C.
y K

= K . I
0
. . L .
Vic o ph hunh quang ch nn tin hnh vi dung dch long C <10
-4

trnh nh hng suy gim cng ca bc x pht ra.
3.1.3.2. My
Ngun sng: thng dng n xenon (to nh sng =200-800nm), laser.
n xenon cho ph bc x khng u nn km nhy hn ngun laser.
82
B n sc: dng cch t
Cuvet: thng dng thch anh (1 x 1cm) c 4 mt trong sut.
3.1.3.3. Chun ho my
My quang ph hunh quang phi c chun ho thng xuyn vi
cht chun.
Tin hnh chun my theo ti liu hng dn ca hng sn xut my.
3.1.3.4. Phng php o
Mu th lun lun c o so snh vi mu chun.
Kim tra vng tuyn tnh ca cng hunh quang v iu chnh
nhy ca thit b vi pha long thch hp ca dung dch chun .
Ghi cng hunh quang ca dung dch th, dung dch chun v cc
mu trng tng ng ca chng.
Tnh ton nng ca dung dch th :
OS S
OX X
S X
I I
I I
C C

= (3.19)
y:
C
X
: nng ca dung dch th
C
S :
nng ca dung dch chun
I
X
: tr s o c ca dung dch th
I
S
: tr s o c ca dung dch chun
I
0X
v I
0S
: tr s o c ca cc mu trng tng ng.
Vng trong cng hunh quang t l thun vi nng ca cht
thng rt hp, cho nn t s (I
X
- I
0X
)/ (I
S
- I
0S
) phi khng c < 0,50 v
khng > 2,0. Nu t s khng nm trong khong trn th phi iu chnh
nng v tin hnh o li.
Trong mt s trng hp, c th s dng cch o so snh vi chun c
nh. Th d: dng knh hunh quang hoc mt cht pht hunh quang khc.
Nu cng hunh quang khng t l nghim ngt vi nng th nn
s dng phng php th chun tng quan gia cng v nng .
nhy ca my c th c kim tra bng mt dung dch bn ca cht
hunh quang khc vi di sng kch thch v pht x tng t nh di ca
cht th, thay th cho cht chun.
V d:
Quinin trong H
2
SO
4
long cho hunh quang mu xanh,
83
Natri fluorescein cho hunh quang xanh l cy,
Rhodamin B cho hunh quang mu .
tinh khit ca dung mi c th nh hng ng k ti cng hunh
quang. Nu cn nn ct li trong my ct bng thu tinh trc khi dng.
S c mt ca cc tiu phn keo c th gy nn s tn x nh sng, cho
nn cn phi loi b chng bng ly tm hoc lc bng mng lc thu tinh xp.
Oxy ho tan trong dung dch lm gim cng hunh quang rt mnh,
cn phi ui n bng cch sc kh tr qua dung dch.
3.2. Phng php sc k lng hiu nng cao
(High- Performance Liquid Chromatography- HPLC)
Sc k lng hiu nng cao i khi cn c gi l sc k lng p sut cao
(High-Pressure) l k thut phn tch da trn c s ca s phn tch cc cht
trn mt pha tnh cha trong ct, nh dng di chuyn ca pha ng lng di
p sut cao. Sc k lng da trn c ch hp ph, phn b, trao i ion hay
loi c l tu thuc vo loi pha tnh s dng. Khi phn tch sc k, cc cht
c ha tan trong dung mi thch hp v hu ht s phn tch u xy ra
nhit thng. Chnh v th m cc thuc khng bn vi nhit khng b phn
hy khi sc k. Sc k thng c hon thnh trong mt thi gian ngn
(khong 30 pht). Ch nhng thnh phn c h s chn lc khc nhau mi c
th phn tch c bng HPLC. Ngy nay HPLC v ang c s dng
nhiu trong lnh vc phn tch ho hc ni chung cng nh trong kim tra
cht lng thuc v phn tch sinh dc hc ni ring.
Trong phn tch thuc bng phng php sc k, phn ln cc dc in
u s dng sc k phn b. Nn chng ti ch yu cp ti phng php ny.
3.2.1. Cc thng s c trng ca qu trnh sc k
H s dung lng k (Capacity factor)








Hnh 3.3. Minh ha cc thng s sc k
84
o
t
o
t
R
t
o
t
R
t
m
Q
S
Q
m
V
s
V
K k

= = = =
'
'
(3.20)
y:
K : h s phn b
V
s
: th tch pha tnh
V
m
: th tch pha ng
Q
s
: lng cht trong pha tnh
Q
m
: lng cht trong pha ng
t
R
: thi gian lu
t
R
: thi gian lu hiu chnh
t
0
: thi gian cht
Cn chn ct, pha ng ... sao cho k nm trong khong ti u : 1 < k< 8.
H s chn lc :
RA
RB
A
B
t
t
k
k
= =
'
'
(3.21)
Qui c y B l cht b lu gi mnh hn A nn >1
tch ring 2 cht thng chn 1,05 < < 2,0

H s i xng ca pic F:
a
W
F
2
= (3.22)
y:
W: Chiu rng pic o 1/20
chiu cao pic,
a: Khong cch t ng vung
gc h t nh pic n mp
ng cong pha trc ti v
tr 1/20 chiu cao pic.


Hnh 3.4: Xc nh h s i xng
85
S a l thuyt v hiu lc ct N:
Hiu lc ct c o bng thng s: S a l thuyt N ca ct

2
2 / 1
2
2
2
54 , 5 16
W
t
W
t
N
R R
= = (3.23)
y:
W: Chiu rng o y pic,
W
1/2
: Chiu rng pic o na chiu cao pic.

phn gii R
s
:
Vi:
t
RB
,

t
RA
: Thi gian lu ca 2 pic lin k nhau (B v A),
W
B
, W
A
: rng pic o cc y pic,
W
1/2B
, W
1/2A
: rng pic o na chiu cao pic.
Cc gi tr: t
RB
,

t
RA
, W
B
, W
A
, W
1/2B
, W
1/2A
phi tnh theo cng mt n v.
Yu cu R
S
> 1, gi tr ti u R
S
= 1,5.

3.24
A B
RA RB
A B
RA RB
S
W W
t t
W W
t t
R
2 / 1 2 / 1
) ( 18 , 1 ) ( 2
+

=
+

=
3.2.2. My HPLC






Hnh 3.5: S nguyn l ca my HPLC.
1- Bnh dung mi 4- Ct 7- Bnh nc thi
2- Bm 5- Detector
3- Tim mu 6- Phn mm x l
86
My sc k lng hiu nng cao gm c cc b phn sau:
Bnh cha pha ng, bm y pha ng qua h thng sc k p sut
cao, h tim mu a mu vo pha ng, ct sc k, detector, my tnh hay
my tch phn hoc my ghi.




H bm
H bm HPLC c chc nng to p sut cao y pha ng t bnh
dung mi qua h thng sc k.
H bm hin i hin nay c iu khin bng my tnh c th lp
chng trnh thay i t l ca cc thnh phn pha ng theo yu cu (sc
k gradient).
Bm HPLC cn phi p ng mt s yu cu :
To c p sut cao 3000-6000 psi (250-500 atm), lu lng bm khong
0,1 n 10ml/pht, khng b n mn vi cc thnh phn pha ng, c tc
bm khng i.
H tim mu
C th dng bm tim tim mu vo u ct. Phng php ph bin l
dng van tim c vng cha mu (sample loop) c dung tch xc nh v chnh
xc, c th thay i vng cha mu vi cc dung tch khc nhau.
Mt s my HPLC hin i c h tim mu t ng c th lp trnh iu
khin th tch mu, s ln tim v chu k ra vng cha mu.
Ct
Ct c dng ph bin lm bng thp khng g, thng thng c chiu di
10-30cm, ng knh trong t 2- 5 mm, ht cht np (packings) c 5-10 m. Ct
c ng knh trong ln hn c dng cho sc k iu ch. Ct c th c lm
nng t hiu qu phn tch tt hn, nhng him khi tin hnh nhit
trn 60
o
C v nhit c th lm suy gim hiu lc ct hoc lm pha ng bay hi.
Cht np thng l silica gel hoc silica gel c bao mt lp mng hu c hoc
lin kt ha hc vi cc nhm chc hu c. Bn cnh silica gel ngi ta cn dng
cc cht liu khc nh: nhm oxyd, polyme xp, nha trao i ion....
Detector
Detector l b phn pht hin v o cc tn hiu sinh ra khi c cht ra
khi ct v cc tn hiu ny c ghi di dng pic trn sc .
Cc loi detector c dng hin nay:
Detector t ngoi v kh kin (UV-VIS) c bc sng tu chn c dng
ph bin nht.
Detector hin i thuc loi ny l detector dy diod (diode array
detector). Detector ny lun lun c t l tn hiu nhiu thp hn so vi
detector UV-VIS bnh thng.
87
Cc loi detector khc :
Detector o ch s khc x
Detector hunh quang, in ho.
3.2.3. Cc k thut HPLC.
C 4 k thut HPLC c bn: Sc k phn b, hp ph, trao i ion v sc
k loi c (ry phn t). Trong phm vi cun sch, ch yu chng ti gii thiu
v sc k phn b, v y l k thut HPLC c s dng nhiu nht trong
kim nghim thuc. Cn cc k thut khc, ch c tnh cht gii thiu tm tt.
Sc k phn b hiu nng cao (Partition Chromatography)
Pha tnh
Pha tnh trong sc k phn b bao gm mt lp mng pha lng hu c
bao trn b mt ca cc tiu phn cht mang silica hoc cc cht liu khc.
Cc tiu phn ny thng c ng knh t 3 n 10 m, c ht c th tng
n 50 m hoc ln hn cho sc k iu ch. Sc k dng loi pha tnh ny
c gi l sc k lng- lng (Liquid - liquid Chromatography - LLC). Nhng
kiu pha tnh ny c nhc im l b ra tri dn theo dng pha ng. Kt
qu, hiu lc ct b gim dn trong qu trnh s dng.
khc phc nhc im trn, ngi ta s dng k thut sc k pha
lin kt (Bonded- Phase Chromatography - BPC). Trong k thut ny, pha
tnh c lin kt ha hc vi cht mang. K thut ny nhanh chng tr
thnh k thut ph bin nht trong HPLC. Trong loi pha tnh ny, cc nhm
chc hu c lin kt vi b mt ca cc tiu phn silica qua cc nhm silanol.
Tnh phn cc ca loi pha tnh ny ph thuc vo tnh phn cc ca cc nhm
chc lin kt, chng trong vng t nhm khng phn cc octadecyl silan n
nhm rt phn cc nitril.
Di y l mt s pha lin kt thng dng:
Octyl = Si- (CH
2)
)
7
- CH
3
C
8
Octadecyl = Si- (CH
2)
)
17
- CH
3
C
18
Phenyl = Si- (CH
2
)
n
- C
6
H
5
C
6
H
5
Cyanopropyl = Si- (CH
2
)
3
- CN CN
Aminopropyl = Si- (CH
2
)
3
- NH
2
NH
2
Diol = Si- (CH
2
)
3
- OCH (OH) - CH
2
- OH
H bao gm pha tnh phn cc v pha ng khng phn cc c gi l
sc k pha thun. Ngc li, pha ng phn cc cn pha tnh khng phn cc,
c gi l sc k pha o.
Khi s dng silica, nhm oxyd hoc polyme xp th cc cht c phn
tch theo c ch hp ph nn c gi l sc k hp ph.
88
Nu pha tnh l nha trao i ion th gi l sc k trao i ion .
Nu pha tnh l polyme xp nh dextran..., ta c sc k loi c (sc k
ry phn t Size Exclution Chromatography).
Pha ng
i lc ca mt thnh phn i vi pha tnh hay ni mt cch khc, thi
gian lu gi ca n trn ct c iu khin bng cch thay i phn cc
ca pha ng. Pha ng c th l dung mi n hay hn hp ca 2, 3 hay 4
thnh phn. Ngi ta c th thay i phn cc ca pha ng bng cch
thay i t l ca cc thnh phn dung mi trong hn hp. K thut thay i
lin tc thnh phn dung mi trong thi gian chy sc k c gi l ra gii
gradient hoc chng trnh ha dung mi.
Tu thuc vo vic s dng pha ng v pha tnh ngi ta chia sc k
phn b thnh 2 loi (sc k pha thun v sc k pha o):
* Sc k pha thun (normal phase):
Trong k thut ny ngi ta s dng cht np ct (packings) gm cc
nhm phn cc lin kt vi cht mang nh nhm alkylnitril [-(CH
2
)
n
CN ] v
alkylamin [-(CH
2
)
n
NH
2
]. Dung mi s dng l dung mi ca sc k hp ph
(sc k lng rn - LSC). K thut ny, s dng pha tnh phn cc hn pha
ng, c gi l sc k phn b pha thun. S phn tch t c bng sc
k pha thun cng tng t nh LSC. Song pha tnh trong sc k pha lin
kt c u im hn pha tnh trong LSC:
. Ct nhanh t cn bng nn rt thun li cho phn tch gradient.
. Ct thch hp vi nhiu dung mi v d ti sinh.
. Ct rt bn.
. Ct alkylamin c th c chc nng nh mt ct trao i anion yu.
Pha ng trong sc k pha thun ni chung khng phn cc, thng
dng hn hp ca cc hydrocarbon mch thng nh pentan, hexan, heptan,
isootan. Cc dn cht halogen dy bo cng c s dng nh: diclorometan,
dicloroetan, butyl clorid v cloroform. C th thm vo cc pha khan nc ny
1- 2% acid acetic hoc phosphoric ngn cn s ion ha ca cc cht nh: cc
phenol, acid carboxylic. Ngc li, s ion ha ca cc base yu c th c km
li bng cch thm mt lng nh amoniac hoc mt amin khc (0,1- 1%).
Ngoi ra cng c th thm vo pha ng nhng cht ci bin hu c (organic
modifier) khc nh cc alcohol, tetrahydrofuran.
Ni chung, vic ui kh ha tan khng thnh vn vi dung mi pha
thun.
Trong k thut sc k pha thun cc cht khng phn cc s c ra
gii sm. Th t ra gii s chm dn theo chiu tng ca phn cc ca cc
thnh phn trong mu th.
89
V d: Tch hn hp steorid bng sc k pha thun.
iu kin sc k:
- Ct: MicroPak NH
2
2,2 x 150 mm, 5 m.
- Pha ng: hexan cha 0,2% isopropanol.
- Tc dng: 1 ml/ pht.
- Detector: UV 254 nm










pht
Hnh 3.6: Sc tch cc steroid
1- Progesterone 5- Andrenosterone
2- Androstendione 6- Cortisone acetat
3-
4
- pregnon-20--ol-one 7- Cortisone
4- 17--hydroxyprogesterone 8- Hydrocortisone
* Sc k pha o (reversed phase) :
Trong kim nghim thuc bng phng php HPLC, sc k phn b pha
o c s dng nhiu nht.
Trong k thut ny pha tnh bao gm cc nhm khng phn cc nh
octadecyl (C
18
), octyl (C
8
) hay phenyl (C
6
H
5
). Pha ng l nhng dung mi
phn cc nh: nc, methanol (MeOH), acetonitril (ACN). Tm li, pha ng
phn cc hn pha tnh.
chn lc ca k thut ny khc bit ng k vi qu trnh tch trn
pha thun. Ngi ta thy rng, mt cht cng tan tt trong nc th n c
ra gii khi ct cng nhanh. Quy tc ra gii ny ngc vi pha thun.
Vi cc cht c ra gii qu sm trong sc k pha thun, chng s rt
kh tch ri khi nhau. Trong khi , pha o cc thnh phn ny s c
ra gii vo gn cui ca qu trnh sc k lm cho vic tch d dng hn (c
ngha l k

ln hn).
90
Khi mt pic nh c ra gii sau mt pic ln th thng rt kh nh
lng bng sc k pha thun. Tuy nhin dy ra gii c th o ngc li bng
cch dng sc k pha o. Khi pic nh c th c xc nh chnh xc hn.
Thnh phn u tin ca pha ng trong sc k pha o l nc. Nhng
dung mi ha ln c vi nc nh methanol, ethanol (EtOH), acetonitril,
dioxan, tetrahydrofuran (THF) v dimethylformamid c thm vo iu
chnh phn cc ca pha ng. Mt s thnh phn khc cng c th c
thm vo pha ng nh: cc acid, base, m , cht din hot.
Cp dung mi c s dng ph bin nht trong sc k pha o l:
H
2
O - MeOH v H
2
O ACN. iu d nhn thy, MeOH c nhc im l
nht cao ca n c th lm gim hiu lc ct. Trong khi ACN c nht
thp hn, thch hp hn vi cc cht khng phn cc v th n thng c
dng nhiu hn MeOH. Thi gian lu s ngn hn nu nng ACN bng
nng MeOH.
Ch : Khi pha ng c thm cc mui v c hoc cc cht hot ng b
mt, nn lc n trc khi dng v c th c cn khng tan trong nc gy bn ct.
Vic ui kh rt quan trng vi pha ng pha o.
V th t ra gii: Trong sc k pha o cc cht phn cc ra trc, cc
cht t v khng phn cc ra sau.
V d: Tch hn hp morphin, codein v heroin bng HPLC pha o
(hnh 3.7).
iu kin sc k:
- Ct: Zorbax Rx, Sb- C 8, 4,6 x 150 mm, 5m.
- Pha ng: ACN/KH
2
PO
4
25 mM, pH 3,5 7/ 93.
- Tc dng: 1,5 ml/pht
- Detector: UV 254 nm






Hnh 3.7: Sc tch morphin, codein, heroin.
Pht
1 - Morphin 2 - Codein 3 - Heroin.
91
Sc k cp ion (Ion-Pair Chromatography IPC)
Sc k cp ion da trn c s s to thnh cp t ion cn phn tch mang
in v tc nhn to cp mang in tri du (i ion).
[ ]
hc n n
XQ Q X = +
+
Nhn chung, IPC thc cht l HPLC pha o.
Dung mi: Chnh l dung mi trong pha o, thng phi hp H
2
O-
MeOH v H
2
O-ACN. im quan trng nht l phi m bo rng thuc th
to cp ion tan ht trong pha ng trnh kt ta trong h thng.
i ion: i ion thng l cc amin bc 4 hoc cc mui sulfonat. Cn
phi iu chnh pH ca pha ng vi m ti gi tr thch hp. ng thi phi
kim tra tan ca m trong dung mi trc khi bm qua h sc k.
Ct: Dng cc cht np pha o vi cc nhm alkyl C
2
, C
8
, v C
18
....
V d: Tch hn hp vitamin tan trong nc bng HPLC cp ion pha o.
iu kin sc k:
- Mu th: Vitamin tan trong nc.
- Ct: Zorbax SB C 8, 4,6 x 150 mm, 5 m.
- Pha ng: ACN/ MeOH/ diethylamin 2%/ acid acetic 2%
(2/ 3/ 4/ 28) v 1,1 g Na heptan sulfonat/ lit.
- Tc dng: 1 ml/ pht.
- Detector: UV 280 nm

Mu
100
88
66
48
28
0
0 1 3 4 5 6
7
8
n
2
DAD1 A, Sig = 280, 4 Ref = 360,100
1
2
3
4
1
7
.
0
5
9

5
.
5
8
3

3
.
2
7
2

2
.
1
5
1










Hnh 3.8: Sc tch hn hp vitamin tan trong nc.
1. Vitamin PP 2. Vitamin B
6
3. Vitamin B
2
4. Vitamin B
1
92

Sc k hp ph hiu nng cao (Liquid Solid Chromatography LSC).
L k thut pht trin sm nht v c dng ph bin. Trong k thut
ny, cht cn phn tch b gi trn b mt pha tnh (cht hp ph) v b dung
mi y ra (phn hp ph).
Pha tnh c th l silicagel, nhm oxyd ... nhng silicagel c dng nhiu
hn. Pha ng thng l nhng dung mi t hoc khng phn cc. Khi ra gii,
cc cht khng phn cc s ra trc, cc cht cng phn cc cng ra chm.
V d: Tch hn hp steroid acetat bng HPLC hp ph
iu kin sc k:
- Mu th: Hn hp steroid acetat
- Ct: LiChrosorb Si 60 4,6 x 250 mm, 7 m.
- Pha ng: Chloroform/ methanol 95,5/ 0,5.
- Tc dng: 1,5 ml/ pht
- Detector: 254 nm









Hnh 3.9: Sc tch hn hp steroid acetat
1. 11- Deoxycorticosteron 4. Hydrocortison
2. Cortison 5. Prednisolon
3. 9 - Fluorohydrocortison acetat
93
Sc k trao i ion (Ion Exchange Chromatography IEC)
Sc k trao i ion c dng phn tch cc thnh phn ion ha tan
trong nc, c phn t lng nh hn 1500. Pha tnh l cc nha trao i ion
di dng bt mn. l nhng hp cht hu c cao phn t c cha nhm
chc c kh nng trao i.
Nha c nhm hot ng mang in tch m, dng tch cc cht
base nh cc amin.
Nha c nhm hot ng tch in dng phn tch cc cht c
nhm mang in m, nh cc nhm phosphat, sulfonat, carboxylat.
pH ca pha ng, nhit , loi ion, nng ion, v cc cht ci bin hu
c nh hng n cn bng, nhng c th iu chnh c thu c kt qu
tch mong mun.
Sc k loi c (Size Exclusion Chromatography SEC)
Phng php SEC c ng dng ch yu phn tch cc cht c phn t
lng (MW) ln hn 2000. Pha tnh l nhng ht xp ca silicagel hay polyme.
Khi sc k, cc phn t ca cc cht phn tch c lc theo c ca khi
lng phn t. Khi cho hn hp cn phn tch qua ct, ta thy:
Cc phn t c MW qu ln, chy nhanh qua ct, chng khng b lu gi.
Cc phn t c MW nh hn th b lu gi trn ct. Khi ra gii chng
s ln lt ra khi ct vi thi gian lu tng theo chiu gim ca khi
lng phn t.
3.2.4. Hng dn chn k thut HPLC
Trong HPLC, c 4 k thut sc k c bn: phn b, hp ph, trao i ion
v sc k loi c. Mun chn mt k thut sc k thch hp phn tch mt
hn hp, phi da vo cc c tnh l, ha hc ca cc cht thnh phn:
+ Tnh tan: tan hay khng tan trong nc.
+ Phn t lng (MW).
+ Tnh phn cc: ion ha hay khng ion ha.
Hnh 3.10. trnh by s chn k thut HPLC.Tt nhin, y cha phi
l mt ch dn tht chnh xc m ch l mt nh hng s b.
Nu phn t lng ln hn 2000: nn s dng sc k ry phn t. Dng
dung mi l nc nu mu th tan trong nc. Nu mu tan trong
dung mi hu c, th dng dung mi hu c lm pha ng.
Pha tnh l Sephadex (dextran) hoc Bondagel Series E th pha ng
l nc. Cn Styragel hoc gel MicroPak TSK th dng pha ng hu c.
Nu phn t lng di 2000: trc ht phi xc nh xem mu c tan
trong nc hay khng.
94
+ Nu mu tan hoc t tan trong nc th c th s dng sc k trao i
ion hoc phn b pha o.
+ Nu tan c tng ln khi thm acid hoc kim th dng k thut
trao i ion.
+ Nu tan khng b nh hng bi acid hoc kim v dung dch nc
ca n hon ton trung tnh th nn chn sc k phn b pha o.
+ Nu mu khng tan trong nc th nn s dng sc k phn b pha
thun hoc LSC.
+ tch cc ng phn, nn dng LSC.
+ tch cc ng ng, nn dng sc k phn b.
Cn phi nhc li rng nhng thng tin dn ch l nhng gi ban
u, khng mang tnh bt buc. Tt nhin, m bo vic phn tch sc k
c thnh cng nh kim nghim cn phi tin hnh kho st bng thc
nghim.

















