T VN

Trong cuc sng hng ngy ca chng ta, thng xuyn dng n ngt nh
cc loi: hoa qu, khoai c, ch, nc ngt, bnh ko...Ngoi tht ngt c bn cht l
protein v cc axit amin th a phn cc v ngt khc u l cc loi ng to ra.
ng sinh hc dng cho chuyn ha t bo l glucose nhng trong thc t
cuc sng chng ta thng a vo c th cc dng ng n, ng a nh fructose,
mantose, saccharose, tinh bt... ly t cc loi hoa, c, qu, thn cy ma, c ci, mt
ong... vn c sn trong t nhin. Cn cc loi ng ha hc ch to v ngt ch khng
chuyn ha c, dng trong vic iu tr cho nhng ngi bnh tha cn hay i
tho ng. Cc ng tng hp nh: saccharin, manitol, acesulfam K, aspartam,
isomalt, sorbitol, sucraloza c php s dng trong ch bin thc phm vi gii hn
ti a v c quy nh ca B Y t.[..]
ng ha hc (hay l cht ngt tng hp) l cht khng c trong t nhin,
thng c v ngt rt cao so vi ng knh saccharose v tuyt nhin khng h c mt
gi tr dinh dng no khc. Do , nhiu c s sn xut v mc ch li nhun nn
thng s dng cc cht ngt ny thay th cho ng ma (saccharose), mc d bit
r nhng iu c hi cho ngi tiu dng khi s dng qu mc qui nh.
Acesulfame K l mt acid hu c sinh ra t acid Acetic.[..] Cht ny khng b
c th phn hy v c ng tiu ha bi xut ra nguyn trng. v c s dng nhiu
trong thc phm ch bin
Aspartam l mt este methyl cu to t 2 acid amin l aspartic acid v
phenylalanine, c ngt cao khong 180 n 200 ln ngt hn ng ma, v c nng
lng khong 4 Kcal/g. Tuy nhin, mt s ngi nhy cm cho rng ng ny lai
trn li mt hu vi khng ngon. V vy, th khc phc thng thng ngi ta trn
aspartam vi cc loi ng khc nh acesulfame K.
Hin nay, trn th trng xut hin kh nhiu sn phm nh: nc gii kht,
bnh ko, nc ngt, ch, sm lnh, sa u nnhv c s dng hai hay nhiu cc
hot cht ny, do gy khng t kh khn cho vic kim tra cht lng cc sn phm
thc hin ang lu thng trn thi trng Tha Thin Hu.
phn tch hm lng cc thnh phn ny , theo TCVN ch nh lng c
hai thnh phn ny ring l trong cc nguyn liu bng phng php chun trong
mi trng khan, hoc mt s phng kim nghim thc phm trong nc thng dng
cc phng php chit vi axit axetic bng, sau xc nh bng phng php chun
in th. Phng php ny khng n nh, mt thi gian, tn km ha cht, cho
nhy km, hn na nu trong sn phm cha hai hay nhiu thnh phn, th phng
php ny t ra khng hu hiu. Chnh v nhng l do ny m hin nay phng php sc
k lng hiu nng cao ang c p dng thay dn phng php ni trn.

T nhng yu cu thc t ca cng tc qun l v sinh an ton thc phm trn

th trng Tha Thin Hu, chng ti thng xuyn phi phn tch cc sn phm loi
ny, ng thi c qui trnh phn tch t yu cu v chnh xc v ng, do
cn phi tin hnh xy dng, ti u ha v nh gi qui trnh phn tch ng thi hai
hot cht trn trong cng sn phm thc phm bng phng php sc k lng hiu
nng cao pha o.
thc hin vn ny chng ti tin hnh trin khai ti: Nghin cu xy
dng qui trnh nh lng ng thi cht ngt tng hp aspatam v acesulpam trong
mt s sn phm thc phm bng phng php sc k lng hiu nng cao nhm gii quyt
cc mc tiu sau:
- Xy dng, ti u ha v nh gi qui trnh phn tch ng thi hai hot cht
trn phng php sc lng hiu nng cao.
- Nghin cu phng php x l mu
- p dng quy trnh xy dng c nh lng mt s sn phm c gi ti trung
tm kim nghim Thuc - m phm - Thc phm - Tha Thin

Chng 1
TNG QUAN TI LIU
1.1. Gii thiu v aspartame v acesulfame-K
1.1.1
Aspartame [1];[9];[7]; [13];[10]
- Tn khc v k hiu ca aspartame:
- Tn: Aspartyl phenylamin metyl ester
K hiu: APM
Tn ha hc : 3-Amino-N-( carbomethoxy-phenyletyl)-acid succinamic
Hay: N-L--aspartyl-L-phenyllamin-1-metyl ester
Cng thc : C14H18N2O5

Khi lng phn t: 294,31

Ngoi quan: Tinh th mu trng, khng mi c v ngt mnh
Chc nng: cht to ngt
Gc quay cc ring: []20D +14,5o- 16,5o
Tnh tan: t tan trong nc v ethanol
pH: 4,5-6,0
* Tnh cht ASPARTAME (E951)[10]
- Aspartame l mt cht to ngt vn c s dng ph bin trong cc loi nc
ung v thc phm dnh cho ngi b bnh tiu ng v ngi mun n king vi
mc ch gim lng ng v nng lng tiu th. Tuy nhin, c nhng lo ngi v tc
dng ph ca n nh c kh nng gy ung th, gy bnh ng kinh, gy u no.
- Aspartame (tn khc l L-aspartyl-L-phenylalanine methyl ester) l mt este
methyl cu to t 2 acid amin l aspartic acid v phenylalanine. Trong t nhin,
aspartic acid v phenylalanine c tm thy trong thc phm c cha protein, nh tht,
ng cc v cc sn phm t sa. Este methyl cng c tm thy trong hoa qu v rau.
- Aspartame c ngt cao, khong 180 n 200 ln ngt hn ng ma. V
ngt ca aspartame kh ging ng. V ngt ny c cm nhn chm hn v ko di
lu hn so vi ng.
- Aspartame c nng lng khong 4 Kcal/g (17 KJ/g). Tuy nhin, ch cn mt
lng rt nh aspartame to ra ngt cn thit. Do nng lng chng ta a vo
c th khi dng thc phm to ngt bng aspartame s khng ng k.
- a s ngi tiu dng c nhn xt l aspartame khng li d v kh chu, tuy
nhin mt s ngi nhy cm cho rng ng ny lai trn li mt hu vi khng
ngon. iu ny c th khc phc bng cch trn aspartame vi cc loi ng khc
(nh acesulfame K hay saccharine). Aspartame rt c a chung trong vic s dng
thay th ng trong cc sn phm thc phm dnh cho ngi n king.
3

