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TRNG I HC CN TH KHOA NNG NGHIP V SINH HC NG DNG

GIO TRNH

THC TP SINH HA
M s mn hc: HS 632

Bin son: Tin s Nguyn Minh Chn Thc s Phan Th Bch Trm Thc s Nguyn Th Thu Thy

NM 2005
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LI NI U
Gio trnh thc tp sinh ha c bin son trn c s k tha v pht huy gio trnh c qu Thy C tin nhim bin son trc y. Gio trnh ny cn b sung v sa i ni dung cho ph hp vi chng trnh ci cch, ph hp vi iu kin hin ti v hng pht trin ca phng th nghim trong tng lai. Mt s phng php c s dng thit b phn tch cng c a vo ngi c tham kho v c th ng dng c trong tng lai khi iu kin phng th nghim c trang b tt hn. Ni dung gio trnh nhm gip cho sinh vin cc chuyn ngnh Trng Trt, Nng Hc, Cng Ngh Thc Phm, Thy Sn, Chn Nui, Mi Trng, Bo V Thc Vt, Hoa Vin Cy King, Khoa Hc t, Cng Ngh Sinh Hc, C Nhn Ha Hc, S Phm Sinh Hc, S Phm Ha Hc v cc ngnh c lin quan hiu c cc kin thc c bn trong th nghim sinh ha v cc phng php th nghim kho st carbohydrate (glucid), lipid, amino acid, enzyme, nucleic acid, vitamin, v cc cht khc. Trn c s ca cc phng php phn tch ny, cc bi thc tp s c la chn ra cho ph hp vi tng chuyn ngnh v iu kin ca tng nm hc. Cc bi thc hnh cn gip lm sng t nhng vn c nu ra trong phn l thuyt. Nhm bin son xin chn thnh bit n C Phm Thu Cc v qu Thy C tin nhim dy cng xy dng gio trnh thc tp trc y v chng ti l nhng ngi tip tc pht huy. Vi nhng iu kin nht nh ca phng th nghim, nhng bi thc hnh chc hn cha p ng ht yu cu nghin cu sinh ha hin i. Chng ti xin chn thnh bit n tt c nhng kin ng gp gio trnh ngy mt hon thin hn.

Thay mt nhm bin son Nguyn Minh Chn

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MC LC
CHNG 1. NHNG KIN THC C BN ..............................................................1 1.1. NI QUI PHNG TH NGHIM .............................................................................1 1.2. K THUT PHNG TH NGHIM ........................................................................1 1.2.1. Cc im cn lu trnh tai nn trong khi lm vic v thc tp trong phng th nghim ..........................................................................................................................1 1.3. K THUT SINH HA............................................................................................3 1.3.1. Cc dng c thng dng trong thc tp sinh ha..................................................3 1.3.2. Cch chun b mt dung dch ha cht....................................................................7 CHNG 2. GLUCID......................................................................................................13 2.1. KHI QUT V GLUCID........................................................................................13 2.2. NH TNH MONOSACCHARIDE V TINH BT ..............................................13 2.2.2. Kho st tinh bt......................................................................................................13 2.2.3. nh tnh monosaccharide (glucose) v tinh bt.....................................................14 2.3. NH LNG NG KH .................................................................................15 2.3.1. nh lng ng kh theo phng php Bertrand ...............................................16 2.3.2. nh lng ng kh theo Hagedorn-Jensen .....................................................18 2.4. PHNG PHP NH LNG NG TNG S ...........................................19 2.5. NH LNG NG SACCHAROSE................................................................20 2.6. NH LNG TINH BT V CELLULOSE.........................................................21 2.6.1 nh lng tinh bt ..................................................................................................21 2.6.2 nh lng cellulose.................................................................................................22 2.7. XC NH HM LNG AMYLOSE ..................................................................23 CHNG 3. LIPID...........................................................................................................25 3.1. KHI QUT V LIPID ............................................................................................25 3.2. KHO ST TNH HA TAN CA LIPID..............................................................25 3.3. CC CH S NH GI LIPID ..............................................................................25 3.3.1. Xc nh ch s x phng ........................................................................................25 3.3.2. Xc nh ch s iod ..................................................................................................26 3.3.3. Xc nh ch s acid ................................................................................................27 3.3.4. Xc nh ch s peroxid...........................................................................................28 3.4. XC NH HM LNG LIPID TH BNG MY SOXHLET.........................29 3.5. XC NH ACID BO BNG SC K KH.........................................................31 3.6. CHIT TCH LECITHIN T LNG TRNG ................................................32 CHNG 4. KHO ST VITAMIN ..............................................................................34
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4.1. KHI QUT ..............................................................................................................34 4.2. NH TNH VITAMIN D .........................................................................................34 4.3. NH TNH VITAMIN B1........................................................................................34 4.3.1. Phn ng to thiocrome...........................................................................................34 4.3.2. Phn ng vi thuc th Diazo .................................................................................35 4.4. NH TNH VITAMIN B2........................................................................................36 4.5. NH LNG VITAMIN C ....................................................................................36 4.5.1. nh lng vitamin C theo phng php Muri.......................................................36 4.5.2. nh lng vitamin C bng enzyme peroxidase .....................................................39 4.6. XC NH VITAMIN BNG PHNG PHP SC K LNG CAO P (HPLC) ..............................................................................................................................39 4.6.1. Phn tch vitamin A v vitamin D ...........................................................................39 4.6.2. Phn tch vitamin E .................................................................................................40 4.6.3. Phn tch vitamin K .................................................................................................40 4.6.4. Phn tch acid nicotinic (vitamin B3)......................................................................41 CHNG 5. KHO ST AMINO ACID V PROTEIN...............................................42 5.1. KHI QUT V AMINO ACID V PROTEIN......................................................42 5.2. PHN TCH HN HP ACID AMIN BNG PHNG PHP SC K TRN GIY .................................................................................................................................42 5.3. CC PHN NG MU C TRNG CA PROTEIN........................................44 5.3.1. Phn ng Biuret .......................................................................................................44 5.3.2. Phn ng Nynhydrin................................................................................................45 5.4. S KT TA PROTEIN........................................................................................... 47 5.4.1. S kt ta thun nghch ..........................................................................................47 5.4.2. S kt ta bt thun nghch .....................................................................................47 5.5. NH LNG PROTEIN BNG PHNG PHP SO MU ...............................48 5.5.1. Khi qut..................................................................................................................48 5.5.2. nh lut Lambert- Beer..........................................................................................49 5.5.3. Phng php nh lng protein theo phn ng biuret...........................................50 5.5.4. nh lng protein theo phng php Lowry.........................................................52 5.6. NH LNG PROTEIN TNG S THEO PHNG PHP KJELDAHL ........53 CHNG 6. ENZYME ....................................................................................................56 6.1. KHI QUT ..............................................................................................................56 6.1. KHI QUT ..............................................................................................................56 6.2. KHO ST HOT NG CA ENZYME AMYLASE T MM LA.............56 6.2.1. H enzyme amylase.................................................................................................56
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6.2.2. S to mu gia iod vi tinh bt v cc chuyn ha ca tinh bt khi thu phn bng amylase .....................................................................................................................57 6.2.3. Ly trch v kho st hot tnh tng i ca amylase mm la ..............................57 6.2.4. Kho st nh hng ca nng c cht ln tc thy gii ca amylase ...........58 6.2.5. Kho st nh hng ca pH ln tc thy gii ca amylase...............................59 6.2.6. Kho st nh hng ca cht hot ha v cht c ch ............................................59 6.3. KHO ST ENZYME UREASE TRONG BT U NNH................................60 6.4. ENZYME HA NU ................................................................................................61 6.4.1. Khi qut v phn ng ha nu ...............................................................................61 6.4.2. Kho st hot tnh tng i ca enzyme ha nu..................................................64 6.5. KHO ST HOT TNH TNG I CA ENZYME -CYANOALANINE SYNTHASE .....................................................................................................................65 6.5.1. Trch enzyme CAS ..................................................................................................65 6.5.2. Kho st hot tnh tng i ca enzyme CAS ......................................................65 CHNG 7. PHNG PHP KHO ST ACID NUCLEIC ......................................66 7.1. KHI QUT ..............................................................................................................66 7.2. PHNG PHP LY TRCH ACID NUCLEIC .......................................................66 7.2.1. Ly trch ADN t t bo vi khun.............................................................................66 7.2.2. Ly trch ARN ...........................................................................................................67 7.3. NH TNH ACID NUCLEIC ..................................................................................68 7.3.1.Tnh tan ca acid nucleic..........................................................................................68 7.3.2. Cc phn ng mu ca acid nucleic ........................................................................68 7.4. NH LNG ACID NUCLEIC..............................................................................69 7.4.1. nh lng ADN ....................................................................................................69 7.4.2. nh lng ARN .....................................................................................................71 CHNG 8. CC PHNG PHP PHN TCH KHC.............................................73 8.1. PHNG PHP XC NH M .....................................................................73 8.2. PHNG PHP XC NH HM LNG TRO.................................................74 8.2.1. Hm lng tro ton phn.........................................................................................74 8.2.2. Xc nh hm lng tro ha tan v khng ha tan trong nc ...............................74

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Gio Trnh Thc Tp Sinh Ha

CHNG 1. NHNG KIN THC C BN


1.1. CC QUI NH CHUNG CA PHNG TH NGHIM 1. Mi nhm thc tp phi chu trch nhim v: trt t, an ton, dng c, ha cht v kt qu th nghim cho bi thc tp ca mnh. 2. Sinh vin phi c mt phng th nghim ng gi qui nh: Sng 7 gi, chiu 13 gi v phi c mt ti phng th nghim sut thi gian thc tp. Sinh vin n tr 10 pht khng c vo phng th nghim. Khi kt thc th nghim sinh vin phi bo co kt qu vi gio vin hng dn trc lc ra v. 3. Sinh vin vng mt phi c giy php v phi xin thc tp b bui khc. 4. Sinh vin phi xem k bi thc tp trc khi vo phng th nghim. 5. Mi nhm thc tp c mt sinh vin i din k nhn mn dng c: kim tra tnh hnh dng c (thiu, hng, b) bo co ngay cho gio vin hng dn. Sinh vin phi ra dng c sch s trc v sau khi thc tp. Kt thc bui thc tp mi nhm phi lau dn, lm sch ch nhm mnh lm th nghim, nu dng c b mt mt, h hng phi bo ngay cho ngi ph trch phng th nghim bit. 6. Mi bui thc tp, nhm trc nht c nhim v: nhc nh cc nhm dn v sinh, kim tra in, nc v ca trc khi ra v. 7. Mi nhm sinh vin lm bi tng trnh kt qu theo yu cu ca tng bi thc tp, np kt qu cho gio vin hng dn vo bui thc tp k tip. Kt thc cc bi thc tp c thi kim tra. 1.2. K THUT PHNG TH NGHIM 1.2.1. Cc im cn lu trnh tai nn trong khi lm vic v thc tp trong phng th nghim 1. Cn thn khi tin hnh th nghim, khng c s dng nhng my mc, dng c khi cha bit r cch s dng. Phi hiu bit r tnh cht ca cc ha cht trnh tai nn ng tic. 2. Tt c chai l ng ha cht u c nhn, khi dng phi c k tn v nng , dng xong phi y ng nt v li ng ch c. Phn ln cc ha cht l c nn phi ht sc cn thn. 3. i vi cc cht kim, acid m c phi lu : - Khng c ht bng ming. - Phi dng ng ong hoc bnh nh git. - Phi acid hoc kim vo nc khi cn pha long chng. - Phi t nghing ming ng nghim hoc cc v pha khng c ngi. - Khi acid b ra ngoi th cho nhiu nc lm long acid. 4. Khi theo di dung dch ang si khng c a mt gn hay khi mt cht lng (cht kim) vo cc phi a ra xa. Khi un mt cht lng trong ng nghim hay cho acid, kim vo phi t ng nghim nghing mt gc 45O. Khi un phi lc u v hng ming ng nghim v pha khng c ngi. 5. Khi lm vic vi cht d chy th tuyt i: Khi s dng cc cht d chy nh ether, xng, benzen, chloroform, natri, kali cn ch : 1
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Gio Trnh Thc Tp Sinh Ha

- Khng dng la ngn v trnh xa la ngn. - Khng cht d chy bn cnh ngun sinh nhit (cht d chy, d bc hi c th lm n hay bt nt, hi bc ra gp ngn la s chy, c khi ngn la xa). - Khi cha chy phi bnh tnh dp tt ngn la bng khn t hay bnh cha chy. 6. Khi lm vic vi dng c thy tinh: - Kim tra k dng c trc khi dng. - Trnh v. - Dng c no dng cho vic . Khi un, ch c un bng dng c thy tinh chu nhit. - Dng c phi c ra sch trc v sau khi s dng. - Khng dng dng c thy tinh, chai l cha cc cht kim mnh hoc acid m c c tc dng b mt n mn thy tinh nh HF. 7. Khi lm vic vi dng c in hoc s dng in tay phi kh, ch lm vic phi kh. Kim tra k ngun in v dy dn in khi s dng. 1.2.2. S cp cu trong phng th nghim S cp cu l bin php tm thi i vi cc trng hp thng tch nh hoc trc khi a bnh nhn n bnh vin nh: 1.2.2.1. Phng a. Phng do nhit (hay vt nng) - Phng nh: Ly vi mng tm dung dch acid picric bo ha p ln mt vt phng. - Phng nng: p nh vi mng tm dung dch acid picric ln vt phng, sau chuyn i bnh vin. b. Phng do ha cht Vic trc tin l ngm vt thng vo chu nc to hoc vt thng di vi nc chy tht nh. Sau mi trung ha ha cht. Ch cc trng hp sau: - Phng do acid: p vi mng tm dung dch bicarbonat natri 8%. - Phng do kim: p vi mng tm dung dch acid picric 3%. 1.2.2.2. Tai nn v mt - Acid hay brom vo mt: Ra mt tc khc nhiu ln bng nc sch, sau tm mt trong dung dch bicarbonat natri 1%. - Cht kim vo mt: X l nh trn ri tm mt bng dung dch acid boric 1%. 1.2.2.3. Ng c Khi b cht c vo ming: - Acid: Xc ming nhiu ln bng dung dch bicarbonat natri 1%. - Kim: Xc ming nhiu ln bng dung dch acid 1%. - Cc ha cht khc: Xc ming nhiu ln bng nc lnh. 2
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Gio Trnh Thc Tp Sinh Ha

1.2.2.4. Nhim hi c a nn nhn ra ni thong kh, ni rng qun o cho d th. H hp nhn to trong lc di chuyn n bnh vin. 1.2.2.5. in git Trc ht ngt mi cu dao in c lin quan n phng th nghim. Ni rng qun o nn nhn sau khi em ra ni thong. H hp nhn to trong khi ch chuyn n bnh vin nu l trng hp nng. 1.2.2.6. Ha hon - Ngn la nh: dp tt bng khn, vi b t hay ct. - La bt u chy qun o: ln vi vng di t dp tt ngn la, trong khi cc bn ly vi t trm ln ch chy v p st cho n khi la tt. Trnh chy hong. - Dng bnh cha chy trc phng th nghim dp la. Lu : Sinh vin phi bo ngay cho nhn vin phng th nghim hoc gio vin hng dn v mi s c trong phng th nghim. 1.3. K THUT SINH HA 1.3.1. Cc dng c thng dng trong thc tp sinh ha 1.3.1.1. Cch ra cc dng c sch ca cc dng c nh hng rt ln n kt qu th nghim, do ra dng c ha hc l mt phn k thut phng th nghim m sinh vin cn phi bit. chn phng php ra dng c trong tng trng hp ring bit thng phi bit tnh cht ca nhng cht lm bn dng c. Sau s dng tnh cht ha tan ca nhng cht bn ny trong nc nng hay trong nc lnh, trong dung dch kim, acid, trong cc mui hay cc dung mi hu c. Thng dng cy c ra hoc dng bn chi ch xt vo cc dng c (dng cy c ra phi ch v ngn cy c c th lm thng y dng c). Cc dng c sau khi ra sch cht bn c ngm vo dung dch sulfo- cromic (hn hp ca K2Cr2O7 10% v H2SO4 m c cng t l th tch) trong mt ngy; sau em ra sch vi nc my v trng mt ln vi nc ct, xong vo t sy kh. Dng c thy tinh c gi l sch khi nc trn thnh khng to thnh nhng git ring m dn mng u. 1.3.1.2. Cc loi dng c v cch s dng 1 a. ng nghim 3 ng nghim thng l hnh tr c th tch khc nhau (Xem hnh 1.1). T khng c un nng ngay ti y ng nghim m ngn la phi c vo thnh Hnh ca ng. Hnh1.1. ng 1 nghim iu kin khi un nng mt dung dch trong ng nghim: - Dung dch khng c nhiu qu 1/3 ng nghim. - ng nghim c gi nghing khong 45O lun lun lc hoc khuy u. 3
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Gio Trnh Thc Tp Sinh Ha

- Ming ng nghim khng c hng vo mt ngi no v n c th gy phng. b. ng ht (pipet) C nhiu loi ng ht thng dng: - Loi c bu an ton: Dng ht nhng dung dch c. - Loi c hai vch: Th tch ghi trn ng l th tch gia hai vch. - Loi bnh thng c phn . i vi cc loi cht lng c, ta dng mt qu bp cao su c bit gn vo u ng ht, qu bp ny c th ht hoc cht lng t do nh mt h thng kha (valve). * Cch s dng: + Trng ng ht bng mt lng nh dung dch s ht. + Ht dung dch ln n bn trn vch ngang. (xem hnh 1.2). + Ly ngn tr bt u trn ng ht li (ngn tr phi sch, kh), lau sch bn ngoi u ng ht bng giy thm. + Nng ng ln cao cho vch chia trn ng ht ngang tm mt, u ng da vo thnh bnh ri cho dung dch chy t t theo thnh bnh n khi ly th tch cn dng cho th nghim th ngng (lc ny cn quan st mc nc cong tip xc vi vch trn ng ht) 2 + Gi ng ht thng ng ri chuyn qua bnh hng, Hnh Hnh 1.2. Pipet t u ng ht chm vo thnh bnh ri bung ngn tr dung dch chy t do (bnh hng phi hi nghing). + Khi dung dch ngng khng chy na, ta xoay u ng ht 2-3 vng trc khi ly ng ht ra khi bnh (khng thi vo ng ht ui git tha cn li trong ng). + Khi c th tch cn ch c theo mt cu lm ca cht lng khng mu hoc trong sut nh nc, c theo mt cu li i vi cht lng c mu sm nh dung dch cha iod. c. Micropipet - Chnh th tch trong khong s dng ca pipet bng cch vn nt pha trn u pipet cng hoc ngc chiu kim ng h cho n khi cc ch s hin r ng th tch cn dng. - Gn u tip ly ha cht vo u pipet sao cho kht vi u pipet. - Gi pipet thng ng ri dng ngn tay ci nhn nt n mc va cng tay u tin. Sau cho u tip ngp di b mt dung dch khong 2-3 mm v nh nhng bung nt ht dung dch. Cn thn nhc pipet ra khi dung dch, chm nh u tip vo thnh dng c ng gt b dung dch tha. - Bm dung dch vo dng c ng bng cch nhn nt ti mc cui cng sao cho khng cn dung dch bm trn thnh tip. * Lu : Cn trng tip mi vi ln bng dung dch sp ht trc khi ly ha cht, c bit khi dung dch cn ly c nht v t trng khc vi nc.