95
Tnh cht
mu th
K thut SK Ct

























tan
mu th
Hexan
C
18
CN
C
8
C
3
Pha o C
18
Alcol
Nc
MW < 2000
MW > 2000
Khng
ion ha
Ion ha
Peptid
- Pha thun
- Hp ph
Pha o
Pha o
Pha o
cp ion
Trao i
ion
Loi c
(ry phn t)
SiCN
C
18
PTH
Amin
C
18

Amin
C
18
- IP
Spherogel
Amin
SAX SCX
C
18
C
8
Tan/
nc
Khng
tan/nc
SEC,
pha nc
SEC,
pha hu c
USG SEC
2000
mu
Th

Hnh 3.10: S hng dn chn k thut HPLC
96
Bng 3.5: iu kin phn tch mt s thuc bng HPLC
Tn thuc Ct Pha ng Detector Mu th
Acenocumarol

RP - 2

ACN/ H
2
O/CH
3
COOH 305 Huyt
tng
Acephyllin C - 18 MeOH/ NaH
2
PO
4
0,01 M

280

Huyt
thanh
Acetaminophen
Guaifenesin
Dextromethorphan
C 18 HCOONH
4
/ MeOH/H
2
O
10/ 450/ 540 (pH 4,2)
254 Siro ho
Acetaminophen
Aspirin
Caffein
Codein
Phenacetin
Salicylamid
C 18

MeOH/ NaH
2
PO
4
0,01 M
19/ 81 pH 2,3

254 Vin nn
Acetaminophen
P aminophenol
Phenyl

TBA phosphat 0,005 M /ACN 85/15 pH 7,5 254 Vin nn
Acetazolamide C - 18 ACN / acetat 0,05 M 254 Huyt
tng,
nc tiu
Acetazolamide Silica

Hexan/CHCl
3
/MeOH/AcOH
65 /25 / 10 / 0,25
254 Nc tiu
Acid aminocaproic C 8 MeOH / H
2
O / H
3
PO
4
40 / 60 / 0,1 345 Huyt
thanh
Acid 5- aminosalicylic C 18 MeOH / H
2
O / H
3
PO
4
40 / 60 / 0,1 345 Huyt
tng
Acyclovir

C 18 NaOAc 0,005 M, Na heptansulfonat 0,0025 M, pH
6,5
300; 418 Huyt
tng
Amoxicilin C 18

H
2
O / MeOH / AcOH
85 / 15 / 0,5
395; 485 Nc tiu
Amoxicilin
Ampicilin
C - 8 KH
2
PO
4
0,067 M (pH 4,6) / MeOH 425 / 75 225 Huyt
tng
Nc tiu
Antipyrin C 18 ACN / AcOH 1% 35/65 254 Huyt
tng
Apomorphin Phenyl MeOH / ACN / m pH 3,5 cha Na laurylsulfat 273 Huyt
thanh
Acid ascorbic C 18 H
3
PO
4
0,8% trong nc 254 Nc tiu
Aspirin Silica Heptan/ AcOH 95/ 5 300 Vin nn
97
Tn thuc Ct Pha ng Detector Mu th
Aspirin
Acid salicylic
Acid salicyluric
Acid gentisic
C- 18 ACN/ AcOH 1% 20/ 80 280 Huyt
tng,
nc tiu

Atenolol Cyano

Acid heptansulfuric/ triethanolamin 0,2 M / MeOH
2/100/ 1900
220 Huyt
tng
Barbiturat C 18 MeOH/ NaH
2
PO
4
0,1 M
60/ 40
240 Huyt
thanh
Benzocain C 18 MeOH/ NaH
2
PO
4
0,1 M
60/ 40
240

Huyt
thanh
Benzocain C 18 MeOH/ ACN/ H
2
O 254 ; 294 Dung dch
Acid benzoic
Acid salicylic
C 18 MeOH/ KH
2
PO
4
10/ 90
pH 6,2
254 M
Benzoyl peroxyd C 18 ACN/ H
2
O 50/ 50 M
Beta methason
Sodium phosphat
C 18 MeOH/ KH
2
PO
4
0,09 M 60/ 40

254 Dung dch
Bromazepam

C 18 MeOH/ ACN/ H
2
O (c cha 20 ml TBAH 10%/ lit
20/ 300/ 70
230 Huyt
tng
Caffein Silica THF/ CH
2
Cl
2
20/ 80

272 Huyt
thanh
Camphor Silica Heptan/ CHCl
3
60/ 40

254 Dng phn
liu
Captopril C 18 MeOH/ K
3
PO
4
(pH 6,5) 52/ 48 340 Huyt
tng
Carbamazepin C - 18 52/ 48
ACN/ H
2
O 50/50
288 Huyt
tng
Chloramphenicol C 8 MeOH/ H
3
PO
4
0,05 %
40/ 60
280 Huyt
tng
Chlodiazepoxide

C 18 ACN/ NH
4
CO
3
0,1% 260 Huyt
tng
Chloroquin

C 18 MeOH/ H
2
O/ AcOH
80/ 19/ 1 Na heptansufonat
340 Huyt
tng
Chlopheniramin

Cyano

ACN/ MeOH/ phosphat 0,015 M (pH 6,6) 25/ 25/ 50

254 Nc tiu
Chlopromazin Cyano Acetat 0,05M(pH 6,5)/ ACN 254 Huyt
tng
98
Tn thuc Ct Pha ng Detector Mu th
Chlorzoxazon C 18 MeOH/ H
2
O 60/40 254 Huyt
tng
Cimetidin C 18 MeOH/ H
2
O 60/40 180 Huyt
tng
Cimetidin C 18 ACN/ H
2
O/ NH
4
OH
1000/ 50/ 1
Huyt
tng
Codein C 18 ACN/ H
2
O 375/ 625 0,005 M Na dioctylsulfonat,
pH 3,3
254 Dng phn
liu
Cortisone v hydrocortison C 18 ACN/ H
2
O 375/ 625 0,005 M Na dioctylsulfonat, pH 3,3 254 Dng phn
liu
Cortisone v hydrocortison Silica Hexan/ ethyl acetat 60/ 50 360 Huyt
thanh
Dexamethason C 18 KH
2
PO
4
0,01 M/ MeOH 50/ 50 254 Dng phn
liu
Dextromethophan C 18 ACN/ m acetat (pH 4,3)
35/ 65
220 Huyt
tng
Diazepam C 18 MeOH/ H
2
O 65/ 35 254 Vin nn
Vitamin D
3
C 18 H
2
O/ MeOH 1/ 9 254 Huyt
thanh
Estrogen C 18 MeOH/ H
2
O 60/ 40 280 Vin nn
Fluorouracil

C 18 MeOH/ acetat 0,01 M (pH 4)
6/ 94
270 Huyt
thanh
Acid folic C 18 H
2
O/ MeOH (pH 7,0) 76/ 24
0,015 M phosphat & 0,3 % TBA hydroxyd
280 Dng phn
liu
Furosemid C 18 ACN/ Na acetat (pH 5,0) 40/60 254 Vin nn
Gentamicin C 18 ACN/ CH
2
Cl
2
/ H
2
O/ MeOH
80/ 10/ 8/ 4
230 Huyt
thanh
Griseofulvin Cyano MeOH/ H
2
O 40/ 60 254 Vin nn
Heptaminol C 8 ACN/ AcNa 0,05% chnh ti pH 6,1 45/ 55 338 Huyt
thanh,
nc tiu
Heroin C 18 ACN/ phosphat 0,015 M
100/ 300 (pH 3,0)
135 Nc tiu
Histamin C 18 NaCl 0,1 M/ MeOH 55/ 45
pH 3,0
350 Huyt
tng
Hydrochlothiazid C 18 MeOH/ AcOH 0,01 M.
15/ 85
228 Vin nn
Hydrocortison Silica CHCl
3
/ isooctan/ MeOH/ H
2
O 48,5/ 48,5/ 2,9/ 0,12 254 Huyt
tng
99
Tn thuc Ct Pha ng Detector Mu th
Ibuprofen C 18 ACN/ AcOH 0,05 M 40/ 60 230 Huyt
tng
Ketoprofen C 8 H
2
O/ MeOH 85/ 15 255 Huyt
tng
Ketoprofen Silica CH
2
Cl
2
/ hexan 60/ 40 254 Huyt
tng
Lidocain Phenyl ACN/ H
3
PO
4
0,006% 30 / 70 200 Huyt
tng
Methyldopa C 18 MeOH/ H
2
O 14/ 86
Na octansulfonat 0,006 M, pH 6,4
GCE
+ 0,5 V
Huyt
tng
Mebendasol C 18 ACN/ phosphat 0,05M
(pH 6) 27/ 73
313 Huyt
tng
Metronidazol

C 18 ACN/ phosphat 10
-5
M
pH 4 8/ 92
322 Huyt
tng,
nc tiu
Morphine C 18 MeOH/ NaH
2
PO
4
60/ 40 254 Dng tim
Acid Nalidixic C 18 MeOH/ H
2
O 70/ 30 313 Huyt
tng
Naphazolin
Tetrahydrozolin
C 18 MeOH/ m citrat pH 2,2
300/ 700
255 Huyt
tng
3.2.5. Chun ho ct sc k lng hiu nng cao
Mc ch:
Mc ch ca vic chun ho ny l kim tra hiu nng ca cc ct
HPLC v xc nh mt cch khch quan xem khi no chng cn phi thay th.
Tt c cc ct pha o (C
18,
, C
8
) v cc ct silica ni chung mi v c
u cn c chun ho.
Cc thng s ca ct HPLC cn chun ho :
+ H s dung lng : k

.
+ Hiu lc ct (s a l thuyt) : N
+ H s i xng : F
i vi cc ct mi: Cn chun ho ngay khi mi nhn khng nh
cht lng ca chng.
i vi ct ang s dng: Nu trong thi gian s dng, nhn thy hnh
dng ca pic b xu hn trc v hiu lc ca ct khng tho mn cho
vic phn tch th nn chun ho li ct v ghi ngy thng thc hin
vo bng theo di.
100
Chun ho ct sc k pha o

Chun b dung dch cc cht chun:
Pha mt hn hp dung dch cc cht chun trong methanol 60%(v/v) vi
cc nng nh sau:
Uracil 0,02 mg/l
Phenol 1,00 mg/l
Anisol 1,50 mg/l
iu kin sc k:
Pha ng : methanol - nc (60 : 40)
Tc dng : 1ml/pht
Th tch tim : 20 L
Detector : UV 254 nm






Hnh 3.11: Sc tch hn hp uracil, phenol, anisol.
1. Uracil 2. Phenol 3. Anisol
Tnh ton:
H s dung lng (k

)
k = (t
A
t
0
)/t
0
t
0
: thi gian lu ca uracil
t
A
: thi gian lu ca anisol
Hiu lc ct (N)
N = 5,54. t
2
A
/ W
2
1/2
t
A
: thi gian lu ca anisol
W
1/2
: chiu rng pic o na chiu cao ca pic anisol
101
H s i xng (F)
F = W
A
/ 2a
y:
W
A
: chiu rng pic anisol o 1/20 chiu cao pic
a: khong cch t ng vung gc h t nh pic n mp ng cong
pha trc ca pic ti 1/20 chiu cao pic.
Chun ho ct sc k pha thun
Chun b dung dch: Ho tan khong 0,1 0.2 ml n-pentan v 20mg
acetophenon trong 100ml hexan c cha 0,5%(v/v) methanol.
iu kin sc k :
Pha ng : hexan - methanol (99,5 : 0,5)
Tc dng : 1,0 ml/pht
Th tch tim : 20 L
Detector : UV 254 nm
Tnh ton tng t chun ho ct pha o .
Vi:
t
AC
: thi gian lu ca acetophenon
t
0
: thi gian lu ca n-pentan
W
1/2
: chiu rng pic o na chiu cao ca pic acetophenon
Bng 3.6. Cc tiu chun chp nhn cho ct
Ct mi Ct s dng
H s dung lng (k

) 2,0 < 0,7
Hiu lc ct (N) 3000 a l thuyt hin nay >10.000 1000 a
H s i xng (F) 0,9 2,0 0,9 2, 0
3.2.6. nh lng bng phng php HPLC
Qu trnh nh lng bng HPLC c th chia thnh 4 bc. Mi bc u
c nh hng trc tip n kt qu nh lng. Cc bc ny l :
Ly mu th
Tin hnh sc k
o tn hiu detector
Phng php nh lng
102


Ly mu
Sai s do ly mu c th tng ln t nht 3 khu: Ly mu i din, bo
qun mu v x l mu.
gim sai s, vic ly mu phi tn trng trit cc nguyn tc
c qui nh i vi tng loi i tng mu c th ly c i din.
Qu trnh bo qun mu cng c th dn n sai s: Vi mu th l cht
bay hi, cht c th phn ng c vi hi nc hoc khng kh.
Cc thnh phn vi lng c th b hp ph hoc phn hp ph bi bnh
cha trong thi gian bo qun.
C th tin hnh cc phn ng ho hc trn mu th to dn cht c
nhy cao hn c th o c bng detector ca my. Cch ny c p
dng i vi nhng cht c p ng rt nh hoc hon ton khng c p ng
vi detector o hp th quang hc.
tin hnh tch sc k
Qu trnh chy sc k cn ch :
C th xy ra s phn hu ca cht th trong khi phn tch.
C th xut hin pic l trn sc do dung mi dng ho tan mu th c
cha tp cht. V vy cn phi kim tra bng sc k cc vt tp .
Chun b mu th:
Dung mi dng ho tan mu th phi c xem xt cn thn . L
tng nht l dng pha ng lm dung mi ho tan n.
Tr trng hp mu th kh tan trong dung mi ny th phi tm dung
mi khc thch hp hn.
Dung mi phi p ng cc yu cu sau :
+ tinh khit cao khng c pic l,
+ C th ho ln c vi dung mi ra gii (pha ng),
+ Cho p ng rt nh vi detector,
+ Dung mi v dung dch th phi c lc qua mng lc 0,45m.
Tim mu: C 2 cch tim mu vo ct:
+ Dng bm tim v
+ Van tim mu th tch xc nh.
Dng van tim mu cho kt qu chnh xc hn (+1%) so vi dng bm
tim ( 1 - 2%).
o tn hiu detector
Detector c dng ph bin nht trong HPLC l detector UV-Vis.

103
Detector s dng phi p ng mt s yu cu sau :
+ Hot ng c vng tuyn tnh ca n,
+ C hp th nh i vi dung mi ( nhiu ng nn thp),
+ Trnh lt khng kh vo ct v detector,
+ Gi sch cell v lm sch n thng xuyn.
Tn hiu detector:
Tn hiu detector o c khi cht ra khi ct sc k c my ghi li
di dng pic.
my sc k c in, vic o din tch hoc chiu cao pic c thc hin
bng tay. Cn cc my sc k hin i vic o c ny c t ng ho nh
my tch phn, my tnh.
3.2.7. Cc phng php nh lng
Tt c cc phng php nh lng bng sc k u da trn nguyn tc:
Nng ca cht t l vi chiu cao hoc din tch pic ca n.
C 4 phng php nh lng c s dng trong sc k :
Phng php chun ngoi (External Standard)
Phng php chun ngoi l phng php nh lng c bn, trong c
2 mu chun v th u c tin hnh sc k trong cng iu kin.
So snh din tch (hoc chiu cao) pic ca mu th vi din tch (hoc
chiu cao) pic ca mu chun s tnh c nng ca cc cht trong mu th.
C th s dng phng php chun ho 1 im hoc nhiu im.
* Chun ho 1 im: Chn nng ca mu chun xp x vi nng
ca mu th.
Tnh nng mu th theo cng thc:



y:
C
X
: nng mu th
C
S
: nng cht chun
S
X
(H
X
): din tch (chiu cao) ca pic mu th
S
S
(H
S
): din tch (chiu cao) ca pic mu chun
S
X
S X
S
S
C c =
104
* Chun ho nhiu im: Tin hnh qua cc bc sau :
Chun b mt dy chun vi cc nng tng dn ri tin hnh sc k.
Cc p ng thu c l cc din tch hoc chiu cao pic mi im chun.
V th chun biu din s tng quan gia din tch S (hoc chiu cao
H) pic vi nng ca cht chun (C), xem hnh 3.12.
S dng on tuyn tnh ca ng chun tnh ton nng ca cht
cn xc nh. C th thc hin vic tnh ton ny theo 2 cch :
p d kin din tch (hoc chiu cao) pic ca cht th vo ng chun
s suy ra c nng ca n .






Hnh 3.12. th phng php chun ngoi
+ Xy dng phng trnh hi qui tuyn tnh m t quan h gia din tch
(hoc chiu cao) pic vi nng ca cht cn xc nh.
Y = a + bC
x
3.25
Trong :
Y: Din tch pic
a : Giao im ca ng chun vi trc tung.
b : dc ca ng chun.
C
x
: Nng ca cht th .
Da vo phng trnh hi quy ny ta tnh c nng cht th .

b
a Y
C
X

=

3.26

Ch : ln ca din tch (hoc chiu cao) pic mu th phi nm trong
on tuyn tnh ca ng chun.
105
Phng php chun ni (Internal standard).
Trong nh lng bng phng php sc k, in di ni chung cng nh
HPLC ni ring, phng php chun ngoi (ESTD) t ra c nhiu nhc im
trong phn tch cc mu phi x l phc tp (chit tch...) v c bit l cc
mu va phc tp va c hm lng thp ca cht cn nh lng. V d nh
cc mu huyt tng trong nghin cu dc ng hc. Phng php chun ni
(ISTD) gip khc phc c nhng nhc im trn ca ESTD. Ngoi ra, n cn
c th gip cc tiu ha c nhng sai s gy nn do my mc v k thut.
K thut chun ni c th c tm tt nh sau: Ngi ta thm vo c
mu chun ln mu th nhng lng bng nhau ca mt cht tinh khit, ri
tin hnh sc k trong cng iu kin. Cht c thm ny gi l chun ni.
T nhng d kin v: din tch (hoc chiu cao) pic v lng (hoc nng
) ca chun, chun ni v mu th, c th xc nh c hm lng ca
thnh phn cn nh lng trong mu th mt cch chnh xc.
C mt s yu cu t ra vi cht chun ni:
Trong cng iu kin sc k, cht chun ni phi c tch hon ton
v c thi gian lu gn vi thi gian lu ca cht cn phn tch trong
mu th .
C cu trc ho hc tng t nh cht th .
C nng xp x vi nng ca cht th .
Khng phn ng vi bt k thnh phn no ca mu th .
Phi c tinh khit cao v d kim .
V rng p ng (response) ca cht chun v chun ni vi detector
khng cng nhy, nn trc ht cn phi xc nh h s p ng (response
factors) hiu chnh trong tnh kt qu.
* Xc nh h s p ng F
X
:
Chun b mt hn hp c cha nhng lng (hoc nng ) bit ca
cht chun v chun ni ri chy sc k. Sc thu c s cho ta bit cc d
liu v din tch ca cc pic.
Trong phng php chun ni, ngi ta thy c mi tng quan gia t
s ca khi lng (hoc nng ) ca chun v chun ni vi t s din tch
ca 2 pic. V h s p ng c tnh theo phng trnh sau:
Tnh theo nng ta c:



C IS
IS C
IS
C
IS
C
X
S m
S m
S
S
m
m
F
.
: = =
) 27 . 3 (
C IS
IS C
X
S C
S C
F
.
=
) 28 . 3 (
106
y:
- m
C ,
m
IS
ln lt l khi lng ca cht chun v chun ni.
- C
C
, C
IS
ln lt l nng ca chun v chun ni.
- S
C
, S
IS
ln lt l din tch pic chun v chun ni.
Cc sai s s c cc tiu ha nu h s F
X
xp x n v, c ngha l
cht chun v chun ni c cng p ng vi detector. Tuy nhin trong thc t
iu ny thng kh t c hon ho.
* nh lng thnh phn trong mu th.
Phng php chun 1 im: Chun ni c thm vo c hai, mu
chun v mu th, ri tin hnh sc k.
Lng hoc nng ca thnh phn trong mu th c tnh nh sau:


Tnh theo nng ta c:


X IS
IS
T
T
F m
S
S
m = ) 29 . 3 (
X IS
IS
T
T
F C
S
S
C =
) 30 . 3 (
Phng php chun ha nhiu im: Chun b mt dy chun c cha
nhng lng (hoc nng ) cht chun khc nhau nhng tt c cng
cha mt lng (hoc nng ) chun ni. Sau khi sc k v thu c cc
d kin din tich, tin hnh v ng chun (hnh 3.13.) biu din s
tng quan gia t s din tch (hoc chiu cao) pic ca chun trn chun
ni (S
S
/ S
is
) vi t s ca nng chun ngoi trn chun ni (C
S
/C
IS
).









S
S
/S
is



S
X
/ S
IS




C
X
/ C
IS
C
S
/C
IS
Hnh 3.13. Phng php ng chun s dng chun ni
107
Song song tin hnh sc k mu th cng c thm chun ni vi lng
(hoc nng ) nh thang chun.
Tnh t s din tch (hoc chiu cao) pic ca cht th trn din tch (hoc
chiu cao) pic chun ni (S
T
/ S
is
) ri da vo ng chun s tm c nng
ca cht th (C
T
).
Phng php thm chun (Standard addition)
K thut ny phi hp phng php chun ngoi v chun ni.
u im ca k thut thm chun l c chnh xc cao v n loi tr
c sai s do cc yu t nh hng, c bit l nh hng ca qu trnh x l
mu (chit xut, tinh ch cc cht t cc dng bo ch...)
K thut so snh tin hnh nh sau:
X l mu th ri tin hnh sc k.
Thm vo mu th nhng lng bit ca cc cht chun tng ng vi
cc thnh phn c trong mu th ri li tin hnh x l mu v sc k trong
cng iu kin. Nng cha bit C
X
ca mu th c tnh da vo s chnh
lch nng C (lng cht chun thm vo) v s tng ca din tch (hoc
chiu cao) pic S theo cng thc:


C th tnh ton nng C
X
ca mu th theo mt cch khc:
Tin hnh sc k mt mu th v mu th c thm chun nh trn.
S dng mt pic khng mun nh lng ca mu th nh l mt chun ni.
Th d: Tin hnh sc k mt mu th c 3 thnh phn, thu c sc
(a), sc ny c 3 pic c nh s l 1; 2 v 3. Gi s khng mun
nh lng thnh phn th nht.
nh lng thnh phn th hai v ba,
chng ta thm nhng lng bit
ca hai thnh phn ny vo hn hp
ri tin hnh sc k li, thu c sc
(Hnh 3.14).


S
C
S c
X X

=
Hnh 3.14: Sc nh lng, phi hp
phng php chun ngoi v ni.


108
Bng cch s dng pic khng mun xc nh ca mu th nh l mt
chun ni trong phng php thm ny, chng ta s cc tiu ho c cc sai
s gy nn trong qu trnh nh lng nh trnh by trn.
Trong trng hp mu th khng cha pic khng mun xc nh th c
th thm mt cht chun ni vo mu th ri tin hnh phn tch nh trn.
Tnh kt qu: Da vo din tch pic (S) hoc chiu cao (h).
Tnh nng ca thnh phn th hai:
R
a
= S
2a
/S
1a
( sc a )
R
b
= S
2b
/S
1b
( sc b )
R
thm
= R
b
- R
a
Y
th
= (Y
thm
). (R
a
)/R
thm
Nng ca thnh phn th ba: Cch tnh tng t .
K thut thm ng chun:
Nguyn tc: Chun b mt dy hn hp gm cc lng mu th ging
nhau v cc cht chun (tng ng vi cc thnh phn cn xc nh) vi lng
tng dn. X l mu ri tin hnh sc k. Dng ng chun tng quan gia
din tch (S) hoc chiu cao (H)ca pic tng (th + chun) vi lng hoc nng
ca cht chun thm (C). Giao im ca ng chun ko di vi trc
honh chnh l nng ca cht cn xc nh.










Hnh 3.15: th phng php thm ng chun
S (H)




dd th
S
X
thm chun

C
X
C chun thm vo
109
phng php chun ho din tch (Area Normalization)
Nguyn tc: Hm lng phn trm ca mt cht trong hn hp nhiu
thnh phn c tnh bng t l phn trm din tch pic ca n so vi tng
din tch ca tt c cc pic thnh phn trn sc .
Phng php ny yu cu tt c cc thnh phn u c ra gii v
c pht hin.
Tt c cc thnh phn u c p ng detector (response) nh nhau.
K thut ny c s dng nhiu trong sc k kh v thng c p ng
nh nhau detector ion ho ngn la (FID).
Trong khi li ch ca k thut ny trong HPLC b hn ch v p ng
nh nhau l iu thiu chc chn.
V d :
Phn tch sc k mt hn hp gm 3 thnh phn : X, Y, Z.
Hm lng phn trm ca X c tnh nh sau :



Trong phng php trn nu xt n p ng khc nhau ca detector th cn
phi xc nh cc h s p ng i vi mi cht hiu chnh s sai khc .
H s p lin h vi cht chun theo cng thc:



y:
S
S
v S
X
: din tch ca pic chun v pic th
C
S
v C
X
: nng ca cht chun v cht th
f
S
: h s hiu chnh ca chun.
Phng trnh tnh hm lng % ca X s l:




Trong mt s trng hp c th ng dng phng php chun ho din
tch pic nh lng cc tp cht trong thuc.