Khi vao c th, bn thn aspartame khng hp th vo mu ma tan ra trong rut thnh
ba cht: aspartic acid (40%), phenylalanin (50%) v methanol (10%). y l c s ca
nhiu kin v tc ng bt li cho sc khe con ngi t aspartame.
-Bn thn methanol l mt cht c c tnh thp. Sau khi c a vo c th,
methanol c xy ha to nn formaldehyde, cht ny li tip tc c xy ha to
nn acid formic (hoc formate, ty theo pH). Acid formic c c tnh rt cao. Chnh
acid formic l nguyn nhn gy nn tnh trng toan chuyn ha (nhim acid) v m la,
nhng tn thng c trng ca nhim c methanol.
- Lo ngi v lng axit aspartic v phenylalanine khi n sn phm c cha
aspartame:
- Phenylalanine cng l mt cht c thn kinh. Qu nhiu phenylalanine gy
ng kinh, thai gy chm pht trin tr c, mt ng Chinh phenylalanine tao ra bnh
phenylketonuria (PKU), la loi bnh m trong mau co qu nhiu axt amin
phenylalanine, lam chm phat trin tri tu nghim trong, va lam tn hai n h thn
kinh. Mt khc, con ngi hp thu cc axt amin cn thit t thin nhin di dng kt
hp vi nhiu cht khc ch khng phi ch ring mt cht c lp.
- Nu d tha, axit aspartic c th gy ra ri lon ni tit (hormone) v cc vn
v th lc. Aspartic acid l mt cht kch thch thn kinh, nh hng n h thng thn
kinh trung ng, gy nn cc chng: nhc u, bun nn, au bng, mt mi, ri lon
gic ng, vn v th lc, trm cm, v bnh suyn, ng kinh
- D sao, quan im ca nhiu t chc c uy tn hin nay ng h vic s dng
aspartame trong hn mc cho php, bao gm T chc Nng lng Quc t (FAO), T
chc Y t Th gii (WHO), y ban Qun l Dc-Thc phm M (FDA). Liu dng
cho php ca aspartame mi ngay (ADI) l nh hn hoc bng 40 mg/kg th trng. y
l mt trong nhng hm lng cho php s dng cao nht trong s cc ph gia thc
phm. Tuy nhin, thng 4 nm 2009, FESA cng t chc mt hi ngh gm cc chuyn
gia c nhng kin thc chuyn su v aspartame. Cc chuyn gia ny xem xt li tt c
nhng nghin cu v d liu v aspartame v nhng nh hng n sc khe (nu c)
ca n. D kin, u nm 2010, hi ng ny s thng bo kt qu.
Ti Vit Nam, aspartame c php s dng cho cc sn phm nh ung, ko, bia,
ru vi mc ch lm to ngt, iu v vi ADI cng t 0 40 mg/kg, ging nh kin
ngh ca cc t chc uy tn trn th gii. Trn nhng sn phm c s dng aspartame s
phi ghi tn aspartame hoc aspartame hay ghi m s 951.
- Vit Nam, theo Quyt nh ca B trng B Y T, S 3742/2001/Q-BYT
v cc cht ph gia c php s dng trong thc phm. c ghi trong bng
sau:
Bng: 1.1. Gii hn ti a cht ph gia aspartame trong thc phm [5] SttNhm thc
phmML

Stt
1
2
3
4
5
6
7
8
9
10
11
12
13
14

Nhm thc phm

ung c sa, c hng liu hoc ln men (VD: sa
s c la, sa cacao, bia trng, sa chua ung, sa c)
Qu thanh trng pasteur ng hp hoc ng chai
Mt, mt c c, mt hoa qu
Hoa qu ngm ng
Rau thanh trng pasteur ng hp, ng chai hoc
ng ti
Bt nho t cacao
Ko cng, ko mm, ko nuga...
Ko cao su
Sn phm dng trang tr thc phm
Du trn, gia v (bao gm cc cht tng t mui)
Nc rau, qu p
Necta rau qu
Bia v nc gii kht ch bin t mch nha
Ru vang

ML
600
1000
1000
2000
1000
3000
10000
10000
5000
2000
2000
2000
600
600

1.1.2. Acesulfame-K [1]; [7]; [4];[8];[10]

Tn ha hc:
Tn: Mui kali 6-metyl-1,2,,3-oxathiazin-4(3H)-mt-2,2-dioxid
Mui kali 3,4-dihydro-6-metyl-1,2,3-oxathiazin-4-mt-2,2-dioxid
Tn khc:
Cng thc: C4H4KNO4S
Khi lng phn t: 201,24
Ngoi quan: Tinh th mu trng, khng mi, c v ngt mnh
Chc nng: cht to ngt, cht iu v
Tnh tan: tan nhiu trong nc, t tan trong ethanol.
*Tnh cht acesulfame-K (E950)
- Acesulfame-K c Clauss v Jensen (hng Hoechst, c) tm ra nm 1967;
c FDA kim nghim v cho a vo s dng t nm 1988, vi cc tn thng mi
khc nhau nh Sunette, Sweet one, Sweet'n safe... Cc sn phm c cha acesulfame-K
cha gy ra bt k s c no cn cnh bo.
- Acsesulfame-K c s dng trong hn 4000 sn phm trn khp th gii...
Ngoi ra, thng c dng kt hp vi aspartame hoc cc loi ng ha hc khc
v n c tc ng h tr, tng cng v duy tr v ngt ca thc n v nc gii kht.
Tnh cht:
- C dng tinh th mu trng vi cu trc ha hc tng t saccharin. V ngt gp 150 200 ln ng saccharose; n nh hn aspartame nhit cao v mi trng acid.