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Gio Trnh Thc Tp Sinh Ha

d. ng chun (Buret) c gn trn gi v c mt kha iu chnh lng dung dch chy ra trn ng c phn . (Hnh 1.3). * Cch s dng: + Kim tra xem kha c bi vaselin trnh chy nc, hoc xem c b qu xt, kh vn khng. + Trng mt ln vi nc ct v mt ln vi dung dch nh dng chun . + y dung dch vo ng ln n mc trn s 0. + Dng tay tri m kha cho dung dch chy t t cho n khi mc dung dch tip xc vi vch 0 (nu mt Hnh Hnh 1.3. 3 Buret git dung dch cn dnh li u ng chun th phi ly ra bng cch chm vo thnh bnh cha). e. ng ong (Cylinder) C dung tch thay i t 5 mL n 2 L, c th c mt y v c phn (hnh 1.4), ty s phn ny ch gn ng nhng th tch ton phn vn ng nht. V th khng nn dng ng ong chia nhng lng qu nh (Hnh 1.4). Hnh 1.5 Hnh 1.4. ng ong f. Bnh tam gic (Erlenmeyer) c s dng rng ri cc th nghim phn tch (chun ). Bnh tam gic c nt mi c gi l Bnh xc nh ch s iod. g. Bnh chit Dng tch ring nhng dung dch lng khng ha tan vi nhau (v d nc v du). Khi lc bnh chit, ngn tay phi gi nt u trn v kha u di bnh (Hnh 1.5). 1.6 Hnh Hnh 1.5. Bnhchi t h. Bnh ht m (Desiccator): L dng c thy tinh c thnh dy v c np, dng lm kh mu t t v bo qun nhng cht d ht hi m t khng kh. (Hnh 1.6) Phn di ca bnh c t nhng cht ht m. Mun m np bnh phi y np v mt pha, trnh nhc np ln cao. 1.7 m Hnh 1.6. Hnh Bnh ht i. Bnh ht chn khng: c s dng khi bm chn khng lc. Bnh c ng nhnh phn trn, ng nhnh ny c ni vi bm chn khng (Hnh 1.7). j. ng sinh hn: L dng c lm lnh v ngng hi (Hnh 1.8). Ty theo iu kin m cht lng c to thnh trong ng sinh hn khi lm lnh hi hoc i sang bnh thu hoc l tr li bnh un nng. S khc nhau v chc nng ca ng sinh hn quyt nh hnh dng v tn gi Hnh 1.7. Bnh ht Hnh1.8 chn khng 5
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Gio Trnh Thc Tp Sinh Ha

ca chng. Khi ni ng sinh hn cn tun theo quy tc: Nc i vo t u thp pha di v i ra t u pha trn.

Hnh 19 Hnh 1.8. ng sinh hn

Hnh 1.9. Bnh nh mc

k. Bnh nh mc: L dng c ti cn thit i vi cc th nghim phn tch. Chng l nhng bnh cu y bng c nt thy tinh mi nhm. Bnh nh mc dng pha long mt dung dch bt k n mt th tch xc nh hoc ha tan mt cht no trong mt dung mi vi th tch xc nh (hnh 1.9). Khi cho dung dch vo bnh nh mc c hp, phi dng phu, xong y np cht v dc ngc bnh nhiu ln trn u. Khi cho nc gn ti vch, cn thn dng ng ht a thm tng git n vch mc. 1.3.2. Cch chun b mt dung dch ha cht 1.3.2.1. Nng ca dung dch Khi ha tan mui vo nc ta c nc mui. + Mui: cht ha tan hay dung cht. + Nc: dung mi. + Nc mui: dung dch. Nng ca dung dch c th c din t nhiu cch khc nhau: 1. Nng phn trm khi lng theo khi lng (% w/w): S gam cht ha tan c trong 100 gam dung dch. V d: Dung dch NH4Cl 5% theo khi lng l trong 100g dung dch c 5g NH4Cl tinh khit. 2. Nng phn trm khi lng theo th tch (% w/v): S gam cht ha tan c trong 100 mL dung dch. V d: Dung dch CuSO4 10% theo th tch l trong 100 mL dung dch c 10g CuSO4 tinh khit. 3. Nng phn trm th tch theo th tch (% v/v): L s mL dung cht c trong 100mL dung dch. V d: Dung dch glycerin 10% theo th tch l trong 100 mL dung dch c 10 mL glycerin nguyn cht. 4. Nng phn t - nng mol (mol/L hay M): L s phn t gam (s mol) trong 1 L dung dch. 5. Nng gam/L (g/l): L s gam cht tan c trong 1 L dung dch. 6
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Gio Trnh Thc Tp Sinh Ha

6. Dung dch nguyn chun (N): Mt dung dch c gi l nguyn chun khi 1 L dung dch y cha mt khi lng cht ha tan c gi l ng lng gam. 1.3.2.2. Cch pha cc nng a. Nng phn trm theo khi lng Th d: Pha 80 gam dung dch NH4Cl 40%. Dung dch NH4Cl 40% ngha l cn c 40g NH4Cl cho 100g dung dch. Vy mun c 80g dung dch th cn mt lng NH4Cl l:
40 x 80 = 32 g 100

Lng nc phi thm cho 80g: 80g - 32g = 48g hay 48 mL Vy ta cn 32g NH4Cl ri ong 48 mL H2O bng ng ong. nc vo ha tan, c dung dch NH4Cl 40%. * Trng hp cc ha cht c ngm nc (CuSO4.5H2O, Na2S2O3.5H2O, NaCO3.10H2O...) khi cn pha cc cht ta phi tnh ti lng nc kt tinh trong chng. V d: Mun pha 500g dung dch CuSO4 20% t tinh th ngm nc (CuSO4. 5H2O): Phn t khi ca CuSO4 khan nc l 160g. Phn t khi ca CuSO4. 5H2O l 250g. Mun pha 500g dung dch CuSO4 20% th ta cn mt lng CuSO4 khan nc l.
20 x 500 = 100 g 100

Mun c 160g CuSO4 khan nc ta cn 250g CuSO4. 5H2O. Vy mun c 100g CuSO4 khan nc th phi cn mt lng CuSO4. 5H2O l:
250 x 100 = 156 gam 160

Lng nc cn thm: 500g - 156g = 344g Vy ta cn 156 g CuSO4.5H2O v thm 344 mL nc ha tan, ta c 500g dung dch CuSO4 20%. b. Nng phn trm khi lng theo th tch Ta ha tan lng cht cn trong nc v thm nc ti th th tch ng. V d: Cn chun b 1 L dung dch NaCl 30% th ta cn mt lng NaCl l:
30 x 1000 = 300 gam 100

ha tan trong nc v thm nc cho th tch 1 L. * Trng hp cc ha cht c ngm nc: Khi cn ta phi tnh thm c lng nc trong phn t nh trng hp trn. * Trng hp cht ha tan l dung dch: Ta cng lm tng t nh trn, ngha l cn cht tan v dung mi em trn ln vi nhau cho u l c. Nhng vic cn 7
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Gio Trnh Thc Tp Sinh Ha

cht lng khng c thun li cho lm nn cn phi a cht lng v n v th tch cho tin theo cng thc: V: Th tch cht lng P P: Trng lng cht lng V = d d: T trng cht lng Mt khc, i vi cht lng thng dng c gii hn ha tan ti a tnh theo %. V d: H2SO4 ha tan ti a 96%, HCl l 37%, H3PO4 l 65%... Cho nn khi cn cc cht lng ny phi tnh c s gam c thc trong dung dch pha cho chnh xc. Nu ta xem HCl l 100% th khi pha dung dch 10% ta ch cn 10g HCl v thm vo 90 mL, trn u l c. Nhng thc ra HCl ch c 37% nn trng lng cn phi l:
x= 100 x 10 = 27,02 g 37

Hay
27 ,02 = 22,7 ml 1,19

V ta thm lng nc: 100g - 27,02g = 72,98g (hay 72,98 mL) c. Nng phn t gam Mol hoc phn t gam l khi lng ca cc cht tnh ra gam bng khi lng phn t ca n. Dung dch phn t gam l dung dch cha 1 phn t gam cht ha tan trong 1 L. chun b dung dch 1M ca cht no , ngi ta tnh khi lng phn t (c gi l tng khi lng cc nguyn t c trong cht) hoc tm tr s ca n trong bng tra cu. Ly lng cn chnh xc em ha tan trong dung mi cho thnh 1 L dung dch (dng bnh nh mc). Khi phi un nng dung dch, hay khi phn ng ta nhit hay thu nhit, phi cho v nhit bnh thng (20OC) ri mi thm ti vch. Cng tng t nh th, ngi ta pha cc dung dch 2-3 M... hay 0,1; 0,01M... bng cch tnh lng tng ng ha tan. V d: Cn 0,5 L dung dch K2C2O7 0,1M K2C2O7 = 294,2 g chun b 1 L dung dch K2C2O7 0,1M cn ly 0,1 phn t gam ngha l 29,42g K2C2O7. chun b 0,5 L ta ch cn 29,42g x 0,5 = 14,71g pha trong bnh nh mc 500 mL. + Trong trng hp cht rn c ngm nc, phn t gam cht phi tnh c khi lng cc phn t nc trong cht . + i vi cht tan l dung dch tinh khit (100%) ta cng tin hnh cn v pha nh i vi cht rn khng ngm nc. 8
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Gio Trnh Thc Tp Sinh Ha

+ i vi cht tan l cht lng c phn trm thp (cha c 100%) ta phi ch ti nng phn trm ti a ca chng tnh ton cho ng. Th d: Pha HCl 1M t HCl 37% (MHCl = 36,5) ta phi cn:
365 x100 = 98,65 gamHCl 37 98,65 = 82,9 83mlHCl 1,19

Hay

Vy ta phi ly 83 mL HCl 37% pha thnh 1 L l c dung dch HCl c nng 1M. d. Nng ng lng gam (N) Dung dch nguyn chun 1N cha 1 ng lng gam cht tan trong 1 L. ng lng gam s c nh ngha theo mi trng hp ring t phng trnh phn ng dng trong lc nh phn. + Trong s nh phn acid hay base: ng lng gam ca acid l khi lng cht c th cho ra trong phn ng 1 ion gam H+. ng lng gam ca mt base l khi lng cht c th cho ra trong phn ng 1 ion gam OHV d: a. H+ + Cl- + Na+ + OH- Na+ + Cl- + H2O 1 phn t gam HCl cho ra 1 ion gam H+ Vy ng lng gam HCl = 1 phn t gam HCl b. 2H+ + SO42- + 2Na+ + 2OH- 2Na+ + SO42- + 2H2O 1 phn t gam H2SO4 cho ra 2 ion gam H+ Vy ng lng gam H2SO4 = 1/2 phn t gam H2SO4 c. Mt phn t gam NaOH cho ra 1 ion gam OHVy 1 ng lng gam NaOH = 1 phn t gam NaOH. - Mt dung dch nguyn chun HCl cha 36,5g HCl trong 1 L - Mt dung dch nguyn chun H2SO4 cha 98g/2 = 49 gam H2SO4 trong 1 lt. Con s 1 hay 2 c dng chia phn t gam trong nhng th d trn c gi l h s nguyn chun . Trong trng hp tng qut s c gi l v M/ c gi l ng lng gam phn ng. + Trong trng hp phn ng oxy ha kh: Mun tm ng lng ca 1 cht trong h thng oxy ha kh, ngi ta em chia phn t gam cho s in t trao i trong phn ng m cht tham gia. Th d: 2Na2S2O3 + I2 Na2S4O6 + 2NaI I2 + 2e 2I 2S 2 O32 2e 2S 4 O62

S in t trao i phn ng ny l 1. Do N=M/1 9


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Gio Trnh Thc Tp Sinh Ha

Cch pha: vic pha dung dch nng ng lng gam (N) cng tng t nh pha nng phn ng gam (M) nhng thay i phn t gam (M) bng ng lng gam (N). 1.3.2.3. Cch xc nh li nng dung dch a. Dung dch chun Trong qu trnh pha ha cht, c nhiu yu t lm sai nng nh: + Vic cn o khng chnh xc. + Cc cht cha tinh khit hay ht nc. + lu b thng hoa hay oxy ha. Do ngi ta phi kim tra nng thc ca cc dung pha da vo cc cht n nh hay nng chnh xc c gi coi l dung dch chun. Cc cht dng trong dung dch chun phi bn vng nng cht phn ng ny khng thay i nhanh chng vi thi gian. Thng thng cc thuc th chun l mt lng cn chnh xc thuc th hoc dung dch thuc th ng kn trn ng thy tinh, trn c ghi 0,1 hay 0,01 ng lng gam ca cht ong trong ng. Khi chuyn ht lng cht c trong ng thy tinh vo bnh nh mc dung tch 1 L ri pha bng nc ct ti vch mc, ta c dung dch chnh xc 0,1 hay 0,01 M. Trong vi trng hp, dung dch chun c th c lm trc tip bng cch cn cht rn v pha long dung dch ti 1 th tch ng. iu ny c th p dng vi 1 vi hp cht bn vng c thnh phn khng thay i nh NaCl, AgNO3, acid oxalic... Cc cht ny c gi l cc cht gc. Tuy nhin, i vi nhng cht khc nh NaOH, HCl v Na2S2O3 cch ny khng thc dng v cc cht ny khng c hiu lc v tinh khit v dng c nh c cn trc tip. V d: NaOH thng b nhim vi 1 lng Na2CO3 thay i v rt d b chy nc, HCl bc hi nhit phng th nghim v Na2S2O3.5H2O r thnh bt n s b mt nc ca tinh th khi ngoi khng kh v v vy c thnh phn khng c nh. trong nhng trng hp ny, iu cn thit l pha 1 dung dch gn ng nng mong mun v sau nh nng chnh xc ca n vi 1 dung dch chun. b. Cch xc nh nng Tr li v d phn ng mt acid v mt base, phn ng c th tm tt: H+ + OH- H2O 1 ha tr gam acid phn ng vi 1 ha tr gam base 1 L dung dch nguyn chun acid s phn ng trn 1 L dung dch nguyn chun base, hai dung dch nguyn chun s trung ho vi nhau cng 1 th tch. Mt cch tng qut, 2 dung dch phn ng vi cng 1 th tch s c cng 1 chun . Khi 1 th tch V1 ca dung dch c chun C1 (C1, N) tc dng ln 1 th tch V2 ca dung dch c chun C2 (C2, N) chng ta c th vit rng s ha tr gam tc dng ln nhau u bng nhau trong 2 trng hp : C1 x V1 = C2 x V2 Ta s dng h thc ny hiu chnh li nng 1 s dung dch chnh xc. Th d: Ta c mt dung dch chun H2SO4 0,1N chnh xc. Mt dung dch NaOH ta pha ly c nng nh pha l 0,1N. em chun ta thy 10 mL H2SO4 0,1N tc dng vi 11 mL NaOH ta pha. Vy nng ca dung dch NaOH ta pha l:
C1 = V2 xC 2 10 x0,1 = = 0,091N V1 11

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Gio Trnh Thc Tp Sinh Ha

H s (10/11 = 0,91) c gi l h s hiu chnh. Ta cng c th p dng cng thc trn thay i nng ca dung dch t m c sang pha long hn. V d: a. Pha 250mL dung dch HCl N/5 t dung dch HCl 1N C1 x V1 = C2 x V2 => N x V1 = N/5 x 250 => V1 = 50 mL Vy ly 50 mL dung dch HCl v thm nc cho 250 mL ta c dung dch HCl N/5. b. Pha 50 mL ethanol 70% t ethanol 95% 50 x 70 = V2 x 95 => V2 = 36,9 mL V vy thm 13,1 mL nc vo 36,9 mL etanol 96% ta s c 50 mL ethanol 70%. c. c th tch bao nhiu khi lm long 25 mL HCl 0,08N sang HCl 0,05N C1 x V1 = C2 x V2 0,08 x 25 = 0,05 x V2 V2 = 40 mL 1.3.2.4. Cch pha v nh chun mt s dung dch thng dng Vi iu kin phng thc tp sinh ha hin nay, chng ta c th pha 2 dung dch chun gc sau y: a. Dung dch acid oxalic (COOH)2 N(=2). Cn tht chnh xc M/2 lng (COOH)2 ha tan vi nc ct thnh 1 L, ta c dung dch chun acid oxalic 1N bo qun trong chai thy tinh nt mi v trong ch khng nh sng. b. Dung dch KIO3 N/10 (=6). Cn tht chnh xc M/6x10 lng KIO3 ha tan vi nc ct thnh 1 lt, ta c dung dch chun KIO3 N/10. Hai dung dch trn dng chun li cc dung dch chng ta pha sau: + Dung dch NaOH 1N. Cn 40g NaOH ha tan thnh 1 lt dung dch vi nc ct. nh li chun NaOH ny vi dung dch (COOH)2 N pha trn: ly 10mL dung dch NaOH mi pha chun vi dung dch (COOH)2 N trn vi s hin din ca thuc th mu phenolphtalein. Nu dung dch NaOH ny c nng >1N hay <1N ta phi tnh thm lng nc hay NaOH thm vo c mt nng ng 1N dng nh chun cc acid sau. + Acid nguyn chun N. Mun pha cc dung dch nguyn chun t acid m c. Nu cha bit ha tan ti a v t trng cc acid m c ta xc nh nguyn chun ca n bng cch ly 1 mL dung dch chun vi NaOH N bit. V d: acid l 30N ta phi pha long 30 ln t hn c acid 1N. Sau hiu chnh li cho ng nng 1N vi NaOH 1N hay Na2CO3 1N. 11
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Dung dch HCl N(HCl m c 12M =12N, =1). Ha 1000/12 = 85 mL HCl m c trong nc ct cho va 1 lt. Xc nh li chun ng vi NaOH N trn (dng thuc th mu phenolphtalein). T dung dch chun HCl 1N, ta p dng h thc C1 x V1 = C2 x V2 pha long thnh cc dung dch HCl N/2, N/5, N/10, N/20. Dung dch H2SO4 1N (H2SO4 m c 18M = 36N, =2). Ha 1000/36 = 29 mL H2SO4 m c vi nc ct cho 1 L. ngui, hiu chnh li cho ng nng 1N vi dung dch NaOH 1N trn (dng thuc th mu phenolphtalein). Dung dch permanganat kali (KMnO4) N/10 (=5). Cn M/5 x 1/10g KMnO4 ha tan trong nc ct cho 1 lt. Hiu chnh nng theo cch sau: Ht 10 mL dung dch (COOH)2 N/10 + 10 mL dung dch H2SO4 N/5. un nng khong 40OC 50OC nh KMnO4 va pha n mu hng nht. Dung dch Na2S2O3 N/10 (=1). Cn M/1 x 1/10g Na2S2O3 ha tan thnh 1lt vi nc ct. nh li nng vi dung dch KIO3 N/10 pha trn theo cch sau: ht 10 mL dung dch KIO3 N/10 thm vo mt t tinh th KI+2 mL HCl m c. nh chun vi dung dch Na2S2O3 va pha n khi ht mu nu ca iod sinh ra (th vi h tinh bt). Dung dch iod I2 N/10 (=1). Cn M/1 x 1/10g + 25,5g KI pha thnh 1 lt vi nc ct, nh phn li nng vi dung dch Na2S2O3 N/10 va hiu chnh.