=
=
+ +
=
n
i
i
X
Z Y X
X
S
S
S S S
S
X
1
100 . 100 . .
%
S X
S X S
X
C S
f C S
F
.
. .
=
Z Z Y Y X X
X X
F S F S F S
F S
X
+ +
=
100 . .
%
110
Ti liu tham kho
1. Trn T An, Nguyn Vn Tuyn (1984). Bi ging kim nghim c
cht. NXB Y hc. Tr. 85 - 90
2. Dc in Vit Nam III . Ph lc 3 , 4 . PL . 75 - PL. 85 .
3. Phm Gia Hu, Trn T An (1998). Ho Phn tch II. i hc Dc
H Ni. Tr. 4-32; 55 104.
4. Vin kim nghim (1995). Tp hun k thut kim nghim thuc
bng phng php phn tch dng c. Tr. .28 30.
5. B.P 2001. Appendix II, Appendix III. A 130 - A.145
6. Bureau of food and drugs (1998). Training course on drug and
cosmetic quality control. BFAD. Philippines . P.p. 95 107.
7. European Pharmacopoeia (1997). P. p. 25 32
8. Indian Pharmacopoeia 1996. P.p .A.72 A77
9. Johnson E. and Stevenson R. (1978). Basic liquid chromatography.
Copyright by Varian Associates.
10. Munson J.W. (1984). High - performance Liquid Chromatography:
Instrumentation and pharmaceutical applications in pharmaceutical
analysis. Marcel Dekker Inc. P.p. 16 - 136.
11. Pharmacopoeia of the peoples Republic of China 1977. P.p. A16 -
A19.
12. Skoog D.A. - West D.M.West, Holler F.J (1988). Fundamentals of
Analytical Chemistry 5
th
. ed. Saunders college Publishing. P.p.
557 - 598.
13. U.S.P. 24 . P.p. 1920 - 1924; 1992 - 1997.
cu hi t lng gi
3.1. a/. Mt dung dch ca mt cht thuc c nng 6,4.10
-5
M, o hp th
ca dung dch ny bc sng 255 nm, dng cuvet 1cm thu c mt
quang A = 0,847.
Tnh h s hp th phn t ca cht 255 nm ?
b/. Cn chnh xc 10,0 mg ca thuc ni trn (c phn t lng Mw 200,0),
em ho tan trong nc va 1 lt. o hp th 255nm vi
cuvet 1cm c A = 0,556.
111
Hy tnh tinh khit ca mu thuc.
p s: a/. = 1,32 . 10
4
b/. 84,2%
3.2. Poldin methylsulfat c cc i hp th 257 nm. Chun b 5 dung dch
ca n trong methanol vi nng di y v o hp th ca mi
dung dch = 257nm, cuvet 1cm.
a/ H ny c tun theo nh lut Lambert-Beer khng ?
b/ Tnh A
1%
1cm
257 nm.
c/ Tnh h s hp th phn t . Bit Mw ca poldin sulfat l 451.
C(mg/ml) A
257
0,1 0,105
0,2 0,207
0,3 0,318
0,4 0,420
0,5 0,529
d/ Ho tan 50,0mg Poldin methylsulfat trong methanol trong mt bnh
nh mc dung tch 1 lt, thm methanol ti vch. o hp th ca
dung dch ny 257nm, dng cuvet 2 cm, c A = 0,946. Gi thit
rng nhng tp cht c mt khng hp th nh sng bc sng ny.
Hy tnh % tinh khit ca mu th.
p s : b/ A
1%
1cm
= 10,5; c/ = 474; d/ 90,2%
3.3. Tolbutamid c Mw 270,4; = 703 262 nm. Nu mt vin nn tolbutamid
c ho tan trong nc v ho long ti 2500ml th hp th ca
n A= 0,520 262 nm, cuvet 1cm.
Hy tnh hm lng ca tolbutamid trong vin ?
p s : 500mg
3.4. S hnh thnh ph hunh quang v ng dng ca phng php ph
hunh quang trong phn tch ?
3.5. ng dng quang ph hng ngoi trong nh tnh cc cht hu c ?
3.6. Tnh nng ca dung dch Viatamin B
12
bit rng khi pha long 200 ln v o
hp th 361 nm c A = 0,450. Cho A (1%, 1cm) ca B
12
361nm = 207.
3.7. Sc k pha thun v pha o l g ?
Cho v d v pha tnh v pha ng dng trong sc k pha thun v pha o.
112
3.8. Thi gian lu tng i ca codein, heroin, methadon, morphin v
propoxyphen l: 1,00; 1,89; 0,55; 1,16 v 5,90.
Bit t
R
ca codein l 6 pht. Tnh thi gian lu ca cc cht khc?
3.9. D kin sau thu c bng sc k trn ct di 40cm.
Tn cht t
R
(pht) W
1/2
(pht)
Cht khng lu gi 1,9 -
A 10,0 0,76
B 10,9 0,82
C 13,4 1,06
Hy tnh :
a/. S a l thuyt trung bnh ca ct ?
b/. Chiu cao ca a ?
c/. phn gii cho:
+ B v A
+ B v C
+ A v C
3.10. Cho ct sc k di 30 cm. Khi chy sc k tch hn hp 2 cht thu c
kt qu:
Cht t
R
(pht) Chiu rng y pic W(pht)
Cht khng lu gi 1,30 -
A 16,40 1,11
B 17,63 1,21
Hy tnh :
phn gii ca ct
S a l thuyt trung bnh ca ct, chiu cao a.
Nu chng ta mun phn gii trong tch 2 cht A v B l R
S(m)
= 1,5 th:
a/. Yu cu ct phi c s a l thuyt l bao nhiu ?
b/. Nu khng thay i 2 pha, chiu di ct s phi l bao nhiu ?
3.11. Khi chy sc k mt hn hp 2 cht A v B thu c thi gian lu ln
lt l: 10,0 v 10,9 trn ct di 40cm. Mt cht khng lu gi qua ct
1,9 pht.
Chiu rng y pic ca A v B l 1,52pht v 1,64 pht.
Cho bit th tch pha tnh V
S
= 19,6ml v th tch pha ng V
M
= 62,6ml.
113
Hy tnh :
a/. H s dung lng k

cho mi cht ?
b/. H s phn b cho mi cht ?
c/. H s chn lc cho A v B ?
3.12. Hy so snh sc k lng hiu nng cao vi sc k ct c in ?
3.13. Trnh by v s khc nhau gia sc k trao i ion v sc k loi c (sc
k trn gel) ?
3.14. Hy trnh by v cc loi bm s dng trong HPLC, u, nhc im ca
mi loi ?
3.15. Nguyn tc hot ng ca detector dn nhit v ion ho ngn la ?
3.16. Hy trnh by v phng php s dng chun ni trong nh lng bng
sc k lng hiu nng cao ?
3.17. Hy ni v phng php thm chun trong nh lng bng HPLC ?
114
Chng 4.
Kim nghim thuc bng phng php sinh hc
Mc tiu hc tp
1. Vit c nhng c im c bn v hnh thi, tnh cht nui cy ca
vi khun, nm mc, nm men p dng trong th nghim vi sinh vt.
2. Bit phng php lm mt mi trng nui cy vi sinh vt v nu
c cc phng php tit trng.
3. Trnh by c mc ch, nguyn tc, phng php th v trng v
m s lng vi sinh vt trong 1 gam (1 ml) dc phm.
Nu c vai tr ca phng php sinh hc trong kim nghim cht
khng sinh, v trnh by c th nghim nh lng cht khng
sinh bng phng php khuch tn.
4.1. M u
Trong ngnh Dc c nhiu phng php khc nhau kim tra cht
lng ca thuc nh phng php ho hc, phng php vt l, phng php
sinh hc.
Phng php ho, l tin hnh nhanh chnh xc nhng ch p dng c
vi cc cht c thnh phn ho hc bit v khng nh gi c bn cht
sinh hc ca mt dc phm. xc nh cc c tnh sinh hc theo yu cu
ca mt s dc phm nh: hot lc tc dng ca cht khng sinh, s v trng,
nhim khun, c tnh bt thng ca mt s loi thuc, hay hiu lc v tnh
an ton ca cc vaccin ... ngi ta phi dng phng php sinh hc.
4.1.1. Nguyn tc
Phng php sinh hc da trn nguyn tc:
So snh hiu lc tc dng hoc cc c tnh ring ca cht th vi cht
chun tng ng trong cng mt iu kin v thi gian th nghim.
Trong lnh vc kim nghim thuc, hai loi th nghim sinh hc c p
dng nhiu nht l:
Kim nghim thuc bng cc phng php th trn ng vt.
Kim nghim thuc bng cc th nghim vi sinh vt.
115
4.1.2. Cht chun
Trong th nghim sinh hc cht chun l mt yu t quan trng nh
gi cht lng cht th. Cht chun c chia lm hai loi:
Cht chun gc: l nhng cht ng nht c tinh khit cao. Cht
chun gc thng c lm cc vin nghin cu quc gia hoc quc t
ring v cht chun sinh hc.
Cht chun th cp: cng l cht c tinh khit cao, c hot tnh sinh
hc c xc nh theo cht chun gc quc t tng ng.
Cht chun phi c bo qun trong cc ng thu tinh nhit thch hp
tu theo mu (thng nhit < 5
o
C) trong iu kin kh, trnh nh sng.
4.1.3. nh gi kt qu
Th nghim sinh hc thng c thi gian th nghim ko di v ph
thuc vo nhiu yu t nh tnh cht p ng ca sinh vt th nghim, ngi
lm th nghim, cc iu kin th nghim. Cc yu t ny thng khng n
nh. V vy kt qu th nghim sinh hc phi c nh gi bng ton thng
k. chnh xc ca php th c th hin bng gii hn tin cy.
4.2. Kim nghim thuc bng phng php th trn ng vt
4.2.1. Nguyn tc
Kim nghim thuc bng cc php th trn ng vt da trn s p ng
ca ng vt th nghim i vi cc ch phm c a vo c th mt liu
lng theo quy nh ca tng th nghim nh gi cht lng ca ch phm
cn th.
4.2.2. ng vt th nghim
ng vt dng trong th nghim phi ng u, thun khit v ni ging,
kho mnh khng nhim bnh, khng c thai v c nui dng y . Mi
th nghim c nhu cu khc nhau v ging, trng lng, tui ca ng vt.
Cht lng ca ng vt quyt nh chnh xc ca php th.
4.2.3. Th in vivo v in vitro
Th nghim c thc hin trn c th ng vt sng gi l th in vivo.
Ngi ta da trn cc thng s nh nhit c th, nhp tim, in tm
, in no , s thay i huyt p, phn x h thn kinh hoc t l
sng, cht ca ng vt th nghim nh gi tc dng v cht
lng ca thuc.
Php th c th c tin hnh trn cc c quan c lp ca ng vt,
nh tim, t cung, rut, mu gi l th in vitro.
116
4.2.4. Liu (Dose)
Liu l lng ch phm th a vo c th ng vt mt ln cho tng
mc ch th nghim. V d: Liu LD
0
, LD
50
, LD
100
, MLD, liu th cht h p,
liu th cht gy st.
4.2.5. Cc th nghim trn ng vt c p dng trong kim nghim thuc
Trong cc dc in thng c cc th nghim sau y c thc hin
bng cc php th trn ng vt nh:
Th c tnh bt thng
Th cht h huyt p
Th cht gy st
nh lng cc hormon: gonadorelin, corticotrophin, insulin, oxytocin,
menotrophin, ...
Kim tra tnh an ton ca vaccin v sinh phm.
Xc nh hiu lc ca cc vaccin v antitoxin.
4.3. Kim nghim thuc bng cc php th vi sinh vt
4.3.1. i cng v vi sinh vt
Vi sinh vt l nhng c th sng c kch thc rt nh m mt thng
khng th nhn thy c. Nu cn quan st hnh dng ca chng phi dng
knh hin vi. Trong t nhin, vi sinh vt tn ti rt phong ph v a dng,
chng ng vai tr tch cc vo vng tun hon vt cht. Nhiu loi vi sinh
vt c ng dng trong cc lnh vc y t, cng nghip, nng nghip nh cc vi
sinh vt c kh nng ln men ru, sinh tng hp khng sinh, vitamin, acid
amin, vi sinh vt c nh m thc vt
Trong qu trnh hot ng sng, bn cnh nhng c tnh c ch, vi sinh
vt cng gy nhiu tc hi cho ngi, ng vt, thc vt nh: lm bin i
cht lng thuc, hng thc phm, mt s c kh nng gy bnh hoc sinh c
t c hi.
phc v cho vic kim nghim thuc bng cc th nghim vi sinh
vt, ta cn tm hiu mt s c im chnh ca hai nhm vi sinh vt l vi
khun v vi nm.
4.3.1.1. Vi khun (Bacteria)
c im:
Vi khun l nhng vi sinh vt n bo c cu to t bo tin nhn
(Procaryote), c kch thc rt nh. ng knh t bo phn ln thay i trong
khong 0,2 2,0m, chiu di t 2 - 8m. Vi khun c nhiu hnh dng khc
nhau, nh hnh cu, hnh que, xon, du phy. Vi khun ch sinh sn v tnh,
117
mt s to bo t. Mi t bo vi khun ch c mt bo t. Mt s vi khun c
kh nng di ng nh s c mt ca mt hoc nhiu lng (flagella).


Phn loi:
Vic phn loi vi khun rt phc tp, phi da vo nhiu c im, hnh
thi, sinh l, sinh ho chia vi khun thnh cc h, chi, loi khc nhau. Vi
mc ch phc v cho cng tc kim nghim thuc, ta khng i su vo nghin
cu phn loi, nhng cn tm hiu vi khun, theo cc nhm da trn hnh th,
tnh cht bt mu thuc nhum Gram v kh nng h hp ca chng.
Theo hnh th:
+ Cu khun (Coccus): Vi khun hnh cu c th ng ring r
(Micrococcus), thnh tng m (Staphylococcus), hoc chui
(Streptococcus), hay xp thnh tng i (Diplococcus).
+ Trc khun (Bacillus): Vi khun hnh que ngn ng ring l hay thnh
chui (Bacillus anthracis) hoc hnh que hai u trn (Escherichia coli).
+ Xon khun (Spirillum): Vi khun hnh l xo nh: Treponema Pallidum.
+ Phy khun (Vibrio): Vi khun hnh du phy nh Vibrio cholerae.
Theo tnh cht bt mu thuc nhum Gram.
+ Vi khun c mu tm sau khi nhum Gram: Vi khun Gram +
+ Vi khun c mu sau khi nhum Gram: Vi khun Gram
Theo c tnh ca qu trnh h hp:
+ S dng oxy t do trong qu trnh h hp: Vi khun hiu kh.
+ Pht trin c c trong iu kin hiu kh v k kh, c qu trnh h
hp nitrat: vi khun k kh khng bt buc.
+ Ch sng trong iu kin k kh, c qu trnh h hp sulfat: Vi khun
k kh bt buc.
Sinh sn ca vi khun:
1
2
3
4
thi gian
log N

Hnh 4.1: ng cong sinh trng ca vi khun
118
Vi khun sinh sn bng cch phn i t bo. S phn chia t bo xy
ra rt nhanh. Trong iu kin mi trng thch hp v khng c cc
yu t km hm th mt t bo vi khun sau 6 gi c th sinh ra
250.000 t bo mi. Tuy nhin, s nhn ln ca vi khun khng phi l
v tn, n cn ph thuc vo nhiu yu t. Trong mi trng nui cy,
s sinh sn ca vi khun sau mt thi gian nht nh s b ngng li v
nhiu nguyn nhn nh: thc n b ht dn, hoc vi khun c th tit
ra nhng cht km hm s pht trin ca chng.
Tc pht trin ca vi khun trong mi trng nui cy tnh thay i
theo thi gian v tun theo mt quy lut nht nh bao gm 4 pha: Pha
lag, pha logarit, pha n nh v pha t vong (Hnh 4.1).
S pht trin ca vi khun trong mi trng lng c th quan st sau
khong 18 - 24 gi nui cy. Chng c th lm c mi trng, to vng
trn b mt hoc lng cn y ng nghim.
Trn cc mi trng c, khun lc ca cc vi khun thng nh hn
khun lc ca vi nm, b mt nhn, bng, t hoc nhn nheo, ... Khun
lc ca vi khun to sc t vi cc mu nh: trng (Staphylococcus
albus), vng (Staphylococcus aureus), hng (Micrococcus), xanh
(Pseudomonas aeruginosa), ... Mi vi khun c c tnh ring v hnh
dng, kch thc, mu sc khun lc. Cc c tnh ny gip cho vic xc
nh vi khun trong qu trnh kim nghim dc phm.
4.3.1.2. Vi nm (Microfungi)


c im:
Vi nm c cu to t bo nhn tht (Eucaryote). T bo vi nm rt nh.
Mun quan st cn dng knh hin vi. Nm khng c cht dip lc, sng hoi
sinh hoc k sinh, sinh sn v tnh hoc hu tnh.
Vi nm bao gm hai loi l nm men v nm mc. Trong kim nghim vi
sinh vt, cn pht hin hai loi ny c trong dc phm.
Nm men (Yeast):
Nm men c cu to n bo, sinh sn ch yu bng ny chi. T bo
nm men c kch thc, hnh dng khc nhau tu loi. Chng c th
hnh cu, bu dc, hnh qu chanh, hnh ng
Khun lc nm men bao gm nhiu c th thng thuc mt loi pht
trin t mt c th m to thnh mt khi. Khun lc nm men thng
to hn khun lc vi khun, b mt c np nhn hoc trn nhn, khng
to si.
Nm men c s dng nhiu trong cng nghip thc phm nh lm
bnh m, bia, ru Nhng nhiu nm men gy bnh hoc lm hng
thc phm, thuc.
119
Nm mc (Mold):

Nm mc c cu to si, sinh sn bng bo t, sng hoi sinh, chng
thng pht trin trn b mt c cht di dng nhng lp hnh si,
mng nhn hoc khi si bng.
Si nm rt nh, ng knh trung bnh 5m, chiu di c th vi chc
centimet. Si nm c vch ngn hoc khng c vch ngn. Ton b si
nm v cc nhnh pht trin t mt bo t nm ri an kt nhau
thnh mt khi gi l h si nm.
Trn mi trng thch nui cy, h si nm pht trin thnh mt khi
c tit din hnh trn hoc gn trn gi l khun lc. Khun lc c
c trng bi mu sc ca si nm v ca bo t. B mt khun lc c
th mt, dng ht, dng si hoc xp
Nm sinh sn bng bo t: Bo t v tnh hoc bo t hu tnh . S
sinh sn v tnh v hu tnh lun an kt nhau trong qu trnh sinh
trng ca nm. V vy, nm pht trin rt nhanh trn b mt cc c
cht. Nm mc thng gy ra nhng bin i v mu sc, mi v, cht
lng ca thuc. Mt s sinh cc c t (Mycotoxin) c hi cho ngi v
ng vt.
4.3.1.3. S nh hng ca cc yu t ngoi cnh i vi qu trnh pht trin
ca vi sinh vt
Sinh trng v trao i cht ca vi sinh vt lin quan cht ch n cc
iu kin ca mi trng bn ngoi. Cc iu kin ny bao gm hng lot cc
yu t khc nhau, tc ng qua li vi nhau. a s cc yu t u c mt c
tnh tc dng chung biu hin 3 im hot ng: Ti thiu, ti u, cc i. Khi
mt yu t c tc dng ti u, vi sinh vt pht trin vi tc cc i. Nu yu
t ny c tc dng cc i , vi sinh vt ngng sinh trng v thng cht.
Cc yu t bn ngoi c nh hng n i sng ca vi sinh vt l vt l,
ho hc v sinh hc, trong cc yu t vt l l ng ch nht. Yu t vt
l bao gm nhit , m v nh sng:
Nhit :
Nhit l yu t quan trng nht i vi i sng vi sinh vt. Mi loi
vi sinh vt c mt gii hn nhit pht trin thch hp. Ni chung i vi vi
sinh vt, nhit pht trin thng t 15 45
o
C.
nhit cao s lm thay i qu trnh trao i cht ca vi sinh vt,
vi sinh vt b cht. Cc t bo sinh dng thng b cht nhit 60
o
C/20
- 30 pht.
Cc bo t ch b tiu dit nhit 120
o
C/ 30 40 pht. Tnh cht ny
c ng dng trong vic tit trng. Nhit thp ch c tc dng km hm s
pht trin ca vi sinh vt (tr vi sinh vt a lnh).
120



m:
Hu ht cc qu trnh sng ca vi sinh vt c lin quan n nc. Khi
thiu nc xy ra hin tng loi nc khi t bo vi sinh vt, trao i cht b
gim, t bo s cht. V vy, bo qun dc phm, dc liu trnh khi tc
ng ca vi sinh vt cn c mt gii hn m nht nh.
nh sng:
nh sng mt tri gm cc tia bc x nh: tia t ngoi, hng ngoi, tia
gamma c tc dng ph hu t bo vi sinh vt, c bit l tia t ngoi. Bc x
UV bc sng khong 260nm, c tc dng dit khun mnh nht. Di nh
hng ca tia UV, vi sinh vt b cht hoc t bin tu theo liu lng.
ngn nga tc hi ca vi sinh vt i vi thuc cc tc nhn vt l
trn cn c vn dng trong qu trnh sn xut v bo qun dc phm,
nhm hn ch ti a s lng vi sinh vt gy nhim ban u. ng thi cc
ch phm dc phi c quy nh gii hn vi sinh vt cho php.
4.3.2. Mi trng nui cy vi sinh vt
Mi trng nui cy l nhng cht dinh dng thch hp nhm m bo
cho vi sinh vt sinh trng v pht trin.
Mi trng cn c 3 iu kin sau: y cht dinh dng theo yu cu
th nghim, c pH trong khong quy nh v phi v trng.
Mi trng gm 3 loi:
Mi trng t nhin: nguyn liu c ngun gc t ng vt hay thc vt
(nh cao tht, cao men, pepton, tinh bt). Thnh phn c th thay i
tu theo ngun gc nguyn liu.
Mi trng tng hp: bao gm cc ho cht thun khit c quy
nh v thng ho tan trong nc.
Mi trng bn tng hp: trong thnh phn mi trng c c cc
nguyn liu t nhin v tng hp.
Pha ch mi trng l mt khu rt quan trng trong cc th nghim vi
sinh vt.
chnh xc ca kt qu th nghim ph thuc rt nhiu vo cht lng
mi trng.
4.3.2.1. Phng php pha ch mi trng
Khi pha ch mi trng cn tin hnh qua 6 bc sau:
Chun b dng c ho cht:
Dng c pha ch mi trng tt nht l bng men hoc thu tinh. Cc
dng c phi ra sch, hoc tit trng nng trc khi s dng.
121
Nguyn liu pha ch mi trng phi m bo cht lng, ho cht phi
tinh khit. Nu l cc dng bt hoc tinh th phi kh, khng i mu, khng
chy nc.





Cn ong nguyn liu:
Cc nguyn liu phi c cn ong chnh xc, nht l nhng ho cht
hoc nguyn t vi lng c th gy c ch vi khun (mui mt, st) phi
c cn bng cn phn tch.
Ho tan nguyn liu:
Thng dng nc ct hoc nc kh khong pha mi trng. Cc ho
cht c ho tan nng hoc lnh tu theo tnh cht ca chng. Mi trng
khng c thch nn ho tan lnh hoc nng nh. Mi trng c thch cn un
cho thch tan hon ton sau mi cho cc thnh phn khc vo.
iu chnh pH:
Khi iu chnh pH ca mi trng nn thc hin nhit 45 - 50
o
C
pH t b thay i sau khi tit trng. Cc dung dch NaOH 1N v HCl 1N
thng c s dng iu chnh pH. Sau khi iu chnh pH, cn b sung
nc cho th tch quy nh.
Lm trong mi trng
Cc mi trng lng (bao gm cc cht ho tan) phi trong d quan
st s pht trin ca vi sinh vt. Sau khi ho tan cc cht, nu mi trng c
cn phi lc qua vi gc hoc giy.
ng ng tit trng:
Mi trng c cho vo ng nghim bnh nn hoc bnh cu, tu theo
yu cu th nghim. Khi ng ng, khng c mi trng dnh vo ming
ng hoc bnh.
Mi trng cn phi c tit trng ngay sau khi ng gi. Nu lu,
tp khun s pht trin lm hng mi trng.
Cc mi trng thng thng c tit trng 110
o
C/30 pht hoc 120
o
C/
20 pht.
Mi trng c cc cht d b ph hu bi nhit, cn tit trng nhit
thp bng phng php Tyndall, Pasteur, hoc dng lc vi khun. Mi trng
c ly ra khi ni hp ngay sau khi tit trng xong. Nu lu trong ni
hp mi trng b chuyn mu v gim cht lng.
Pha ch mi trng t hn hp bt mi trng ch sn:
nhiu nc trn th gii c cc loi mi trng di dng bt kh cha
y cc thnh phn theo yu cu, cc mi trng ny c lm t nguyn
liu, ho cht tinh khit nn cht lng mi trng m bo v n nh. Khi
lm th nghim mi trng c pha vi nc theo t l quy nh, nhng phi
122
dng nc mi ct hoc nc kh khong, trung tnh pha ch. Nu bt mi
trng c cn phi kim tra li pH sau khi lm mi trng.
4.3.2.2. Bo qun mi trng
Mi trng bt kh c gi 10 - 12
o
C trong iu kin kh, trnh nh
sng.
Mi trng pha ch c bo qun 4 10
o
C trong 1 2 thng tu
theo thnh phn mi trng.
4.3.2.3. Cc phng php tit trng
Tit trng l mt qu trnh lm cho mt vt hoc mt sn phm khng cn vi
sinh vt sng c. Tit trng c thc hin bng phng php vt l, ho hc.
Chn phng php tit trng ph thuc vo tnh cht l ho v bn
vng ca mi trng.



Tit trng bng nhit kh:
Phng php ny dng tit trng cc dng c th nghim bn vi
nhit nh bng, bng, vi, gc, dng c thu tinh
iu kin tit trng l: 180
o
C/ 30 pht hoc 170
o
C/ 1 gi, hoc 160
o
C/2 gi.
Cc dng c thu tinh ng mi trng phi c tit trng kh trc
khi dng.
Tit trng bng hi nc:
Phng php dng nhit t thng c dng tit trng mi trng
nui cy v cc dng c phu thut.
Mi trng thng c tit trng bng ni hp 121
o
C/ 15 pht.
Cc mi trng d b hng bi nhit nh mi trng c ng, sa, bia,
mu, albumin cn tit trng nhit thp bng cc phng php sau:
Tit trng gin on (phng php Tyndall):
Mi trng c hp 3 - 4 ln nhit khng qu 100
o
C trong 30 - 40
pht, cch nhau 24 gi. Gia hai ln hp cho mi trng vo 28 -
32
o
C/ 24 gi cho bao t ny mm. Cc bo t sng st ny mm s b tiu
dit ln hp tip theo.
Kh trng nhit thp (phng php Pasteur):
un cch thu mi trng 60
o
C/ 30 pht, hoc 73
o
C/ 15 pht sau
lm lnh t ngt di 10
o
C. Phng php ny khng dit c bo t.
Phng php lc:
Phng php lc c dng tit trng cc cht d b ph hu bi
nhit. Cho cht lng chy qua mng lc c kch thc l lc 0,22m. Phn
123
chy qua phu c ng trong cc dng c v trng. Thit b lc v mng lc
phi c tit trng trc khi dng.