- Acesulfam-K khng cung cp nng lng cho c th v n khng tham gia qu trnh
trao i cht v c thi ra ngoi theo nc tiu m khng c bt k s bin i ha
hc no.
- Thm u im l gi thnh r; song iu d l c d v hi ng...
- Vit Nam, theo Quyt nh ca B trng B Y T, S 3742/2001/Q-BYT v cc
cht ph gia c php s dng trong thc phm. c ghi trong bng sau:
Bng: 1.2. Gii hn ti a cht ph gia aceslfame-K trong thc phm SttNhm thc
phmML

Stt
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

18
19

Nhm thc phm

ung c sa, c hng liu hoc ln men (VD: sa
s c la, sa cacao, bia trng, sa chua ung, sa c)
Sa ln men v sa c cht rennet (nguyn cht)
khng k ung t sa
Thc n trng ming c sa (VD: kem, sa lnh, bnh
puting, sa chua hoa qu hoc c hng liu...)
Qu thanh trng pasteur ng hp hoc ng chai
Mt, mt c c, mt hoa qu
Hoa qu ngm ng
Sn phm cacao, s c la (VD: s c la sa, s c la
trng)
Ko cng, ko mm, ko nuga...
Ko cao su
Sn phm dng trang tr thc phm
Bnh nng v cc loi sn phm bnh nng thng
thng
Bnh c sa, trng
Nc chm dng sa (VD: nc st mayonne, nc
st salt
Nc chm khng c sa (VD: tng c chua, tng
t, nc chm c kem)
Nc qu thanh trng pasteur ng hp hoc ng
chai
Nc rau p thanh trng pasteur ng hp hoc ng
chai
Nc gii kht c hng liu, bao gm nc ung
dnh cho th thao, nc ung c hm lng khong
cao v cc loi nc ung kht
C ph, ch, nc ung c dc tho v cc loi
ung t ng cc, khng k nc ung t cacao.
Ru vang

ML
500
GMP
1000
500
1000
500
1000
350
5000
500
GMP
350
1000
350
600
600
600

GMP
350

1.1.3 Tnh cht ca hn hp aspartame v acesulfame-K

- ngt cao gp 200 ln so vi ng t nhin, do vy, ch cn mt lng rt
nh aspartame cng cho ngt tng ng nh s dng ng bnh thng. V
ngt ca aspartame chm hn lc u, nhng li ko di lu hn - nn nn aspartame
thng c dng kt hp vi acesulfame-K cho v ngt ging (v ngt hn) ng t
nhin. Khng li d v ho cht hoc v kim loi kh chu nh mt s cht lm ngt
khc, d bo qun v s dng, gi thnh r hn nhiu so vi vic s dng ng t
nhin nn hin nay aspartame c s dng rng ri ti hn 125 quc gia trn th gii
v hin din trong khong 6.000 sn phm khc nhau - trong bnh ko, nc hoa qu,
trong cc sn phm "khng ng" hoc sn phm "light", "diet"..., ko cao su, sa
chua, ng cho ngi n king v ngay c trong mt s thuc, vitamin, thc phm b
sung...
- Aspartame c thng mi ho di mt s tn nh Canderel, Equal,
NutraSweet, Sanecta, Tri-Sweet, Aminosweet, Spoonful, Sino sweet...
- Acesulfame-K - cn c gi l Sunette, Sweet One, Sweet 'n an ton
Acesulfame-K c pht hin vo nm 1967 v l 150-200 ln ngt hn ng.
Acesulfame-K l mt rt n nh, kt tinh cht ngt vi mt cu trc ha hc tng t
nh saccharin.
- Acesulfame-K thng c s dng kt hp vi aspartame hoc cht ngt khc
v n c mt tc dng hip ng tng cng v duy tr v ngt ca thc phm v
ung. l nhit n nh n c th c s dng trong cc sn phm nng. N
khng cung cp calo v c th khng chuyn ha n v n c bi tit trong nc tiu
m khng b thay i.
- Acesulfame-K c tm thy trong

Chng 2
NI DUNG V PHNG PHP NGHIN CU
2.1. Ni dung nghin cu
(1). La chn cc iu kin sc k tch ng thi hai cht acesulfame-K v
aspartame trong cc mu nc gii kht bao gm:
- Qui trnh chit mu nc gii kht: loi mu, th tch dung mi chit.
7

- Cc iu kin tin hnh sc k: dung mi pha ng, t l pha ng, tc

dng, nng pH.
(1). nh gi tin cy ca phng php phn tch qua cc i lng nh
lp li, ng, gii hn pht hin v gii hn nh lng.
(3). p dng thc t: p dng phng php la chn phn tch mt s mu
c cha acesulfame-K v aspartame trong cc nc gii kht hin ang lu hnh trn
th trng Tha Thin Hu
2.2. Thit b, dng c v ha cht

2.2.1. Thit v dng c

- Cn phn tch AUW220D (0,1 mg) ca hng Shimadzu Nht Bn.
- My sc k lng hiu nng cao Series 20A ca hng Shimadzu Nht Bn.
- Cc thit b khc nh: T sy, my lc, my ct nc hai ln, thit b lc nc
siu sch.
- Cc dng c thy tinh nh: Bnh nh mc cc loi, pipet vch v pipet bu
cc loi, cc...
- Micropipet cc loi: 0,5-10L; 10-100L; 100-1000L...
- Mng lc 0,45m
- Ct sc k pha o C18, 150x5mm, 4,5m
- B lc ht chn khng

2.2.2. Ha cht
-

Nc ct hai ln lc qua mng lc 0,45m.

Cht chun acesulfame- K
Cht chun aspartame v cc ha cht khc loi PA
KH2PO4 Merck
Acetonitril (ACN) Merck
Etylacetat ca PA
NaCl ca PA
NaHCO3 ca PA
H3PO4

2.2.3. Cc dung dch gc:

- Dung dch gc 5000ppm: cn mi loi 0,5g, lc cho tan hon ton v nh
mc n 100mL.
- T dung dch gc, pha cc dung dch chun lm vic: 1ppm; 5ppm; 10ppm;
20ppm; 50ppm; 100ppm; 200ppm.
2.3. Phng php nghin cu
2.3.1. X l mu:
8

+ Cc sn phm cht lng trong (i vi nc chanh, nc cola, cc loi ung)