Gio Trnh Thc Tp Sinh Ha

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Gio Trnh Thc Tp Sinh Ha

CHNG 2. KHO ST CARBOHYDRATE (GLUCID)


2.1 KHI QUT V CARBOHYDRATE Cc hp cht carbohydrate gm 3 loi: - Monosaccharide: l n v cu to ca carbohydrate, khng b thy phn. V d: Glucose v fructose. - Oligosaccharide: c t 2 n 10 monose gm: di, tri hay tetrasaccharide V d: Saccharose (ng ma), lactose (ng sa), maltose (ng mch nha). - Polysaccharide: gm 2 loi: + Polysaccharide thun: gm nhng monomer cng loi. V d: tinh bt, cellulose v glycogen. + Polysaccharide tp: Gm nhng monomer khc loi hay dn sut ca monomer hoc c thm cht bo. V d: Heparin, acid hyallycoric. Tnh cht - Monosaccharide: C tnh kh, d b mt nc bi acid v c mnh (H2SO4, HNO3 m c) cho ra dn sut furfural. Cht ny d kt hp vi cc loi phenol (resorcinol, - naphthol, orcinol...) cho phn ng mu. - Oligosaccharide: B thy phn bng acid hoc enzyme thch hp cho ra cc thnh phn cu to tng ng. Trng hp disaccharide, ty cch kt hp cc monomer s c tnh kh hay khng. Kh nng kh ca oligosaccharide yu hn monosaccharide. Trong c th sinh vt carbohydrate ng vai tr ln, n l thnh phn cu to nn cc c quan v dch gian bo. N cung cp phn ln nng lng cho t bo sng. 2.2 NH TNH MONOSACCHARIDE V TINH BT 2.2.1 Kho st tinh bt Tinh bt l cht d tr ca thc vt, l ngun thc n v ngun cung cp nng lng chnh cho ngi v ng vt. Tinh bt khng tan trong nc lnh. Khi un nng vi nc th trng ln v to thnh h (h tinh bt), cho mu xanh (i khi mu tm ) vi iod, khng c tnh kh. Tinh bt b thu phn bi acid m c (HCl, H2SO4).
CH2OH O CH2OH O CH2OH O CH2OH O

OH

u kh

Cu to mt on amylose ca tinh bt 13
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Gio Trnh Thc Tp Sinh Ha

Chun b tinh bt Khoai lang ra sch, mi trn bn mi t trn ry sn trong chu thy tinh. Mi nh tay cho nhuyn, xong vt tht k b khoai lang vi 200 mL nc ct, chuyn nc bt t chu thy tinh vo cc 250 mL, yn cho bt lng. Khi bt lng k, gn b phn nc trn ri tip tc cho nc ct vo khuy u, lng tip tc ri li gn b phn nc trn. Lp li vi ln cho n khi tinh bt trng v sch. Khuy tinh bt vi vi mL nc ct ri cho vo 100 mL nc ct ang si th ta c dung dch h tinh bt. Ly dung dch h tinh bt va nhn c lm cc th nghim sau. Tin hnh a. Cho mu vi iod Tin hnh: Cho vo ng nghim 5 mL dung dch h tinh bt, cho tip 2 git iod. Nhn xt kt qu. Sau xem ng nghim un cch thy khong 5 pht - Nhn xt. ngui - Quan st, nhn xt v gii thch. b. Phn ng thy phn Tin hnh: Dng ng nghim ln cho vo 15 mL dung dch h tinh bt. Cho tip vo 5 mL HCl m c, khuy u. un cch thy, c sau 1 pht ly 1 git dung dch ang thy phn nh ln 1 git iod sn trn a knh ng h. Th nh vy cho n khi khng cho mu vi iod. Nhn xt mu thay i khi th dung dch thy phn vi iod. Kt lun. Lu : Gi dung dch thy phn lm cc phn ng sau. 2.2.2 nh tnh monosaccharide (glucose) v tinh bt a. Phn ng Molish Nguyn tc: Cc loi carbohydrate u cho phc mu tm vi dung dch naphthol trong acid H2SO4 m c.
CH2OH O

H2SO4 -3H2O

O HOCH2 CHO

-D-glucose

5-hydroxymethyl furfural
OH OH - H2O OH

furfural

O HOCH2 CHO +2 Napthol

5-hydroxymethyl furfural

CH C CH O C CH CH2OH

Phc mu tm 14
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Gio Trnh Thc Tp Sinh Ha

Tin hnh Chun b cc ng nghim theo bng sau: ng nghim Ho cht Dung dch Thuc th Molish H2SO4 m c 1 Glucose 1% 2 mL 3 git 2 H tinh bt 1% 2 mL 3 git 3 H tinh bt thy phn 2 mL 3 git

K thut: cho t t thnh dng chy lin tc theo thnh ng nghim, khng lc, n khi xut hin mu tm th dng li, c th tch dung dch acid dng.

Nhn xt tc phn ng ca mi ng nghim. Gii thch v nu ngha ca phn ng Molish. b. Phn ng vi thuc th Fehling Nguyn tc: Trong mi trng kim un nng, monosaccharide dng enoldiol khng bn, d dng kh cc kim loi nng nh Cu2+, Ag+, Hg2+. Monosaccharide s kh ng II trong dung dch Fehling thnh ng I c kt ta gch. Cc disaccharide c nhm -OH glycoside t do u c tnh kh:
CHO (HCOH)n CH2OH O CH COONa COOH H (HCOH)n O CH COOK CH2OH O O CH CH COONa COOK

+ Cu

+ Cu2O + o gach

Tin hnh ng nghim Ho cht Glucose 1% Tinh bt thu phn (phi trung ho) Tinh bt khoai lang Fehling (A+B t l 1:1) 1 2 mL 0 0 3 mL 2 0 2 mL 0 3 mL 3 0 0 2mL 3 mL

un cch thy 3 ng nghim trong khong t 3- 5 pht. Quan st hin tng xy ra 3 ng nghim, gii thch v vit phng trnh phn ng. 2.3 NH LNG NG KH Cc phng php ha hc xc nh ng kh u da trn kh nng kh cc hp cht khc nhau ca chng. Trong thc t c rt nhiu phng php xc nh, ty thuc vo hm lng nhiu hay t ngi ta c th s dng mt trong cc phng php thng dng sau y:

15

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Gio Trnh Thc Tp Sinh Ha

2.3.1 nh lng ng kh theo phng php Bertrand Nguyn tc Trong mi trng kim cc ng kh (glucose, fructose, maltose...) d dng kh ng II thnh ng I theo phn ng Fehling. Kt ta ng I oxyt c mu gch, c kh nng kh vi mui Fe3+ thnh mui Fe2+ trong mi trng acid: Cu2O + Fe2(SO4)3 + H2SO4 2CuSO4 + 2FeSO4 + H2O Fe2+ sinh ra c tnh kh li tc dng vi KMnO4 l cht oxy ha nn dng KMnO4 chun Fe2+ trong mi trng acid: 10FeSO4 + 2KMnO4 + 8H2SO4 K2SO4 + 2MnSO4 + 5Fe2(SO4)3 + 8 H2O Da vo lng KMnO4 s dng ta c th tnh c lng Cu2O v t tnh c lng ng kh trong dung dch bng cch tra bng t l gia dung dch KMnO4 v ng kh ca Bertrand. Ha cht - Thuc th Fehling = Fehling A+Fehling B t l 1:1 - Fehling A: 40 g CuSO4.5H2O trong 1 lt nc ct - Fehling B: 20g natri kali tartrate (C4H4O6NaK.4H2O) v 150g NaOH trong 1 lt nc ct. - Dung dch Fe2(SO4)3 trong H2SO4: 50g Fe2(SO4)3 v 20g H2SO4 thm nc n 1 lt. - Dung dch KMnO4 1/30N. - Dung dch acetate ch 30% - Dung dch Na2SO4 bo ho Tin hnh Chun b nguyn liu Cn v cho vo ci s 5-10 gram nguyn liu ti, nghin nh, thm khong 50ml nc ct vo bnh tam gic 250 ml. em un cch thy 80oC trong 15 pht, thnh thong lc u trong khi un chit tch ng. Sau ly ra, ngui n nhit phng. Tin hnh kh tp cht bng cch: + Thm 7 mL dung dch acetate ch 30% vo dch chit ng cho vo bnh nh mc 100mL, lc u v lng 5 pht. Nu thy xut hin mt lp cht lng trong sut bn trn lp cn th vic kh tp cht xong. +Cho tip 20 mL dung dch Na2SO4 bo ho vo loi ch tha. Lc u v ta lng xung. +Thm nc ct va n vch 100 mL, lc u v lc qua giy lc kh. Dch lc dng tin hnh nh lng. Tin hnh nh lng Cho 10 mL dung dch ng cn kho st v 10mL thuc th Fehling vo bnh tam gic 250 mL. y bnh bng nt thy tinh v un trn bp c li aming. un si khong 3 pht, kt ta xut hn trong bnh. Ly bnh ra ngui. Ra kt ta vi ln vi nc m un si cho n khi dch ra khng cn phn ng kim trn giy qu. Qu trnh ra c tin hnh trn phu lc chn khng vi giy lc xp G4 v ch l phn ln kt ta trn phu lc v trong bnh lun c ph mt lp nc Cu2O khng b oxy ha bi oxy khng kh. 16
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Gio Trnh Thc Tp Sinh Ha

Ha tan kt ta Cu2O vo bnh tam gic bng cch cho t t khong 5mL dung dch Fe2(SO4)3 trong H2SO4 v dng a thy tinh khuy tht cn thn ha tan hon tan kt ta trn phu. Trng cn thn bnh v phu lc 3-4 ln bng nc nng cho vo bnh tam gic 100mL. Chun dung dch thu c bng KMnO4 1/30N cho n khi xut hin mu hng nht bn trong 20-30 giy. T lng KMnO4 1/30N dng chun , tra bng 2.1 suy ra lng ng c trong mu phn tch. Song song lm th nghim i chng bng cch thay dung dch ng bng nc ct. Tnh kt qu Hm lng ng kh tnh theo cng thc:
X= a x Vx 100 V1 x m x 1000

trong : X: hm lng ng kh tnh theo % a: s mg glucose tm c khi tra bng ng vi s mL KMnO4 0,1N dng chun mu phn tch tr i s mL KMnO4 1/30N dng chun mu khng. V: th tch pha long mu (100mL) V1: th tch mu ly em xc nh ng kh m: lng mu em phn tch 1000: h s i gam thnh mg Bng 2.1. T l gia KMnO4 1/30N v lng ng kh KMnO41/30N(mL) 0.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Glucose(mg) 0,0 0,8 1,8 2,8 3,9 5,0 6,1 7,2 8,3 9,3 10,4 11,5 12,6 13,7 14,8 15,9 17,0 18,1 17 KMnO41/30N(mL) 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Glucose(mg) 19,2 20,3 21,5 22,7 23,8 25,3 26,2 27,4 28,6 29,9 31,2 32,5 33,8 35,1 36,3 37,6 38,9 40,2
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Gio Trnh Thc Tp Sinh Ha

2.3.2. nh lng ng kh theo Hagedorn-Jensen (b tc bi Isskuts v Both) Nguyn tc: Da vo tnh kh ca monosaccharide, nh lng glucose theo phn ng oxy ha - kh. Glucose l cht kh; fericyanua kali l cht oxy ha. Phn ng xy ra trong mi trng kim un nng.
CHO 2K3Fe(CN)6 + (HC OH)n + 3Na2CO3 + H2O CH2OH COONa (HC OH)n CH2OH + 3NaHCO3 + 2K3NaFe(CN)6

(1)

Glucose s kh mt phn fericyannua kali. y l phn ng thun nghch, phn ng xy ra mt chiu, ferocyanua kali c lm kt ta dng ha hp km khng tan: 2K3NaFe(CN)6 + 2ZnSO4 2K2ZnFe(CN)6 + Na2SO4 + K2SO4 (2) Phn fericyanua kali tha c nh phn gin tip qua phng php nh phn iod bng thiosulfit natri (Na2S2O3) K3Fe(CN)6 + KI K4Fe(CN)6 + 1/2I2 (3) 2Na2S2O3 + I2 Na2S4O6 + 2NaI (4) Tin hnh + Dng dung dch glucose c nng bit trc 2 mg/mL pha thnh 5 dung dch c nng khc nhau t 0,4 mg/mL n 2 mg/mL theo bng sau: ng nghim
Ho cht

1 2 5 0 0 15 10

5 0,4 1 0 4 15 10

6 X 0 5 0 15 10

7 0 0 0 5 15 10

Nng dung dch glucose (mg/mL) Th tch dung dch glucose 2 mg/mL (mL) Th tch dung dch glucose nh phn Xmg/mL Th tch nc ct (mL) Pha long bng nc ct (mL) Fericianua kali K3Fe(CN)6 0,02N (mL)

1,6 1,2 0,8 4 3 2 0 0 0 1 2 3 15 10 15 10 15 10

Lu : Nn kt hp cho nc ct mt ln trnh sai s. V d: ng 1 cho thm 15mL nc ct, ng 2 cho 16 mL, ng 3 cho 17 mL... + un cch thy cc ng nghim trn 30 pht + un xong ngui (c th lm lnh) ri thm vo mi ng 10 mL ZnSO4.KI + Quan st mu mi ng nghim (khng c khuy) + Ghi nhn xt, gii thch v kt lun. Chun b nh phn + Ra sch buret bng nc ct, trng qua bng Na2S2O3 0,02 N. Cho tip Na2S2O3 0,02 N n vch 0 ri bt u nh phn. 18
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Gio Trnh Thc Tp Sinh Ha

+ nh phn mu i chng trc (ng 7), chuyn dung dch trong ng qua cc 250 mL. Cho vo ng nghim 10 mL CH3COOH 9% trng ng nghim ri chung vo cc 250 mL. Quan st hin tng, gii thch, vit phng trnh phn ng xy ra. + Cch nh phn: cho Na2S2O3 0,02N buret xung cc t t v lc nh cc cho n khi dung dch chuyn thnh mu vng rm rt lt. Thm vo 1-2 git h tinh bt, dung dch trong cc chuyn sang mu xanh, cho tip Na2S2O3 0,02N t t tng git mt cho n khi mu xanh t ngt bin mt v tr thnh mu trng sa c l c. Ghi nhn th tch Na2S2O3 0,02N trn buret. + Tip tc nh phn t ng nghim 1 n ng nghim 6, tng t nh trn. Lu : - Khi nh phn cho dung dch Na2S2O3 0,02N chy t t v lc u. - Theo di dung dch trong cc nh phn n mu vng rm rt lt, nu khng s b sai s nhiu. + Kt qu nh phn lp bng: ng nghim Nng dd glucose (mg/mL) Th tch dd Na2S2O3 0,02N V=V0-Vi (mL) 1 2 V1= 2 1,6 V2= 3 1,2 V3= 4 0,8 V4= 5 0,4 V5= 6 X V6= 7 0 V0=

V1= V2= V3= V4= V5= V6= 0

V ng biu din V = f(C) mg/mL ng bin din c dng y = ax. + Da vo th, suy ra nng glucose nh phn (tc nng dung dch trong ng 6) 2.4. PHNG PHP NH LNG NG TNG S S nh phn ny cn bn da trn phn ng mu c trng cho ng v nhiu cht hu c vi s hin din ca acid sulfuric (H2SO4). S chnh xc ca kt qu ny ty thuc vo: + sch cc dng c. + tinh khit ca thuc th, nht l H2SO4 + Nhit phi c nh trong thi gian un Ha cht - Alcol 90o, 80o - Phenol 5% - H2SO4 m c - Saccharose 0,1% Tin hnh Cch trch ng Ly 1-2 gam nguyn liu ti nghin nh cha khong 5-50 mg ng (cn bng cn phn tch) cho vo cc thy tinh 50ml v thm 10 ml alcol 90O vo (nu dng nguyn liu kh th cn ly t mu hn). Sau cc un trn ni cch thy cho si 3 ln. Khuy u bng a thy tinh, sau khi ngui lc khng tro (khi lc ch nn gn ly phn alcol) ng cn trn lc. 19
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Gio Trnh Thc Tp Sinh Ha

Sau li cho 10ml alcol 80O vo cc ng b, khuy u un 2 ln ti si trong ni cch thy. ngui li lc tip. Chit rt nh vy khong 3 ln, xong a b ln lc v ra sch 2-3 ln bng alcol 80O nng (ra tng t mt), alcol qua lc c bay hi trong phng hoc ni cch thy. un nh sau khi cho bay hi alcol, mu c th lu trong bnh ht m. Cn kh trong cc c pha long thnh 50ml vi nc ct (dng bnh nh mc). Nu c cn th lng xung. Khi em lm hin mu, dung dch ny c th pha long thm 5-10 ln ty theo nng ng nhiu hay t. Sau c th to phn ng mu ca dung dch ng theo phng php sau: Dng Phenol Ht 1ml dung dch ng c khong 10-70g ng cho vo ng nghim ri cho thm 1ml dung dch phenol 5%. Sau , cho vo ng nghim 5ml H2SO4 m c. Tuyt i khng vy acid vo thnh ng nghim. ngui 10 pht ri lc v gi trn ni cch thy 20 pht 30OC xut hin mu. Mu bn vng trong vi gi, em o hp th bc sng 490nm. Xy dng th chun Pha dung dch saccharose gc nng 100 g/mL. T dung dch gc, pha dy dung dch c nng t 0- 70g/mL. Thm cc ho cht vo 8 ng nghim theo bng sau:
ng nghim Nng dung dch saccharose (g/mL) Th tch dung dch saccharose gc (L) Th tch nc ct (L) Th tch dung dch phenol 5% (mL) H2SO4 m c (mL)
1 2 3 4 5 6 7 8