Phng php dng tia bc x:
Tia t ngoi (UV) c dng nhiu nht tit trng cc bung pha ch,
t cy vi sinh vt.
n t ngoi phi c chiu trc tip, thng gc vi ni lm th nghim
v liu lng chiu phi vi din tch bung.
Tia UV t c tc dng dit nm, v vy khi kh trng bung pha ch cn
phi hp thm phng php dng ho cht kh nm.
4.3.3. Th v trng
4.3.3.1. Mc ch
Th v trng nhm mc ch pht hin s c mt ca vi khun, vi nm
trong cc ch phm nh dch tim truyn, mt s loi thuc tim, thuc tra
mt v cc dng c y t m theo tiu chun ring cn phi v trng.
4.3.3.2. Nguyn tc
Vi sinh vt c trong ch phm th s pht trin trn cc mi trng dinh
dng thch hp, chng lm c mi trng lng to vng trn b mt hoc
lng cn y ng nghim. Trn mi trng c vi khun, vi nm mc thnh
cc khun lc c trng.
4.3.3.3. Mi trng
Trong nhiu dc in thng dng mi trng Thioglycolat lng pht
hin vi khun hiu kh, k kh v mi trng Casein u tng lng pht
hin vi khun, vi nm. Tuy nhin, c th dng cc mi trng khc cho th
nghim, vi iu kin cc mi trng ny thch hp cho s pht trin ca loi
vi sinh vt cn pht hin. V d c th dng mi trng canh thang cao tht -
pepton pht hin vi khun hiu kh; mi trng Wilson - Blair pht hin vi
khun k kh; mi trng Sabouraud lng cho s pht hin vi nm.
Kim tra cht lng mi trng:
v trng:
Ly 1-2 ng (hoc bnh) mi trng mi lm 30 - 35
o
C t nht trong 4
ngy i vi mi trng pht hin vi khun, v 25 - 28
o
C t nht trong 7 ngy
vi mi trng pht hin vi nm. Sau thi gian nui cy, cc ng mi trng
khng c c vi sinh vt mc (c th cc ng mi trng song song vi cc
ng c cy cht th)
Kh nng dinh dng:
Cy vo mi ng mi trng thch hp khong 100 t bo cc vi sinh vt sau:
124
+ Vi khun hiu kh:
Staphylococcus aureus
Bacillus subtilis
Pseudomonas aeruginosa
+ Vi khun k kh:
Clostridium sporogenes
+ Vi nm:
Candida albicans
Aspergillus niger
ng th vi khun c nui cy 30 - 35
o
C/ 3 ngy, ng th vi nm 25
- 28
o
C/ 5 ngy.
Sau thi gian nui cy vi sinh vt phi pht trin tt trn cc mi trng.
4.3.3.4. Ly mu th
Trong th v trng, c th coi ton b s ng hoc l thuc c kh
trng hoc c phn b v trng trong cng mt iu kin l mt l thuc.
Thng thng, khi mt l c t hn 100 n v, ly 3 n v kim nghim.
Nu nhiu hn th c 50 n v ly thm mt n v nhng khng qu 10.
Tuy nhin, khi ly mu cng cn phi da vo tiu chun ngnh hoc
tiu chun c s ca tng sn phm ly mu cho thch hp.
4.3.3.5. Kim tra tc dng c ch vi sinh vt ca ch phm th
Mt s thuc trong qu trnh sn xut c thm cc cht bo qun.
Nhng cht ny c th nh hng n s pht hin vi sinh vt c trong
ch phm.
Mt ch phm cha bit c tc dng c ch hay khng th cn phi kim
tra tc dng c ch i vi cc vi sinh vt sau:
Ly t nht 2 ng ca mi loi mi trng pht hin vi khun hiu kh,
k kh, vi nm, cy vo mt trong hai ng ch phm cn th.
Cy khong 100 t bo (0,1 ml nh dch vi sinh vt c pha khong
nng thch hp) Staphylococcus aureus (vi khun hiu kh), Clostridium
sporogenes (vi khun k kh), Candida albicans (vi nm) vo c hai ng ca
cc mi trng tng ng. Nui cy 30 - 35
o
C/4 ngy i vi vi khun v 25 -
28
o
C/7 ngy i vi vi nm.
Trong thi gian nui cy, nu vi sinh vt pht trin ging nhau (mc
nhanh v phong ph) trong ci ng chng v ng kim tra, ch phm th c
coi l khng c tc dng c ch.
125
Nu cc ng c cht th, vi sinh vt pht trin yu hoc khng pht trin
so vi ng khng c cht th, ch phm c cht c ch.
Tc dng c ch ca cht th phi c loi b bng cch pha long,
trung ho, hoc dng phng php mng lc.
4.3.3.6. Phng php th
Th v trng c th c thc hin theo hai phng php tu theo tnh
cht ca mu th:
Phng php lc qua mng lc vi khun .
Phng php nui cy trc tip.



Phng php dng mng lc:
Thit b lc thng bng thu tinh, thp khng r hoc nha gm hai b
phn c th tho ri, gia c li mng lc. Mng lc c thnh phn l
nitrat celleulose thng dng lc nc, du, v dung dch alcol yu. Mng
ecetat cellulose lc cc dung dch alcol mnh. L mng lc c nhiu kch
thc khc nhau, trong th v trng thng dng mng c ng knh khong
50mm v ng knh l mng lc 0,45m.
Thit b lc v mng lc phi c tit trng trc khi th nghim .
Dung dch cht th chy qua mng lc, cc vi sinh vt c gi li
trn mng, cy mng lc vo cc mi trng thch hp pht hin vi
khun, vi nm.
Phng php mng lc kim nghim c cc thc c tc dng c ch
vi sinh vt, c bit l thuc khng sinh, nhng i hi thit b tt v
iu kin v trng cao.
Phng php nui cy trc tip:
Phng php nui cy trc tip c k thut n gin, nhng kh nng
pht hin vi sinh vt gim khi s lng vi sinh vt c t v phn phi trong mt
th tch cht th ln. Phng php ny khng thc hin c vi cc ch phm
c tc dng c ch v cc cht khng sinh, v khi th nghim cht th c cy
trc tip vo cc mi trng nui cy thch hp cho cc vi khun, vi nm.
Lng mu th dng trong th nghim:
Lng mu th cn cy vo cc mi trng tu thuc vo tng loi mu
(bng 4.1.).
Cht lng l du hay dung dch du phi thm vo mi trng nui cy
1% tween 80 hoc cc cht nh ho khc vi nng thch hp.
Mu th l dng m hay kem c ho long vo dung dch pepton
0,1% v trng theo t l 1/10 trc khi cy vo mi trng, (dung dch
pepton, cn thm tween 80 vi t l 1ml/ 1lt).
126
K thut th:
Mu th l dc phm:
Dng dng c v trng cy trc tip ch phm th vo cc mi trng
pht hin vi khun, vi nm theo s lng quy nh. Cht rn l dng bt c
th cho trc tip vo mi trng hoc lm thnh dung dch hay nh dch 1%
sau cy vo mi trng.
Bng 4.1. Lng mu th dng cho th nghim nui cy trc tip
Lng ch phm trong
mt n v ng gi
Lng ti thiu cho
mt mi trng nui cy
Th tch
mi trng (ml)
- Cht lng:
Th tch V < 1ml
1 ml V< 4 ml
4 ml V < 20ml
20ml V < 50ml
50ml V< 100ml
V 100ml
- Cht rn:
Khi lng P< 50mg

50mg < P< 200mg

P 200mg

Ton b mt ng
1/2 ng
2ml
5ml
10ml
Thng 10%

Ton b mt n v ng gi
1/2 khi lng ca mt n v
ng gi
100mg

10
15
20
40
80
100

20

40

80
Mu th l bng gc, ch khu phu thut nu kch thc v hnh dng
cho php, nhng ton b mu th vo 100ml mi trng.
Mu th l dy truyn dch: Cho dung dch pepton 0,1% v trng chy
qua thu c t nht 15ml v cy vo 100ml mi trng.
Mi trng pht hin vi khun c nui cy 30 - 35
o
C t nht trong 4
ngy, v 25 - 28
o
C t nht trong 7 ngy i vi vi nm.
(Mi loi mi trng lm 2 - 3 ng th song song).
Nhn nh kt qu:
Mu th c coi l v trng nu sau thi gian nui cy khng c vi
khun, vi nm pht trin.
Nu c 1 hoc nhiu ng c vi sinh vt mc cn lm li ln th hai (Trc
khi lm li th nghim ln 2 cn phn lp xc nh c tnh hnh thi ca vi
sinh vt cc ng th dng tnh):
127
Nu khng c vi sinh vt mu v trng.
Nu c vi sinh vt ging vi ln mt mu th khng v trng.
Nu c vi sinh vt khc vi ln mt th nghim c lm li ln 3 vi
s lng mu gp i:
+ Khng c vi sinh vt pht trin mu v trng.
+ C vi sinh vt mc mu khng v trng.
4.3.4. Th gii hn vi sinh vt
Cc dc phm trong qu trnh sn xut thng b nhim vi khun, vi
nm do cc nguyn nhn nh:
Do bn cht nguyn liu, nu nguyn liu c ngun gc t ng vt,
thc vt th mc nhim khun cao hn nhiu so vi cc ho cht.
Cc t dc nh tinh bt, ng, mt l mi trng cha nhiu vi sinh
vt, v vy d gy nhim bn cho thuc.
C s sn xut, trang thit b, bao b ng gi, ngi sn xut cng l
nguyn nhn gy nhim khun.
Dng bo ch nh vin hon mm c m cao thng to iu kin
cho vi sinh vt pht trin hn vin nn, vin hon cng.
V vy, th gii hn vi sinh vt l mt th nghim bt buc cho cc dc
phm t nguyn liu n thnh phm khng c tit trng trong qu trnh
sn xut.
4.3.4.1. Mc ch
Th nhim vi sinh vt nhm mc ch xc nh gii hn ti a ca s
lng vi khun hiu kh, vi nm c trong 1g (hoc 1ml) ch phm th. ng
thi pht hin cc vi khun ch im v sinh quy nh khng c c trong
thuc l:
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, cc
loi Salmonella, vi khun k kh Clostridia.
4.3.4.2. Nguyn tc
Php th c da trn nguyn tc:
m s vi khun hiu kh, nm mc, nm men c trong dc phm c
th hin bng cc khun lc c trng trn a thch dinh dng thch hp.
Cn c vo cc c tnh hnh thi, sinh l, sinh ho ca tng loi vi khun
xc nh vi khun gy bnh. Trn c s kt qu th nghim nh gi cht
lng ca thuc theo tiu chun dc in hoc tiu chun c s.
128
4.3.4.3. Phng php th



Mi trng:
a s cc dc in thng dng mi trng thch casein u tng
m vi khun hiu kh, mi trng thch Sabouraud - khng sinh m vi
nm. Cng c th dng mi trng thch thng th vi khun hiu kh v
mi trng ny rt thch hp cho s pht trin ca a s vi khun hiu kh
(theo Dc in Trung Quc 1997)
Chun b mu th:
Mu th theo quy nh chung c ly 10g (hoc 10 ml) th nghim.
Mu th l cht rn hay cht lng c th lm thnh dung dch hay nh
dch trong nc, c pha long vo dung dch m phosphat pH =7,2
hoc dung dch NaCl 0,9% c nng 10
-1
sau pha cc nng
tip theo 10
-2
, 10
-3
tu theo yu cu th nghim.
Mu th l cht lng khng ho ln vo nc nh dng du, kem, hoc
thuc m. Cn ch to nh dch bng cch thm mt lng cht nh
ho v trng thch hp nh tween 20, tween 80, lm nng nh 45
o
C
to mt nh dch ng nht.
Kim tra cht c ch:
Kt qu th nghim s khng c gi tr nu trong mu th c cht bo
qun hoc cc thnh phn c nh hng n s pht trin ca vi sinh vt. V
vy, cn kim tra cht c ch trc khi m s lng vi sinh vt.
Vi sinh vt ch th:
Staphylococcus aureus (i din vi khun G +).
Escherichia coli (i din vi khun G -).
Candida albicans (i din vi nm).
Cc chng trn c nui cy trong cc mi trng dinh dng thch hp
sau 18 - 24 gi (i vi vi khun) v 24 n 48 gi i vi vi nm, c lm
thnh nh dch c khong 100 t bo/ ml.
Cch tin hnh:
a th 1: Cho 1ml cht th nng thch hp.
a th 2: 1 ml cht th + 1ml nh dch vi sinh vt.
a chng: 1ml nc ct v trng + 1ml nh dch vi sinh vt
Cho vo mi a 15 - 20 ml mi trng dinh dng thch hp
ngui di 45
o
C.
30 - 35
o
C/ 24 - 48 gi i vi vi khun v 25 - 28
o
C/48 - 72 gi i vi C.
albicans.
129
Nhn xt kt qu:
m s khun lc vi sinh vt trn cc a th nghim.
Gi A l s khun lc a 1
Gi B l s khun lc a 2
Gi C l s khun lc a chng
Nu B A + C mu th khng c cht c ch.
Nu B A hoc B << A + C mu th c cht c ch vi sinh vt. Cht
c ch phi c x l bng cch tng th tch mi trng, trung ho cht c
ch bng cc cht trung ho thch hp nh tween 20 4%. Nu mu c cht c
ch qu mnh phi th bng phng php mng lc.
* Kim tra cht c ch cng c th c thc hin theo cch sau:
Cy khong 100 t bo cc vi khun vo cc ng mi trng chn lc
khng c mu th v c mu th nng thch hp cho E. coli Salmonella,
Staphylococcus aureus, Pseudomonas aeruginosa.
Mu th khng c tc dng c ch nu vi khun mc tt nh nhau c
hai ng mi trng.
m s lng vi sinh vt:

i vi vi khun:
Cho vo mi a petri 1ml cht th nng thch hp sao cho khng qu
300 khun lc trong 1ml. Thm 15 - 20ml mi trng thch casein - u
tng hoc mi trng thch thng ngui di 45
o
C, xoay nh a
cht th trn u vo mi trng. Nui cy 30 - 35
0
C trong 1 - 2 ngy.
i vi vi nm:
Cho vo mi a petri 1ml cht th nng thch hp sao cho khng
qu 100 khun lc vi nm trong 1ml. Thm 15-20ml thch Sabouraud
+ khng sinh.
Nui cy 25 - 28
o
C trong 4 - 5 ngy.
Th nghim c thc hin vi 1 hay 2 nng pha long cui cng.
Mi nng c lm 2 - 3 a th ly gi tr trung bnh. Sau thi
gian nui cy, cc a c s khun lc vi khun nh hn 300 v vi nm
nh hn 100 c m tnh s lng.
Tnh kt qu:
Tng s vi khun hiu kh, vi nm trong 1g (1ml) c tnh theo cng thc:
2
k A k A
X
2 2 1 1
+
=
(Php th thc hin vi 2 nng ).
130
A
1
: S khun lc vi sinh vt trung bnh nng pha long k
1
.
A
2
: S khun lc vi sinh vt trung bnh nng pha long k
2
.
k
1
, k
2
: pha long.
Cc dc in c quy nh: mu th c t hn 10 vi sinh vt/1g (1ml) nu
khng c khun lc mc trn a th nng 10
-1
.
4.3.5. Xc nh hot lc cht khng sinh bng cc php th vi sinh vt
4.3.5.1. Mc ch
Trong lnh vc kim nghim, nh lng khng sinh ngi ta c th
s dng cc phng php khc nhau. Mt s cht khng sinh c th
c nh lng bng phng php ho hc, phng php ha l. Tuy
nhin, phng php sinh hc vn ng vai tr quan trng trong vic
nh gi hot tnh sinh hc ca thuc v kim tra c s gim hay
mt hot lc ca cht khng sinh m cc phng php l, ho khng
thc hin c.
Cc cht khng sinh c cu trc phc tp hoc thnh phn c tc dng
gm mt hn hp nhiu thnh phn, thng phi dng th nghim vi
sinh vt xc nh hot lc ca thuc.
Php th trn vi sinh vt cn cho bit nhy cm ca vi khun gy
bnh i vi cc cht khng sinh c th, v mc khng thuc ca
chng. Trn c s c th chn khng sinh thch hp cho bnh nhn
trong iu tr.
4.3.5.2. Nguyn tc
Hot lc ca mt cht khng sinh c xc nh bng cch so snh kh
nng c ch ca chng vi sinh vt ch th, bi nhng nng bit ca
khng sinh th (cha bit hot lc) v nng bit ca khng sinh chun
( bit r hot lc).
4.3.5.3. Chng ch th
Chng ch th l chng vi sinh vt thun khit, nhy cm i vi mt
cht khng sinh, chng ch th thng c phn lp t cc bo tng ging
Quc gia, c bo qun trong ng ng kh hoc trong mi trng thch hp
4 - 10
o
C. Tu theo dc in ca mi nc, mi cht khng sinh c th c mt
vi chng ch th khc nhau.
Ch to nh dch vi sinh vt khng c bo t:
Cc chng vi khun:
Staphylococcus aureus
Sarcina lutea
131
Escherichia coli
Micrococcus luteus
Klebsiella faecalis.
Chng vi nm:
Saccharomyces cerevisiae.
i vi cc chng vi sinh vt khng c bo t, khi th nghim cn s
dng chng sau 16 - 24 gi nui cy c cc chng ch th ang giai on
pht trin logarit.
Chng ch th l vi khun trc khi s dng c cy sang mi trng
thch dinh dng (pepton 10g, cao tht 1,5g, cao men 3g, glucose 1g, thch
15g, nc ct 1000 ml). Sau 16 - 18 gi nui cy 35 - 37
o
C cc vi khun c
lm thnh mt nh dch c nng thch hp vi NaCl 0,9% to vng v
khun r rt trong phng php khuch tn v c c thch hp trong
phng php o c.
Chng ch th l vi nm c nui cy trn mi trng thch Sabouraud
28 - 30
o
C trong 24 gi v cng c lm thnh mt nh dch c nng
thch hp.


Ch to nh dch bo t:
Cc chng ch th c bo t:
Bacillus subtilis
Bacillus pumilus
Bacillus cereus
Nu chng ch th c bo t nn ch to dng bo t s dng, v dng
bo t c bo qun di 4
o
C c th tn ti trong mt thi gian rt di.
Cc chng vi khun trn c cy vo bnh mi trng thch dinh dng
c b mt thch rng (pepton 6g, cao tht 1,5g, cao men 3g, glucose 1g,
K
2
HPO
4
3,68g, KH
2
PO
4
1,32g, thch 15g, nc ct 1000ml)
Mi trng ny c thm MnSO
4
vi hm lng 0,001g/lt thc y
s hnh thnh bo t. Nui cy 35 - 37
o
C trong vng 7 ngy vi B. cereus nui
cy 30
o
C. Dng nc ct v trng ra lp bo t trn b mt mi trng
to mt nh dch. un cch thy nh dch ny 70
o
C/ 30 pht, pha long nh
dch n nng 10
7
- 10
8
bo t trong 1ml.
4.3.5.4. Cht chun v n v hot lc
Cht chun:
Cht chun l nhng cht c tinh khit cao, c th dng cht chun
gc hoc cht chun th cp c hot lc c xc nh theo mu chun quc t
132
tng ng. Cht chun c ng trong ng thy tinh hn kn bo qun kh,
trnh nh sng, di 0
o
C.


n v hot lc:
Hot lc ca mt cht khng sinh c tnh bng n v hot lc quc t
(vit tt l U hoc UI, IU).
Mt n v hot lc l hot lc c trng ca mt lng nht nh cht
khng sinh chun sinh hc quc t hoc ch phm i chiu sinh hc vi
chun quc t. V d 1U gentamicin l hot lc ca 0,0056mg gentamicin.
n v hot lc ca cc cht khng sinh c xc nh bi cc vin
nghin cu ln ca t chc y t th gii v cht chun sinh hc.
Hot lc ca cc cht khng sinh c th c th hin bng g hot lc.
i vi cht khng sinh hon ton tinh khit, trong a s cc trng hp g
hot lc tng ng vi g khi lng ca cht khng sinh. Khi cht khng
sinh cha hon ton tinh khit, hoc l hn hp ca nhiu thnh phn tng
t nhau v ho hc nhng khc nhau v hot tnh sinh hc th g hot lc
khng nht thit tng ng vi g khi lng ca cht khng sinh.
4.3.5.5. Mi trng v dung mi
Ty theo cht khng sinh v vi sinh vt ch th chn cc mi trng
thch hp cho th nghim.
Dung mi v cc cht pha long khng c nh hng n s pht
trin ca vi sinh vt. Cc cht pha long nh: nc ct, dung dch HCl,
ethanol, methanol, dung dch m c pH khc nhau c s dng tu
thuc vo tan ca cc cht khng sinh. Cc vn ny cn tham
kho dc in.
4.3.5.6. nh lng cht khng sinh bng phng php khuch tn
C hai phng php vi sinh vt nh lng cht khng sinh l:
Phng php o c: (Turbidimetric method)
Phng php khuych tn: (Diffusion method)
Phng php khuych tn thng c s dng nhiu trong nh lng
khng sinh.
Nguyn tc:
Cht khng sinh khuych tn vo mi trng dinh dng c cy vi
sinh vt ch th, to cc vng c ch vi sinh vt c ng knh t l thun vi
logarit nng tng ng. Hot lc ca cht th c so snh vi cht chun
theo phng php thng k.
133


Pha dung dch chun v dung dch th:
Pha chun: Cn mt lng cht chun thch hp ho vo dung mi c
mt dung dch gc nng khong 1000 UI/ ml. T dung dch gc pha thnh 3
nng cui cng theo cp s nhn h s hai l s
1
, s
2
, s
3
hoc hai nng s
1
, s
2

(s
1
< s
2
< s
3
).
Cht th c pha nh cht chun vi gi nh cht th c hot lc tng
ng cht chun. Cht th c 3 nng cui cng l t
1
, t
2
, t
3
(hoc t
1
, t
2
).
Phng php khuch tn ch cho kt qu tt khi cht chun v cht th c
cng bn cht ha hc. S khng ng nht gia chun v th lm thay i
tnh song song ca hai ng thng biu th s tng quan gia ng knh
vng c ch v logarit nng . V hai cht c hai h s khuch tn ring. V vy
khng so snh cht khng sinh cc dng mui khc nhau nh erythromycin
stearat v erythromycin propionat, oxytetracyclin v clotetracyclin.
Khi pha dung dch chun v dung dch th cn lu : nu chn khong
nng cui cng nh lng khng thch hp, hoc hot lc gi nh ca
th qu xa hot lc ca chun, th s lm thay i tnh cht ca s tng
quan gia ng knh vng c ch v logarit nng . Kt qu nh lng s
khng chnh xc.
Tin hnh th nghim:
vo cc a Petri mt lng mi trng dinh dng c cy
chng ch th to mt lp ng nht c dy t 2-5mm. Chiu dy lp
thch mng hay dy qu, hoc khng ng u c th lm cho quan h gia
ng knh vng c ch v logarit nng khng cn l ng thng. Cng c
th lm 2 lp mi trng, nhng ch lp trn c cy vi sinh vt ch th.
Chng ch th l loi khng c bo t, phi cy vo mi trng c ngui
di 45
o
C, nu l dng c bo t nhit cho php khi cy chng l 65 - 70
o
C.
Cn cy vo mi trng mt lng vi sinh vt ch th sao cho c vng c ch r
nt vi kch thc ng knh thch hp tng ng vi cc nng khng sinh
chn. Nu lng chng qu t, vng c ch s qu to, vi sinh vt mc tha
tht, kh xc nh c vng c ch. Nu lng chng qu nhiu, vng c ch
nh s kh o v km chnh xc.
Cc a mi trng c kh nhit phng th nghim trong 30
pht trc khi dng. B mt thch t s lm cho vng c ch b nhe.
i vi th nghim 3 liu: dng 6 ng tr bng thp khng r c kch
thc nh nhau (chiu cao khong 10mm, ng knh trong khong 6mm).
t cc ng tr ln b mt a thch theo s , cho cht th v chun vo cc
ng tr vi mt lng bng nhau. C th thay ng tr bng cch c cc l
trn a thch c ng knh t 6 - 8mm hoc dng cc khong giy tm
khng sinh c dy thch hp, ng knh khong 6 mm.
134
to vng c ch to nht cn thc hin
giai on tin khuch tn bng cch cc a
th nhit phng trong 2 gi cho khng
sinh khuych tn vo mi trng (i vi
colistin, polymyxin B thi gian khuych tn
di hn t 3 4 gi). Sau thi gian khuych
tn, cc a th c nhit thch hp
trong 16 18 gi. Trong qu trnh nhit
phi n nh v phi ng u mi ni. Cc
a th nn mt lp trong t m sao cho v
tr ca cc nng t v s c s khc nhau t
nht v ngun nhit.