Pha long mu vi nc ng trong bnh nh mc 100ml. Lc dung dch qua
b lc c l 0,45 m trc khi bm.
+ Cc sn phm dng c(nc qu, ung t sa c hng v)
Pha long 20ml mu ng ha vi 50ml nc ct, nh mc va 100ml.
Lc qua giy lc gp np, loi b 10ml dch lc u tin. Cho phn dch lc qua b lc
mng c l 0,45m trc khi bm.
tnh n th tch cht kt ta,nu cht ha tan khng cha cht boc trong
mu (trng hp ny l 20ml) vt qu 3g, th li tm hn hp mu lm trong 10
pht vi tc 5000 vng/pht, trc khi lc sang bnh nh mc 100ml. Ra phn
cht lng hai ln vi nc ct v cho li tm li, thu ly phn ni pha trn sang bnh
nh mc 100 ml ri pha long dung dch bng nc ct n vch. Nu lng cht
khng ha tan nh hn 3 g th cng c th thc hin qui trnh ny.
2.3.2. Phng php phn tch
* iu kin tin hnh th nghim
Cc th nghim c tin hnh trn h thng sc k lng hiu nng cao ca
SHIMAZDU, detectr diotarray c bc sng t 190 n 800 nm, ct tch pha o RP
C18:
-

Pha tnh v chiu di ct: ht hnh cu 3m i vi ct di 100mm, n 10m

i vi ct di 300 mm

ng knh trong: 4,0mm

Tc dng : t 0,8 ml/min n 1ml/min

Th tch tim: 10l n 20l

Pht hin bc sng: = 217nm i vi aspartame; = 227nm i vi

acesulfame-K; hoc = 220 nm i vi c hai cht.

T l pha ng:
+ dung dch m phosphat + axetonitril [80: 20] (v/v)
+ dung dch m phosphat + axetonitril [85: 15] (v/v)
+ dung dch m phosphat + axetonitril [90: 10] (v/v)
+ dung dch m phosphat + axetonitril [95: 5] (v/v)
* nh tnh v nh lng

- nh tnh: Da trn vic so snh thi gian lu ca cc cht c trong mu phn

tch vi thi gian lu ca cc cht chun khi tin hnh trong cng iu kin sc k.
9

- nh lng:
+ Xc nh theo phng php ngoi chun: tnh tch phn cc din tch
pc/chiu cao v so snh cc kt qu vi gi tr tng ng ca cc cht chun.
+ Phng php ng chun: bm mt lng thch hp cc cc dung dch
chun c cc nng thch hp. V din tch pic/chiu cao ca dung dch chun tng
ng vi nng mg/l . Kim tra tuyn tnh ca ng chun.
* Tnh kt qu:
+ Phng php ngoi chun: Hm lng cc cht to ngt (w), c trong dung
dch th c tnh mg/l, theo cng thc sau:
w( mg / l ) =

A1 V1 C % F
1000
A2 V2

(1)

Trong :
A1: din tch pic ca cht to ngt thu c dung dch mu th
A2: din tch pic ca cht to ngt thu c dung dch chun
V1: th tch ca dung dch mu, tnh bng mililit (ml)
V2: th tch ca dung dch mu, tnh bng mililit (ml)
C%: nng phn trm ca cht to ngt trong dung dch chun
+ Phng php ng chun:
Hm lng cc cht to ngt (w), c trong dung dch th c tnh mg/l, theo
cng thc sau:
w(mg / l ) =

V1 C F
m0

(2)

Trong :
C : hm lng cht to ngt c lin quan trong dung dch th, c c
t ng chun, tnh bng mg/l.
F,V1, m0 xem trong cng thc (1)
2.3.3. nh gi tin cy ca phng php phn tch
2.3.3.1 lp li
nh gi lp li trn c s xc nh lch chun v sai s tng i RSD%
ca 5 php th song song trn mi i tng nghin cu.
RSD (%) =

10

S
.100
X tb

Trong : S : lch chun

X tb : gi tr trung bnh

2.3.3.2. ng
ng ca phng php phn tch c nh gi qua thu hi (Rev) khi
phn tch mu thc t c them chun (spiked sample)
Re v(%) =

x 2 x0
.100
x1

Trong :
x0 : l nng cht phn tch trong mu
x1: l nng cht phn tch them chun vo mu
X2: l nng cht phn tch xc nh c trong mu them chun
2.3.3.2.Gii hn pht hin (LOD) v gii hn nh lng (LOQ)
LOD xc nh bng cch phn tch lp li t 8 10 ln mu trng hoc mu
chun. T kt qu phn tch lp li, tnh lch chun S, LOD v LOQ tnh theo cng
thc sau:
LOD = t * S
LOQ = 10* S
Trong : t l gi tr chun student vi bc t do f (f = n-1, n l s ln o lp
li), P = 0,99
S : l lch chun
2.3.3.3. Phng php x l s liu th nghim
Cc s liu thc nnghim c x l theo phng php thng k ( s dng cc
phn mm Excel v Origin 8.0)
2.3.3.4. nh gi kh nng ng dng thc tin ca phng php nh lng nghin cu
S dng phng php nh lng nghin cu vo vic nh lng
acesulfame-K v aspartame c trong cc sn phm nc gii kht... c trn th trng
Tha Thin Hu.

11

CHNG 3
KT QU V BN LUN
3.1.La chn cc iu kin tin hnh sc k phn tch ng thi hai hot cht
acesulfame-K v aspartame trn h thng HPLC
la chn cc iu kin phn tch ng thi aspartame v acesulfame-K,
chng ti tin hnh bm vo h thng HPLC hn hp aspartame v acesulfame-K c
nng 20 mg/l cho mi cht theo cc iu kin kho st khc nhau (bng 3.1). Sau
da trn s phn gii cc pic, tn hiu v thi gian hon thnh php phn tch la
12

chn iu kin phn tch ph hp. kt qu c trnh by bng 3.2. Sc k ca hn

hp theo cc iu kin khc nhau c trnh by hinh 3.1. v cc ph lc .
Bng 3.1. Cc iu kin phn tch hn hp trn h thng HPLC
K hiu

Tc dng
(ml/pht)

T l pha ng
(Phosphat + axetonitril)