0 0
1000

10 100 900 1 5

20 200 800 1 5

30 300 700 1 5

40 400 600 1 5

50 500 500 1 5

60 600 400 1 5

70 700 300 1 5

1 5

ngui cc ng nghim 10 pht, lc u. em un cch thy 20 pht 30OC xut hin mu. Mu bn vng trong vi gi, em o hp th bc sng 490nm. V th tng quan gia hp th v nng saccharose ri tnh hm lng ng tng s c trong mu theo cng thc sau: Hm lng ng tng s % = X x V x 100/m X: nng saccharose c suy ra t th chun (g/mL) V: th tch pha long sau khi ly trch mu. m: khi lng mu em phn tch. chnh xc ca phng php ny l 2% 2.5. NH LNG NG SACCHAROSE ng saccharose cng vi mt s ng kh c nhiu trong cc loi tri cy, rau, c... Saccharose khng c tnh kh nn khng th xc nh trc tip bng cc phng php Bertrand. c th xc nh c saccharose bng phng php ny phi thy phn n thnh cc ng kh glucose v fructose. 20 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

Nguyn tc: Khi thy phn dung dch saccharose bng acid ta thu c hn hp ng glucose v fructose. nh lng ng kh to thnh cho php tnh c lng sucrose c trong mu phn tch.
C12H22O11 saccharose

H2O

H+

C6H12O6 + glucose

C6H12O6 fructose

Ha cht - Dung dch HCl 5% - Dung dch Na2CO3 bo ha - Methyl 0,02% (0,02 g methyl trong 60mL cn v 40mL nc ct) - Cc ha cht xc nh ng kh theo phng php Bertrand Tin hnh Ly 10 mL dung dch mu phn tch ( loi b protein v tp cht nh phn chun b ng kh mc 2.3.1 cho vo bnh tam gic 250mL. Cho thm 5mL HCl 5%. un cch thy qua ng sinh hn trong 30-40 pht, lm ngui nhanh. Trung ha hn hp bng dung dch Na2CO3 bo ha n pH 6,5-7,0 c ch th methyl (3 git). Thm tng git kim vo cho n khi dung dch chuyn t sang vng. em hn hp nh lng ng theo phng php Bertrand. Tnh kt qu Hm lng saccharose trong mu th nghim c tnh theo cng thc sau: X = (a-b) x 0,95 trong : X- hm lng saccharose tnh theo % a- hm lng ng kh theo glucose ca dch ng sau khi thy phn bng acid (%) b- hm lng ng kh ca dch ng trc khi thy phn (%) 0,95- h s chuyn i t glucose sang saccharose 2.6. NH LNG TINH BT V CELLULOSE 2.6.1. nh lng tinh bt Nguyn tc Da trn s thy phn hon ton tinh bt bng acid thnh glucose. Sau dng mt trong cc phng php nh lung glucose to thnh ri nhn vi h s 0,9 xc nh c hm lng tinh bt. n C6H12O6 (C6H10O5)n + n H2O 162,1 180,12 F=
162,1 = 0,9 180,12

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Gio Trnh Thc Tp Sinh Ha

Tin hnh Cn chnh xc 1-2 gam bt (cha khong 200-250 mg tinh bt) nghin nh v sy kh trc khi cn, cho vo bnh tam gic dung tch 100mL, thm vo 50 mL nc ct, lc u yn 30-45 pht. Lc qua giy lc, ra cn bng nc ct 2-3 ln. Chc thng giy lc v chuyn bt vo bnh tam gic c cha 25mL HCl 5%. em un cch thy qua ng sinh hn trong 3-5 gi. Sau khi tinh bt thy phn hon ton, lm lnh dung dch. Trung ha hn hp bng dung dch NaOH 0,5 % n pH 5,6-6,0 (c th th bng giy qu). Chuyn hn hp vo bnh nh mc 100mL. Kh tp bng Pb(CH3COO)2 30% v loi lng mui ch tha bng 20mL dung dch Na2SO4 bo ha. Thm nc ct n vch, lc u v lc. nh lng ng glucose trong dung dch bng phng php Bertrand, t tnh c hm lng tinh bt. Tnh kt qu Hm lng tinh bt trong mu phn tch c tnh theo cng thc sau:
X= a x Vx 100 x 0,9 V1 x m

trong : X- hm lng tinh bt tnh bng % a- s mg glucose tm c khi tra bng ng vi s mL KMnO4 dng chun mu phn tch tr i s mL KMnO4 dng chun mu khng. V1: th tch mu ly em xc nh ng kh V : th tch pha long mu (100mL) m: lng mu em phn tch 0,9: h s i glucose thnh tinh bt Ch : phng php da trn c s thy phn tinh bt bng acid s khng cho kt qu chnh xc nu trong mu c cha hemicellulose, pectin v mt s polysaccharide c trng lng phn t thp. Do khi dng phng php thy phn bng acid, trc tin cn phi tch tinh bt ra khi nguyn liu thc vt, sau mi tin hnh thy phn v xc nh hm lng glucose. 2.6.2. nh lng cellulose Nguyn tc: nh lng cellulose da trn tnh cht bn ca cellulose i vi tc dng ca acid mnh v kim mnh, khng b phn hy di tc dng ca acid yu. Cc cht khc thng i km theo cellulose nh hemicellulose, lignin, tinh bt ...t bn hn i vi tc dng ca acid v kim nn b oxy ha v phn gii sau tan vo dung dch sau khi x l nguyn liu. Ha cht - Dung dch NaOH 0,5% - Dung dch NaCl 0,5%, - Dung dch HCl 10% - Dung dch nc javen (natri hypochlorite) 22
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Gio Trnh Thc Tp Sinh Ha

Tin hnh Cn chnh xc 1-2 gam mu cho vo bnh tam gic 200mL dung dch NaOH 0,5%, lp vo ng sinh hn un hon lu trong 30 pht k t lc si. Ch ng bt tro ln. Lc qua giy lc bit trng lng hoc ly tm. Ra cn cn li vi dung dch NaOH 0,5% nng. Tip tc cho cn tc dng vi 10mL HCl 10%. Thm vo 10mL dung dch natri hypochlorite tng git mt, va cho va khuy u. yn trong 5 pht ri lc qua giy lc bit trng lng hoc ly tm. Cho cn li tip tc tc dng tr li vi NaOH 0,5% nhit 40oC. yn vi pht v ly tm. Lm nh th 1-2 ln na c cellulose tht trng. Sau cng ra sch tht k bng nc si. Sy kh v cn. Tnh kt qu Hm lng cellulose c tnh theo cng thc sau: X% =
ax100 m

Trong :

a: trng lng cellulose (g) m: trng lng mu th nghim (g) 2.7. XC NH HM LNG AMYLOSE Nguyn tc: Amylose v amylopectin l 2 polysaccharide chim 96,1-97% trong thnh phn ca tinh bt. T l hai polysaccharide s quyt nh n kh nng do ca tinh bt. Hin nay, phng php ph bin nh lng amylose v amylopectin da trn s to phc mu xanh ca amylose vi iod trong mi trng acid. Sau , em o hp th bc sng 620nm. T hm lng amylose s suy ra c hm lng amylopectin. Ha cht - Amylose tinh khit - Dung dch trichloro acetic acid (TCA) 0,6% - Dung dch NaOH 1N - Methanol - Dung dch Iod 0,01N Tin hnh *Xy dng ng chun vi cc nng amylose khc nhau + Pha dung dch amylose gc nng 2mg/mL trong dung dch NaOH 1N. T dung dch gc, pha dy dung dch c nng t 0- 2mg/mL. Thm cc ho cht vo 7 ng nghim theo bng sau:
ng nghim Nng dung dch amylose (mg/mL) Th tch dung dch amylose gc (L) Th tch nc ct (L) Th tch dung dch TCA 0,6% (mL) Th tch dung dch Iod 0,01N (L) 1 0 0 100 5 50 2 0.4 20 80 5 50 3 0.8 40 60 5 50 4 1 50 50 5 50 5 1.4 70 30 5 50 6 1.8 90 10 5 50 7 2 100 0 5 50

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Gio Trnh Thc Tp Sinh Ha

+ Lc u cc ng nghim, yn 20 pht ri o bc sng 620nm. V th tng quan gia hp th v nng amylose. * Chun b mu phn tch Xc nh m ca mu go bng my o m. Cn chnh xc 15mg mu go. Thm 5mL dung mi methanol ly trch bo. un cch thy 600C trong 30 pht. Ly tm 6000 vng trong 15 pht, ly phn cn lng. Lp li bc ly trch bo mt ln na. Phn cn lng thm 2ml NaOH 1N v 4mL nc. un cch thy 950C trong 30 pht. * Tin hnh o mu Dng micropipet ht 100 l dung dch mu va chun b trn cho vo ng nghim, thm vo 5 ml dung dch TCA 0,6% v 50 l dung dch Iod 0,01N. Lc u cc ng nghim v yn 20 pht. o bc sng 620nm Tnh kt qu Hm lng amylose % = X x 6 x 100/m x: nng amylose c suy ra t th chun (mg/mL) m: khi lng thc ca mu go m = 15 x (100- H )/100 H: m ca mu go

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Gio Trnh Thc Tp Sinh Ha

CHNG 3. KHO ST LIPID


3.1. KHI QUT V LIPID Lipid thuc nhm hp cht hu c khng ng nht, khng tan trong nc, nhng d tan trong dung mi hu c khng phn cc nh: cloroform, ether, benzen ... trong alcol cht bo tan c mc . Trong phn t lipid c t nht 1 chc ester ca acid bo cao phn t vi alcol. Khi thy phn lipid trong mi trng kim mnh nh NaOH (KOH) trong alcol un nng cho x phng v glycerin (gi l s x phng ha). Trong c th sinh vt, lipid c phn gii nh enzyme thy phn lipase to thnh cc acid bo tng ng v alcol. 3.2. KHO ST TNH HA TAN CA LIPID Lipid khng tan trong nc, nhng tan trong cc dung mi hu c khc th khc nhau. Tin hnh Ly 5 ng nghim, cho vo mi ng du n v cc dung mi theo bng sau: ng nghim Dung dch Du n Ether Cloroform Acetone Alcol Nc ct 1 5 git 1 mL 0 0 0 0 2 5 git 0 1 mL 0 0 0 3 5 git 0 0 1 mL 0 0 4 5 git 0 0 0 1 mL 0 5 5 git 0 0 0 0 1 mL

Lc k. Quan st ha tan ca du v nhn xt, gii thch, kt lun. Sau em ng nghim th 4 un cch thy 5 pht. Ghi nhn kt qu, gii thch v kt lun. 3.3. CC CH S NH GI LIPID 3.3.1. Xc nh ch s x phng nh ngha: Ch s x phng l s miligam KOH cn thit trung ha tt c nhng acid bo t do v kt hp c trong 1 gam cht bo. Nguyn tc: Cho cht bo cn phn tch kt hp vi mt lng KOH tha cht bo chuyn ht thnh x phng. Phn KOH tha c nh phn bng dung dch acid chun vi phenolphtalein lm ch th mu. Ho cht - KOH 0,5N trong alcol - HCl 0,5 N trong alcol - Thuc th phenolphtalein 25
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Gio Trnh Thc Tp Sinh Ha

Tin hnh Cn chnh xc khong 0,3 gam du vo bnh tam gic 250 mL, cho vo 6mL KOH 0,5N trong alcol (ng thi tin hnh song song vi bnh th khng: 0,3 mL nc thay v du). Lc u. em 2 bnh un cch thy qua ng sinh hn, cho si nh trong 45 pht. Nh vy cht bo trong bnh thy phn hon ton (phn ng x phng ha kt thc). cho bnh ngui, thm vo 2 mL nc ct, 2-3 git phenolphtalein v chun bng HCl 0,5N trong alcol cho n khi mu hng mt. Lu : Cn chun mu th khng trc. Tnh kt qu Chng ta bit rng 1mL KOH 0,5N tng ng vi 28,05mg KOH Ch s x phng c tnh theo cng thc sau:
Cxp = 28,05 x (a-b) m

Trong :

- Cxp: Ch s x phng - a: S mL HCl 0,5N dng chun bnh th khng - b: S mL HCl 0,5N dng chun bnh th tht (bnh c mu). - m: S gam cht bo ly lm th nghim

3.3.2. Xc nh ch s iod nh ngha: Ch s iod l s gam iod kt hp vi 100 gam cht bo Nguyn tc Trong nhng iu kin th nghim xc nh nhng ni i -CH=CH- cho vi halogen phn ng cng ch khng cho phn ng th. Cho mt lng tha ICl (iodine monochloride) tc dng vi cht bo cn phn tch trong bng ti phn ng cng xy ra hon ton. Lng iod phn ng c xc nh da vo phn ng chun lng iod gii phng ra (sau khi thm KI) bng dung dch Na2S2O3 chun vi tinh bt lm ch th. Nh vy ch s iod xc nh tng qut cc acid bo khng no trong cht bo. Cc phng trnh phn ng c trnh by nh sau:
C H C H

ICl

H C Cl

H C I

ICl + KI KCl + I2 Na2S2O3 + I2 2 NaI + Na2S4O6 Ho cht - Dung dch ICl 0,2M - Dung dch KI 0,1% - Dung dch Na2S2O3 0,1N - Cloroform - Dung dch h tinh bt 1% 26
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Gio Trnh Thc Tp Sinh Ha

Tin hnh Cn chnh xc 0,2 gam du cho vo bnh tam gic 250 mL (c nt nhm), thm vo 10 mL cloroform, tip tc cho thm 25 mL dung dch ICl 0,2M. Lc k bnh, yn 1 gi trong bng ti. Tin hnh chun b mu i chng tng t nh mu tht. Trng np v c bnh tam gic vi khong 50 mL nc ct, thm 10 mL dung dch KI 0,1% (w/v) v nh lng iod gii phng ra bng dung dch Na2S2O3 0,1N n khi c mu vng sm ri thm 1 mL h tinh bt 1%. Nu c mu xanh xut hin th tip tc chun cho n khi dung dch mt mu. Tnh kt qu Ta bit 1mL Na2S2O3 0,1N tng ng 0,01269 g I2
Ci = 0,01269 x 100 x (a - b) m

Trong :

- Ci: Ch s iod - a: S mL Na2S2O3 0,1 N dng chun bnh th khng - b: S mL Na2S2O3 0,1 N dng chun bnh th nghim - m: Lng du dng th nghim (tnh bng gam) - 100: tnh trong 100 gam cht bo

3.3.3. Xc nh ch s acid nh ngha: Ch s acid l s miligam KOH cn thit trung ha ht nhng acid bo t do c trong 1 gam cht bo. Nguyn tc: Ngi ta dng KOH 0,01N trung ha cc acid bo t do c trong cht bo dng phn tch vi phenolphtalein lm ch th mu. Ha cht - KOH 0,01N trong alcol - Alcol tuyt i - Ether ethylic Tin hnh Dng 1 bnh tam gic 100 mL cho vo 5 mL alcol tuyt i v 5 mL ether (theo t l 1:1) cho thm vo bnh ny 2-3 git phenolphtalein v dng KOH 0,01N trong alcol trung ha hn hp n khi xut hin mu hng lt. Sau thm vo hn hp va trung ha 0,5 gam du. em hn hp ny trung ha bng KOH 0,01N trong alcol cho n khi c mu hng bn vng sau 30 giy. c th tch KOH dng trn buret. Tnh kt qu Ta bit 1 mL KOH 0,01N tng ng vi 0,56 mg KOH Ch s acid c tnh theo cng thc sau:

Ca =
Trong :

a x 0,56 m

- Ca: Ch s acid - a : S mL KOH 0,01N dng trung ha hn hp c du - m: Khi lng du ly lm th nghim (tnh bng gam) 27
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Gio Trnh Thc Tp Sinh Ha

3.3.4. Xc nh ch s peroxid nh ngha: Ch s peroxid l s gam iod c gii phng ra bi peroxid c trong 100 gam mu. Nguyn tc: Trong khng kh, cc acid bo c trong cht bo, c bit l cc acdi bo khng no d dng b oxy ha mt phn to thnh peroxid, gy ra hin tng i ha cht bo. Xc nh ch s peroxid da trn phn ng sau:
R1 H C O H C O R2 + KI + 2 CH3COOH R1 H C O H C R2

+ I2 + 2 CH3COOK + H2O

Lng iod gii phng ra c chun bng dung dch Na2S2O3 vi tinh bt lm ch th mu. Na2S2O3 + I2 2 NaI + Na2S4O6 Ho cht - Acid acetic m c - Cloroform - Dung dch KI bo ha - Dung dch Na2S2O3 0,1 N - Dung dch h tinh bt 1% Tin hnh Cn chnh xc khong 2 gam du cho vo bnh tam gic 250 mL, thm vo 10 mL dung dch hn hp acid acetic : cloroform (t l 2: 1) v 1 mL dung dch KI bo ha mi pha. y np, lc k bnh v yn trong bng ti 10 pht. Tin hnh chun b mu i chng tng t nh mu tht, dng 2 mL nc ct thay v du. Cho thm 25 mL nc ct v tin hnh nh lng iod gii phng ra bng dung dch Na2S2O3 0,1N n khi c mu vng sm ri thm 1 mL h tinh bt 1%. Nu c mu xanh xut hin th tip tc chun cho n khi dung dch mt mu. Tnh kt qu Ta bit 1mL Na2S2O3 0,1N tng ng 12,69 mg I2 Ch s peroxid c tnh theo cng thc sau:
Cp = 0,01269 x ( a - b) x 100 m

Trong :

- Cp: Ch s peroxid - a: S mL Na2S2O3 0,1 N dng chun bnh th khng - b: S mL Na2S2O3 0,1 N dng chun bnh th nghim - m: Lng du dng th nghim (tnh bng gam) - 100: tnh trong 100 gam cht bo