T
1
T
2
T
3
S
1
S
2
S
3

o ng knh vng c ch to bi cc nng chun v th bng thc
o c chnh xc n 0,1mm.
Mt th nghim c tin hnh vi 10 a th song song.
Tnh kt qu:
(Th nghim 3 liu)
T l phn trm hot lc ca khng sinh th so vi chun c tnh theo
cng thc:
) , ( log log 95 0 P I
B
A
3
4
2 Anti R =

+ =
A = (T
1
+ T
2
+ T
3
) (S
1
+ S
2
+ S
3
)
B = (T
3
T
1
) + (S
3
S
1
)
T
1
, T
2
, T
3
, S
1
, S
2
, S
3
l tng gi tr ng knh vng v khun tnh bng
mm ca cc nng t
1
, t
2
, t
3
,

s
1
, s
2
, s
3 .
I l t s ca hai nng pha long k
tip nhau, I = 2. Vy

+ =
B
A
Anti R . 2 log
3
4
2 log

+ =
B
A
. 4013 , 0 2 log Anti R
Hot lc ca mu th =
100
chun lc hot R

135
Mun php th c gi tr, th nghim phi t c cc iu kin sau:
Hot lc ca cht th c gi thit gn st vi thc t kt qu c
c nm trong phm vi ng cong chun.
T
a
< t
a
.V
dc ca ng biu din logarit nng vi ng knh vng v
khun phi c ngha.
T
b
>> t
b
.V
Cc ng biu din logarit nng ca cht th v cht chun vi
ng knh vng v khun phi song song.
T
ab
< t
ab
.V
Trong khong nng chn s ph thuc gia logarit ca nng
vi ng knh vng v khun tng ng l tuyn tnh.
T
c
< t
c
.V
T
ac
< t
ac
.V
V: l tng ca cc hiu gi tr ng knh ln nht v nh nht ca mt
nng .
T
a
= (T
1
+ T
2
+ T
3
) (S
1
+ S
2
+ S
3
)
T
b
= (T
3
+ S
3
) (T
1
+ S
1
)
T
ab
= (S
1
+ T
3
) (S
3
+ T
1
)
T
c
= (S
1
+ S
3
+ T
1
+ T
3
) 2 (S
2
+ T
2
)
T
ac
= (2S
2
+ T
1
+ T
3
) (2T
2
+ S
1
+ S
3
)
t
a
, t
b
, t
c
, t
ab
, t
ac
l cc h s tra bng theo s vng v khun (n) do c ca
mt nng .
Gi tr t:
Gi tr t (P = 0,95)
S vng
v khun (n)
t
a
t
b
t
c
t
ab
t
ac
5
6
7
8
9
10
0,80
0,80
0,81
0,82
0,83
0,84
0,66
0,66
0,66
0,67
0,68
0,68
1,14
1,14
1,14
1,16
1,17
1,19
0,66
0,66
0,66
0,67
0,68
0,68
1,14
1,14
1,14
1,16
1,17
1,19
136
Xc nh gii hn tin cy:
lch chun:
2
2 2
M
B
B 5 1 A n V Hn 666 2
S
) , ( . . , +
=
Hn: H s vng o ph thuc vo n.
n: S vng v khun o c ca mt nng .
Gii hn tin cy:
M 2 1
S 301 0
B
A
4013 0 M , ,
,
=
Phn trm gii hn tin cy trn:
R
1
= Antilog (2 + M
1
)
Phn trm gii hn tin cy di;
R
2
= Antilog (2 + M
2
)
Sai s th nghim:
100
2R
2
R
1
R
e%

=
Kt qu nh lng c gi tr vi e 5%.
Gi tr H
n
N 3 4 5 6 7 8 9 10
H
n
1,27 1,03 0,90 0,81 0,75 0,69 0,65 0,65
Ti liu tham kho
1. B y t (2002). Dc in Vit Nam III. NXB Y hc, H Ni
2. Nguyn Ln Dng v cng s (1997). Vi sinh vt hc. H Ni
3. British Pharmacopoeia 2001.
4. Collins C.H. and Patricia M.L (1976). Microbiological methods, London.
5. Pharmacopoeia of the Peoples Republic of China (1997).
6. The United States Pharmacopoeia XXIV (2000).
137
Cu hi t lng gi
4.1. Trnh by nhng c im chnh v hnh thi v tnh cht nui cy ca vi
khun, nm mc, nm men?
4.2. Nu cch phn loi vi khun theo hnh th v c tnh h hp?
4.3. Phn tch nhng yu t ngoi cnh nh hng n s pht trin ca vi
sinh vt?
4.4. Trnh by phng php lm mi trng nui cy vi sinh vt?
4.5. M t cc phng php tit trng?
4.6. Trnh by th nghim th v trng bng phng php nui cy trc tip?
4.7. Nu mc ch, nguyn tc ca th nghim th v trng v th gii hn vi
sinh vt?
4.8. Trnh by th nghim m s lng vi sinh vt trong 1g (1ml) dc phm
bng phng php a thch?
4.9. Vit tn nhng chng ch th c s dng th cht c ch trong th
nghim th v trng v th gii hn vi sinh vt. Nu s khc nhau v
chng ch th trong hai php th v gii thch?
4.10. Vai tr ca phng php sinh hc trong kim nghim cht khng sinh?
4.11. Trnh by phng php chun b nh dch vi sinh vt trong nh lng
khng sinh.Ti sao i vi nhng chng ch th khng c bo t trc khi
s dng cn phi cy truyn vo mi trng thch hp?
4.12. Cch pha dung dch chun v dung dch th trong nh lng khng
sinh bng phng php khuych tn. Nu nhng im cn ch trong
qu trnh pha cc dung dch ny?
4.13. M t cch tin hnh nh lng cht khng sinh bng phng php
khuych tn?
4.14. Nu nhng nguyn nhn trong cc giai on lm th nghim nh hng
n s chnh xc ca phng php khuych tn?
4.15. Nu nhng iu kin th nghim cn t c trong nh lng khng
sinh php th c gi tr?
4.16. Vit cc cng thc tnh kt qu nh lng khng sinh vi th nghim ba
liu v cng thc xc nh gii hn tin cy?
138
4.17. Trnh by phng php xc nh s lng ti a vi khun hiu kh, vi
nm trong mt gam sp bi mi Lip Ice. Cho bit mu th c cht bo
qun ring ca nh sn xut?
4.18. Trnh by phng php nh lng bt Gentamicin sulphat bng th
nghim vi sinh vt( phng php khuych tn), vi ch th Bacillus
pumilus NCTC 8241.
Cho bit: Gentamicin sulphat chun c 680 U/ ml, dung mi pha long l nc.
Ba nng l: S1= 4 U/ ml; S2=8 U/ ml; S3=16 U/ ml.

