1a

0,8

80 : 20

1b

0,8

90 : 10

2a

1,0

80 : 20

2b

1,0

90 : 10

3a

1,2

80 : 20

3b

1,2

90 : 10

4a

1,5

80 : 20

4b

1,5

90 : 10

Vi cc iu kin phn tch trn, sc k thu c c s phn tch tng i

tt, khng c hin tng chng pc. Tuy nhin, theo iu kin phn tch (2b) sc k
thu c c phn gii tt, nhng tn hiu (chiu cao pc) ca aspatam b hn so vi
cc iu kin khc, hn na h s i xng ca pc ra sau (S b2 = 1,33 > 1,000) c th
nh hng ti vic xc nh cng tn hiu (din tch).... Khi phn tch iu kin
(3b), mc d thi gian phn tch ngn d b nhiu cc tp cht, phn gii gia hai
pc khng c tt s gy kh khn khi tin hnh mu tht c bit cc mu nc gii
kht c mu tng hp, cht bo qun v lipid.
Theo iu kin (4a), phn gii ca hai pic tt hn so vi (3b), c tn hiu pic
cao, thi gian phn tch khng qu di ( khong 5 pht). Nh vy, vi mc tiu la
chn cc iu kin tin hnh sc k sao cho tng nhy ca phng php, gim thi
gian phn tch (gim chi ph).vn cho kt qu phn tch tin cy. Do , chng ti
chn iu kin (4a) tin hnh nghin cu tip theo.

13

Hnh 3.1. sc hn hp chun iu kin 2b

14

Hnh 3.2. sc hn hp chun iu kin 3b

Hnh 3.3. sc hn hp chun iu kin 4a

15

Tm lai, cc iu kin phn tch dung dch hn hp chun: aspartame v

acesulfame-K trn h thng HPLC nh sau:
-

Ct pha o : RP C18 (5m,150 mm; 4,0 mm)

Tc dng : 1,5 ml/min

Th tch tim: 20l

Pht hin bc sng: = 220 nm i vi c hai cht.

T l pha ng:
+ (dung dch m phosphat + axetonitril) - [80:20] (v/v)
Bng 3.2. Thi gian lu (tR), din tch (S), phn gii (R), v
tng thi gian phn tch(T) theo iu kin 4a vi nng 20 mg/l
i kin
Hot
Cht

4a
tR

Acesunfan

3,3

1334798

Aspatam

7,2

1372789

11,98

3,67

3.2. Kho st v chn la quy trnh x l mu

Thc t, mt s mu nc gii kht c nn mu phc tp, mun phn tch chnh
xc i hi phi c quy trnh x l mu thch hp khng mt cht cn phn tch, do
cn phi loi tp cht trc khi nh lng. Trong qui trnh chun b mu phn tch
aspartame v acesulfame trong nc gii kht, giai on nh hng ln nht l giai
on chit cht phn tch ra khi mu ban u (loi cc cht cn tr nh: cht bo qun,
phm mu tng hp, cht to hng hoc mt s nc sa gii kht c lipid v cc cht
hu c km phn cc, cc vitamin.) Theo kt qu tng hp cc ti liu cng b v
phng php chun b mu phn tch aspatam v acesulfame-k trong nc gii kht
[.]; [..], chng ti nhn xt rng:
- Giai on lm sch mu (elean-up): lm sch ngi ta dng k thut chit
pha rn (SPE: Soil Phase Extraction) vi pha rn thng dng l florisil.Trong trng
hp ny, thm 2ml dch lc lm trong vo ct, trc hot ha 3 ml methanol
v 20 ml nc. Cht phn tch c ra gii bng 20 ml dung dch pha ng, sau
dch lc i qua b lc mng c kch thc l 0,45m trc khi bm vo h thng
HPLC. Tuy nhin, cng c tc gi ch lm sch dch chit bng thuc th carrez I, II,
sau tch trn ct sc k pha o ca HPLC [].

16

R rng, c nhiu phng php x l mu khc nhau cho mi giai on ca qu

trnh x l mu. Vi mc ch xy dng qui trnh x l mu n gin, chi ph thp, ph
hp vi iu kin hin ti phng th nghim ca teung tm, nhng vn m bo
chnh xc ca kt qu phn tch. Do , chng ti xut 2 quy trnh x l mu phn
tch ng thi aspartame v acesulfame-K trong mt s nc gii kht theo s hnh
3.4; 3.5.

17

Mu nc gii
kht (10-40ml)

+20ml NaCl bo ho
+ H3PO4 ; pH= 1-2ml

Chit Asp+Ace
bng 275ml
ethylacetat

+30ml NaCl bo ho
+ Lc k

Dch chit ethyacetat

c chit 2 20ml
natrihydrogen
carbonat

C quay ui
ht dung mi
ethyacetat

Loi
Nc

Thm 2ml TBA-Br 0,1M,

nh mc

HPLC

Hnh 3.4. S quy trnh x l mu, chit soxhlet bng dung mi thch hp

18

10ml -20 ml mu,

pha long nc ct
va 100 ml

CT SPE
( c hot ha vi 3ml
metanol v 20 ml nc)

+ ra gii bng
20 ml pha ng

Dch lc

Lc qua mng
0,45m

HPLC

Hnh 3.5. S tch tp cht dng ct chit pha rn v ra gii bng pha ng

19

3.2.1. La chn bin php chit mu

Nh ta cp trn 2 hnh 3.2 ; .3.3 chng ti thc hin 2 bin php chit mu
khc nhau:
1/ Mu c chit qua dung mi ethylacetat v chit ethylacetat bng dung mi
natrihydrogen carbonat , sau loi nc v c quay chn khng n cn 2ml, thm 2ml
TBA-Br 0,1M, nh mc, lc qua mng 0,45m ri bm vo HPLC
2/ Mu c lm sch qua ct chit pha rn, c ra gii pha ng, v lc qua
mng 0,45m sau bm vo HPLC
Hiu sut ca vic chit cht phn tch ra khi mu ban u ph thuc vo
nhiu yu t khc nhau nh k thut chit mu, dung mi s dng, cc iu kin tin
hnh th nghim. Khi cc iu kin c kim sot, th thnh phn ca mu li l
yu t quyt nh n s thnh cng ca qu trnh chit (hay l hiu sut chit). Do vy,
trong nghin cu ny la chn bin php chit ph hp,chng ti tin hnh th
nghim trn mu thc t mhu sau: Vi mi bin php chit mu, chng ti tin hnh
song song trn cng mt mu:
+ 1 th nghim trn mu khng thm chun
+ 1 th nghim trn mu c thm hn hp aspatam v acesunfan (thm vo mu
nc gii kht 1ml chun hn hp aspatam v acesunfan 100mg/l , mi cht)
Sau tnh ton thu hi cho tng bin php x l mu. thu hi l c s
chng ti la chn bin php chit mu thch hp.
Do iu kin b gii hn, chng ti ch tin hnh th nghim thm chun mc
100 % . Tuy nhin, cc th nghim c tin hnh cn thn nn kt qa thu c c
m bo tin cy.
Di y m t c th 02 bin php chit mu:
Bin php 1:
- Ly khong 10- 20 ml cho vo ng ly tm hoc phu chit.
- Thm vo 20ml dung dch NaCl bo ho, thm H3PO4 c pH=1-2
- Chit bng 2x 75ml ethylacetat.
- Thm 30ml mui bo ho vo ton b dch chit ethylacetat thu c.
- Lc k b lp nc
- Chit lp ethylacetat bng 2 x 20ml natrihydrogen carbonat
- Ct quay ui ht dung mi ethylacetat
- Thm vo 2ml TBA-Br 0,1M
- nh mc ti vch v chy HPLC.
Bin php 2:
20