3.4. XC NH HM LNG LIPID TH BNG MY SOXHLET Nguyn tc

Da trn kh nng ha tan ca lipid trong dung mi hu c khng phn cc, dng dung mi hu c trch lipid ra khi nguyn liu c nghin nh. Trong qu trnh trch, cc hp cht tan c trong cht bo nh cc sc t, cc vitamin tan trong cht bo... cng b tch ra khi nguyn liu. Do c ln cc tp cht khc, nn thnh phn trch ly c gi l lipid tng s hay lipid th. 28 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

C 2 phng php xc nh: - Phng php trc tip: Trch lipid ra khi nguyn liu v cn lng lipid c trch ly. - Phng php gin tip: Xc nh chnh lch khi lng nguyn liu kh trc v sau khi chit xut lipid ra khi nguyn liu, t suy ra khi lng lipid trong mu phn tch.
Dng c, ha cht

- Bp cch thy - T sy - Cn phn tch - Ether ethylic hoc ether du ha. - B Soxhlet [gm: bnh cu (a), tr chit (b) v ng sinh hn (c)]. (Xem hnh 3.1)

sinh hn nng g sinh ha n (c)(c)

Tru chi Tr chi t t (b) (b)

Dung mi Dung mi
Bnh c (a) Bnh cu u (a)

Hnh 3.1. H thng Soxhlet Tin hnh - Sy kh nguyn liu c nghin nhuyn n khi lng khng i. Cn chnh xc khong 5 gam nguyn liu (s dng cn phn tch), cho vo ti giy lc c sy kh v bit khi lng (Ti giy phi c ng knh nh hn ng knh tr chit v chiu di ngn hn chiu cao ca ng chy trn). - t ti giy c cha mu vo tr chit. - Lp tr chit vo bnh cu ( c sy kh v xc nh khi lng) v lp ng sinh hn. - Cho dung mi vo tr chit sao cho mt lng dung mi chy xung khong bnh cu v cn mt lng trn phu chit cn ngp mu. - M nc vo ng sinh hn. - t h thng Soxhlet ln bp cch thy v iu chnh nhit sao cho chu k hon lu ca dung mi t t 5 n 8 ln trong mt gi. Chit trong 8 12 gi cho n khi trch ly hon ton cht bo. Th thi im kt thc qu trnh trch bng cch ly vi git ether t u cui tr chit cho ln a knh ng h sch. Sau khi dung mi 29
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Gio Trnh Thc Tp Sinh Ha

bay hi ht, trn mt knh ng h khng li vt du loang th xem nh lipid c chit hon ton. - Sau khi chit xong, ly bnh cu v ti ng mu ra, lp ng sinh hn vo bnh cu mi v ct thu hi ether. Tnh ton kt qu Sy bnh cu c cha lipid n khi lng khng i, cn xc nh khi lng. Nu chit bng ether ethylic th sy kh nhit 60- 70oC trong 30 pht, nu chit bng ether du ha th sy nhit 80- 90oC trong 45- 50 pht. Hm lng lipid c trong 100gam nguyn liu c tnh theo cng thc sau:
X= (a - b) x 100 m

Trong :

- X: hm lng lipid tnh theo % - a: khi lng bnh c cha cht bo (g) - b: khi lng bnh cu ban u (g) - c: khi lng mu khan nc ban u (g)

3.5. CHIT TCH LECITHIN T LNG TRNG Lecithin l lipid c cha gc phosphate (phospholipid), trong thnh phn c nhm phn cc alcol amincholine. Cng thc cu to ca lecithin nh sau:
O CH2 O C O CH O C O CH2 O P O
-

(CH2)14

CH3

(CH2)7

CH

CH

(CH2)7 CH3

CH3

CH2 CH2

CH3 CH3

Tin hnh Dng 2 mL lng trng nh nhuyn cho vo 1 ng nghim 25 mL, thm vo ng nghim 10 mL alcol tuyt i, khuy u. t ng nghim vo ni un cch thy 75 - 80OC, tip tc khuy u trong 10 pht (l thi gian rt tch lecithin) (Nu lng alcol b bc hi th ta cho tip vo ng th tch ban u). Lc hn hp trn qua ng nghim kh, dng dung dch chit tin hnh th nghim sau: ng nghim Dung dch Dch chit Aceton Nc ct

1 1 mL 2 mL 0 mL 30

2 1 mL 0 mL 2 mL
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Nhn xt hin tng, gii thch v kt lun.

Gio Trnh Thc Tp Sinh Ha

CHNG 4. KHO ST VITAMIN


4.1. KHI QUT Vitamin l nhng phn t hu c cn cho c th sinh vt vi mt lng rt nh. Tn gi vitamin xut hin ln u tin vo nm 1911 khi thiamine (vitamin B1) c nhn dng. Vitamin c chia thnh 2 nhm chnh: - Vitamin tan trong nc: Vitamin nhm B, vitamin C, vitamin H - Vitamin tan trong cht bo: Vitamin nhm A, D, E, K, Q... 4.2. NH TNH VITAMIN D Cc vitamin D l dn sut ca cc sterol. Khi c chiu tia t ngoi cc sterol s c hot tnh ca vitamin D. Nguyn tc: Khi un nng hn hp vitamin D vi hn hp anilin v acid HCl m c thu c cht lng c mu . Tin hnh: Cho vo ng nghim dung dch trch t 2 vin du c, thm vo 2 mL hn hp anilin : HCl m c (theo t l th tch 15:1). un si mu na pht trn n cn. Quan st s chuyn i mu, gii thch, kt lun. 4.3. NH TNH VITAMIN B1 Chun b dung dch vitamin B1: Gi 1 vin vitamin B1, pha vi 5 mL nc ct, lc qua giy lc kh. Ly dung dch lc tin hnh 2 th nghim sau. 4.3.1. Phn ng to thiocrome Nguyn tc: Khi oxy ha cn thn trong mi trng kim, vitamin B1 s bin i thnh thiochrome, trong qu trnh bin i c dng keto ca thiamine, coi nh l sn phm trung gian.
CH2 N
+

CH3 K3Fe(CN)6 S CH2 CH2 OH NaOH

N H3C N

NH2

Thiamine
CH3 N H3C N CH2 O NH2 N -H2O S CH2 CH2 OH H3C N N CH2 N CH2 CH2 OH CH3

Thiochrome

Thiocrome c tch bng ru isoamyl alcohol (isoamylic, isobutylic) to vng hunh quang mu xanh tm pht quang khi chiu tia t ngoi. Cng ca hunh quang t l vi hm lng ca thiocrome. Do phn ng ny cn c s dng nh lng vitamin B1 theo phng php hunh quang. Ha cht - Dung dch NaOH 15% 31
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Gio Trnh Thc Tp Sinh Ha

- Dung dch K3Fe (CN)6 1% - Isoamylic Tin hnh Chun b cc ng nghim theo bng sau: ng nghim Ha cht Dung dch vitamin B1 Nc ct Dung dch NaOH 15% K3Fe (CN)6 isoamyl alcohol 1 2 mL 0 1 mL 0,5 mL 2 mL 2 0 2 mL 1 mL 0,5 mL 2 mL

yn khong 5 pht ri quan st. Sau tip tc quan st di nh sng mt tri. Ghi nhn hin tng v kt lun. 4.3.2. Phn ng vi thuc th diazo Nguyn tc: Trong mi trng kim thiamine phn ng vi thuc th diazo (hn hp acid sulfanilic v natri nitric) s cho mu vng cam hoc . Mu l mt hp cht phc tp hnh thnh gia thiamine v thuc th diazo. Ho cht - Dung dch acid sulfanilic 1% - Dung dch natri nitric 5% - Dung dch natri carbonate 10% Tin hnh Cho cc ho cht ln lt vo ng nghim theo trnh t sau: 0,5 mL dung dch vitamin B1 + 0,5 mL dung dch acid sulfanilic 1% + 0,5 mL dung dch natri nitric 5% + 10 git dung dch natri carbonate 10%. Lc u ng nghim, quan st mu, gii thch v kt lun. 4.4. NH TNH VITAMIN B2 Nguyn tc: Vitamin B2 l dn xut ribitol ca isoalloxazine, n c kh nng phn ng oxy ha kh thun nghch. Vitamin B2 dng oxy ha (riboflavin) c mu vng, dng kh (dihydroriboflavin) th khng mu. Ngi ta s dng phn ng ny pht hin vitamin B2. Phng trnh phn ng c trnh by nh sau:
CH2 CH2 H3C H3C N (CHOH)3 N NH N O O +2H -2H CH2OH H3C H3C N (CHOH)3 CH2OH H N NH N H O O

Riboflavin (mau vang)

Dihydroriboflavin (khng mau)

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Khi cho HCl m c (hoc acid acetic m c) v km vo dung dch vitamin B2 th hydro bay ln v dung dch mu vng s mt mu. Tin hnh Gi mt vin B2 cho vo 5 mL nc ct v lc. Dng 2 mL dung dch lc cho vo ng nghim, cho thm 1 mL HCl m c v mt t bt km cho n khi dung dch mt mu - Vit cc phng trnh phn ng xy ra. - Gii thch v kt lun. 4.5. NH LNG VITAMIN C 4.5.1. nh lng vitamin C theo phng php Muri Vitamin C (acid ascorbic) C6H8O6 c 2 dng ng phn quang hc D v L, dng D khng c hot tnh sinh hc. Bi ny kho st v L- acid ascorbic, acid ny c th thy dng kh v oxy ha. O C O C

HO HO H HO

C C C C

- 2H +2H

O O H

C C C C

HO

CH2OH
L acid ascorbic (dng kh)

CH2OH
L acid dehydroascorbic (dng oxy ha)

Nguyn tc: nh lng vitamin C da trn tnh kh ca n i vi thuc th 2,6 dichlorophenol indophenol (DIP). Dng oxy ha ca thuc th DIP c mu xanh b kh bi acid ascorbic c trong dch chit ca nguyn liu thc vt thnh dung dch khng mu. im cn bng tt c acid ascorbic th thuc th mu d tha khng b kh c mu hng. OH O C O O C Cl Cl Cl Cl O C HO C O O +HCl + O C + HO C NH N H C H C
HO C H O-(Na+) HO C H OH

CH2OH

CH2OH

L acid ascorbic (dng kh)

2,6 - dichlorophenol indophenol sodium (mu xanh) 33

2,6 - dichlorophenol indophenol (khng mu)


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Gio Trnh Thc Tp Sinh Ha

Trong mi trng acid th thuc th 2,6 dichlorophenol indophenol c mu hng.


O Cl Cl Cl O Cl

HCl

NaCl

O-(Na+)

OH

Ho cht - Dung dch DIP 0,001N - Dung dch acid oxalic 1% - Dung dch HCl 1% Tin hnh Cn khong 5g tri cy (tri cc, bi...) c cha acid ascorbic c thi nh, chuyn sang ci s cng vi 20 mL HCl 1%, cht ly dch ngm gi li trong cc, em phn tht nghin mn, xong chuyn sang bnh nh mc 100 mL cng vi dung dch HCl 1% va chit ra. Ra ci v trng dng c t nht 3 ln, mi ln vi mt t acid oxalic 1% v cng dn vo bnh nh mc. Dng acid oxalic a th tch ln vch 100 mL. Lc k, chuyn qua cc kh 100 mL, yn 15 pht ri lc qua giy lc kh. - Tin hnh nh phn mu i chng: Ly 8 mL acid oxalic 1% 2 mL HCl 1% cho vo bnh tam gic dung tch 100 mL, dng microburet vi DIP 0,001N chun n lc xut hin mu hng bn sau ba mi giy. Lu : Cn tin hnh nh phn mu i chng v mu tht vi 3 ln lp li ly kt qu trung bnh. - Chun mu tht: Dng pipet ly 10 mL dch lc cha vitamin C cho vo bnh tam gic dung tch 100 mL, tin hnh chun nh mu i chng. Tnh kt qu S mg vitamin C trong 100g mu vt c tnh nh sau:
X =
(a b) x0,088 xVx100 vxm

a: S mL trung bnh khi nh chun mu vt b: S mL trung bnh khi nh chun mu i chng. 0,088: s mg acid ascorbic tng ng vi 1 mL dung dch chun DIP 0,001N c nh chun bng acid ascorbic chun. V: Th tch dch chit ban u ( y l 100 mL) v: Th tch dung dch chit ly nh chun (10 mL) m: Trng lng mu vt cn lc u (tnh bng gam). Lu : - Khi chun b mu vt, cn mu vt, ct mu vt (bng dao inox) v nghin mu vt cn tin hnh nhanh chng trong mt ci s HCl 1% (hoc vi HPO3 34 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

CH3COOH). V vitamin C rt d b oxy ha trong khng kh, nht l khi c s hin din ca cc ion kim loi (Fe, Cu). - Sn phm c cha Fe2+, Sn2+, Cu2+ s cho ta nhng kt qu v s lng acid ascorbic cao hn s lng tht nu ta dng phng php ny. Ta c th trc nghim n gin xem nhng ion c tnh kh ny c hin din vi nhng s lng lm sai kt qu thc nghim : + Thm 2 git dung dch xanh methylene 0,05% (ha tan trong nc) vo 10 mL ca hn hp mi iu ch gm mt th tch dung dch mu vt (cha acid ascorbic) v mt th tch dung dch HCl 1% (hoc H2PO3 - CH3COOH). Lc u, nu dung dch xanh methylene b mt mu trong 5-10 giy cho thy c s hin din ca cc cht c tnh kh trn. + Sn2+ khng cho phn ng vi trc nghim ny v c th c th nghim nh sau: Cho 10 mL dung dch HCl: nc theo t l 1:3 vo 10 mL dung dch mu vt. Thm vo 5 git dung dch carmin indigo 0,05% (pha vi nc). Khuy, nu hn hp dung dch b mt mu trong 5-10 giy cho thy s hin din ca Sn2+ hay nhng cht c tnh kh khc ni trn. 4.5.2. nh lng vitamin C bng enzyme peroxidase Vitamin C c th b oxy ha bi enzyme peroxidase. Da trn nguyn l ny c th pht trin mt phng php nh lng vitamin C bng enzyme peroxidase. 4.5.2.1. Trch vitamin C Cn 10g tri cy hoc rau ci v d nh chanh ri cho vo trong 20 mL dung dch m (pH 6,8) metaphosphoric acid 5%, sau nghin mn v lc. Dung dch cha vitamin C sau c nng ln n 100 mL trong bnh nh mc bng dung dch m trn. 4.5.2.2. nh lng vitamin C - Chun b dung dch enzyme: 0,01g enzyme peroxidase c pha trong 5 mL dung dch m pH 6,8 v gi trong nc . - Chun b dung dch vitamin C chun 5mg/ 100 mL: 10 mg vitamin C (tinh khit) c pha trong dung dch m pH 6,8 v nng ln 200 mL trong bnh nh mc. Sau c pha long thnh 1, 2, 3, 4 mg/ 100 mL thit lp ng chun. Trc khi tin hnh o mu chun v mu th tht, mu khng s c o bng dung dch m vi bc sng 265 nm. Mu chun c chun b t 1, 2, 3, 4, v 5 mg/100 mL. Mu dch trch c th c pha long thnh 10, 20 hoc 50 ln d kho st. Phn ng c tin hnh theo trnh t cho cc cht vo cuvette nh sau: - Tin hnh o: Cho vo ng nghim 2,5 mL dung dch m + 0,5 mL dung dch vitamin C chun hoc mu + 0,1 mL dung dch enzyme peroxidase v o hp th bc sng 265 nm ta c gi tr Ai. Khi kt qu th hin s n nh trn ng biu din th thm vo 0,1 mL H202 v cho n nh sau 1 pht, tip tc o c gi tr Af. hp th ca mu khi o l: A265 = Ai - Af Hm lng mu tht s c tnh theo ng chun v suy ra gi tr tht.

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CHNG 5. ACID AMIN V PROTEIN

5.1. KHI QUT V ACID AMIN V PROTEIN Protein l nhng hp cht cao phn t c cu to t cc acid amin lin kt nhau bng lin kt peptide. Khi thy phn protein, ta thu c khong 20 loi acid amin. Protein c th phn lm 2 loi: - Protein n gin: trong thnh phn phn t ch gm cc acid amin lin kt vi nhau bng lin kt peptide - Protein phc tp: trong thnh phn ngoi cc acid amin cn c nhng hp cht khc khng phi acid amin . Protein c rt a dng. Ty thuc vo cc cu trc ca chng m cc tnh cht l ha hc, sinh hc ca protein s khc nhau. + nh tnh v nh lng acid amin, protein ta dng phn ng mu vi nynhydrin v phn ng Biuret. + tch cc acid amin khi hn hp, ta dng phng php sc k trn giy, sc k trao i ion, in di... + Gc R khc nhau ca acid amin s cho nhng phn ng mu ring bit ca mi acid amin: phn ng xanthoproteic, phn ng millon... + Phn ng Biuret xc nh lin kt peptide c trong phn t protein. + Khi a pH ca dung dch protein v im ng in (pI) bng dung dch m thch hp th cc protein b trung ha in tch. Mi trng c pH gn bng pI ca protein th protein s b kt ta nhiu nht. + Do kch thc phn t ln nn protein khng i qua c cc mng bn thm. Da vo tnh cht ny ngi ta dng phng php thm tch loi mui v nhng phn t nh khc khi dung dch protein. 5.2. PHN TCH HN HP ACID AMIN BNG PHNG PHP SC K TRN GIY Nguyn tc Trong nhng iu kin th nghim nht nh (dung mi, nhit , loi giy sc k, bo ha ca dung mi trong bnh sc k...), tc di chuyn ca cc acid amin khc nhau th khc nhau v c trng bi mt h s Rf. Nh tnh cht ny, sau khi sc k cc acid amin trong hn hp tch ri nhau. Sau dng thuc hin mu thch hp nhn bit cc acid amin. Hn hp dung mi sc k l mt hn hp khng trn ln thng gm nc v mt vi dung mi hu c khc. Trong nc c i lc mnh vi giy sc k nh c lin kt cu hydro s gi vai tr pha ng yn, cn dung mi hu c l pha di ng. Cc acid amin khc nhau s b li cun bi pha di ng vi vn tc di chuyn khc nhau v s tch dn khi hn hp acid amin. H s Rf c dng nh gi s di chuyn ny. Rf: l h s gia khong cch t im xut pht ti tm im ca vt v c tnh nh sau: 36
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Gio Trnh Thc Tp Sinh Ha