139
Chng 5.
kim nghim cc dng bo ch
Mc tiu hc tp:
1. Trnh by c cc yu cu k thut v phng php th nh gi
cht lng cc dng bo ch: Thuc bt, thuc vin nn, vin nang,
thuc tim, thuc tim truyn, thuc nh mt, thuc m, thuc ung
dng lng, thuc n, thuc trng.
2. nh gi c kt qu kim nghim i vi mt mu kim nghim
thnh phm c th ca cc dng bo ch trn.
Khi tin hnh kim nghim mt mu ch phm thuc mt dng bo ch
c th (thuc bt, vin nn, vin nang, thuc tim, ...) theo chuyn lun ring
ca ch phm trong Dc in, thng thng yu cu ghi phn u ca
chuyn lun l ch phm phi p ng cc yu cu chung ca dng bo ch
v cc yu cu ring ca dc cht, ca ch phm nh tnh cht, nh tnh,
nh lng, tp cht (nu c), ... Cn nu tiu chun kim nghim l tiu
chun ca nh sn xut (TCCS) th trong tiu chun kim nghim c y
yu cu k thut v phng php th c th cho ch phm , mt s tiu ch
c trng cho dng bo ch ca ch phm c tin hnh th theo hng
dn ca Dc in.
Trong ti liu ny, chng ti cp ti yu cu k thut v phng php
th ca cc dng bo ch thng dng nh thuc bt, thuc vin nn, vin
nang, thuc tim, thuc tim truyn, thuc nh mt, thuc m, thuc ung
dng lng, thuc n, thuc trng. Ti liu tham kho chnh c s dng l
Dc in Vit Nam III, ng thi chng ti cng s dng Dc in ca mt
s nc khc nh Dc in Anh, Dc in M, Dc in Trung Quc v
mt s ti liu kim nghim cc dng bo ch khc.
5.1. Kim nghim thuc bt
5.1.1. Yu cu k thut v phng php th
5.1.1.1. Tnh cht
Bt phi kh ti, khng b m, vn, mu sc ng nht. Mi v ty theo
tng loi ch phm ca cc nh sn xut.
Cch th: Tri mt lng bt va thnh mt lp mng trn mt t
giy trng mn. Quan st mu sc bng mt thng, di nh sng t nhin.
140
5.1.1.2. m
Cc thuc bt khng c cha hm lng nc qu 9,0%, tr ch dn khc.
Cch th: theo Xc nh mt khi lng do lm kh - Ph lc 5.16 -
DVN III.
5.1.1.3. mn
Thuc bt phi t mn quy nh trong chuyn lun ring.
Nu khng c ch dn khc, php th ny dng cho tt c cc thuc bt
kp, cc thuc bt dng p, thuc bt dng pha ch thuc dng cho
mt, tai.
Cc c bt c quy nh da vo cc s ca ry. Ry c li ry v li
ry c th lm bng si kim loi hoc si cc vt liu thch hp khc c dt
thnh nhng mt vung. Li ry dng ry bt thuc c phn loi bng
nhng con s biu th kch thc l ry, quy nh tnh bng m theo bng 5.1.
Cch th: Chn c ry thch hp theo qui nh ca tiu chun. Cn mt
lng thuc bt, em ry qua ry c c qui nh.
i vi bt th hoc na th: ly 25g -100 g bt. Cho vo ry thch hp,
lc ry theo chiu ngang quay trn t nht 20 pht v ry ti khi xong. Cn
chnh xc s lng cn li trn ry v s thu c trong hp hng.
i vi bt na mn, mn hay rt mn: ly khng qu 25 g bt, cho vo ry
thch hp, lc ry theo chiu ngang quay trn t nht 30 pht ri ry ti khi
xong. Cn chnh xc s lng cn li trn ry v s thu c trong hp hng.
i vi cht c du hay bt c xu hng bt mt ry th trong qu trnh
ry thnh thong chi cn thn mt ry, tch ri nhng ng t li khi ry.
Thuc bt t tiu chun v mn nu:
Khi quy nh dng 1 ry xc nh c bt th khng c c di 97%
khi lng thuc bt qua c c ry .
Khi quy nh dng 2 ry xc nh c bt th mt ry ln trn ry
kia v tin hnh ry; khng c c di 95% khi lng thuc bt qua
ry c s ry cao hn v khng c qu 40% khi lng thuc bt qua
ry c s ry thp hn.
141
Bng 5.1. Qui nh s ry
S ry (m) C mt ry (mm) ng knh si ry (mm)
1400 1,400 0,710
710 0,710 0,450
355 0,355 0,224
250 0,250 0,160
180 0,180 0,125
125 0,125 0,090
90 0,090 0,063
Cc k hiu quy nh c bt:
Bt th (1400/355)
Bt na th (710/250)
Bt na mn (355/180)
Bt mn (180/125)
Bt rt mn (125/90)
5.1.1.4. ng u khi lng
Nhng thuc bt khng quy nh th ng u hm lng th phi
th ng u khi lng. Ring thuc bt pha tim hoc truyn tnh
mch, nu khi lng nh hn hoc bng 40 mg th khng phi th ng
u khi lng nhng phi t yu cu ng u hm lng.
Cch th: Cn tng n v trong s 5 n v ng gi nh nht c ly
bt k. Khi lng thuc phi nm trong gii hn cho php theo bng 5.2.
Bng 5.2. Gii hn cho php chnh lch khi lng i vi thuc bt
Khi lng ghi trn nhn Phn trm chnh lch
Di hoc bng 0,50g
Trn 0,50 - 1,50g
Trn 1,50 - 6,00g
Trn 6,00g
10
7
5
3
Tt c cc n v phi t qui nh trong bng trn.
Nu c mt n v c khi lng lch ra ngoi quy nh ny th th li vi
5 n v khc, nu ln th li c qu mt n v khng t th l thuc khng
t yu cu.
142
i vi cc ch phm ng gi trong hp, l th sau khi cn c v phi b
ht thuc ra, dng bng lau sch thuc, cn v ri tnh theo lng thuc trong
tng hp hoc l.
chnh lch c tnh theo t l phn trm so vi khi lng trung
bnh bt thuc trong mt n v ng gi.
5.1.1.5. ng u hm lng
Tr khi c ch dn khc, php th ny p dng cho cc thuc bt ung,
tim c trnh by trong cc n v ng gi mt liu c cha mt hoc
nhiu hot cht, trong c cc hot cht c hm lng di 2 mg hoc di
2% (kl/kl) so vi khi lng thuc mt liu.
Php th ng u hm lng c tin hnh sau php th nh lng
v hm lng hot cht trong gii hn quy nh.
Cch th: Ly 10 n v ng gi nh nht bt k, xc nh hm lng
hot cht tng gi theo phng php nh lng ch dn trong chuyn lun.
Cch nh gi:
Ch phm em kim tra t yu cu php th nu khng qu mt n v
c gi tr hm lng nm ngoi gii hn 85 - 115% ca hm lng trung bnh v
khng c n v no nm ngoi gii hn 75 - 125% ca hm lng trung bnh.
Ch phm khng t yu cu php th nu qu 3 n v c gi tr hm
lng nm ngoi gii hn 85 - 115% ca hm lng trung bnh hoc 1 n v
tr ln nm ngoi gii hn 75 - 125% ca hm lng
Nu hai hoc ba n v c gi tr hm lng nm ngoi gii hn 85 -115%
nhng trong gii hn 75 - 125% ca hm lng trung bnh th th li trn 20
n v khc, ly ngu nhin. Ch phm t yu cu, nu khng qu 3 n v
trong tng s 30 n v em th c gi tr hm lng nm ngoi gii hn 85 -
115% ca hm lng trung bnh v khng c n v no c gi tr hm lng
nm ngoi gii hn 75 - 125% ca hm lng trung bnh.
5.1.1.6. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu
chun, thuc bt phi cho cc phn ng ca cc hot cht c trong ch phm
5.1.1.7. nh lng
Ly thuc ca 5 n v ng gi nh nht bt k, trn u. Tin hnh
nh lng theo cc phng php c qui nh trong tiu chun, hm lng
ca tng hot cht trong ch phm phi nm trong gii hn cho php theo
bng 5.3.
143
5.1.1.8. Gii hn nhim khun
Thuc bt c ngun gc dc liu phi t yu cu Gii hn nhim khun.
Cch th: Nu khng c qui nh ring th tin hnh th v nh gi
theo "Th gii hn vi sinh vt" - phn 4.3.4, trang 119-121.
Bng 5.3. Gii hn cho php v hm lng hot cht i vi thuc bt
Lng ghi trn nhn Phn trm chnh lch
Ti 100 mg
Trn 100 mg ti 1g
Trn 1 g n 5 g
Trn 5 g
15
10
5
1
5.1.2. Cc loi thuc bt
5.1.2.1. Thuc bt ung
Thuc bt ung phi p ng cc yu cu cht lng chung ca thuc bt.
Thuc bt si bt phi t thm yu cu v tan.
Cch th: Th mt lng thuc bt tng ng vi mt liu vo mt
cc thu tinh c cha 200 ml nc nhit 15 -20
o
C, xut hin nhiu bt
kh bay ra. Khi ht bt kh bay ra, thuc phi tan hon ton. Th nh vy
vi 6 liu n. Mu th t yu cu nu mi liu th u tan trong vng 5
pht, tr khi c ch dn ring.
5.1.2.2. Thuc bt dng ngoi
Thuc bt dng ngoi phi p ng cc yu cu cht lng chung ca
thuc bt. Ngoi ra, phi t cc yu cu ring sau:
v khun: thuc bt p, dng cho vt thng rng hoc trn da
b tn thng nng, thuc bt dng cho mt phi v khun.
Cch th: Nu khng c qui nh ring th tin hnh th v nh gi
theo "Th v trng" - phn 4.3.3, trang 115-118.
5.1.2.3. Thuc bt pha tim
Thuc bt pha tim p ng cc yu cu cht lng chung ca thuc bt
v yu cu i vi thuc tim, thuc tim truyn dng bt nh: v khun,
cht gy st ...
Cch th: Theo cch th chuyn lun thuc tim.
V d: Thuc tim benzylpenicilin - DVN III trang 32 - 33.
144
5.2. Kim nghim thuc vin nang
5.2.1. Yu cu k thut v phng php th
5.2.1.1. Tnh cht
Nang cng hoc nang mm cha bt hoc cm hoc cht lng.
Cch th: Th bng cm quan.
5.2.1.2. ng u khi lng
Cn khi lng ca mt nang: Vi vin nang cng th tho ri hai na v
nang thuc ra, dng bng lau sch v ri cn khi lng ca v; vi vin
nang mm, ct m nang, bp thuc ra ht ri dng ether hoc dung mi hu c
thch hp ra sch v nang, kh t nhin ti khi ht mi dung mi, cn khi
lng v. Khi lng thuc trong nang l hiu gia khi lng nang thuc v v
nang. Lm nh vy vi 19 nang khc c ly bt k. chnh lch khi lng
ca tng vin vi khi lng trung bnh phi t theo bng 5.4.
Nu c yu cu th ng u hm lng th khng phi th ng
u khi lng.
Bng 5.4. Gii hn cho php chnh lch khi lng i vi vin nang
Khi lng trung bnh nang Phn trm chnh lch
Nh hn 300 mg
Ln hn hoc bng 300 mg
10
7,5
5.2.1.3. ng u hm lng
Nghin mn bt thuc ring tng vin v tin hnh th v nh gi nh
i vi thuc bt.
5.2.1.4. r
Nu khng c qui nh ring th tin hnh th v nh gi theo "Php
th r vin nn v vin nang" - phn 5.10, trang 154 - 157.
Vin khng cn th r khi php th ha tan c thc hin.
5.2.1.5. ha tan
Trong DVN III, nhiu ch phm thuc vin yu cu nh gi ha
tan. Khi c yu cu s ch dn trong chuyn lun ring.
Cch th: Tin hnh theo Phng php th tc ha tan ca vin nn
v vin nang - phn 5.9, trang 160 - 165.
145
5.2.1.6. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu
chun, vin nang phi cho cc phn ng ca cc hot cht c trong ch phm
5.2.1.7. nh lng
Cn thuc trong 20 vin, tnh khi lng trung bnh vin, lm ng nht
bng cch nghin (i vi vin nang cha bt hoc cm) hoc trn u (i vi
nang cha cht lng). Tin hnh nh lng theo cc phng php c qui
nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi
nm trong gii hn cho php theo bng 5.5.
Bng 5.5. Gii hn cho php v hm lng i vi thuc vin nang, vin nn
Lng ghi trn nhn Phn trm chnh lch
Ti 50 mg
Trn 50 mg ti 100 mg
Trn 100 mg
10
7,5
5
5.2.1.8. Tp cht (nu c)
Khi c yu cu s ch dn trong chuyn lun ring.
5.2.2. Cc loi vin nang
Cc loi vin nang phi tun theo yu cu chung ca thuc vin nang v
yu cu ring i vi ty tng loi.
Thuc nang cng phi t cc yu cu cht lng chung ca vin nang.
5.2.2.1. Thuc nang cng
Thuc nang cng c v nang gm hai phn hnh tr lng kht vo nhau,
mi phn c mt u kn, u kia h. Thuc ng trong nang thng dng
rn nh bt hoc cm.
V d: Vin nang Cephalexin - DVN III. Trang 53 - 54.
5.2.2.2. Thuc nang mm
Thuc nang mm c v nang l mt khi mm vi cc hnh dng khc nhau.
Thuc ng trong nang thng dng dung dch, hn dch hoc nh tng.
V d: Vin nang mm vitamin A - DVN III. Trang 291 - 292.
146
5.2.2.3. Thuc nang tan trong rut
Thuc nang tan trong rut l cc nang cng hoc nang mm c v nang
bn vng vi dch d dy, ch tan trong dch rut; hoc cc nang c ng cm
c bao lp ch tan trong dch rut.
5.2.2.4. Thuc nang gii phng hot cht c bit
Thuc nang gii phng hot cht c bit l cc nang cng hoc nang
mm c v nang hay thuc trong nang hoc c v nang v thuc trong nang
c bo ch c bit, kim sot hay chng trnh ho tc hoc v tr gii
phng hot cht trong c th.
Thuc nang gii phng hot cht c bit phi t cc yu cu cht lng
chung ca vin nang, ring r khng phi th v bt buc th ha
tan theo yu cu ring ca tng chuyn lun.
5.3. Kim nghim thuc vin nn
5.3.1. Yu cu k thut v phng php th
5.3.1.1. Tnh cht
Vin nn thng c dng hnh tr dt, hai y phng hoc cong, c th
khc ch, k hiu hoc rnh. Cnh v thnh vin lnh ln. Mu sc ng nht.
Cch th: Bng cm quan.
5.3.1.2. r
Nu khng c qui nh ring th tin hnh th v nh gi theo "Php
th r vin nn v vin nang" - phn 5.10, trang 154 - 157.
Vin khng cn th r khi php th ha tan c thc hin.
5.3.1.3. ng u khi lng
Cn chnh xc 20 vin bt k v xc nh khi lng trung bnh ca vin.
Cn ring khi lng tng vin v so snh vi khi lng trung bnh, tnh
lch theo t l phn trm ca khi lng trung bnh t tnh ra khong gii
hn ca gi tr trung bnh. Khng c qu 2 vin c khi lng chnh lch
qu khong gii hn ca khi lng trung bnh v khng c c vin no c
chnh lch qu gp i lch tnh theo t l phn trm, theo bng 5.6.
Bng 5.6. Gii hn cho php chnh lch khi lng i vi vin nn
Khi lng trung bnh vin Phn trm chnh lch
Ti 80 mg
Trn 80 mg n 250 mg
Trn 250 mg
10
7,5
5
147
Nu c yu cu th ng u hm lng th khng phi th ng
u khi lng.
5.3.1.4. ng u hm lng
Nghin mn ring tng vin v tin hnh th v nh gi nh i vi
thuc bt.
5.3.1.5. ha tan
Ch thc hin khi c yu cu c ch dn trong chuyn lun ring.
Nu khng c qui nh ring th tin hnh th v nh gi theo "Php
th ha tan ca vin nn v vin nang" - phn 5.9, trang 149 - 154.
5.3.1.6. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu
chun, vin nn phi cho cc phn ng ca cc hot cht c trong ch phm.
5.3.1.7. nh lng
Cn 20 vin, xc nh khi lng trung bnh vin. Nghin mn. Tin
hnh nh lng theo cc phng php c qui nh trong tiu chun, hm
lng ca tng hot cht trong ch phm phi nm trong gii hn cho php,
theo bng 5.5.
5.3.1.8. Tp cht (nu c)
Khi c yu cu s ch dn trong chuyn lun ring.
5.3.2. Cc loi vin nn
Cc loi vin nn phi tun theo yu cu chung ca thuc vin nn v
yu cu ring i vi ty tng loi.
5.3.2.1. Vin nn khng bao (Uncoated tablets)
Gm cc vin nn c bo ch bng cch nn cc ht gm dc cht, t
dc. Vin nn loi ny khng cha mt thnh phn no thay i s gii
phng hot cht trong ng tiu ha. Khi b gy vin nn mt lp v quan
st bng knh lp th thy ng nht, khng c lp bao ngoi.
5.3.3.2. Vin bao (Coated tablets)
Vin bao c b mt nhn, thng c mu, c nh bng. Ly mt phn
vin b gy, quan st di knh lp, thy r nhn c bao bng mt lp
hay nhiu lp.
148
5.3.2.3. Vin bao bn vi dch v d dy (Gastro - resistant tablets)
Vin c bao bng lp bao bn vi dch v d dy nhng li tan c
trong dch rut do s dng cc cht bao l cellacephate (cellulose acetat
phtalate) v cc polymer.
5.3.2.4. Vin nn si bt (Effervescent tablets)
L vin nn khng bao c cha cc acid v carbonat hoc hydrocarbonat,
chng phn ng vi nhau rt nhanh khi c mt ca nc v gii phng CO
2
,
ng thi ho tan hay phn tn hot cht trong nc trc khi dng.
5.3.2.5. Vin ngm (Tablets for use in the mouth)
Vin ngm thng l vin nn khng bao c iu ch cc thnh
phn v hot cht gii phng dn v tc dng ti ch, gii phng v hp th
di li hoc cc phn khc trong ming.
5.3.2.6. Vin nn tan trong nc (Soluble tablets)
Vin nn tan trong nc l vin nn khng bao, ha tan trong nc.
Dung dch to thnh c th hi c nh.
5.3.3.7. Vin nn phn tn trong nc (Dispersible tablets)
Vin nn phn tn trong nc l vin nn khng bao phn tn trong nc
thnh cc tiu phn.
5.3.2.8. Vin nn thay i gii phng hot cht (Modified release tablets)
Thng l vin bao hoc khng bao, c iu ch bng cch thm cc t
dc c bit hoc iu ch theo phng php c bit nhm thay i tc
gii phng hoc v tr gii phng ca thuc.
Vin nn thay i gii phng hot cht thng khng yu cu th r
m phi th tc ha tan.
V d: Vin nn acid ascorbic - DVN III trang 6
Vin bao ibuprofen - DVN III trang 141.
5.4. Kim nghim thuc tim, thuc tim truyn
Ch phm dng tim (parenteral preparations) c 5 loi l thuc tim
(injections), thuc tim truyn (infusions), dung dch m c dng tim hoc
truyn (concentrates for injections and infusions), thuc bt dng tim hoc
truyn (powders for injections and infusions), thuc cy (implants).
Trong phm vi ca chng trnh i hc, chng ti ch cp ti yu cu
k thut v phng php kim nghim thuc tim v thuc tim truyn.
149
5.4.1. Tnh cht
Dng dung dch nc hoc du, nh dch hoc dch treo, bt... ty theo
tng ch phm.
Cch th: Bng cm quan.
5.4.2. trong
Dung dch tim khi kim tra bng mt thng iu kin qui nh phi
trong v hu nh khng c tp c hc - Theo ph lc 8.9, mc B - DVN III.
Dung dch thuc tim truyn tnh mch c liu truyn t 100ml tr ln
phi p ng yu cu v gii hn kch thc v s lng cc tiu phn - Theo
ph lc 8.9, mc A - DVN III.
Nh tng phi khng thy du hiu ca s tch lp.
Hn dch tim c th lng cn nhng phi phn tn ngay khi lc u
v gi c s ng u khi ly liu ra khi ng thuc tim.
5.4.3. Mu sc
Khng mu hoc c mu do hot cht ty theo tng chuyn lun. Tin
hnh so vi mu mu - Ph lc 5.17 - DVN III.
5.4.4. pH
pH phi nm trong gii hn qui nh.
Tin hnh o pH bng my o pH - Ph lc 5.9 - DVN III.
5.4.5. v khun
Thuc tim phi v khun.
Cch th: Nu khng c qui nh ring th tin hnh th v nh gi
theo "Th v trng" - phn 4.3.3, trang 115 - 118.
5.4.6. Ni c t vi khun
Php th ni c t vi khun thc hin khi c yu cu c qui nh
trong chuyn lun ring. Th theo Ph lc 10.3 - DVN III.
Khi th ni c t vi khun th khng phi th cht gy st, tr khi c
qui nh khc.
5.4.7. Cht gy st
Thuc tim phi khng c cht gy st. Th theo Ph lc 10.5-DVN III.
Php th ny c tin hnh i vi:
150
Thuc tim ng liu n l c th tch t 15 ml tr ln v khng c qui
nh php th ni c t.
Thuc tim ng liu n l c th tch nh hn 15 ml nhng trn nhn
c ghi "khng c cht gy st" v khng c qui nh php th ni c t.
5.4.8. Th tch (i vi thuc tim, tim truyn dng lng)
Cch th: Thuc tim c thng bng vi nhit phng v phi
c phn tn ng u trc khi th.
i vi thuc tim c th tch nh hn hoc bng 5 ml:
+ Ly 6 ng thuc, trng bm tim bng 1 ng, th trn 5 ng.
+ Kim tra bng cm quan 5 ng thuc, thy cc ng th cha th tch
ngang nhau.
+ Dng bm tim kh sch c dung tch khng ln hn 2,5 ln so vi th
tch cn o, c gn kim tim thch hp. Ly thuc sao cho trong bm
tim khng c bt kh v trong kim tim vn cha y thuc tim.
Ln lt ly thuc ca tng ng tim theo cch .
Tnh kt qu th tch trung bnh ca 5 ng.
i vi thuc tim c th tch ln hn 5 ml, thuc tim truyn:
+ Ly 4 ng thuc, trng bm tim bng 1 ng, th trn 3 ng.
+ Cch tin hnh nh i vi thuc tim c th tch khng ln hn 5 ml.
Tnh kt qu th tch trung bnh ca 3 ng.
i vi thuc tim nhiu liu trong 1 l th th tch phi ln hn so vi
s liu qui nh c ly ra.
Th tch ca ch phm phi nm trong gii hn cho php theo bng 5.7.
Bng 5.7. Gii hn cho php v th tch ca thuc tim, thuc tim truyn
Th tch ghi trn nhn (ml) Phn trm chnh lch
Ti 5 ml
T 5ml ti 50 ml.
Trn 50 ml
+ 15
+ 10
+ 5
5.4.9. ng u khi lng (i vi ch phm dng bt)
Loi b ht nhn, ra sch v lm kh bn ngoi, loi b ht cc nt v
cn ngay khi lng c thuc v v; ly ht thuc ra, dng bng lau sch, nu
cn th ra bng nc, sau ra bng ethanol 96% ri sy 100 - 105
0
C
trong 1 gi; nu v ng khng chu c nhit th lm kh iu kin
151
thch hp v cn. Hiu s khi lng hai ln cn l khi lng ca thuc. Lm
nh vy vi 9 n v khc ly bt k.
Cho php khng qu mt n v c khi lng chnh lch khi bng 5.2.
5.4.10. ng u hm lng
Tr khi c ch dn khc, ch phm c hm lng hot cht di 2 mg
hoc di 2% (kl/kl) so vi khi lng thuc ca ton b. Thuc tim, thuc
tim truyn dng bt ng gi c khi lng nh hn hoc bng 40 mg.
Php th ng u hm lng c tin hnh sau php th nh lng
v hm lng hot cht trong gii hn quy nh.
Cch th: Ly 10 n v ng gi nh nht bt k, xc nh hm lng
hot cht tng ng theo phng php ch dn trong chuyn lun.
Cch nh gi: Ch phm t yu cu php th nu hm lng u nm
trong gii hn 85 - 115% ca hm lng trung bnh.
Ch phm khng t yu cu nu c qu 1 n v c gi tr hm lng
nm ngoi gii hn 85 - 115% ca hm lng trung bnh hoc c mt n v c
hm lng nm ngoi gii hn 75 - 125% ca hm lng trung bnh.
Nu mt n v c gi tr hm lng nm ngoi gii hn 85 -115% nhng
trong gii hn 75 - 125% ca hm lng trung bnh th th li trn 20 n v
khc, ly ngu nhin. Ch phm t yu cu, nu khng qu 1 n v trong
tng s 30 n v em th c gi tr hm lng nm ngoi gii hn 85 -115%
ca hm lng trung bnh v khng c n v no c gi tr hm lng nm
ngoi gii hn 75 - 125% ca hm lng trung bnh.
5.4.11. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu
chun, thuc tim v thuc tim truyn phi cho cc phn ng ca cc hot
cht c trong ch phm theo tng chuyn lun.
5.4.12. nh lng
Ly thuc trong s l thuc nh i vi php th th tch (ch phm
dng lng) hoc nh i vi php th ng u khi lng (ch phm
dng bt), trn ng nht. Tin hnh nh lng theo cc phng php c
qui nh trong tiu chun, hm lng ca tng hot cht trong ch phm phi
nm trong gii hn cho php theo bng 5.8.
Bng 5.8. Gii hn cho php v hm lng i vi thuc tim, thuc tim truyn
Lng ghi trn nhn Phn trm chnh lch
Dng dung dch
Dng bt
5
10
152
5.5. Kim nghim thuc nh mt
5.5.1. Tnh cht:
Th cht, mu sc ty theo tng chuyn lun.
Cch th: Bng cm quan.
5.5.2. trong
Thuc nh mt dng dung dch phi trong, khng c cc tiu phn khi
quan st bng mt thng v t yu cu v trong theo tng chuyn lun.
Thuc nh mt dng hn dch c th lng ng, nhng khi lc phi phn
tn d dng v ng nht trong ton khi.
Cch xc nh: trong c xc nh bng cch so snh cc dung dch
vi cc hn dch mu i chiu. Th theo ph lc 5.12 - DVN III.
5.5.3. Th tch
Th tch thuc nh mt phi nm trong gii hn 100 - 110% so vi th
tch ghi trn nhn i vi mi th tch ng gi.
Cch th: Ly 5 n v ng gi bt k. Xc nh th tch ca tng n
v bng bm tim chun hoc ng ong chun sch, kh, c chnh xc ph
hp (Th tch khng ln hn 2,5 ln th tch cn xc nh).
Th tch mi n v phi nm trong gii hn cho php.
Nu c mt n v khng t th phi kim tra li ln th hai ging nh
ln u. Nu ln th hai c qu mt n v khng t th l thuc khng t
yu cu.
5.5.4. pH
pH phi nm trong gii hn qui nh.
o bng my o pH - Ph lc 5. 9 - DVN III.
5.5.5. v khun
Thuc nh mt phi v khun.
Cch th: Nu khng c qui nh ring th tin hnh th v nh gi
theo "Th v trng" phn 4.3.3, trang 115 - 118.
5.5.6. Gii hn cc tiu phn
Th nghim ny ch yu cu i vi thuc nh mt dng hn dch.
Cho mt th tch ch phm thch hp vo cc o hay vt knh ca knh
hin vi. Quan st bng knh hin vi mt din tch tng ng 10 g pha rn.
153
Kt qu:
- Khng c c qu 20 tiu phn c kch thc ln hn 25 m.
- Khng c c qu 2 tiu phn c kch thc ln hn 50 m.
- Khng c c tiu phn no c kch thc ln hn 90 m.
5.5.7. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun,
thuc nh mt phi cho cc phn ng ca cc hot cht c trong ch phm.
5.5.8. nh lng
Ly thuc ca 5 n v ng gi nh nht bt k, trn u. Tin hnh
nh lng theo cc phng php c qui nh trong tiu chun, hm lng
ca tng hot cht trong ch phm phi nm trong gii hn cho php qui nh
trong chuyn lun.
5.6. Kim nghim thuc ung dng lng
5.6.1. Yu cu k thut v phng php th
5.6.1.1. Tnh cht
Th cht, mu sc, mi v ty theo tng chuyn lun.
Cch th: Bng cm quan.
5.6.1.2. trong
Thuc ung dng dung dch phi t yu cu v trong theo tng
chuyn lun.
Cch xc nh: Ging nh i vi thuc nh mt.
5.6.1.3. Th tch
Cch xc nh: Ging nh i vi thuc nh mt.
Th tch thuc phi nm trong gii hn cho php theo bng 5.9.
5.6.1.4. pH
Mt s ch phm c yu cu th pH phi nm trong gii hn qui nh.
o bng my o pH - Ph lc 5.9 - DVN III.
154
Bng 5.9. Gii hn cho php th tch thuc ung dng lng
Dng thuc Th tch ghi trn nhn (ml) Phn trm chnh lch
Thuc nc ung Ti 20
Trn 20 - 50
Trn 50 - 150
Trn 150
+ 10
+ 8
+ 6
+ 4
Siro Ti 100
Trn 100 - 250
Trn 250
+ 10
+ 8
+ 6
5.6.1.5. nhim khun
nhim khun phi t gii hn qui nh cho ch phm theo tng
chuyn lun.
Cch th: Nu khng c qui nh ring th tin hnh th v nh gi
theo "Th gii hn vi sinh vt" - phn 4.3.4, trang 119-121.
5.6.1.6. T trng
T trng ca ch phm phi nm trong gii hn qui nh theo tng
chuyn lun.
Cch th: Theo Xc nh t trng cht lng - ph lc 5.15 - DVN III.
5.6.1.7. ng u hm lng
Mt s ch phm yu cu th phi tin hnh php th ny.
Cch th v nh gi ging nh i vi thuc bt.
5.6.1.8. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu chun,
ch phm thuc phi cho cc phn ng ca cc hot cht c trong ch phm.
5.6.1.9. nh lng
Ly thuc ca 5 n v ng gi nh nht bt k, trn u. Tin hnh
nh lng theo cc phng php c qui nh trong tiu chun, hm lng
ca tng hot cht trong ch phm phi nm trong gii hn cho php theo
bng 5.10.
5.6.2. Cc dng thuc ung dng lng
Thuc ung dng lng phi t yu cu tiu chun k thut chung i
vi thuc ung dng lng v yu cu ring ty tng chuyn lun.
155
Bng 5.10. Gii hn cho php v hm lng i vi thuc ung dng lng
Dng thuc Phn trm chnh lch
Thuc c A, B
Thuc thng
5
10
5.6.2.1. Cn thuc
Cn thuc l nhng ch phm lng c iu ch bng cch ngm chit
dc liu thc vt, ng vt hoc bng cch ha tan cao thuc, dc cht theo
t l qui nh trong ethanol cc nng khc nhau.
5.6.2.2. Siro
Siro l dung dch m c (khng di 60%) ca ng trng (Saccharose)
trong nc, c cha cc dc cht hoc cc dch chit t dc liu v cc cht
lm thm.
5.6.2.3. Potio
Potio l dng thuc nc c v ngt, cha mt hay nhiu dc cht, dng
ung tng tha. Potio c th dng hn dch hoc nh dch.
Dung mi ca potio c th l nc, nc thm, nc hm hay nc sc
dc liu. Potio thng cha 20% siro.
5.6.2.4. Dung dch ung (Oral solution)
Dung dch ung l nhng ch phm lng trong sut, cha mt hay nhiu
dc cht ha tan trong mt dung mi hay hn hp nhiu dung mi thch hp
dng bng cch ung.
Dung mi ca dung dch c th l nc, du, ethanol, glycerin...
5.6.2.5. Thuc ung git (Oral drop)
Thuc ung git l thuc ung c dng vi th tch nh bng dng c
phn liu l ng nh git.
5.6.3. Dung dch thuc dng ngoi dng lng
Thuc dng ngoi dng lng l cc ch phm lng ng nht, thng l
dung dch, cn thuc, hn dch hoc nh dch ca mt hay nhiu hot cht
trong cht dn thch hp dng ngoi.
Cc thuc dng ngoi dng lng phi t theo cc yu cu k thut
chung ca thuc ung dng lng v ring tng dng thuc c nhng yu cu
ring. Ring yu cu " nhim khun" khng phi lm.
156
5.7. Kim nghim thuc m
5.7.1. Yu cu k thut v phng php th
5.7.1.1. Tnh cht
Th cht: thuc m phi mn, ng nht, khng cng li hoc tch lp
iu kin thng, khng chy lng nhit 37
o
C v phi bt dnh
c trn da hay nim mc khi bi.
Mu sc, mi: ty tng ch phm.
Cch th: Bng cm quan.
5.7.1.2. ng u khi lng
Cch th v nh gi: Ging nh i vi thuc bt. chnh lch khi
lng phi t theo bng 5.11.
Bng 5.11. Gii hn cho php chnh lch khi lng i vi thuc m
Khi lng ghi trn nhn (gam) Phn trm chnh lch
Di 10,0
T 10,0 - 20,0
Trn 20,0 - 50,0
Trn 50,0
15
10
8
5
5.7.1.3. ng nht
Cc tiu phn phi phn tn ng u.
Cch th: Ly 4 n v ng gi, mi n v khong 0,02 -0,03 g, tri ch
phm ln 4 tiu bn, bn trn t mt phin knh. y mi phin knh bng
mt phin knh khc v p mnh cho n khi to thnh mt vt c ng knh
khong 2 cm. Quan st vt thu c bng mt thng, 3 trong 4 tiu bn
khng c nhn thy cc tiu phn. Nu c cc tiu phn nhn thy trong
phn ln s cc vt th phi lm li vi 8 n v ng gi. Trong s cc tiu bn
ny, cc tiu phn cho php nhn thy khng c vt qu 2 tiu bn.
5.7.1.4. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu
chun, thuc m phi cho cc phn ng ca cc hot cht c trong ch phm.
5.7.1.5. nh lng
Cn thuc trong 5 n v ng gi nh nht, tnh khi lng trung bnh,
trn ng nht. Cn mt lng ch phm nh ch dn trong chuyn lun,
157
tin hnh nh lng. Hm lng hot cht phi nm trong gii hn qui nh
theo bng 5.12.
Bng 5.12. Gii hn cho php v hm lng i vi thuc m
Nng hm lng ghi trn nhn Phn trm chnh lch
Ti 200 mg
Trn 200 mg - 1 g
Trn 1 - 5 g
Trn 5 g
15
10
7,5
3
5.7.2. Thuc m tra mt
t cc yu cu ca thuc m v nhng yu cu ring ca thuc m tra
mt nh sau:
5.7.2.1. v khun
Thuc m tra mt phi v khun v khng c c Staphylococcus
aureus v Pseudomonas aeruginosa.
Cch th: Nu khng c qui nh ring th tin hnh th v nh gi
theo "Th v trng" - phn 4.3.3, trang 115-118.
5.7.2.2. Cc phn t kim loi
S lng cc phn t kim loi phi nm trong gii hn cho php.
Cch th: Ly 10 ng thuc, bp ht thuc cha bn trong vo tng a
petri ring c ng knh 6 cm, y bng, khng c cc vt xc v cc phn t
l nhn thy c. y cc a, un nng n 80 -85
o
C trong 2 gi v cho
thuc m phn tn ng u. Lm ngui v lm ng lnh thuc m, lt ngc
mi a t ln bn soi ca knh hin vi thch hp; chiu sng t trn xung
bng mt ngn n chiu t gc 45
o
so vi mt phng ca bn soi. Quan st
v m cc phn t kim loi sng bng, ln hn 50 m bt k kch thc no.
Khng c c qu mt ng trong 10 ng thuc em th cha nhiu hn 8
phn t v khng c qu 50 phn t tm thy trong 10 ng. Nu ch phm
khng t ln th th nht th lm li ln th hai vi 20 ng thuc khc.
Mu th c coi l t yu cu nu khng c qu 3 ng cha qu 8 phn t
trong mi ng v tng s khng qu 150 phn t trong 30 ng th.
5.7.2.3. Gii hn kch thc cc phn t
Khng c c phn t no ca thuc c kch thc ln hn 75 m.
Cch th: Tri mt lng nh ch phm thnh mt lp mng trn bn soi
ca knh hin vi, ph phin knh ln trn v soi.
158
5.8. Kim nghim thuc n, thuc trng
5.8.1. Tnh cht
Thuc n c hnh nn hoc hnh con sut, khi lng 1 - 3 g, di 3- 4 cm.
Thuc trng c hnh cu hoc hnh tri xoan, khi lng 2 - 4 g.
Th cht, mu sc, mi t theo chuyn lun ring.
Cch th: Bng cm quan.
5.8.2. ng u khi lng
Cch th: Nh i vi vin nn
nh gi kt qu: Khi lng ca mi vin ( mi khi lng) phi nm
trong khong khi lng trung bnh 5%.
Cho php khng qu 2 n v lch ra ngoi gii hn, nhng khng c
c n v no lch gp hai ln gii hn cho php.
5.8.3. r
r ca thuc n v thuc trng l kh nng tan r hoc mm ra ca
thuc n v thuc trng trong mi trng th, bng thit b th r
c qui nh trong thi gian nht nh c qui nh tng chuyn lun.
Cch tin hnh: Th trn 3 vin, theo ph lc 8.5 - DVN III.
Mu th c coi l t yu cu v r
i vi thuc n:
Ha tan hon ton; hoc
Phn tn thnh nhng tiu phn v tp trung trn mt nc (cc cht
m), chm xung y (bt khng tan) hay ha tan trong nc (thnh
phn ha tan c); hoc
Tr nn mm ra km theo bin dng m khng nht thit phi b phn
tn hon ton thnh nhng tiu phn, nhng khi dng a thy tinh
chc vo th phi khng c c nhn rn.
Thuc n c t dc thn m phi r trong thi gian di 30 pht.
Thuc n tan trong nc phi r trong thi gian di 60 pht.
i vi vin nang t trc trng:
V nang gelatin v ra v gii phng cc cht cha bn trong.
Thi gian r phi trong vng 30 pht.
i vi thuc trng:
V nang gelatin v ra v gii phng cc cht cha bn trong.
159
Thi gian r phi trong vng 30 pht.
i vi vin nn t m o:
Khng c cn cn trn a kim loi; hoc
Nu cn cn trn a kim loi th ch l khi mm hay rng xp, khi
dng a thy tinh chc vo th phi khng c c nhn rn.
Thi gian r phi trong vng 30 pht.
5.8.4. nh tnh
Tin hnh nh tnh theo cc phng php c qui nh trong tiu
chun, thuc n v thuc trng phi cho cc phn ng ca cc hot cht c
trong ch phm.
5.8.5. nh lng
Cn thuc trong 20 vin, tnh khi lng trung bnh vin, lm ng nht.
Tin hnh theo phng php qui nh trong tiu chun, hm lng ca tng
hot cht trong ch phm phi nm trong gii hn cho php nh bng 5.5.
5.9. php Th ho tan ca vin nn v vin nang
(Dissolution test for tablets and capsules)
5.9.1. Khi nim
ho tan ca mt ch phm l t l hot cht c gii phng ra khi
dng thuc theo thi gian vi cc iu kin qui nh trong tng chuyn lun.
Vi mi ch phm c cc qui nh c th v thit b th, mi trng ha
tan, thi gian th nghim v phn trm hot cht c gii phng (Ch s Q).
5.9.2. Thit b
Ty thit b c th c mt bnh hoc 6 - 8 bnh th.
5.9.2.1. Thit b kiu gi quay (Basket apparatus)
Thit b bao gm:
1. Mt cc hnh tr C bng thu tinh borosilicat hoc bng cht liu
trong sut thch hp, c y hnh bn cu v c dung tch 1000ml, hnh 5.1 v
hnh 5.2. Ming bnh c vin rng, c y bng np c mt s l nh, trong
c mt l tm ca np.
2. Mt ng c vi b phn iu tc c kh nng duy tr tc quay ca
gi trong phm vi 4% tc t. ng c ny gn vi mt b phn khuy bao
gm trc quay A v gi hnh ng tr B. Tr kim loi phi quay c mt cch
nh nhng, khng b lc l nhiu trong khi quay. B phn gi gm c 2 phn:
160
Phn np trn c mt l thot nh c gn vi trc. Np ny c lp
3 ci nhp n hi, hoc bng cch thch hp c th gi chc chn
phn di ca gi ng trc vi trc ca bnh trong khi quay v c th
tho phn di ra d dng khi cn cho ch phm th vo gi.
Phn di tho lp c l mt gi hnh ng tr c lm bng vi ry
kim loi, mp khu c hn lin, ng knh si dy l 0,254 mm, c
cnh hnh vung ca l ry l 0,381 mm; gi hnh ng tr ny c vnh
kim loi bao quanh y trn v y di. Gi c th c m mt lp
vng kim loi dy 2,5 m khng b hng khi th trong mi trng
acid. Khong cch t gi n y trong ca bnh lun c gi trong
khong 23 - 27 mm trong qu trnh th.
3. Mt chu cch thu duy tr mi trng ho tan nhit 37
0
C 0,5
0
C.

Hnh 5.1. Thit b th ha tan ca vin nn, vin nang ca Pharma TEST
1. B iu nhit 4. a y
2. Cc ng mi trng ha tan 5. Trc khuy
3. Cnh khuy 6. B phn iu khin
5.9.2.2. Thit b kiu cnh khuy (Paddle apparatus)
Thit b ny ging nh thit b gi quay m t trn, ch khc l gi c
thay bng cnh khuy D hnh 5.1 v hnh 5.3. Cnh khuy c lp t sao
161
cho i qua tm ca trc v cnh di ca n ngang bng vi mt y ca trc.
Trc cnh khuy c lp t v tr sao cho khng lch qu 2 mm so vi trc
ca bnh v cnh y ca cnh khuy cch mt y trong ca bnh t 23 - 27
mm. Thit b c vn hnh sao cho cnh khuy quay trn mt cch nh
nhng v khng c rung ng r rt.