- Ly khong 10 20 ml mu ng ha k, cho vo bnh nh mc 100ml,

pha long nc ct va , lc. Ly 4 ml dch lc c lm trong vo ct chit pha
rn c hot ha vi 3 ml methanol v 20 ml nc v ra gii vi 20 ml pha ng.
Cho phn dch lc mng c kch thc l 0,45 m v bm trc tip vo my HPLC.
Kt qu nh lng trn my HPLC s dng tnh ton thu hi cc cht
phn tch cho tng bin php x l mu. Sau da trn thu hi tnh ton c
la chn bin php x l mu.
Kt qu tnh thu hi vi 2 bin php x l mu khc nhau c trnh by
bng 3.3.
Bng 3.3. thu hi aspatam v acesulpam vi hai bin php chit mu khc nhau
thu hi %

Tn cht

Bin php 1

Acesulpam
Aspatam

Bin php 2

105
65

98

Kt qu thu c cho thy: Bin php bin php 2 cho thu hi cc cht phn
tch cao ( t 89 n 105 %), sc k c ng nn thp. Cn bin php 1 hiu sut
thu hi km: aspatam < 70%. Nh vy, bin php x l mu c la chn l lm sch
mu qua ct chit pha rn v ra gii bng pha ng.

3.3.nh gi tin cy ca phng php phn tch

21

3.3.1. lp li
Tin hnh o lp li 3 ln hn hp dung dch chun 2

Mu c chit bng dung mi ethylacetat trong mi trng

acid phosphoric (pH =1-2 ). Sau ng saccarin v
acesulpham K li c chit bng dung dch natri hydrogen
carbonat. Dch chit c ct quay ui ht ethylacetat v
thm vo 2ml dung dch TBA-Br, nh mc v bm vo h
thng HPLC xc nh bi detector PDA bc sng 210 v
230nm.
Phm vi p dng: Phng php ny p dng cho cc sn phm
nc gii kht, mai, bnh mt ko...

i vi mu trng thi rn

c xay, nghin nh v ng

nht k
- Mu trng thi lng phi trn u
- Cn khong 10- 40 g cho vo ng ly tm hoc phu chit.
- Thm vo 20ml dung dch NaCl bo ho, thm H3PO4 c
pH=1-2
- Chit bng 2x 75ml ethylacetat.
- Thm 30ml mui bo ho vo ton b dch chit
ethylacetat thu c.
- Lc k b lp nc
- Chit lp ethylacetat bng 2 x 20ml natrihydrogen
carbonat
- Ct quay ui ht dung mi ethylacetat
- Thm vo 2ml TBA-Br 0,1M
- nh mc ti vch v chy HPLC.

22

Da vo trng lng phn t, cu trc ha hc, tnh cht ha tan, tnh phn
cc ca aspartame v acesulfame-K, kt hp vi vic tham kho tng quan ti liu ca
hai cht aspartame v acesulfame-K: aspartame v acesulfame-K u l cht phn cc
v cng c lin kt nn c hp th t ngoi vng 200nm-300nm (aspartame hp th
bc sng cc i max=214nm nm v acesulfame-K hp th bc sng cc i
max =237 nm). Do chng ti s dng k thut sc k pha o ct C18 v
detector dioaray phn tch ng thi hai hot cht ny.
Bc sng pht cng c la chn trn c s u tin ti bc sng cc i ca cht
c nng b (210nm). Pha ng c kho st v la chn trong cc dung mi v
hn hp theo t l ca cc dung mi: Methanol; nc; ACN; dung dch m.
Ct: S dng ct Lichrosorb RP 18 (150x4,6mm; 3m). Ct t yu cu s
dng ca hng sn xut.
Detector: Chn bc sng pht hin sao cho tp cht v cc cht ph khng
gy cn tr, cng ca peak phi ph hp vi php phn tch ng thi c hai cht
aspartame v acesulfame-K trong nc gii kht. Cc bc sng th ?, u tin chn
bc sng cc i ca cht c hm lng b hn l bc sng 210nm.
Dung mi pha mu: Dung mi pha mu l dung mi ha tan tt cht cn phn
tch nhng hn ch tan cc tp cht khc c trong mu, ng thi cng phi c thnh
phnh tng t vi thnh phn pha ng gim hin tng gy nhiu ng nn
trong qu trnh sc k.
Th tch bm mu: 20L (bung tim c nh ca my) v vy cn phi a
ra nng cht phn tch thch hp. Qua mt s ln bm mu thm d, chng ti thy
nng cc cht phn tch c pha di y cho kt qu chp nhn c (din tch
peak nm trong khong o ca my, peak cn i, tch r rng).
-Nng dung dch chun aspartame: cn chnh xc 0,050g aspartame vo
bnh nh mc 100mL, thm dung dch NaHCO 3 0,1M lc cho tan ri nh mc n
vch bng dung dch NaHCO3 0,1M, lc u (dung dch A). Ly 20mL dung dch A
cho vo bnh nh mc 100mL, thm dung dch NaHCO3 0,1M cho n vch ri lc
qua mng lc milipor 0,45m (dung dch A').
Dung dch chun acesulfame-K: Cn chnh xc 0,050g acesulfame-K vo
bnh nh mc 100mL, thm dung dch NaHCO3 0,1M lc cho tan ri nh mc n
vch bng dung dch NaHCO3 0,1M, lc u (dung dch B). Ly 20mL dung dch B
cho vo bnh nh mc 100mL, thm dung dch NaHCO3 0,1M cho n vch ri lc
qua mng lc milipor 0,45m (dung dch B').
-Dung dch mu th: Ly chnh xc 50,0mL ch phm vo bnh tam gic,
acid ha bng acid phosphoric cho n pH khong 23. un nng trn bp cch thy
khong 15 pht, chit hai ln bng dung mi ethylacetat (mi ln l 25mL). Ra chit
23