Rf = a/ b Vi: a: khong cch t im xut pht ti tm im ca vt b: khong cch t im xut pht ti vch cui ca dung mi. Sau khi sc k, cc acid amin c nhn din bng cch lm hin mu v so snh Rf ca n vi Rf ca cc acid amin chun trong cng iu kin th nghim. Thuc th - Sakaguchi I: Napthol 0,01 gam + 5 gam urea trong 1000 mL C2H5OH 95o - Sakaguchi II: 2 gam brom trong 100 mL NaOH 5% - Isatin: 100 mg isatin v 50 mL butanol c cha 5% acid acetic m c - Nynhydrin: 0,2% trong aceton - Dung mi sc k: Butanol: nc: acid acetic theo t l 25:15:10 Tin hnh th nghim a. Chun b giy sc k nh hnh v sau:

I Arg A Arg

II M M O Pro Pro Val 1 cm li g B 6 cm

Leu M III r = 1,5 cm R = 7,5 cm AOC = manh I COB = manh II AOB = manh III

b. Chm dung dch acid amin ln giy - Dng micropipet chm ln lt mt git nh, gn t dung dch acid amin chun tng ng ghi sn v hn hp acid amin M trn giy sc k. Mi ln chm tng git nh gn trnh s lan rng ti v tr cc acid amin khc. Sau khi chm xong mt lt cc acid amin cn sy ngay. Tip tc chm acid amin ln th hai tng t nh ln u. 37 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

- Manh giy hnh ch nht nh c gp lm 3 v ct xo gc. l li g. - Cm phn trn ca li g c gn xuyn qua khe tm ca giy sc k, t giy sc k cn i ln beaker sao cho 2/3 b cao ca li g nhng su vo hn hp dung mi sc k trong beaker. y kn bnh sc k. - Theo di giy sc k trong khong 1 gi 30 pht, khi dung mi cch g ca giy sc k 0,5 cm th cn thn m np bnh ly giy ra, b li g, nh du vch dung mi bng bt ch v sy kh giy sc k. c. Lm hin mu: Ct giy sc k lm 3 manh nh I, II, III (nh hnh v). + Manh I: lm hin mu bng thuc th sakaguchi I, II: u tin dng bnh xt m manh I bng thuc th sakaguchi I, sy tht kh. Sau li xt m tip bng thuc th sakaguchi II ri sy kh. Nhn xt mu ca acid amin chun v acid amin trong hn hp M. + Manh II: Lm hin mu bng thuc th isatin. Xt m manh II bng dung dch isatin. Sy kh. Nhn xt mu ca acid amin chun v acid amin trong M. + Manh III: Lm hin mu bng thuc th nynhydrin. Xt m manh III bng dung dch nynhydrin. Sy kh. Nhn xt mu ca acid amin trn manh ny tng t nh hai manh I v II. + Tnh Rf ca mi acid amin trong hn hp v mi acid amin chun. + Kt lun v cc acid amin trong hn hp M. Lu :- Lun gi giy sc k tht sch. - Bnh sc k lun gi c nh ti mt v tr v phi c y tht kn bo m mi trng bo ha dung mi trong sut thi gian chy sc k. Khi m v y bnh sc k: khng c nhc thng np bnh sc k m y nh np bnh v pha trc hay v pha mnh ang ng. - Nhit sy kh khng qu 60OC. 5.3. CC PHN NG MU C TRNG CA PROTEIN Dung dch protein cho phn ng mu vi mt s thuc th, cc phn ng ny c p dng nh tnh v nh lng protein. 5.3.1. Phn ng Biuret Trong mi trng kim vi s c mt ca Cu2+ cc cht c cha cc nhm sau:
CO NH

CS

NH

C NH

NH2

s phn ng cho hp cht c mu tm. Nguyn tc: Trong phn t protein c mt nhm - CO - NH - nn c th to phc biure vi Cu2+ trong mi trng kim. C ch phn ng c th trnh by nh sau:

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Gio Trnh Thc Tp Sinh Ha

R2 H2N CH C R1 O NH CH C O NH CH C R3 H bin R2 H2N CH C R1 OH N CH C OH N CH C R3 2NaOH Cu(OH)2 R2 N C R1 CH NH2 O


-

R4 NH CH C O

O OH

R4 N CH C OH

O OH

CH O Cu

C O

N CH R3 C N O
-

2Na

2H2O

CH

R4

Ha cht - Dung dch NaOH 10% - Dung dch CuSO4 0,2% Tin hnh th nghim Dng 1 ng nghim cho 2 mL dung dch lng trng trng +1 mL dung dch NaOH 10% + 2 git CuSO4 0,2%. Lc k - Ghi nhn kt qu. 5.3.2. Phn ng Nynhydrin Nguyn tc: Dung dch protein hay acid amin khi un nng vi dung dch nynhydrin 2% s cho mu xanh tm hoc mu vng (i vi acid amin prolin). Nynhydrin l mt cht oxy ha nn c th to phn ng carbonyl ha acid amin vi nc cui cng cho ra CO2, NH3 v aldehyde. Nynhydrin b kh to thnh li tc dng vi NH3 va c phng thch ra v kt hp vi 1 phn t nynhydrin th hai to thnh sn phm ngng kt mu xanh tm. Phng trnh phn ng c trnh by nh sau:

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Gio Trnh Thc Tp Sinh Ha


Giai oan kh amin hoa va kh carboxyl :
O NH2 R CH COOH C OH C C O OH -H2O O C R CHO + NH3 + CO2 OH C C O H

Nynhydrin oxyhoa Giai oan tao phc mau :


O C C C O O O C C C O H N C C O C +NH3 C O ONH4 C C N C C O O C H OH H H N H HO O C C HO C O -3H2O

Nynhydrin-kh

Phc mau xanh tm

Ha cht - Dung dch nynhydrin 2% trong acetone - Dung dch acid amin 0,1% - Dung dch proline 0,1% Tin hnh th nghim: Dng 3 ng nghim, cho vo mi ng cc ha cht theo bng sau:
ng nghim Dung dch Lng trng trng Acid amin 0,1% Proline 0,1% Nynhydrin 2% 1mL 0 0 1 mL 0 1mL 0 1mL 0 0 1mL 1mL 1 2 3

Lc u cc ng nghim, em un cch thy. Ghi nhn kt qu. 5.4. S KT TA PROTEIN Nguyn tc Dung dch keo protein bn vng do cc tiu phn protein mang lp o nc b ngn cch nhau v do s tch in cng du nn chng y nhau. Nu lm mt mt trong hai yu t trn th s dn ti s kt ta cc tiu phn protein . 40
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Gio Trnh Thc Tp Sinh Ha

a. Lm mt lp o nc bng cch: thm cc cht in gii hoc kh nc nh NaCl, (NH4)2SO4, alcol, aceton... b. Trung ha in tch ca protein bng cch: - Thm cht in gii nh NaCl, (NH4)2SO4... - Hoc a pH ca mi trng cha protein v gn im ng in pI ca protein. Ngoi ra khi lm bin tnh protein bng nhit cao hay di tc dng ca cc cht nh acid, kim mnh hoc mui ca kim loi nng... cng dn ti kt ta protein. Ho cht - (NH4)2SO4 tinh th - Dung dch (NH4)2SO4 bo ha - H2SO4 m c - NaOH m c - Dung dch acid sulfosalicilic 10% - Dung dch NaCl 30% bo ha 5.4.1. S kt ta thun nghch (Phng php thu nhn protein) nh ngha: S kt ta thun nghch l sau khi kt ta, protein kt ta c th li c ha tan tr li trong dung mi thch hp. Trong s kt ta thun nghch, protein vn gi c cc tnh cht l ha hc v sinh hc. Cc cht NaCl, (NH4)2SO4, dung mi hu c (ether, alcol, aceton...) c th gy kt ta thun nghch protein. Tin hnh th nghim Dng 1 ng nghim ly 4 mL dung dch lng trng trng, thm 4 mL dung dch (NH4)2SO4 bo ha. Lc u, lc kt ta qua giy lc. + Phn ta em ha tan bng dung dch NaCl 30% bo ha. Nhn xt. Kt lun. + Phn nc c thm tip (NH4)2SO4 tinh th ti khi bo ha (ngha l (NH4)2SO4 tinh th khng tan na th ngng). Lc b phn ta, dung dch qua giy lc em thc hin phn ng biuret. Nhn xt v gii thch qua ln ta th 2 v kt lun. 5.4.2. S kt ta bt thun nghch (bin tnh protein) nh ngha: S kt ta bt thun nghch protein thng lin quan n s bin tnh protein hay ni cch khc, cu trc khng gian 2, 3, 4 ca phn t protein b ph v vnh vin, khng ti lp d iu kin no. Cc yu t gy ta khng thun nghch thng l acid hu c (trichloroacetic, acid sulfosalycilic...), acid v c m c (H2SO4, HCl, HNO3...), mui kim loi nng, nhit ... a. Kt ta bng nhit Phn ln cc protein b kt ta khi un nng, ty thuc vo bn cht v bn m n s b kt ta bt thun nghch (bin tnh) mt nhit nht nh. Th d: Protein trng b ta nhit 80 - 90OC, cu trc khng gian bc 2,3,4 b ph v ti nhit ny. Cho vo ng nghim 2 mL dung dch lng trng trng. un cch thy 20 pht, thm vo 2 mL dung dch NaCl 30% bo ha. Khuy u. Quan st s ha tan ca kt ta. Nhn xt - Kt lun. 41
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Gio Trnh Thc Tp Sinh Ha

b. Kt ta protein bng acid hu c Cho vo ng nghim kh 2mL dung dch lng trng trng, thm 5-8 git acid sulfosalycilic 10%. Lc u, sau lc ly kt ta. Phn ta thm 2 mL dung dch NaCl 30% bo ha. Lc u, quan st s ha tan ca kt ta. Nhn xt, kt lun. c. Kt ta bng acid v c mnh
1 ml dd trng H2SO 4 pH = pI Dung dch H 2SO 4 co tua pH pI Tua tan ra Thc hin Trung hoa bng NaOH phan ng Biure

Cho 1mL dung dch lng trng trng vo ng nghim, nh t t tng git H2SO4 m c vo cho n khi thy xut hin kt ta. Tip tc cho H2SO4 m c n khi ta tan. em trung ha dung dch trn bng NaOH m c v th phn ng Biuret. Quan st hin tng, gii thch cch tin hnh v kt lun. 5.5. NH LNG PROTEIN BNG PHNG PHP QUANG PH 5.5.1. Khi qut Mt trong nhng k thut phn tch hiu qu nht trong sinh ha l phng php phn tch so mu. Trong phng php ny, nng ca hp cht mu c xc nh bng cch o cng mu ca dung dch cha hp cht mu . Phng php so mu l mt phn ca phng php quang ph hp th, mt trong cc phng php quang hc. Cc phng php o quang ph thng c o trong di bc sng t 220- 800 nm. Di quang ph ny c chia ra: - Vng t ngoi (UV- ultra violet): 200 nm < < 400 nm - Vng kh kin (VIS- visible): 400 nm < < 800 nm - Vng hng ngoi (IR- infrared): > 800 nm nh sng c bc sng trong vng kh kin (nh sng trng) thng c dng trong phng php so mu. Khi cho nh sng trng i qua dung dch mu, mt s bc sng nh sng s b hp th (ty theo bn cht dung dch), trong khi nhng bc sng khc th gn nh khng b hp th. Mu ca dung dch m ta quan st c l mu ca nhng bc sng khng b hp th. S lin h gia mu dung dch v nh sng b hp th c trnh by bng 4.1 sau: Bng 4.1: Tnh cht mu dung dch trong vng nh sng trng nh sng trng ti , vng & xanh da tri nh sng hp th Vng & xanh da tri Xanh da tri Xanh da tri & & vng Vng nh sng khng hp th vng Vng Vng & xanh da tri Xanh da tri Xanh da tri & 42 Mu ca dung dch Da cam Vng Xanh l cy Xanh da tri Tm
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Gio Trnh Thc Tp Sinh Ha

Cng mu ca dung dch c th c xc nh bng cch o cng nh sng hp th bi hp cht mu c trong dung dch. Cng mu ca cht hp th t l vi cng cht trong dung dch, v th ta c th p dng phng php o cng mu trong phng php phn tch nh lng. 5.5.2. nh lut Lambert- Beer Khi chiu mt chm tia sng n sc c bc sng , cng I0 qua mt dung dch c chiu dy lp dung dch l, nng C. Ta c: Cng tia nh sng ti bng tng cng nh sng b hp th, nh sng b phn x v cng nh sng i ra khi dung dch (cng tia l). I0 = Ih + Ip + I Vi: - I0 : Cng tia nh sng ti - Ih: Cng nh sng b hp th - Ip: Cng nh sng phn x - I: Cng tia l V lng nh sng phn x rt nh, c th b qua, nn ta c: I0 = Ih + I nh lut Lambert- Beer m t mi lin h gia cc yu t trn nh sau: Khi nh sng n sc i qua dung dch cha cht hp th, cng nh sng b hp th s ph thuc vo nng dung dch v chiu di ca ng truyn nh sng qua dung dch. log (I0/I) = x C x l Trong : - I0 : Cng tia nh sng ti - I: Cng tia l - : h s hp th phn t - C: Nng dung dch - l: chiu di ca ng truyn nh sng qua dung dch (cm) Gi tr log(I0/I) c gi l hp th (A - absorbance) hay mt quang (OD - optical density). A

Ax

Cx

C (mg/mL) 43
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Trong cng mt iu kin, khi v l khng i, th hp th s t l thun vi nng cht mu. V vy ta c th xy dng ng chun s ph thuc ca hp th vo nng dung dch chun. T ta c th xc nh c nng ca dung dch protein cn phn tch khi bit c hp th ca dung dch ny. ng chun c xy dng bng s ph thuc ca gi tr hp th theo nng dung dch. Thng thng, c kt qu phn tch vi chnh xc cao, ngi ta chn iu kin sao cho hp th nm trong khong 0,1 1, tt nht l t 0,2 0,5. 5.5.3. Phng php nh lng protein theo phn ng biuret Nguyn tc Trong mi trng kim, cc hp cht c cha nhm CO-NH-, -CS-NH-, C(NH)NH2 s c phn ng vi Cu2+ to hp cht phc mu tm. Trong th nghim ny, protein c cho phn ng vi Cu2+ trong mi trng kim to phc mu tm; cng mu t l vi nng protein trong dung dch. Do ta c th nh lng protein theo phng php so mu bc sng 550 nm. Vt liu-Thuc th a. Dung dch protein chun: Dung dch albumin huyt thanh b (BSA bovine serum albumin) 20 mg/mL pha trong dung dch NaCl 0,9 %. b. Dung dch lng trng trng (dung dch phn tch) Cn chnh xc khong 4g lng trng trng, cho vo bnh nh mc 50 mL. Dng dd NaCl 0,9% a ln n vch. c. Thuc th Biuret 1,5g - CuSO4.5H2O - K, Na tartrat 6g - NaOH 30g (Pha trong 1lt dung dch) Tin hnh a. Chun b dy dung dch protein chun Chun b dy dung dch protein chun c nng tng dn t 0 20 mg/mL t dung dch protein gc (BSA 20 mg/mL) ng nghim Nng dung dch (mg/mL) Th tch dd protein chun (L) Th tch dd NaCl 0,9% (L) Th tch nc ct (mL) Thuc th Biuret (mL) 1 0 0 2 4 3 8 4 12 5 16 6 20 0 1 3,5

Gio Trnh Thc Tp Sinh Ha

100 200 300 400 500

500 400 300 200 100 1 3,5 1 3,5 1 3,5 1 3,5 1 3,5

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Gio Trnh Thc Tp Sinh Ha

b. Chun b dung dch phn tch: Cho vo ng nghim: - 500 L dung dch protein trng (dung dch phn tch) - 1 mL nc ct - 3,5 mL thuc th biuret Sau khi chun b cc dung dch trn, lc u, yn 20 pht. Quan st dy dung dch v o hp th bc sng 550 nm. V th biu din s ph thuc ca hp th A vo nng dung dch protein chun. T gi tr hp th ca dung dch phn tch, c th suy ra nng protein trong mu da vo ng chun. Tnh kt qu: T kt qu c c trn my, ta xc nh c nng protein trng trong dung dch phn tch: x mg/mL. Hm lng protein trong lng trng trng c tnh theo cng thc sau: C (mg/100g) = (50 x Trong :

x x 100)/a

x: nng protein trng trong dung dch phn tch c c trn my


(mg/mL). a: s gam lng trng trng em phn tch. 5.5.4. nh lng protein theo phng php Lowry Nguyn tc C s ca vic nh lng protein theo phng php Lowry l da vo phn ng mu ca protein vi thuc th Folin to thnh phc cht c mu xanh hp thu cc i bc sng 750nm. Cng mu ca dung dch t l thun vi nng protein. Phng php ny c nhy cao, cho php xc nh dung dch mu cha vi chc microgam protein. Ho cht - Dung dch BSA chun 200g/mL pha trong dung dch NaCl 0,9%. - Dung dch A: Na2CO3 2% trong NaOH 0,1N - Dung dch B: CuSO4 0,5% trong kali natri tartrat 1% - Dung dch C: Pha dung dch A v B theo t l th tch 49:1 trc khi dng. - Thuc th Folin 1N: 100g + Natri tungstate (Na2WO4.2H2O): 25g + Natri molypdate (Na2MoO4.2H2O): + Nc ct: 700mL Cho vo bnh cu un cho tan ri b sung thm: + Acid phosphoric 85%: 50mL + Acid chlohydric m c: 100mL 45
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Gio Trnh Thc Tp Sinh Ha

un si hn hp trong ng sinh hn trong 10 gi. Dung dch tr thnh mu vng hay xanh l cy thm. Tip tc cho thm: 150g + Liti sulphate LiSO4: + Brom: 5 git + Nc ct: 50mL un si trong 15 pht. Dung dch thu c c mu vng sng. Nu dung dch c mu xanh th phi x l vi brom ln th hai. Sau khi lm ngui thm nc ct n 1 lt. Xc nh li nng thuc th Folin bng cch chun vi NaOH 1N vi ch th phenolphtalein. Cha dung dch trong chai nu, bo qun trong t lnh. Tin hnh a. Chun b dy dung dch protein chun Chun b dy dung dch protein chun c nng tng dn t 0 200 g/mL t dung dch protein gc (BSA 200 g/mL), sau cho thm cc ho cht vo 6 ng nghim theo bng sau: ng nghim Nng dung dch (g/mL) Th tch dd protein chun (L) Th tch dd NaCl 0,9% (L) Th tch dung dch C (mL) Thuc th Folin (L) b. Chun b dung dch phn tch: Cho vo ng nghim: - 1000 L dd dung dch phn tch (Tnh ton sao cho nng protein trong dung dch t 20 200 g/mL) - 4 mL dung dch C - 0,5 mL thuc th Folin Sau khi chun b cc dung dch trn, lc u, yn trong 30. Quan st dy dung dch v o hp th bc sng 750 nm. V th biu din s ph thuc ca hp th A vo nng dung dch protein chun. T gi tr hp th ca dung dch phn tch, c th suy ra nng protein trong mu phn tch da vo ng chun. 5.6. NH LNG PROTEIN TNG S THEO PHNG PHP KJELDAHL Nguyn tc Khi un si lu mu vt c cha nitrogen (m) trong H2SO4 m c vi s hin din ca cht xc tc thch hp th tt c cc hp cht hu c b oxy ha cn NH3 c gii phng ra lin kt vi H2SO4 to thnh (NH)2SO4. 46
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1 0 0 500 2,5 250