Hnh 5.2. Thit b kiu gi quay H. 5.3. Thit b kiu cnh khuy
(kch thc tnh bng mm) (kch thc tnh bng mm)
5.9.3. Cch th
5.9.3.1. Chun b mi trng ha tan
Mi trng ho tan c ch dn trong tng chuyn lun ring. Nu l
dung dch m th pH phi iu chnh sai khc khng qu 0,05 n v. Mi
trng ho tan phi loi kh trc khi dng.
Cho mt th tch quy nh mi trng ho tan ui kh vo bnh (th
tch qui nh 1%). Lm m mi trng ho tan n nhit 37 0,5
o
C.
5.9.3.2. Cho vin vo thit b th
Nu khng c ch dn g khc th th ng thi trn 6 vin, cho mi vin
vo mt bnh tr hoc mt gi quay.
Khi dng thit b gi quay: Cho vin nn hay vin nang vo trong gi
kh. H thp gi vo ng v tr ri cho gi quay.
162
Khi dng thit b kiu cnh khuy: Cho vin nn hay vin nang chm
xung y bnh trc khi cho cnh khuy quay; c th dng mt dy
xon bng kim loi hay thu tinh gi cho vin thuc nm ngang
di y bnh. Cn loi tr bt khng kh khi b mt vin.
5.9.3.3. Vn hnh thit b
Vn hnh thit b ngay tc quay c ch dn trong chuyn lun ring.
5.9.3.4. Ly mu
Tu theo ch dn chuyn lun m ly mu ra th pht th 45 hoc
sau nhng khong thi gian quy nh, hoc ly mu ra lin tc; cho php
chnh lch so vi thi gian qui nh l 2%. V tr ht mu khong gia b
mt mi trng ho tan v mt trn ca gi quay hay cnh trn ca cnh
khuy; im ny phi cch thnh bnh t nht 10 mm. Tr trng hp phn
tch mu lin tc, o mu t ng khi mu ly ra sau khi phn tch li tr
v bnh ho tan, cng nh trong trng hp ly mu phn tch mt ln; cn
cc trng hp khc phi thm mt th tch mi trng ho tan bng th tch
mu th ly nhit 37 0,5
o
C, hoc c th dng php tnh hiu chnh.
5.9.3.5. Xc nh lng hot cht
Mu th ly ra c th c lc bng giy lc, mng lc hoc thit b thch
hp. Dung dch thu c c th c dng ring bit hoc gp li xc nh
phn trm hot cht gii phng.
Xc nh lng hot cht cha trong dch lc bng phng php c ch
dn trong chuyn lun ring.
5.9.3.6. nh gi kt qu
Lng hot cht c gii phng ca c 6 vin th phi khng di
70% lng hot cht qui nh, tr khi c ch dn trong chuyn lun
ring. Nu c 1 vin khng t th th vi 6 vin khc, ln ny c 6
vin u phi t yu cu (theo DVN III).
Hoc phn trm dc cht gii phng so vi hm lng ghi trn nhn
c nh gi theo bng 5.13 v bng 5.14 (theo USP 24).
Nu v vin nang nh hng ti kt qu phn tch, ly t nht 6 vin
nang, loi b ton lng thuc trong nang, ho tan v nang rng trong
mt th tch mi trng ho tan c ch dn trong chuyn lun ri
tnh hiu chnh. H s hiu chnh khng c ln hn 25% hm lng
ghi trn nhn.
163
Bng 5.13. Gii hn v phn trm dc cht gii phng i vi vin nn,
vin nang nh gi theo tng vin (Theo USP 24 - trang 1943)
Ln
S vin
th
Yu cu kt qu
(Phn trm hot cht ha tan)
Kt lun - Gii php
1 6 Mi vin phi Q + 5%
t: Mu th t yu cu v ha tan.
Dng th nghim.
Khng t: Lm ln 2
2 6
Trung bnh ca 12 vin phi Q
v khng c vin no < Q - 15%
t: Mu th t yu cu v ha tan.
Dng th nghim.
Khng t: Lm ln 3
3 12
Trung bnh ca 24 vin phi Q
v c di hai vin < Q - 15% v
khng c vin no < Q - 25%
t: Mu th t yu cu v ha tan.
Khng t: Mu th khng t yu cu v
ha tan.
Bng 5.14. Gii hn v phn trm dc cht gii phng i vi vin nn, vin nang
nh gi theo cch gp dch lc ca cc vin th (Theo USP 24 - trang 1943)
Ln
S vin
th
Yu cu kt qu
(Phn trm hot cht ha tan)
Kt lun - Gii php
1 6 Trung bnh ca 6 vin phi Q + 10%
t: Mu th t yu cu v
ha tan. Dng th nghim.
Khng t: Lm ln 2
2 6
Trung bnh ca 12 vin phi Q + 5%

t: Mu th t yu cu v
ha tan. Dng th nghim.
Khng t: Lm ln 3
3 12 Trung bnh ca 24 vin phi Q .
t: Mu th t yu cu v
ha tan.
Khng t: Mu th khng t yu
cu v ha tan.
5.9.4. Th tc ha tan ca vin nn v vin nang bao tan rut
Tin hnh cc bc nh trn. Ring mi trng v thi gian nh sau:
Giai on 1: dng 1000 ml dung dch acid hydrocloric 0,1 M lm cht
lng. Th trong 2 gi.
Giai on 2: 1000ml dung dch m phosphat pH 6,8. Th trong 45 pht.
nh gi kt qu:
+ Lng hot cht c gii phng ca c 6 vin th phi khng di
70% lng hot cht qui nh, tr khi c ch dn trong chuyn lun
ring. Nu c 1 vin khng t th th vi 6 vin khc, ln ny c 6
vin u phi t yu cu (theo DVN III).
Hoc
Trong mi trng acid hydrocloric 0,1 M: theo bng 5.15.
Trong mi trng m pH 6,8: nh gi nh bng 5.13 v 5.14.
164
Bng 5.15. Gii hn v phn trm dc cht gii phng i vi vin bao, vin nang
tan trong rut (mi trng th l HCl 0,1M) - Theo USP 24 - trang 1947
L
S vin
th
Yu cu kt qu
(Phn trm hot cht ha tan)
Kt lun - Gii php
1 6
Khng c vin no vt 10% so vi hm
lng ghi trn nhn
- t: Mu th t yu cu. Dng
th nghim.
- Khng t: Lm ln 2
2 6
Trung bnh ca 12 vin phi nh hn 10%
so vi hm lng ghi trn nhn
- t: Mu th t yu cu. Dng
th nghim.
- Khng t: Lm ln 3
3 12
Trung bnh ca 24 vin phi nh hn 10%
so vi hm lng ghi trn nhn
- t: Mu th t yu cu.
- Khng t: Mu th khng t yu
cu v ha tan.
V d:


Vin nang Cephalexin (DVN III, trang 53 - 54)
Thit b: Gi quay
Mi trng ha tan: 1000 ml nc
Tc : 100 vng/ pht
Thi gian: 45 pht
Ly v x l mu: Lc dung dch sau khi ha tan. Pha long dch lc
bng nc c dung dch cephalexin c nng 25 g/ml.
o hp th ca dung dch thu c bc sng 262 nm vi mu
trng l nc.
Song song lm mt mu chun cephalexin c cng nng .
Yu cu: Khng c t hn 80% lng cephalexin (C
16
H
17
N
3
O
4
S) ghi trn
nhn c ha tan trong 45 pht.
Vin bao Ibuprofen (DVN III, trang141)
Thit b: Cnh khuy
Mi trng ha tan: 900 ml dung dch m phosphat pH 7,2 (Phi hp
250 ml dung dch KH
2
PO
4
0,2 M vi 175 ml dung dch NaOH 0,2 M, thm nc
va 1000 ml).
Tc : 50 vng/ pht
Thi gian: 60 pht
Ly v x l mu: Lc dung dch sau khi ha tan. Pha long dch lc vi
dung dch m phosphat pH 7,2 c dung dch ibuprofen c nng
thch hp.
o hp th ca dung dch thu c bc sng 221 nm trong cuvet
thch anh c chiu dy 1 cm vi mu trng l dung dch m phosphat pH 7,2.
Tnh hm lng ibuprofen theo A (1%, 1cm) l 449 bc sng 221 nm.
165
Yu cu: Khng c t hn 85% lng ibuprofen (C
13
H
18
O
2
) ghi trn
nhn c ha tan trong 60 pht.
5.10. Th r ca vin nn v vin nang
(Disintegration test for tablets and capsules)
5.10.1. Khi nim
r ca vin nn, vin nang l kh nng tan r ca chng trong mi
trng th theo qui nh (thng l nc), bng thit b th r c qui
nh (My th r Erweka hay mt thit b tng ng) trong thi gian
nht nh c qui nh tng chuyn lun.
5.10.2. Thit b
Thit b bao gm: (Hnh 5.4, hnh 5.5)
1. Mt gi ng th thch hp, 6 ng thy tinh hnh tr c chiu di
75,0 - 80,0 mm, ng knh trong 21,5 mm v chiu dy thnh ng khong 2 mm.
2. Mt a hnh tr y trn mi ng, c ng knh 20,55 - 20,85 mm v
chiu dy 9,35 - 9,65 mm, c lm bng cht do trong sut c t trng 1,18 -
1,20. a c 5 l hng, ng knh mi l l 2 mm, 1 l gia cn 4 l cn li
t cch u nhau trn mt vng trn c bn knh 6 mm t tm a. C 4 rnh
cch u nhau c ct bn cnh a sao cho mt trn a, rnh c chiu
rng 9,5 mm, chiu su 2,55 mm; mt di ca a, rnh l hnh vung c
cnh 1,6 mm.
3. Cc ng c gi v tr thng ng trong 6 l trng ca 2 tm nha
cng trong sut c ng knh 90 mm v chiu dy 6 mm. Cc l cch u
nhau v cch u tm. Mt di ca tm nha pha di c gn 1 tm li
an bng cc si thp khng g c ng knh 0,635 mm v kch thc mt l
l 2 mm.
4. Hai tm nha c c nh v cch u nhau 77,5 mm bng cc thanh
kim loi c cht thng ng pha ngoi v mt thanh kim loi c gn
cht vo tm tm nha pha trn, sao cho gi ng th c th lp rp vi b
phn c vn hnh thit b chuyn ng ln xung mt khong t 50 - 60
mm vi tn sut 28 - 32 ln/ pht.
5. Gi ng th c nhng chm trong mt bnh thch hp, thng l
cc thy tinh c dung tch 1000 ml cha cht lng c qui nh. Th tch
cht lng phi sao cho khi gi ng th v tr cao nht th li kim loi
phi di b mt cht lng t nht 15 mm v khi gi ng th v tr thp
nht th li kim loi cch y cc thy tinh t nht 25 mm v ming cc ng
thy tinh phi trn b mt cht lng.
6. Mt thit b ph hp (thng l chu cch thy) duy tr nhit
ca cht lng l 37
0
C 0,5
0
C.
166









Hnh 5.4. Thit b o r ca vin nn, vin nang (chi tit)

7
6
3
2
4
5 1
Hnh 5.5. Thit b o r ca vin nn, vin nang
1. Ngun cung cp nhit 2. B iu nhit 3. Cc ng mi trng th
4. a y 5. ng hnh tr 6. Gi 7. B phn iu khin
167
5.10.3. Cch th
5.10.3.1. Chun b
Cho mt th tch thch hp mi trng th (thng l nc) vo cc.
Vn hnh my iu nhit nhit ca mi trng th t 37
0
C 0,5
0
C.
Cho vo mi ng th mt vin nn hoc mt vin nang ri y a cht
do vo tng ng.
5.10.3.2. Vn hnh thit b
Nhng gi vo trong cc ng cht lng v vn hnh thit b theo thi
gian qui nh.
5.10.3.3. nh gi kt qu
Sau thi gian qui nh hoc khi thy cc vin r ht, ly gi ng
th ra khi cc cht lng.
Mu th c coi l t yu cu v r khi khng cn cn, tr nhng
mnh v nang hoc v bao khng tan ca vin nn, cn li trn mt
li ca thit b th hoc dnh vo b mt di ca a y. Nu cn
cn th n ch l mt khi mm, khng c c nhn kh rn s thy
c.
Kt qu:
Nu c 6 vin u r ht: Mu th t yu cu v r.
Nu cn di hai vin cha r ht: Th li trn 12 vin na.
Ch phm t yu cu v r khi 16 trong s 18 vin th t r theo
qui nh.
5.10.4. Th r ca vin nn v vin nang bao tan rut
Tin hnh theo cc bc nh trn. Ring mi trng v thi gian nh sau:
Giai on 1: mi trng th l acid hydrocloric 0,1M; trong 120 pht.
Tt c cc vin th phi cn nguyn vn, khng th hin s gii phng
hot cht.
Giai on 2: mi trng th l dung dch m phosphat pH = 6,8; trong
60 pht.
Nu c vin dnh a th th li vi 6 vin khc.
nh gi kt qu: Nh i vi vin nn v vin nang.
Cch pha m phosphat pH 6,8: Trn dung dch acid hydrocloric 0,1N v
dung dch natri phosphat 0,2N theo t l 3: 1. iu chnh pH n 6,8 0,05
bng dung dch acid hydrocloric 2N hoc dung dch natri hydroxyd 2 N.
168
Ti liu tham kho
1. B Y t (2002). Dc in Vit Nam III. NXB Y hc. Tr. 6; 141 - 142;
254; PL. 15; PL. 18 -20; PL.131 - 133; PL. 136 -138.
2. ng Vn Ha (2001). Gio trnh kim nghim thuc. Trng H Y -
Dc TP H Ch Minh.
3. European Pharmacopoiea (2003), 4
th
edition, suppl. 4.3; 4.4. P.p. 3247
- 3250; 3367 - 3376.
4. Pharmacopoeia of The Peoples Republic of China (PPRC)1997, p.p.
A5; A7; A51 - A56.
5. The Bristish Pharmacopoiea 2001, p.p. 1775 - 1824; A167; A233;
A235; A236; A250; A252; A317.
6. The United States Pharmacopeia XXIV(2000), p.p. 1939; 1941 - 1951;
2000 - 2002; 2107 - 2118;
7. The United States Pharmacopeia XXVII (2004), p.p. 2108 2111;
2302 - 2312; 2577 - 2589.
8. Umesh Banakar. Pharmaceutical dissolution testing. Marcel Dekker
1992, p.p. 65 - 66; 71 - 72.
9. WHO, The International Pharmacopoiea (2003), 3
rd
edition, Vol. 5.
P.p. 7- 28.
10. Brossard D. (1992). Les formes dermiques et les suppositoires dans
L'analyse pratique du mdicament. Editions Mdicales
Internationales, Paris. P.p. 971- 981.
11. Postaire E., Brossard D. (1992). Les formes liquides dans L'analyse
pratique du mdicament. Editions Mdicales Internationales, Paris.
P.p. 954 - 969.
Cu hi t lng gi
5.1. Trnh by tiu chun v k thut kim nghim chung ca thuc bt?
5.2. Trnh by tiu chun v k thut kim nghim chung ca vin nang?
5.3. Phn loi cc loi vin nang? Cho bit nhng yu cu ring cho tng
loi nu c?
5.4. Trnh by tiu chun v k thut kim nghim chung ca thuc vin nn?
5.5. Phn loi cc loi vin nn? Cho bit cch th r i vi tng loi?
169
5.6. Trnh by tiu chun v k thut kim nghim chung ca thuc tim
v thuc tim truyn?
5.7. Trnh by tiu chun v k thut kim nghim chung ca thuc nh mt?
5.8. Trnh by tiu chun v k thut kim nghim chung ca thuc m
v thuc m tra mt?
5.9. Trnh by cch th tc ha tan v nh gi kt qu ca vin nn
paracetamol 500 mg; vin nang cephalexin 500 mg v vin bao
diclofenac 50 mg?
5.10. Anh (ch) hy cho bit cch th r v nh gi kt qu ca vin
nn acid ascorbic 100 mg; vin nang cloxacilin 250 mg v vin bao
cimetidin 400 mg ?
5.11. Anh (ch) hy xut tiu chun v qui trnh kim nghim cho vin
nn A c hm lng hot cht l 1,5 mg/ vin.
5.12. So snh phng php th r ca vin nn ca Dc in Vit
Nam I - tp 1 v Dc in Vit Nam III.
5.13. Ngi ta tin hnh nh lng paracetamol trong vin nn
paracetamol 500 mg nh sau: Cn 20 vin c khi lng 14,6853g.
Nghin mn. Cn 0,2205 g bt vin, ha tan trong va 200 ml
dung mi. Lc u, lc. Pha long 1,00 ml dch lc thnh 100,0 ml
trong bnh nh mc bng dung mi. Mt quang ca dung dch
ny 257 nm l 0,501.
Hi ch phm c t yu cu v hm lng khng? Bit
paracetamol c A (1%, 1cm) bc sng 257 nm l 715.
5.14. Th ha tan ca vin nn ciprofloxacin 250 mg theo DVN III,
sau khi dng khuy, pha long chnh xc mi trng ha tan trong
mi cc th 50 ln. o hp th ca cc dung dch ny theo iu
kin cho c kt qu ln lt tng ng vi cc cc 1; 2; 3; 4; 5 v
6 l 0,435; 0,346; 0,462; 0,378; 0,489 v 0,408. Hi ch phm c t
yu cu v ha tan khng? Bit dung dch ciprofloxacin chun
nng 5,5 g/ ml c hp th l 0,505 o trong cng iu kin.




170
Chng 6.
n nh v tui th ca thuc
Mc tiu hc tp
1. Trnh by c mc tiu v tiu chun nh gi n nh ca thuc.
2. Gii thch c nguyn tc xc nh n nh v cch tnh tui th
ca thuc.
Mt trong nhng yu cu quan trng nht i vi thuc phng v cha
bnh l n nh v cht lng trong sut qu trnh bo qun t khi xut
xng n khi ht hn dng. n nh ca thuc lin quan n qu trnh
sn xut, phn phi v bo qun thuc.
Khi nghin cu trin khai thuc mi hoc hon thin nng cao hiu lc
ca mt thuc c x dng trong lm sng, nh sn xut cn nh gi
n nh ca thuc trong iu kin bo qun xc nh, t tnh ra tui th
ca n. Ngi phn phi, lu gi thuc phi duy tr c iu kin bo qun
thuc qui nh m bo hn dng ca thuc.
Vic nghin cu n nh ca thuc l mt qu trnh hon thin
phng php v cho ti gn y, T chc Y t th gii (WHO) mi c vn bn
chnh thc hng dn nghin cu n nh ca thuc. Dc in ca mt s
nc c chuyn lun v vn ny.
6.1. Qu trnh pht trin nghin cu n nh
Ngi ta c nhng ghi nhn ban u lin quan n bn vng ca
thuc. Mt s ch phm kh bo qun , d phn hy nh aspirin, procain,
mt s vitamin nh A, C. Ngc li, mt s ch phm khc li kh bn vng
nh sulfonamid. T y khi nim v n nh ca thuc dn dn c
hnh thnh.
Nm 1948, tp ch Cng ngh Dc v M phm ca M ng mt kt
qu nghin cu l th v vitamin A: bo qun vitamin A trong 5 tun l
42
0
C s cho mt lng vitamin ny b phn hu tng ng nh khi bo
qun trong hai nm nhit phng. Kt qu nghin cu gi mt cch
nh gi n nh. E.R. Garrett (1955) gi y l phng php th nghim
cp tc. Cng trong thi gian ny ngi ta tin hnh mt php th n
nh ca thuc (gang - testing): Mt nm ngi ta ly mu ngu nhin cc
nh thuc mt ch phm no , tin hnh nh lng hot cht, nh gi s
suy gim ca hm lng. Trn c s kt qu nghin cu, cc nh bo ch M
khuyn co i vi cc ch phm thuc cn:
171
Ghi hn s dng trn nhn thuc,
t tiu chun dc in trong thi gian lu hnh.
u nhng nm 70 M, ngi ta thc hin mt chng trnh nghin
cu n nh ca thuc, c nh gi thng k x l s liu. n nm 1975
Dc in M c a ra hn dng ca thuc. Tuy nhin Dc in ny cha
qui nh phng php xc nh hn dng. Mi n nm 1984, c quan qun l
thuc v thc phm M (FDA) mi c vn bn u tin v th n nh ca
thuc. Vn bn ny c b sung trong ln xut bn th hai (1987) v hon
chnh trong ln xut bn th ba (1994).
Nm 1993 Hi ngh Quc t bn v hi nhp cho xut bn ti liu
hng dn chi tit v th n nh ca thuc (Harmonized Tripative
Guideline, Geneva). Vn bn hng dn ca FDA xut bn nm 1994 c ni
dung tng t vi vn bn ho nhp 1993.
Nm 1994, WHO xut bn ln u trn ti liu hng dn nghin cu
n nh ca thuc. Ti liu ny c b sung ti bn nm 1998.
Mt iu cn ghi nhn l: vic nghin cu n nh ca thuc ngy
cng c y mnh v thu c kt qu kh quan l nh cc phng php
phn tch xc nh hot cht v tp cht lin quan hoc tp cht phn hu c
tin cy cao trong qu trnh sn xut cng nh bo qun v lu thng thuc.
Bc t ph u tin l x dng quang ph UV-VIS nh lng hot cht
v sc k lp mng pht hin tp cht phn hy. Tip n my sc k kh
ri my sc k lng ra i gip cc nh phn tch xc nh c lng cht
di ppm, phn tch nh lng c nhiu thnh phn trong hn hp, mt
loi hnh phn tch hay gp trong nghin cu n nh ca thuc.
Gn y vic p dng cc phng php phn tch hin i nh: nhiu x
tia X, nhit vi phn,.... to ra nhiu thun li trong nghin cu n nh.
Ni dung ca chng ny c bin son da ch yu vo ti liu hng
dn ca WHO nm 1998, c tham kho thm mt s Dc in v mt vi ti
liu khc.
6.2. i cng v n nh ca thuc
6.2.1. nh ngha
Theo WHO, n nh ca thuc l kh nng ca nguyn liu hoc ch
phm c bo qun trong iu kin xc nh c th gi c nhng c tnh
vn c v ho l, vi sinh, sinh dc hc..... trong nhng gii hn nht nh.
n nh ca thuc ph thuc vo nhiu yu t. C th phn chia cc
yu t ny ra hai nhm:
Cc yu t lin quan n mi trng nh: nhit , m, chiu
sng, hm lng oxy cng cc yu t bn ngoi khc tc ng ln thuc.
Cc yu t lin quan n thuc. l:
172
Tnh cht l ha ca hot cht v t dc c dng bo bo ch thuc.
V d: dng tinh th, hm lng nc, tp cht trong nguyn liu...
Dng bo ch ca thuc,
Qui trnh sn xut thuc
Nguyn liu cho ng, bao b, ng gi.
Nh vy n nh ca thuc ph thuc ch yu vo c im ca
nguyn liu, qui trnh sn xut v iu kin mi trng. Vic a vo cng
thc bo ch cc cht lm tng n nh ca thuc ch c chp nhn trong
trng hp hu hn v phi c chng minh v mt khoa hc v thc t.
6.2.2. Mt s thut ng lin quan.
Cc ti liu hng dn nghin cu n nh ca thuc cp n nhiu
khi nim v thut ng. y s tm tt mt s thut ng chnh:
Cc php th n nh (stability testings)
l tp hp cc php th c thit k nhm thu c nhng thng tin
v n nh ca ch phm. Trn c s nh ra tui th, hn dng iu
kin ng gi v bo qun xc nh.
Php th n nh di hn (long term (real time) stability testing)
l nhng nghin cu thc nghim nh gi s thay i cc tnh cht
ha l, sinh hc, sinh dc hc .... ca mt ch phm thuc trong qu trnh bo
qun iu kin xc nh. Trn c s kt qu nghin cu ny xc nh hn
dng v khuyn co iu kin bo qun.
Php th n nh cp tc (accelerated stability testing)
y l nghin cu thc nghim c b tr lm tng tc phn hy
ha hc v thay i trng thi vt l ca thuc nh s thay i ca nhit ,
m, chiu sng,... Thng nghin cu c tin hnh nhit cao 35 -
40
0
C, c khi ti 50
0
C, m tng i 80 - 90% hoc hn.
L sn xut (batch N
0
)
l lng xc nh ca mt sn phm c sn xut theo mt qui trnh
v c coi l ng nht. Trong trng hp sn xut lin tc, l tng ng vi
mt phn nht nh ca qu trnh sn xut v c c trng bi tnh ng
nht ca sn phm.
Hn dng thuc (expiration date)
y l thi im ht hn x dng ca thuc, c ngha l sau thi im
ny ch phm thuc khng cn gi c cc tnh cht nh ng k trong
tiu chun cht lng. Hn dng c xc nh theo l sn xut.
Hn sn xut (manufacture date)
y l thi im kt thc qu trnh sn xut l thuc. Hn ny c ghi
thnh thng v nm cho tng l sn phm. C th tnh hn sn xut l thi
173
im thuc c a vo lu thng vi iu kin l khong thi gian gia thi
im bt u sn xut v thi im a vo lu thng khng vt qu 1/ 20
ca thi hn bo qun.
Thi hn bo qun (expiration dating period)
l khong thi gian bo qun thuc trong iu kin xc nh m thuc
vn gi c cc tnh cht ng k trong tiu chun cht lng. Da vo thi
hn bo qun v hn sn xut tnh ra hn dng ca thuc. Ngi ta cng
dng tui th (shelf life) ca thuc ng ngha vi thi hn bo qun thuc.
iu kin bo qun chun ha (controlled storage condition)
l iu kin bo qun trong kho thong kh, nhit trong khong 15
- 25
0
C. Tu vng kh hu nhit c th ln ti 30
0
C. Trong kho khng c mi
l, khng c cht nhim, khng c nh sng trc tip chiu vo.
Nhit ng hc trung bnh - NT (mean kinetic temperature)
y l tr s nhit trung bnh c tnh ton cho tng vng kh hu
da trn s phn b ca nhit theo thi gian. NT c dng nh gi
tc ng ca nhit ln ng hc ca qu trnh phn hu ha hc lm gim
n nh ca thuc. Tr s NT thng ln hn tr s trung bnh s hc.
6.2.3. Mc tiu nh gi n nh
Theo hng dn ca WHO, nghin cu n nh ca thuc nhm 4 mc
tiu chnh (bng 6.1)
Giai on pht trin sn phm
giai on ny, cc php th cp tc c thc hin nhm la chn cng
thc bo ch thuc, qui trnh sn xut v bao gi thch hp. Sau khi c sn
phm nh sn xut tip tc dng cc th nghim cp tc d bo n nh, s
b nh gi tui th trong iu kin bo qun nh. Mt khc cc th nghim
di hn cng bt u c trin khai phc v cho giai on tip theo.
Bng 6.1. Bn mc tiu cho nghin cu n nh
TT Mc tiu
Phng php
nghin cu
Giai on
p dng
1 Xy dng cng thc, k
thut pha ch v bao gi
Th nghim cp tc Pht trin sn phm
2 Xc nh tui th v iu
kin bo qun
Th cp tc v di hn Pht trin sn phm v lp h s
ng k
3 Khng nh bng thc
nghim tui th thuc
Th di hn Lp h s ng k
4 Thm nh n nh
lin quan n cng thc
v qui trnh sn xut
Th cp tc v di hn Thuc lu hnh trn th trng
174
Giai on lp h s ng k
Trn c s kt qu thc nghim cp tc v di hn, nh sn xut xc
nh tui th v hn dng ca thuc iu kin bo qun v bao gi thch
hp. Cc thng tin ny c ghi r trn nhn thuc v bao gi. Nh sn
xut trnh vi c quan qun l thuc ton b h s khc xin php lu hnh
thuc trn th trng.
Giai on thuc lu hnh trn th trng
Khi thuc lu hnh trn th trng, nh sn xut tip tc theo di
n nh khng nh tui th ca thuc trong iu kin bo qun xut.
bo m an ton v cht lng thuc lu hnh trn th trng, cc c
quan qun l cng theo di n nh ca thuc thng qua vic thanh tra,
kim tra qui trnh sn xut, ly mu thuc kim nghim.
i vi mt ch phm c cp php lu hnh, nu c s thay i v
cng thc, v qui trnh sn xut hoc qui cch ng gi,... nh sn xut phi
nghin cu b sung v n nh v bo co vi c quan qun l thuc.
6.2.4. Tiu chun nh gi n nh
C 5 tiu chun nh gi n nh ca thuc
n nh ha hc
Cc tnh cht ha hc (thnh phn nh tnh v nh lng) ca (cc)
hot cht c mt trong ch phm nm trong mt gii hn cho php theo tiu
chun cht lng.
n nh vt l
Cc c im vt l ca nguyn liu lm thuc nh: mu sc, trng thi
tinh th, tan, im chy.... khng thay i. Cc c im ca ch phm nh
mu sc, cng, r, ho tan dao ng trong khong gii hn cho php
ca tiu chun cht lng.
n nh vi sinh
v trng hoc gii hn nhim khun ca ch phm phi p ng yu
cu ca tiu chun. Nu ch phm c cha cht khng khun th hm lng
ca n khng vt qu gii hn cho php.
n nh iu tr
Tc dng iu tr ca ch phm khng thay i
n nh c tnh
c tnh ca ch phm khng c tng ln trong sut qu trnh bo
qun v lu hnh trn th trng.
175
6.2.5. Phn vng kh hu
Tui th ca thuc ph thuc vo vng kh hu m thuc lu hnh.
nghin cu n nh ngi ta chia th gii ra 4 vng kh hu:
Vng 1: Kh hu n ho. l cc nc bc u, Anh, Canada, Nga.
Vng 2: Kh hu nhit i c th c m cao nh M, Nht bn, cc
nc nam u (Hy lp, B o Nha).
Vng 3: Kh hu nng kh nh Iran, Irac, Sudan.
Vng 4: Kh hu nng m. l mt s nc nam M (Brazil,
Nicaragua,....), ng nam (Vit Nam, Philippin, Indonesia,...).
Bng 6.2. tm tt gi tr trung bnh bn thng s ca 4 vng kh hu.
Bng 6.3. cho ta iu kin bo qun ch phm trong nghin cu n
nh di hn. iu lu l thng s nhit c chn ti thiu l 21
0
C mc
d vng c nhit thp hn tr s ny.
Bng 6.2. Nhit v m ca 4 vng kh hu
Tr s o c ngoi tri Tr s o c trong kho
Vng
Nhit (
0
C) m (%) Nhit (
0
C) m (%)
1 10,9 75 18,7 45
2 17,0 70 21,1 52
3 24,4 39 26,0 54
4 26,5 77 28,4 70
Bng 6.3. Thng s kh hu tnh ton v iu kin bo qun cho th di hn
Thng s kh hu tnh ton iu kin bo qun
Vng
Nhit NT m ** m bar * Nhit m **
1 20,0 20,0 42 9,9 21 45
2 21,5 22,0 52 13,5 25 60
3 26,4 27,9 35 11,9 30 35
4 26,7 27,4 76 26,6 30 70
* p sut hi nc ring phn trong kh quyn
** m tng i tnh bng %
WHO khuyn co cc nh sn xut:
Nu ch phm c lu hnh vng 1 nn nghin cu n nh trong
iu kin bo qun vng 2.
Nu ch phm c lu hnh vng 3 hoc vng 4 nn nghin cu
n nh trong iu kin bo qun vng 4.
176
Cn lu l vi mt s ch phm vic nghin cu n nh phi c
tin hnh nhit di 0
0
C, hoc iu kin lnh (+2
0
C n 8
0
C). Vi mt s
khc i khi phi xem xt tc ng ca nh sng.
6.3. ng ha hc dung dch
Di tc ng ca iu kin bn ngoi, nguyn liu v ch phm dn n
b phn hu, hm lng hot cht gim dn theo thi gian. Qu trnh phn
hu ng hc ny rt phc tp, bi v:
Nhiu yu t tc ng ng thi,
Nhiu loi tng tc c th din ra: tng tc vt l, phn ng ha hc,
cc qu trnh sinh hc,
Ch phm tn ti nhiu dng khc nhau: dung dch nc, dung dch
du, nh dch, hn dch, bt rn,....
Ch phm thng c nhiu thnh phn: mt hoc nhiu hot cht, cc
loi t dc.
Chnh v vy n nh ca thuc khng ng ngha vi ng ha hc.
Trong qu trnh bo qun c th c mt s phn ng ha hc phn hu dc
cht c m t bng phng php ng ha hc. V vy trc khi nghin
cu n nh ca thuc chng ta n tp vi vn c bn ca ng ha hc
dung dch.
6.3.1. Bc ca phn ng
Tc ca mt phn ng c xc nh bng ba thng s:
Nng ca cc cht phn ng,
Nhit xy ra phn ng,
S c mt ca cht xc tc,
Cho mt phn ng:
A + B P (6.1)
Tc v ca phn ng c xc nh theo nh lut Vant Hoff:
) 2 . 6 ( ] [ ] [
) (
n m
n m
B A k
dt
dC
v
+
= =
C l nng ca cht nghin cu, cc mc vung l ch nng ca A v B,
k l hng s tc (c lp vi nng C),
n v m l bc ring ca A v B tng ng,
n + m = p l bc tng cng ca phn ng.
177
Trong nghin cu n nh ngi ta quan tm n bc tng cng p. N
c th l bc khng, bc nht hoc bc hai.
6.3.1.1. Phn ng bc khng
v =
0
k
dt
dC
= (6.3)
Phng trnh tc cho phn ng bc khng
Tch phn phng trnh ta c
C = C
0
k
0
t (6.4)
hoc C
0
(1 a) = k
0
t
a
(6.5)
y a l phn s mol cn li thi im t
a
.
thi im t
1/ 2
, tc l C
t
= 0,5C
0
ta c
t
0
0
1/2
2k
C
t = (6.6)
Thi gian bn hu t
1/2
ph thuc vo nng ban u C
0
. Th nguyn
ca hng s k l mol/ n v t.
Trong thc t c rt t phn ng ng ha hc bc khng.
6.3.1.2. Phn ng bc nht
) 7 . 6 ( C k
dt
dC
1
=
Tc phn ng ph thuc vo nng
) 8 . 6 ( ln
1
0
t k
C
C
=
Dng tch phn
) 9 . 6 ( ln
1 a
t k a =
Phn s mol a cn li thi im t
a
Trong nghin cu ng hc phn hu thuc bc nht ngi ta lu tm
thm thi im t
0,9
. l thi im m t s [C/ C
0
] = 0,9
kt
1/2
= - 0,693 (6.10)
kt
0,9
= -0,105 (6.11)
Th nguyn ca hng s k l t
-1
178
Khi nghin cu n nh cn kim tra nng hot cht hoc tp cht
phn hu. Nu trong h ch c mt tp cht phn hy, vic xc nh t s C/
Co kh n gin. Ngi ta c th xc nh nng hot cht hoc tp cht
thi im t tnh hng s tc . Vn tr nn phc tp hn khi h to ra
hai hay nhiu sn phm phn hu. Hai trng hp sau c th gp trong thc
t nghin cu n nh .
Phn ng bc nht k tip
C trng hp phn ng phn hu ban u khng n nh. Ta c s
phn ng sau:
A B C (6.12)
k
1
k
2
Nu c ba cht trong h c xc nh th mi thi im tng s mol
ca ba cht bng s mol ban u ca A.
Cc phn ng din ra theo qui lut ng hc bc nht, ta c cc phng
trnh tc :
) 13 . 6 ( ] A [ k
dt
] A [ d
1
=
) 14 . 6 ( ] A [ k ] B [ k
dt
] B [ d
1 2
+ =
) 15 . 6 ( ] B [ k
dt
] C [ d
2
=
Gii cc phng trnh vi phn ny ta c
[A] = A
0
exp(k
1
t) (6.16)
[B] = A
0
[k
1
/ (k
2
k
1
)][exp(k
1
t) exp(k
2
t)] (6.17)
) 18 . 6 ( ) t k exp(
k k
k
) t k exp(
k k
k
1 A ] C [
2
1 2
1
1
1 2
2
0