hai ln bng dung dch NaHCO3 0,1M (mi ln 25mL). un trn bp cch thy ui
dung mi hu c, chuyn ht vo bnh nh mc 100mL dng dung dch NaHCO3 nh
mc n vch, lc qua mng lc milipre 0,45m
3.1.1. Kho st la chn dung mi pha ng
Kho st cc h dung mi phn cc khc nhau: MeOH- nc; MeOH- m;
ACN- m. Tc duy tr l 1mL/pht. Kim tra s n nh ca my sau mi ln thay
i pha ng, tin hnh bm 20L vo ct. Sc k thu c ng vi mi h dung
mi hnh 3.1; 3.2; 3.3 V bng 3.1
Nhn xt:
Kt qu cho thy khi thay i h dung mi pha ng th thi gian lu,
phn gii v h s i xng ca peak thay i theo
Vi h dung mi pha ng l MeOH-nc, chng ti thy chng trnh sc k ang
kho st khng cho php xc nh aspartame v acesulfame-K v c hai peak b don
v khng tch ri nhau.
Vi h dung mi pha ng l MeOH-m, chng ti thy chng trnh sc k ang
kho st khng cho php xc nh aspartame v acesulfame-K v peak b chn bi
mt peak ca cht khc.
Vi h dung mi ACN- m (t l 10:90), chng ti thy chng trnh sc k
ang kho st cho php tch hn hp aspartame v acesulfame-K hon ton ring l
(R=6,734). Tuy nhin h s i xng Sa1= 3,959; Sa2=3,275 u ln hn hai.
Qua qu trnh kho st la chn dung mi pha ng chng ti quyt nh
chn h dung mi ACN- m. c h s i xng tt chng ti tip tc kho st t
l gia hai thnh phn ACN v m.
3.1.2. Kho st la chn t l dung mi pha ng
Khi xc nh c h dung mi pha ng l ACN- m, chng ti tin hnh kho st
h dung mi ACN- m theo cc t l 5:95; 10:90; 15:85; 20:82, trong cc iu kin:
-S dng ct RP18 (150x4,6mm; 3m)
-Th tch bm mu 20L
-Tc dng: 1mL/pht
-Nhit ct: nhit phng
-Detector: UV bc sng 210nm
Sc k thu c ng vi mi t l ca ACN- m hnh 3.4; 3.5; 3.6; 3.7
v bng 3.2.
Nhn xt:
Kt qu cho thy khi thay i t l dung mi pha ng th thi gian lu,
phn gii v h s i xng ca peak cng thay i theo.
Khi t l ACN thay i t 5% n 20%, tR2 gim xung 2,22 ln. iu ny
ph hp vi l thuyt v y l qu trnh sc k pha o, dung mi cng t phn cc kh
nng ra gii cng mnh. phn cc ca pha ng lm gim thi gian phn tch.

24

Khi t l ACN l 5%, dung mi pha ng phn cc mnh, thi gian lu qu

ln do thi gian phn tch tng.
Khi t l ACN l 20%, dung mi t phn cc, s ra gii xy ra qu nhanh
dn n hin tng cc peak b chp v peak th 2 b don.
Khi t l ACN l 10%, hai peak tch nhau hon ton nhng h s i xng
ln Sa1= 3,959; Sa2= 3,275, nn khng ph hp vi qu trnh phn tch.
Khi t l ACN l 15%, hai peak tch nhau hon ton, cc h s i xng
(Sa1=1,561; Sa2= 1,095) u tha mn cho qu trnh phn tch. Thi gian lu
tR2=7,271l thi gian ngn do t tn km dung mi. Vi kt qu trn, chng ti
chn t l ACN l 15% l thch hp cho qu trnh phn tch.
Bng 3.2. Gi tr trung bnh cc thng s c bn ca qu trnh tch hn hp
aspartame v acesulfame-K cc t l dung mi pha ng khc nhau (n= 5)
T l ACN:m
5:95
10:90
15:85
20:80
Cc thng s
Thi gian lu tR1
5,067
3,608
3,493
5,284
Thi gian lu tR2
13,295
6,865
7,271
5,981
H s i xng Sa1 2.574
3,959
1,561
1,700
H s i xng Sa2 1,948
3,275
1,095
2,174
phn gii R
12,325
6,734
13,452
2,247
3.1.3. Kho st tc dng
Sau khi kho st thnh phn v t l dung mi pha ng chng ti nhn thy
rng vi h dung mi ACN-m theo t l 15:85 l thch hp cho chng trnh tch sc
k, chng ti tip tc kho st s nh hng ca tc dng cc tc 0,8mL/pht;
1mL/pht; v 2mL/pht. Dung mi pha ng l ACN: m l 15:85. Kim tra s n
nh ca my sau mi ln thay i tc dng, tin hnh bm 20L vo ct. Sc k
thu c ng vi mi tc dng hnh3.8; 3.9; 3.10 V bng 3.3
T kt qu thu c chng ti nhn thy tc dng c nh hng ng k
n thi gian lu. Khi tc dng tng, thi gian lu gim (tc dng tng t
0,8mL/pht n 2mL/pht, thi gian lu gim 1,87 ln) do lm gim thi gian phn
tch.
S thay i tc dng c nh hng hn ch n phn gii, khi tc dng tng
t 1mL/pht n 2mL/pht th phn gii gim t gi tr 13,452 n 4,510.
Tc dng tng lm cho tc di chuyn ca cc phn t qua ct sc k
tng, sc k ca cc peak gn v cn i hn (h s i xng gim).
-Vi tc dng l 0,8mL/pht th c hai h s i xng u ln hn 2
(Sa1=3,968; Sa2=3,278), c hai peak u b don.
-Vi tc dng l 1mL/pht, c phn gii ln (R=13,452). Cc yu t:
tR1=3,493; tR2=7,271 l khong thi gian ngn nn t tn km dung mi; h s i