2 40 100 400 2,5 250

3 80 200 300 2,5 250

4 120 300 200 2,5 250

5 160 400 100 2,5 250

6 200 500 0 2,5 250

Gio Trnh Thc Tp Sinh Ha

H2SO4 Xuc tac, tO Hp cht cha N

(NH4)2SO4

Ta c th xc nh lng m ny khi cho chng tc dng vi NaOH, chng s c li cun bng hi nc v c ct qua bnh hng c cha dung dch acid sulfuaric ( H2SO4) hay acid boric v hn hp thuc th. (NH4)2SO4 + 2 NaOH = 2 NH4OH + Na2SO4
NH4OH

to

NH3 +

H2 O

NH3 + H2SO4 (NH4)2SO4 Hoc NH3 + 4H3BO3 (NH4)2B4O7 Sau nh lng amoni tetraborate to thnh bng dung dch H2SO4 0,005N theo phn ng sau : (NH4)2B4O7 + H2SO4 + 5H2O (NH4)2SO4 + 4H3BO3 Ha cht v dng c * Dng c - My ct m bn t ng Gerhardt - H thng v c ha Gerhardt - Bnh Kjeldahl 250 mL - Cc dng c thy tinh thng thng trong phng th nghim * Ha cht - Dung dch NaOH 10% - K2SO4 - CuSO4 - Dung dch H2SO4 0.01 N - Dung dch acid boric 2% - Selenium - H2SO4 m c - Dung dch bromoncresol green v methyl trong alcol Tin hnh a. S v c ha Cn chnh xc 1 gam mu vt nghin nhuyn hay 1 mL (nu mu vt dng dung dch), chuyn mu vt cn vo bnh Kjeldahl c th tch 50-100mL, sau thm 5 mL H2SO4 m c. rt ngn thi gian v c ha, ta thm vo bnh mt lng cht xc tc cn thit l 0,5 gam. Hn hp cht xc tc ny gm K2SO4: CuSO4: Se (100:10:1). Sau khi cho cht xc tc vo bnh, em un si trong my v c ha Gerhardt c ni vi h thng hp thu kh c cho n khi dung dch trong bnh Kjeldahl trong sut. ngui, ta kim tra kt thc s v c ha bng cch cho vo bnh mt lng nh nc ct (thng bng bnh tia) ri lc nh trng thnh bnh, thy khng cn nhng ht mi en li ti l c. Nu cn ta phi un tip tc cho n ht. 47
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Gio Trnh Thc Tp Sinh Ha

Lu : Khi v c ha ta cn thc hin vi 3 bnh th tht (c mu vt) v 3 bnh th khng (khng c mu vt). b. Ct m Sau khi v c ha kt thc, mu c em ct m trong my ct bn t ng Gerhardt. Chun b my: cm in, bt my, mn hnh s hin ln ch H, m vi nc lm lnh, ch n khi mn hnh hin ch P, my sn sng lm vic. Cho 20 mL acid boric 2% vo bnh tam gic 100 mL, thm 2 git thuc th, lp vo v tr hng mu trn my. Ch nhng ngp ng vo dung dch. Lp ng Kjeldahl cha mu v c ha vo h thng. Ci t chng trnh cho my nh sau: - Nhn RESET - Nhn PROGRAM - Step 1: 01 (ng vi 10 mL NaOH 40%) - Nhn PROGRAM - Step 2: 05 (ng vi thi gian phn ng l 5 giy) - Nhn PROGRAM - Step 3: 300 (ng vi thi gian sc hi nc l 5 pht) - Nhn PROGRAM - Step 4: 60 (ng vi hiu sut lm vic ca my) - Nhn PROGRAM c mn hnh vi ch P. - Cho my chy bng cch nhn nt RUN Kt thc mt ln th nghim mn hnh s hin ln ch End, ta thay ng phn ng mi v tip tc nhn RUN. c. nh phn Ly bnh tam gic ra khi my sau khi trng nc ct ly ht mu bm trn ng. nh phn bng dung dch H2SO4 0,1N cho n khi dung dch chuyn t mu xanh l sang mu hng nht. c th tch trn buret. Cch tnh kt qu: * Mu rn: Hm lng nit (gam) c trong 100 gam mu c tnh theo cng thc sau:
N% = (a - b) x NH2SO4 x 1,4 m

Trong : - a: th tch dung dch H2SO4 chun dng chun mu tht (mL) - b: th tch dung dch H2SO4 chun dng chun mu i chng (mL) - NH2SO4 : Nng ng lng ca dung dch acid chun - m: Khi lng mu em phn tch (g) * Mu lng: Hm lng nit (gam) c trong 1 lt mu c tnh theo cng thc sau:
N% = (a - b) x NH2SO4 x 1,4 V

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Gio Trnh Thc Tp Sinh Ha

Trong : - a: th tch dung dch H2SO4 chun dng chun mu tht (mL) - b: th tch dung dch H2SO4 chun dng chun mu i chng (mL) - NH2SO4 : Nng ng lng ca dung dch acid chun - V: th tch mu em phn tch (mL)

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Gio Trnh Thc Tp Sinh Ha

CHNG 6. KHO ST ENZYME


6.1. KHI QUT Enzyme l nhng cht c bn cht l protein, ng vai tr l cht xc tc sinh hc, xc tc cho cc phn ng sinh ha xy ra trong c th. Enzyme c tnh c hiu cao v c hiu lc xc tc ln. Enzyme c trong t bo ca cc c th sng, nh s hin din ca enzyme m cc phn ng sinh ha trong c th xy ra rt nhanh, nhy iu kin sinh l bnh thng ca c th sng. 6.2. KHO ST HOT TNH CA ENZYME AMYLASE T MM LA 6.2.1. H enzyme amylase Amylase l h enzyme rt ph bin trong th gii sinh vt. Cc enzyme ny thuc nhm cc enzyme thy phn (hydrolase), chuyn xc tc s phn gii cc lin kt glycoside trong phn t polysaccharide vi s tham gia ca nc. - C cht tc dng ca amylase l tinh bt v glycogen. - Sn phm ca s thy phn do amylase xc tc l cc thnh phn n gin hn nh dextrin, maltose, glucose. - Enzyme amylase c t nhiu ngun nh: trong nc bt, trong dch tiu ha ca ngi v ng vt, trong ht ny mm, nm mc, nm men v vi khun. - Ty theo tnh cht v kh nng tc dng, ngi ta phn bit: - amylase, amylase v - amylase. - Amylase (EC 3.2.1.1) - Khng ch thy phn h tinh bt m n thy phn c ht tinh bt nguyn, song vi tc rt chm. - C kh nng phn ct cc lin kt 1-4 glycoside nm pha trong phn t c cht (tinh bt, glycogen) mt cch ngu nhin. - Sn phm ca s thy phn tinh bt nh -Amylase l cc dextrin c phn t lng thp (khng c mu vi iod), mt t maltose v rt t glucose. - pH ti thch cho -amylase mm la l 5,3 - Nhit ti thch: 58-60OC - -Amylase c hot ha bi Ca2+, Cl- v b c ch bi Cu2+, Ag+, Hg2+. - Amylase (EC 3.2.1.2) - Ch thy phn mnh m h tinh bt, c th -amylase phn gii 100% amylose thnh maltose v 54-58% amylopectin. - C kh nng phn ct lin kt -1-4 glycoside t cc u khng kh ca cc nhnh ngoi cng, s phn ct s dng li 1 vng gn nhnh (lin kt 1-6) trong phn t amylopectin. - pH ti thch 4,5 - Nhit ti thch: 50OC - - Amylase c kch thch bi Na+, b c ch bi Cu2+, Hg2+, urea, iodoacetamide, iod, ozon. 50 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

- Amylase (EC 3.2.1.3) - C kh nng xc tc thy phn c lin kt -1-4 ln -1-6 glycoside tun t tng gc glucose. - Sn phm thy phn l glucose. - pH ti thch l 3,5 - 5,5 - Cht kch thch -Amylase l Ca2+, Na+, Cl- v cht c ch l Cu2+, Hg2+. - Km bn di tc dng ca ru ethylic, aceton. Trong bi th nghim chng ta dng phc h amylase ca mm la kho st s thy phn dung dch h tinh bt. 6.2.2. S to mu gia iod vi tinh bt v cc chuyn ha ca tinh bt khi thy phn bng amylase Phc h amylase ca mm la c kh nng thy phn tinh bt ln lt thnh cc sn phm sau: Tinh bt (mu xanh vi iod) Amylodextrin (mu tm vi iod), Erythrodextrin (mu tm vi iod) Acrodextrin (khng kt hp vi iod nn khng cho mu) Maltodextrin (khng kt hp vi iod) Maltose v glucose (khng kt hp vi iod). Da vo s bin i mu ny n khi sn phm thy phn khng cho mu vi iod, ta c th kt lun phn ng thy phn kt thc. Ha cht - H tinh bt 1% - Dung dch m pH t 2-8 - Dung dch iod 1% - Dung dch CuSO4 2% - Dung dch CaCl2 1% 6.2.3. Ly trch v kho st hot tnh tng i ca amylase mm la 6.2.3.1. Ly trch amylase Dng khong 100 ht la ny mm cho vo ci s, nghin nhuyn vi 3-5mL nc ct. Tip tc nghin v thm dn n ht 50 mL nc ct ha tan enzyme. Lc dung dch qua ry bng giy lc hoc ly tm 5.000-10.000 vng/pht trong 15 pht. Thu dch lc c cha enzyme amylase. Gi li tin hnh th cc nghim sau. 6.2.3.2. Kho st hot tnh tng i ca dch chit amylase mm la Dung dch amylase thu c thng c hot tnh xc tc t n nh. N thay i ty theo vo iu kin th nghim v cc yu t mi trng. Do ta cn phi kho st hot tnh tng i ca dung dch enzyme amylase v chn nng thch hp cho cc th nghim sau.

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Gio Trnh Thc Tp Sinh Ha

Chun b dung dch th nghim theo bng sau:


ng nghim 1
Dung dch cho vo H tinh bt 1% (mL) Nc ct (mL) Dung dch m pH 5 (mL)

2 1 0,6 2

3 1 0,8 2

4 1 1,0 2

5 1 1,2 2

6 1 1,4 2

7 1 1,6 2

8 1 1,8 2 0,2

1 0,4 2

n nh 50OC Th tch dch enzyme (mL) Thi gian kt thc thy phn 1,6 1,4 1,2 1,0 0,8 0,6 0,4

Sau khi pha xong dung dch, n nh nhit b nc nng 50oC, sau cho cc ng nghim t 1 n 4 vo n nh 5 pht. Ht 1,6 mL dch enzyme cho vo ng 1, ng thi ghi nhn thi im . Dng pipet loi 1 mL khuy nh dung dch v ly ra 1 git th mu vi iod. Khi mu ca dung dch iod khng thay i th s thy phn tinh bt c coi l kt thc. Ghi li thi im kt thc. Ln lt tin hnh cc ng cn li tng t nh ng 1. Lu : Sau khi kt thc ng nghim 1, ly ra khi b nc nng ta a ng 5 vo n nh tip tc 50oC. - Ghi nhn thi gian, lp bng kt qu, bnh lun v v th. - Chn th tch enzyme ng nghim no c thi gian thy phn tinh bt trung bnh khng nhanh khng chm ( 1 pht) cho cc th nghim k tip. Lu : * Cn ly dung dch chnh xc, trnh nhm ln. * Lun gi nhit n nh 50OC khi tin hnh th nghim. 6.2.4. Kho st nh hng ca nng c cht ln tc thy gii ca amylase Chun b dung dch th nghim theo bng sau: ng nghim Dung dch cho vo 1 2 3 0,6 1,4 2 3 4 0,8 1,2 2 4 5 1 1,0 2 5 6 1,2 0,8 2 6 7 1,4 0,6 2 7 8 1,6 0,4 2

Nng tinh bt tng ng (mg/mL) 2 H tinh bt 1% (mL) 0,4 Nc ct (mL) 1,6 Dung dch m pH 5 (mL) 2 Th tch dch enzyme (mL) Thi gian kt thc thy phn

n nh 50OC Th tch chn th nghim 1

Tin hnh th nghim tng t nh trn. Ghi nhn thi gian, lp bng kt qu, bnh lun v v th. 52
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Gio Trnh Thc Tp Sinh Ha

6.2.5. Kho st nh hng ca pH ln tc thy gii ca amylase Enzyme rt nhy vi s thay i ca pH mi trng, mi enzyme ch hot ng mnh nht mt vng pH xc nh, gi l pH ti thch. Chun b dung dch th nghim theo bng sau: ng nghim Dung dch cho vo H tinh bt 1% (mL) Nc ct (mL) Gi tr m pH Th tch dung dch m pH (mL) Th tch dch enzyme (mL) Thi gian kt thc thy phn 1 2 3 4 5 6 7 1 1 8 2

1 1 1 1 1 1 1 1 1 1 1 1 7 6 5 4 3 2 2 2 2 2 2 2 O n nh 50 C Th tch chn th nghim 1

Lu : * Cn n nh cc ng nghim 3, 4, 5 (tng ng vi pH 4, 5, 6) trc ng 1, 2, 6, 7. Ghi nhn thi gian kt thc phn ng thy phn ca mi ng nghim. * Lp bng kt qu, v th v nhn xt. 6.2.6. Kho st nh hng ca cht hot ha v cht c ch Cht hot ho l nhng cht c tc dng lm cho enzyme t trng thi khng hot ng tr thnh hot ng, t hot ng yu tr thnh hot ng mnh hn... Cc cht hot ho thng c bn cht ho hc rt khc nhau: cc cht hu c phc tp, cc cht c tc dng phc hi nhng nhm chc ca trung tm hot ng enzyme, mt s cation, anion. Cht c ch l cht c kh nng lm yu hoc lm chm dt hon ton tc dng ca enzyme. Cc cht c ch c bn cht ho hc rt khc nhau c th l cc ion kim loi, hoc cc cht hu c phc tp... Sau y ta s kho st nhng nh hng trn vi dung dch A (CaCl2) v B (CuSO4) l nhng dung dch c cha sn mt s ion c kh nng gy kch thch hoc c ch s xc tc ca amylase. Cch pha dung dch c trnh by trong bng sau: ng nghim Dung dch cho vo H tinh bt 1% (mL) Nc ct (mL) Dung dch m pH=5 (mL) Cht gy nh hng A (mL) Cht gy nh hng B (mL) Th tch dch enzyme (mL) Thi gian kt thc thy phn 1 2 3

1 1 1 0 0 1 2 2 2 0 1 0 1 0 0 O n nh 50 C Th tch chn th nghim 1

Ghi nhn thi gian, nhn xt, kt lun.


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53

Gio Trnh Thc Tp Sinh Ha

6.3. KHO ST ENZYME UREASE TRONG BT U NNH Nguyn tc: Urease l enzyme c th thy gii urease theo phn ng sau: (NH4)2CO3 (NH2)2CO + H2O Nu thm formol vo mi trng, s c phn ng to thnh hexamethylene tetramine v acid carbonic theo phn ng sau: 2 (NH4)2CO3 + 6 HCHO (CH2)6N4 + 6 H2O + H2CO3 Acid to thnh c th c nh phn bng kim v xc nh lng urea b thy gii, t suy ra hot tnh ca enzyme urease. Ha cht - Dung dch N/20. - Dung dch urea 2% (w/v). - Formol trung ha. - Dung dch urease: Khuy 3 gam bt u nnh trong 100 mL nc ct. yn trong 15 pht. Nc trn cha nhiu urease. em lc thu ly dch lc. - Dung dch phenolphtalein 1% trong alcol. Tin hnh Chun b cc ng nghim theo bng sau: ng nghim Th tch dung dch urea 2% (mL) Th tch dung dch enzyme urease (mL) Th tch dd enzyme urease un si (mL) 1 5 5 0 2 5 0 5

cc ng nghim trn trong nc m 40oC trong 20 pht. Cho vo mi ng 5 mL formol c trung ha, lc u trong vi pht. Ln lt cho cc dung dch trong ng ra bnh tam gic 100 mL, trng ng vi mt t nc ct. nh phn H2CO3 c gii phng ra bng dung dch NaOH N/20 vi phenolphtalein lm ch th mu. Kt qu Hot tnh enzyme urease c biu th bng s mol urea b thy gii bi lng enzyme c trong 1 gam bt u nnh trong thi gian 1 pht 40oC, hot tnh c tnh theo cng thc sau:
(V1 - V2) x A 20 x 103 x 2 x t x a x m

mol/ g/ pht

Trong : - V1: S mL NaOH N/20 dng nh phn ng 1 - V2: S mL NaOH N/20 dng nh phn ng 2 - a: Th tch enzyme cho vo mi ng (mL) - A: Tng th tch enzyme thu c t m gam bt u nnh (mL) - t: Thi gian thy phn (pht) 54 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

6.4. ENZYME HA NU 6.4.1. Khi qut v phn ng ha nu C 4 loi phn ng ha nu trong sn phm t thc vt l phn ng Maillard, s caramel ho, s oxi ho acid ascorbic v phn ng ho nu do enzyme. Trong phn ng ho nu do enzyme th cc hp cht phenol thc vt thng c to thnh sau khi thu hoch rau qu, do thc vt b tn thng v vt l hoc do qu trnh ch bin. Thc vt cha cc enzyme oxy ha khc nhau v chng to ra s chuyn ha cc hp cht phenol cng khc nhau. y cp n hai enzyme quan trng trong phn ng ha nu l enzyme polyphenol oxidase (EC 1.10.3.1) v enzyme peroxidase (POD) (EC 1.11.1.7). 6.4.1.1. Enzyme polyphenol oxidase Enzyme polyphenol oxidase hay phenolase tn ti hai dng t do v lin kt rt hot ng, trong ch yu dng lin kt. Enzyme polyphenol oxidase chu trch nhim khi u phn ng ha nu. Phn ng c thc hin khi c nhm ngoi ca ng v oxygen, ng c th l ha tr I trong trng hp phenolase ca nm hay ha tr II trong trng hp ca khoai ty. Enzyme polyphenol oxidase c khong hot ng pH 5 - 7 v b bt hot hon ton khi pH thp hn 3. * C ch phn ng
Phenolase