=
A
0
l nng mol ban u ca A. tnh C, trong nhiu trng hp
ngi ta tnh [C] mt thi im theo phng trnh
[C] = A
0
[A] [B] (6.19)
Bng 6.4 minh ho s quang phn ca cefotaxim bng hai phn ng bc
nht k tip (Lerner 1988).
179
Bng 6.4. Quang phn ca Cefotaxim
Thi gian (gi) Cefotaxim A (% mol) B (% mol) C (hiu s)
0,00 100 0 0
0,25 82 10 8
0,50 70 15 15
0,75 55 18 27
1,00 43 19 38
1,50 28 18 54
2,00 20 15 65
3,00 10 10 80
4,00 5 5 90
Ct C cui cng tnh theo phng trnh (6.19).
Phn ng bc nht song song
Nu A phn hu to ra hai sn phm ng thi l B v C
A B A C (6.20)
k
1
k
2
Phng trnh tc c dng:
d[A]/ dt = k
1
[A] k
2
[A] (6.21)
ln[A/ A
0
] = (k
1
+ k
2
)t (6.22)
thi im bt k t l ca hai sn phm to thnh B v C lun khng i
[B] / [C] = k
1
/ k
2
(6.23)
Trong thc t i khi cc phn ng k tip v song song din ra khng ng
thi. Trong dung dch nc mui Cefotaxim natri phn hu theo s sau:
Cefotaxim Lacton sn phm cui
k
2
k
3
Cefotaxim sn phm cui
k
1

180
Kt qu nghin cu ny (Fabre 1984) l mt v d minh ho s phc tp
ca nghin cu ng hc phn hu thuc.
6.3.1.3. Phn ng bc hai
Cho phn ng A + B C (6. 24)
Tc phn ng
v = d[A]/ dt = k
2
[A][B] (6. 25)
Nu nng ban u ca A v B l a v b, lng C to thnh thi im
t l x th
dx/ dt = k
2
(a x)(b x) (6. 26)
hoc di dng tch phn:
) 27 . 6 (
) x b ( a
) x a ( b
ln t k ) b a (
2

=
a
b
ln
x b
x a
ln t k ) b a (
2
+

=
R rng th nguyn ca k
2
l (thi gian x nng )
-1
.
Nu khi phn ng din ra a>>b, lng A tham gia phn ng coi nh
khng ng k so vi lng ban u nn [A] coi nh khng i v phng
trnh (6.25) c dng
v = k
2
[A][B] = k
2
[B] (6. 28)
Trong trng hp ny, ta gi l phn ng bc nht gi.
6.3.2. nh hng ca nhit
Hng s k trong phng trnh ng hc ph thuc vo nhit . Khi nhit
tng, hng s k tng theo. Kt qu thc nghim cho thy: khi nhit tng
ln 10
0
C hng s tc k tng ln xp x 2 ln.
nh lut Arrhenius
Nm 1889 Arrhenius nhn thy kt qu thc nghim c th c biu
din bng h thc kinh nghim:
ln k = C
1
C
2
/ T (6.29)


181

C
1
v C
2
l hai hng s v C
2
lun lun dng. Phng trnh (6.29) ging
phng trnh Vant Hoff di dng tch phn:
ln k = C H
0
/ (RT) (6.30)
H
0
l i lng khng ph thuc vo nhit .
t C
2
= E
0
/ R, c th vit phng trnh Arrherius di dng
ln k = C
1
E
0
/ (RT) (6.31)
E
0
l nng lng hot ha c xc nh bng cch v th ln k theo 1/T
(T l
0
K =
0
C + 273,15).
C th vit phng trnh (6.31) di dng hm s m
k = A exp(E
0
/ RT) (6. 32)
y ln A = C
1
6.4. Xc nh n nh ca thuc
Khi nh gi n nh cn xem xt n cc yu t:
Tnh cht v c im ca dc cht trong ch phm,
Vng kh hu cho thuc lu hnh,
Ti liu cng b lin quan n n nh ca thuc cn nghin cu.
Nhng yu t ny s quyt nh s mu th, thi gian v tn s th nghim.
6.4.1. Ly mu
Vi dc cht tng i n nh, cn ly hai mu hai l sn xut
khc nhau. Nu dc cht km bn hoc c t ti liu cng b, cn ly ba
mu ba l khc nhau. Cc mu ny cn ly i din cho qui trnh sn xut.
i vi nghin cu n nh mt cch lin tc qui m cng nghip
vic ly mu cc l c tin hnh theo chng trnh nh trc, v d:
Vi cng thc n nh, c hai nm ly mu trn 1 l.
Trong trng hp ngc li, mt nm ly mu trn 1 l.
i vi cng thc nghin cu xong n nh, thng c 3 n 5
nm s kim tra li n nh mt ln.
182
6.4.2. Phng php th cp tc
iu kin th
Bng 6.5. tm tt iu kin th cho vng II v vng IV
Bng 6.5. iu kin th cp tc
Vng Nhit (
0
C) m (%) Thi gian th (thng)
II 40 2 75 5 3
IV 40 2 75 5 6
Vi cc ch phm c hot cht km bn, hoc c t ti liu nghin cu
c cng b, thi gian th ko di hn 3 thng so vi qui nh.
Ngi nghin cu c th la chn nhit cao hn trong thi gian ngn
hn, v d 45 n 50
0
C trong 3 thng vi m 75%.
Nu trong qu trnh nghin cu, ch phm c nhng thay i quan trng
th cn thc hin cc php th nghim b sung iu kin n ho hn, v d 30
2
0
C, m (60 5)%. Nhng du hiu chng t c s thay i quan trng l:
Gim hm lng hot cht t 5% tr ln so vi tr s ban u.
C sn phm phn hu vi lng cao hn tr s cho php.
pH nm ngoi gii hn qui nh.
Tc ho tan ca 12 vin nn hoc vin nang thp hn gi tr ca
tiu chun.
Thay i c tnh vt l ca thuc nh: bin mu, tch pha, kh r,...
Vic th cp tc thng c tin hnh trong bung vi kh hu c th
kim sot c nhit ( 2
0
C) v m ( 5%). Mt s ch phm khng thch
hp vi th nghim cp tc nh: ch phm sinh hc, thuc n, thuc trng,...
V d:
Nghin cu mt ch phm c hm lng theo cng thc l 100 mg. Th
cp tc thi gian 9 thng ba nhit 35
0
C, 45
0
C v 55
0
C. Kt qu c bng
6.6. Tnh hng s tc v tui th ca thuc khi bo qun 30
0
C.

183
Bng 6.6. S thay i hm lng (mg) ca ch phm
Hm lng
Thi gian
(thng)

35
0
C

45
0
C
Hm lng
Thi gian
(thng)

55
0
C
0 105 105 0 105
2 103 102 1 102
4 102 100 2 99
6 101 97 3 97
9 99 94 4 94
5 91
Gii:
V th lnC
t
t(thng). Tnh hi qui bc nht, ta c hng s tc :
- 35
0
C: k
35
= 0,00623 thng
-1
(r = 0,993)
- 45
0
C: k
45
= 0,0123 thng
-1
(r = 0,997)
- 55
0
C: k
55
= 0,0280 thng
-1
(r = 0,997)
tnh tui th nhit 30
0
C cn tnh k
30
V th lnk 1/T (
0
K) theo phng trnh (6. 31)
Nhit (
0
C) k ln k (1/ T). 1000
35 0,0062 5,083 3,246
45 0,0123 4,398 3,145
55 0,0280 3,576 3,049
Tnh hi qui bc nht
ln k = C
1
E
0
/ RT = 19,7 7642 / T
Tnh hng s k
30
theo phng trnh (6.32)
ln k
30
= 19,7 7642 / (30 + 273,15) = 5,52
k
30
= 0,00411
184
Tui th ca thuc l khong thi gian hot cht cn li 90% so vi tr
s ban u (phng trnh 6.11), nn:
t
0,9
= 0,105/ k
30
= 25,5 thng
y l mt v d th n nh cp tc d bo tui th ca thuc. Tr
s ny cn c khng nh bng phng php th di hn.
6.4.3. Phng php th di hn
Theo hng dn ca WHO th iu kin thc nghim trong phng php
th di hn phi gn vi iu kin bo qun thc t ca thuc, tc l vng kh
hu m thuc d kin c lu hnh (bng 6.3). Tuy nhin mt s tc gi
xut iu kin th di hn l nhit 25 2
0
C, m tng i 60 5%.
Nghin cu n nh di hn cn c tin hnh trong sut thi hn
bo qun thuc.
Thi gian kim tra hm lng trong mu:
Nm u 3 thi im 0, 6 v 12 thng,
T nm th 2: mt ln cho mt nm.
Vi cng thc rt n nh ch cn kim tra hai ln: ln u sau 1
nm; ln th 2 cui hn dng.
Vi ch phm km n nh, s ln kim tra nhiu hn:
Nm th nht: 3 thng/ ln
Nm th 2: 6 thng/ ln
T nm th 3: 12 thng/ ln
i vi ch phm c yu cu bo qun c bit nh: ch phm vaccin,
hormon, thuc c hot cht rt khng n nh,... ngi nghin cu cn chn
iu kin thch hp.
Kt qu thc nghim c chp nhn nu:
Khng c s thay i cc tnh cht vt l, ha hc, sinh hc.
Ch phm vn p ng ng yu cu ca tiu chun.
Phng php th di hn mt nhiu thi gian, nhng cho kt qu tin
cy. Php th ny gip ngi nghin cu khng nh tui th d bo theo
php th cp tc.
6.4.4. Phng php phn tch nh gi kt qu
La chn phng php phn tch
Vic la chn phng php nh lng hot cht v (hoc) tp cht phn
hu ph thuc vo:
185
Tnh cht l ha ca hot cht, tp cht,
Hm lng ca chng trong ch phm,
Yu cu ca th nghim.
Cc phng php phn tch cn c thm nh: kim tra ng,
lp li, ti hin, gii hn nh lng. Ring phng php xc nh tp cht
lin quan, tp cht phn hu cn c kim tra tnh c hiu v nhy.
X l s liu
S liu nghin cu cn c x l thng k thu c thi hn bo
qun tm thi. Thi hn ny c ngoi suy trn c s th n nh cp tc.
Mt thi hn bo qun tm thi 24 thng c chp nhn khi:
Hot cht c coi l n nh,
Th nghim cp tc khng c thay i r rt,
S liu hin c chng minh rng nhng cng thc tng t c thi hn
bo qun 24 thng hoc hn.
Nh sn xut s tin hnh th n nh di hn trong qu trnh bo
qun xut.
Sau khi nh gi n nh, nh sn xut cn ghi r iu kin bo qun
trn nhn thuc:
Bo qun iu kin thng,
Bo qun trong khong + 2
0
C n 8
0
C (t lnh),
Bo qun di 8
0
C (t lnh),
Bo qun trong khong 5
0
C n 20
0
C (t ),
Bo qun di 18
0
C (t ).
iu kin thng theo WHO l: nhit 15 n 25
0
C, c th n 30
0
C,
kh ro, khng c nh sng trc tip chiu vo. iu kin thng c th c
qui nh theo tng quc gia.
iu lu l nhng li ch gii nh: bo qun ni kh ro, bo qun
trnh nh sng khng c s dng nhm mc ch che du s n nh km
ca thuc.
6.5. Cc dc cht km bn vng
Nm 1986 WHO thc hin ti :Nghin cu n nh cp tc ca cc
dc cht thng dng trong iu kin nhit i. iu kin th cp tc:
Bo qun 30 ngy 50
0
C, m 100%.
Nu khng c du hiu phn hu: tng nhit ln 70
0
C trong thi
gian tip theo 3 n 7 ngy.
186
Kt qu cho thy khong 100 dc cht cn c lu v n nh:
chng c coi l cc cht km bn vng (bng 6.7)
Bng 6.7. Cc dc cht km bn vng
TT Tn dc cht TT Tn dc cht
1 Acid acetyl salicylic 50 Hexyl resorcinol
2 Acid ascorbic 51 Hydralazin, HCl
3 Acid undecylenic 52 Hydrocortizon (Na succinat)
4 Aminophyllin 53 Hyoscyamin sulfat
5 Amitryptilin, HCl 54 Lidocain, HCl
6 Amoni clorid 55 Melarsoprol
7 Amphotericin 56 Metrifonat
8 Ampicillin (mui natri) 57 Naloxon, HCl
9 Ampicillin trihydrat 58 Natri calci edetat
10 Antimon tartrat 59 Natri lactat
11 Bc nitrat 60 Natri nitrit
12 Bacitracin 61 Natri p. aminosalicylat
13 Bacitracin (mui km) 62 Natri stibogluconat
14 Benzathin benzylpenicillin 63 Neomycin sulfat
15 Benzylpenicillin (mui Na, K) 64 Nystatin
16 Bephenium hydronaphtoat 65 Orciprenalin sulfat
17 Calci gluconat 66 Oxytetracyclin, HCl
18 Calci p.aminosalicylat 67 Paromomycin sulfat
19 Carbenicillin (mui Na) 68 Penicillamin
20 Cephalexin 69 Pethidin, HCl
21 Cloral hydrat 70 Phenobarbital (mui Na)
22 Cloramphenicol (Na succinat) 71 Phenoxymethyl penicillin (mui Na hoc K)
23 Clorphenamin hydrogeno- maleat 72 Phentolamin mesilat
24 Clorpromazin, HCl 73 Phenyl butazon
25 Clortetracyclin, HCl 74 Pilocarpin, HCl
26 Cloxacillin (mui Na) 75 Pilocarpin nitrat
27 Codein phosphat 76 Procain amid, HCl
28 Colecalciferol 77 Procain benzyl penicillin
187
TT Tn dc cht TT Tn dc cht
29 Dapson 78 Procain, HCl
30 Dexametason (dng Na phosphat) 79 Procarbazin, HCl
31 Dicloxacillin (mui Na) 80 Promazin, HCl
32 Dietyl carbamazin (dihydrogeno
citrat)
81 Prometazin, HCl
33 Doxycylin hydrat 82 Pyridoxin , HCl
34 Ephedrin 83 Quinin, bisulfat
35 Ephedrin sulfat 84 Quinin, 2HCl
36 Epinephrin 85 Retinol (vitamin A)
37 Epinephrin hydro-geno tartrat 86 Salbutamol sulfat
38 Ergocalciferol 87 Sulfacetamid (mui Na)
39 Ergometrin hydrogenotartrat 88 Sulfadiazin (mui Na)
40 Ergotamin maleat 89 Sulfadimidin (mui Na)
41 Ergotamin tartrat 90 Tetracain , HCl
42 Etho suximid 91 Tetracyclin, HCl
43 Ethyl morphin, HCl 92 Thiamin, HCl
44 Fe(II) sulffat 93 Thiamin mononitrat
45 Fluphenazin decanoat 94 Thiopental (mui Na)
46 Fluphenazin, HCl 95 Thu ngn oxyd vng
47 Formaldehyd dung dch 96 Tolbutamid
48 Gentamycin sulfat 97 Warfarin (mui Na)
49 Guanethidin sulfat
ti liu tham kho
1. Bureau of Food & Drug (1998)
Training course drug and cosmetic quality control,
Filinvest Corporate city, Philippines.
2. Carstensen J.T. (1995)
Drug stability
Principles & Practices, 2
nd
edition, Marcel Dekker Inc. , USA.
188
3. Didier R. : Chimie minerale
Technique et Documentation, Lavoisier, V
e
edition, Paris.
Bn dch ting Vit, tp II, NXB Gio dc, H ni 1997.
4. OMS (1998)
Assurance de la qualit des produits pharmaceutiques,
Vol 1, Genve.
5. Skoog D. A. , West D. M. , Holler F. J (1988)
Fundamental of Analytical Chemistry, 5
th
edition, chapter 22, pp 578- 597
Saunders College Publishing.
cu hi t lng gi
6.1. Ti sao cn t vn nh gi n nh ca thuc.
6.2. Gii thch cc yu t tc ng n n nh ca thuc.
6.3. Phn tch php th n nh di hn v n nh cp tc. Ti sao
cn tin hnh c hai php th ?
6.4. Nhng ch phm no khng thch hp vi php th cp tc? Ti sao?
6.5. Ti sao phi phn thnh 4 vng kh hu khi nghin cu tui th ca
thuc. Theo anh (ch) vng kh hu no nh hng nhiu nht n n
nh ca thuc?
6.6. Gii thch vai tr ca ng ha hc trong nh gi tui th ca thuc.
6.7. Trnh by nguyn tc s dng phng trnh Arrhenius tnh tui
th ca thuc trong phng php th cp tc.
6.8. Nhng phn ng no thng xy ra lm gim n nh ca thuc?
Cho v d.
6.9. Khi xc nh tui th ca thuc da vo s liu hm lng hot cht, cn
thm nh: ng, chnh xc v gii hn nh lng. Gii thch?
6.10. Gii thch l do ti sao khi xc nh tp cht lin quan, tp cht
phn hy cn kim tra tnh c hiu v nhy ca phng php
thc nghim?
6.11. nh gi vai tr ca cc k thut phn tch trong nghin cu n
nh ca thuc.
6.12. Ngi ta nh gi n nh ca mt ch phm vin nn hm
lng 200 mg bng phng php lo ha cp tc trong thi hn 6
thng hai nhit 40
0
C v 60
0
C. S liu thc nghim c trnh
by bng sau:
189
Hm lng (%)
Thi gian (thng)
40
0
C 60
0
C
0 102,1 102,1
1 101,5 101,6
2 101,1 101,1
3 100,6 99,5
4 100,1 98,9
5 99,5 98,3
6 98,8 97,6
Hy tnh tui th ca vin nn 200 mg.
190