25

xng (Sa1=1,561; Sa2=1,095) u nh hn 2, rt thch hp cho qu trnh sc k m

chng ti ang kho st.
-Vi tc dng l 2mL/pht, s ra gii xy ra qu nhanh dn n hin
tng xut hin peak tp v hin tng chp peak. H s i xng (Sa2=2,04) ln hn
2 nn khng ph hp cho qu trnh sc k v peak ny b don, ngoi ra, h s phn gii
(R=4,510) l b nht trong ba h s phn gii ca cc tc dng ny.
- Vi kt qu kho st chng ti chn tc duy tr hp l l 1mL/pht.
Bng 3.3 Gi tr trung bnh cc thng s c bn ca qu trnh tch hn hp
aspartame v acesulfame-K cc tc dng khc nhau (n= 5)
Tc dng
Cc thng s
Thi gian lu tR1
Thi gian lu tR2
H s i xng Sa1
H s i xng Sa2
phn gii R

0,8 (mL/pht)

1
(mL/pht)
3,493
7,271
1,561
1,095
13,452

3,608
6,876
3,968
3,278
6,735

2
(mL/pht)
1,688
3,675
1,459
2,046
4,510

* T cc kt qu kho st trn chng ti chn iu kin sc k

-Ct RP18 (150x4,6mm; 3m)
-Th tch bm mu 20L
-Tc dng: 1mL/pht
- Dung mi pha ng: ACN: m (15:85)
-Nhit ct: nhit phng
-Detector: UV bc sng 210nm
Vi iu kin sc k trn, cc sc k thu c:
-2 chun n 3.11; 3.12
-1 chun hh 3.13
-1sk ca nc gii kht 3.14
3.2. XC NH N NH CA H THNG SC K
Vi mc ch kim tra h thng thit b v dung mi ha cht phi t tiu
chun trc khi s dng. Tin hnh tim 6 ln mu vo h thng sc k lng cao p
cc iu kin chn. Ghi din tch peak thu c. n nh ca h thng sc k
c biu th bng sai s tng i (RSD) ca kt qu sau 6 ln tim ca mu.
Pha mt hn hp mu chun cha:
-Dung dch chun gc cha aspartame 500ppm v acesulfame-K 500ppm:
Cn chnh xc mi loi 0,05g cho vo bnh nh mc 100ml, cho dung dch NaHCO3
0,1M lc tan hon ton ri cng dng NaHCO3 nh mc ti vch.
-T dung dch gc pha long thnh dung dch 50ppm ri lc qua mng lc
0,45m tim HPLC.
26

Bng 3.4. Kt qu lp li ca h thng sc k

Din tch peak
Aspartame

Acesulfame

1
2
3
4
5
6

Kt qu cho thy sai s tng i RSD% thu c vi hai cht u nh hn 1%, iu

chng t h thng sc k m chng ti tin hnh l ph hp v m bo s n nh
ca phng php xy dng.
3.3 XC NH KHONG TUYN TNH CA PHNG PHP NH LNG
Kt qu kho st khong tuyn tnh gia nng v din tch peak ca
aspartame v acesulfame-K:
-Chng ti tin hnh xc nh khong ph thuc tuyn tnh gia nng
aspartame v acesulfame-K vi din tch trn sc k . Php xc nh ny c tin
hnh trn cc dung dch chun.
-Tin hnh chun b cc dung dch chun nh sau:
+ Dung dch chun gc cha aspartame 500ppm v acesulfame-K 500ppm:
Cn chnh xc mi loi 0,05g cho vo bnh nh mc 100ml, cho dung dch NaHCO3
0,1M lc tan hon ton ri cng dng NaHCO3 nh mc ti vch.
-T dung dch gc pha ra cc dung dch chun lm vic 0,25; 0,5; 0,1; 0,2;
0,5; 10,0; 20,0; 25,0; 50,0; 100,0; 150,0; 200,0 g/mL, lc qua mng lc 0,45m, sau
khi my n nh tin hnh o iu kin chn, tim 20L cc dung dch vo ct.
Din tch cc peak c xc nh t ng cho tng nng (bng chng trnh tch
phn ci t trong phn mm ca my tnh). Kt qu c th hin bng 3.5.
Chn nng aspartame v acesulfame-K theo t l thch hp vi cc sn
phm ph bin trn th trng.
Bng 3.3. Phng trnh tuyn tnh ca aspartame v acesulfame-K
Cht

Acesulfame

Nng dung dch Din tch peak

chun (ppm)
2,5
5
12,5
25
37,5

64044,333
129309,666
326980,000
654045,333
980866,333
27

Phng trnh hi
quy v h s tng
quan
y= 26740x-24686
R2=0,9996

50,0
1306740,333
62,5
1635948,000
100,0
2618562,333
125,0
3275844,333
187,0
4917224,000
250,0
6748100,666
Aspartame
2,5
144902,333
y=55916x+115715
5
293316,666
R2=0,9993
12,5
742418,666
25
1485414,666
37,5
2226078,000
50,0
2961666,000
62,5
3690586,333
100,0
5853287,333
125,0
7270801,000
187,0
10684803,000
250,0
13867141,000
Qua cc phng trnh tuyn tnh ca tng cht chng ti nhn thy c mi tng quan
tuyn tnh rt cht ch gia nng v din tch peak tng ng thu c trn sc k
. Trong khong nng kho st h s tng quan R2=0,9996 i vi acesulfamK, v R2=0,9993 i vi aspartame.
3.4. GII HN PHT HIN V GII HN NH LNG
3.4.1. Gii hn pht hin
L lng cht thp nht ca cht cn phn tch c th pht hin c v mt
nh tnh. Trong phng php HPLC gii hn ny c nh gi bng:
: nhiu ca ng nn, trong HPLC dng detector DAD = 0,04.
b: h s gc ca ng tuyn tnh
Vy, gii hn php hin ca acesulfame-K l
Tng t, gii hn php hin ca aspartame l
3.4.2. Gii hn nh lng
L lng thp nht ca cht cn phn tch c trong mu th c th pht hin
c v mt nh lng vi ng v chnh xc thch hp.
Gii hn nh lng c xc nh theo cng thc:
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