CH2 HO

CH-COOH NH2 Chm

+O

HO HO

CH2

CHCOOH Nhanh NH2

+O

O O

CH2

CH-COOH NH2

Tyrosine

(1)

Dopa

(2)

Dopa Quinone
Nhanh

HO HO NH

+ CO2

C - COOH
NH

+O

HO

C -COOH
NH

Ch m

Nhanh HO

5,6-Dihydroxyindole
Nhanh +O

Dopachrome

Leuco Compound

O O NH Indole 5,6-Quinone

+O

HN

O O

Tng i chm

NH Melanin

C ch phn ng ha nu do enzyme phenolase 55 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

Phn ng ho nu do enzyme t xy ra m cn nguyn v c cht phenol v enzyme phenolase b tch ri. Phn ng ho nu xy ra nhanh chng mt ct ngang ca tri cy v rau ci bi s oxy ho phenol thnh orthoquinine v sau chuyn thnh cc hp cht mu hoc melanin. Hai loi phn ng lin quan n s xc tc phenolase l s hydroxy ha v oxy ho. Tyrosine v acid chlorogenic l hai c cht chnh ca phenolase v tc phn ng ca chng xy ra tng i nhanh, bn cnh cn cc c cht khc nh catechol, acid caffeic , acid protocatechuic ... i vi phn ng (1) tc cht l monophenol v i vi phn ng (2) tc cht l diphenol. Theo sau phn ng (2) l s chuyn hydrogen to thnh dopachrome (acid 5,6-quinine indole-2-carboxylic) c mu . Dopachrome to thnh cht melanin mu nu bi phn ng polymer ha. Cng nh tyrosine, catechol l mt ortho diphenol, d dng b tn cng bi enzyme phenolase. OH O OH O phenolase + [O]

Catechol

O-Benzoquinone

S to thnh o-quinone do phenolase S to thnh quinon ty thuc vo enzyme v oxygen. Khi phn ng ny xy ra, cc phn ng tip theo xy ra khng cn c s hin din ca phenolase hay oxygen. Phn ng u tin l hydroxyl ha o-quinone hay o-diphenol. OH OH O OH O OH H 2O hay OH
Catechol O-Benzoquinone Trihydrobenzen

S hydroxyl ha o-quinone. Sn phm trihydrobenzen tip tc phn ng vi o-quinine thnh lp hydroxyl quinine. O O O OH O O OH OH + hay
OH OH Phn ng thnh lp hydroxyquinone O

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Gio Trnh Thc Tp Sinh Ha

Hydroxyquinone xy ra s a phn ha to thnh hp cht polymer mu nu v cui cng xut hin melanin mu nu. 6.4.1.2. Enzyme peroxidase Enzyme peroxidase tn ti hai dng t do v lin kt, enzyme ny hin din trong nhiu thc vt, ng vt v vi sinh vt. Hot tnh tng i ca enzyme peroxidase trong dch trch th khng thay i khong t nht l 5 thng khi 4C v gim 2-3% trong 8 gi phn ng 25C. Enzyme peroxidase xc tc s oxy ha cc hp cht phenol thc vt nhng khng s dng trc tip c oxy phn t ca khng kh m phi da vo s phn gii H2O2 s dng oxy nguyn t, oxy nguyn t di chuyn n phn t cht nhn, cht thch hp l phn t hu c guaiacol. Phn ng ny xy ra nhanh khi c enzyme peroxidase. kho st hot tnh tng i ca enzyme peroxidase ngi ta thng dng guaiacol. Mt n v guaiacol c nh ngha nh lng enzyme oxy ha 1 mol ca guaiacol trong 1 pht 25C vi pH 7. Enzyme POD xc tc phn ng gia guaiacol vi H2O2 to thnh hp cht tetraguaiacol c mu tm nu. Da vo cng mu ta xc nh c hot tnh tng i ca enzyme bng my o quang ph bc sng 470 nm. Phn ng c th nh sau:
OCH3 OH Peroxidase + 4Guaiacol 4 H2O2 O O OCH3 OCH3 + 8 H2O OCH3 O O OCH3

Tetraguaiacol (tm nu)

Phn ng thnh lp tetraguaiacol Sn phm tetraguaiacol to thnh sau phn ng xc tc ca enzyme s c kho st bi s chuyn mu ca dung dch sang mu nu tm do s oxy ha guaiacol. Di s xc tc ca peroxidase, guaiacol b oxy ha dn v mu tm nu m dn theo thi gian. Nng tetraguaiacol c to thnh th hin qua cng mu ca dung dch sau phn ng. 6.4.2. Kho st hot tnh tng i ca enzyme ha nu 6.4.2.1. Kho st hot tnh tng i ca enzyme polyphenol oxidase * Trch enzyme polyphenol oxidase Gt sch v khoai ty, ra sch v bo mn, sau cn khong 10 g ri cho vo 50 mL dung dch m pH 6,8 (pha 100 mL dung dch m phosphate 0,1 M trong 0,4g NaF, chnh n pH 6,8). Hn hp sau c trn u bng my lc v lc bng giy lc hoc ly tm lnh thu c dch trch enzyme. Ch gi mu k trong nc lc thao tc. 57 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

* o hot tnh tng i ca enzyme polyphenol oxidase Cho vo cuvette 2,5 mL dung dch DOPA 4mM (DOPA: 3,4-dihydro-L phenylalanine trong m phosphate pH 6,8) (c th dng tyrosine) sau cho thm 0,5 mL dch trch enzyme v quan st phn ng ha nu 37C bng cch o hp th vi my quang ph 475 nm trong 2 pht. Ch : C th dng cc vt liu khc so snh v hot tnh ca enzyme ha nu trch t khoai ty, hoc thay i iu kin x l nhit hoc pH nh gi hot tnh ca enzyme ny. 6.4.2.2. Kho st hot tnh tng i ca enzyme peroxidase * Trch enzyme peroxidase Ht sen sau khi bc v v ly tm sen s c mi mn v cn 10 g pha trong 100 mL dung dch m phosphate pH 6. Hn hp sau c trn u bng my lc v lc bng giy lc hoc ly tm lnh thu c dch trch enzyme. * o hot tnh tng i ca enzyme peroxidase Phn ng ha nu c thc hin bng cch cho ln lt vo ng nghim 2,8 mL dung dch m phosphate pH 6, sau cho thm 50 L guaiacol 27 mM v 50 L H2O2 0,8%, 100 L dch trch enzyme c cho vo sau cng quan st phn ng ha nu. Hot tnh tng i ca enzyme ha nu POD c tnh bng tc to thnh sn phm tetraguaiacol c mu nu tm thng qua s gia tng hp thu sau mi pht c o bng my o quang ph bc sng 475 nm. 6.5. KHO ST HOT TNH TNG I CA ENZYME CYANOALANINE SYNTHASE Nm 1963, BlumenthalGoldschmidt v cng tc vin m t s xc tc ca enzyme -cyanoalanine synthase (CAS) (EC 4.4.1.9) ln phn ng gia L-cysteine v HCN to thnh -cyanoalanine v H2S. Ngy nay phn ng ny ang c quan tm nh l phn ng ca qu trnh chuyn ha m.
SH CH2 H2N CH
L-cysteine -cyanoalanine synthase

CN CH2 H2N CH CO2H + H2S

CO2H + HCN

-cyanoalanine

6.5.1. Trch enzyme CAS CAS c th c trch d dng t la v cc loi cy h u. Sau khi c nghin mn trong nit lng, 1 g vt liu nghin s c trch bng 2,5 mL dung dch tris buffer lnh 0,1 M (pH 8,5). Sau khi ly tm 10000 g/ 10 pht, dung dch enzyme thu c c th sn sng c dng cho sinh trc nghim. 6.5.2. Kho st hot tnh tng i ca enzyme CAS kho st hot tnh tng i ca enzyme CAS, cho vo ng nghim 0,2 mL dch trch enzyme, ri thm vo 0,8 mL dung dch tris buffer (0,1 M, pH 8,5) cha 25 mM sodium cyanide v 3 mM L-cysteine. Hn hp c y kn bng np cao su 58
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Gio Trnh Thc Tp Sinh Ha

mm, v lc u 35C trong 30 pht. Hot tnh ca enzyme c quan st bng phn ng hin mu xanh methylene. Sau khi H2S c phng thch t L-cysteine, 100 L N, N-dimethyl-p-phenylene diamine 20 mM trong HCl 7,2 N v 100 L FeCl3 30 mM trong 1,2 N HCl c thm vo bng cch bm qua np ng nghim. Mu ca xanh methylene xut hin do s hin din ca H2S c xc nh bng my o quang ph bc sng 650 nm, s dng Na2S lm cht chun.

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CHNG 7. KHO ST ACID NUCLEIC

7.1. KHI QUT Acid nucleic l cc polymer ca nhiu mononucleotide kt hp vi nhau bng lin kt phosphodiester. Acid nucleic gm hai nhm ln: Acid deoxyribonucleic (ADN) v acid ribonucleic (ARN). ADN c khi lng phn t ln hn ARN, gm hai si polynucleotide xon ngc chiu nhau nh cc lin kt hydro gia cc base nit. ARN c cu to 1 si polynucleotide. Acid nucleic l cc polyanion, d ha tan trong dung dch kim hoc mui long, da vo tnh cht ny chit tch chng ra khi cc ngun nguyn liu sinh vt. Di tc dng ca cc enzyme nuclease hoc un nng acid nucleic vi acid, kim nhit cao, acid nucleic b thy phn thnh cc mononucleotide. Mi mononucleotide c cu to gm cc base nit, ng pentose v acid phosphoric. Base nit gm hai loi: base nit purine (Adenine v Guanine), base nit pyrimidine (Cytosine, Thymine v Uracil). Cc base nit l dn xut ca cc hp cht vng thm, c kh nng hp th nh sng vng t ngoi vi bc sng t 250nm n 280nm. ng pentose gm hai loi: ribose v deoxyribose. Cc phn ng mu c trng ca ribose (phn ng orcinol) v deoxyribose (phn ng Feugel hoc diphenylamin), l c s pht hin ADN v ARN. Trong t bo acid nucleic thng c kt hp vi protein di phc hp nucleoprotein, nn trc khi ly trch acid nucleic cn phi dng cc cht ty ra v cc cht lm bin tnh protein ADN hoc ARN d dng phng thch ra mi trng. 7.2. PHNG PHP LY TRCH ACID NUCLEIC 7.2.1. Ly trch ADN t t bo vi khun Nguyn tc Phng php ly trch c bn gm ba bc: + Ph v mng t bo v mng nhn + Loi b protein + Lm kt ta acid nucleic Ha cht - T bo vi khun ( E.Coli hoc B.Subtilus) - Dung dch mui-EDTA ( NaCl 0,15M v EDTA 0,1M, pH = 8) - Sodium dodecyl sulphate (SDS) 25% - Dung dch lysozyme 10mg/mL - Natri perchlorate 5M - Chloroform: isoamylic : 24:1 - Ethanol 95% - Dung dch m (NaCl 150mM v citrate 15mM, pH 7) 60
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Gio Trnh Thc Tp Sinh Ha

Tin hnh Nghin 2-3 gam t bo vi khun trong 25 mL dung dch mui- EDTA, thm vo 1 mL lysozyme v nhit 37oC trong 30 pht. Sau cho vo hn hp trn 2 mL SDS 25%, lc u v em un cch thy 60oC trong 10 pht. Lm lnh hn hp n nhit phng, thm vo 9 mL dung dch natri perchlorate 5M, sau dn thm nc sao cho t n nng mui 1M. Lc u, thm vo 1 th tch chloroform- isoamyl alcol 24:1 bng vi th tch dch chit (khong 40-45mL). Lc u hn hp trong 20-30 pht. Ly tm dch huyn ph trong 5 pht vi tc 13.000 vng/pht. Ht cn thn phn nc ni cha ADN trn vo 1 ng nghim khc, cho vo thnh ng nghim 2 ln th tch ethanol 95%, ly tm hn hp 3000 vng/pht trong 10 pht . Ly phn ta, tip tc cho vo 10 mL dung dch mui - EDTA ha tan ta, ra li 1 ln na vi chloroform- isoamyl alcol 24:1. Ly tm ly phn nc ni v kt ta 1 ln na vi ethanol 95%. Ly phn ta cha ADN ha tan trong 10 mL dung dch mui - citrate dng cho cc th nghim sau. 7.2.2. Ly trch ARN Nguyn tc ARN khng bn nn d b phn hy bi cc ARNase. Do khi ly trch ARN cn phi thn trng, thao tc trong iu kin v trng, eo bao tay. Ly trch ARN gm cc bc tng t nh ly trch ADN. Ho cht - Men bnh m - Phenol - Ethanol tuyt i lnh - Ethanol 70% lnh - Kali acetate - Nc ct v trng Tin hnh Cn khong 0,3 gam men bnh m trong ng eppendorf 2 mL. Ra sinh khi t bo bng cch huyn ph t bo vi 1,5mL nc ct. Trn mu u bng my mixer, ly tm 5000 vng/pht thu nhn sinh khi. Huyn ph ha sinh khi trong 0,4 mL nc ct c lm m n 37oC. nhit ny trong 15 pht. Thm vo eppendorf 0,6 mL phenol. Vortex c th nhiu ln sao cho tng thi gian vortex khong 20-30 pht, ly tm 5000 vng/pht trong 10 pht iu kin lnh. Thu ly dch cha ARN vo mt eppendorf. Ly tm tc trn 7000 vng/pht trong lnh ta protein, thu ly dch lp trn vo mt ng eppendorf khc, thm 10mg kali acetate, ha tan bng cch o v lc nh, thm vo 1mL acetate p lnh. o ng vi ln. yn 1 gi trong nc lnh. Ly tm trn 7000 vng/pht trong 15 pht iu kin lnh. Ht b dch ni thu nhn cn ta cha ARN. Ra ta ARN bng cch thm nh nhng 0,5mL ethanol 70%. Ly tm nh trn thu nhn ARN. Lm kh ARN trong bn ht chn khng. Bo qun trong t lnh di 0oC. 61
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Gio Trnh Thc Tp Sinh Ha

7.3. NH TNH ACID NUCLEIC 7.3.1.Tnh tan ca acid nucleic Acid nucleic tan tt trong mi trng kim, khng tan trong nc v dung dch acid acetic long. Trong nc acid nucleic to thnh dung dch keo, do d dng b kt ta bi cc cht ho nc. Ho cht - Dung dch ARN 0,1% nm men. - Dung dch 0,1% ADN t vi khun - HCl 0,1N. - NaOH 0,1N. - Ethanol 96% - Isopropanol Tin hnh Ly hai ng nghim, cho ln lt vo mi ng 0,5 mL ADN 0,1%; 0,5 mL ARN 0,1%, thm vo mi ng 0,5 mL HCl 0,1N, kt ta trng ca acid nucleic c to thnh. Thm tng git NaOH 0,1N kt ta ha tan tr li. Gii thch kt qu. Ly hai ng nghim, cho ln lt vo mi ng 1 mL dung dch ADN 0,1%; 1 mL ARN 0,1%, thm vo mi ng 2 mL ethanol 96% lnh hay 0,6 mL isopropanol, kt ta trng ca acid nucleic xut hin. 7.3.2. Cc phn ng mu ca acid nucleic a. Phn ng vi xanh methylene Nguyn tc: Trong mi trng acid, acid nucleic kt hp vi xanh methylene to kt ta mu xanh. Ho cht - Acid nucleic 0,1% - Acid acetic 0,1N - Dung dch xanh methylene 0,1% Tin hnh Cho vo ng nghim 1 mL dung dch acid nucleic 0,1%, thm tng git acid acetic 0,1N vo n khi dung dch hi vn c. Cho khong 5-6 git dung dch xanh methylene 0,1% to thnh kt ta xanh da tri. b. Cc phn ng mu phn bit ADN v ARN Cc phn ng ny da trn s khc bit gia hai loi ng deoxyribose v ribose ca ADN v ARN. * Phn ng ca ADN vi diphenylamine Nguyn tc: Deoxyribose c trong thnh phn ADN tc dng vi diphenylamine trong mi trng acid to thnh hp cht mu xanh hp th bc sng 595nm. Phn ng ny l c s ca phng php nh lng ADN bng phng php so mu. Ho cht: - ADN, ARN thng mi - Dung dch m (mui NaCl 150mM v natri citrate 15mM, pH 7) 62
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Gio Trnh Thc Tp Sinh Ha

- Thuc th diphenylamine (1g diphenylamine trong 100 mL acid acetic m c, thm 0,25mL acid sulfuric m c). Tin hnh: Ha tan 1mg acid nucleic trong 5mL dung dch m mui Cho vo hai ng nghim, mi ng 1mL dung dch ADN hoc ARN, thm vo mi ng 2 ml thuc th diphenylamine. Lc u, em 2 ng nghim un cch thy ang si trong 10 pht. Quan st s hnh thnh mu hai ng nghim. Gii thch. * Phn ng ca ARN vi thuc th orcinol Nguyn tc: y l phn ng ca ng ribose c trong thnh phn ARN s to thnh furfural khi un nng vi acid chlohydric m c. Sau orcinol s phn ng vi furfural vi s xc tc ca clorua st III to thnh hp cht mu xanh lc. Phn ng ny l c s ca phng php nh tnh v nh lng ARN. Ho cht - Dung dch ADN v ARN chun b nh trn - Thuc th orcinol (0,1 gam FeCl3.H2O pha trong 100 mL HCl m c, thm 3,5 mL orcinol 6% trong alcohol). Tin hnh Cho vo hai ng nghim, ln lt mi ng 1 mL dung dch ADN hoc ARN, thm vo mi ng 1,5 mL thuc th orcinol. Lc u, em 2 ng nghim un cch thy ang si trong 20 pht. Quan st s hnh thnh mu hai ng nghim. Gii thch. 7.4. NH LNG ACID NUCLEIC 7.4.1. nh lng ADN 7.4.1.1. nh lng ADN bng cch o hp th nh sng bc sng 260nm Nguyn tc Trong thnh phn ADN cha cc base nit c kh nng hp th nh sng vng t ngoi v hp th cc i bc sng 260nm, hp th A t l vi nng ADN trong dung dch. Ha cht - Dung dch ADN chun 50g/mL - Dung dch ADN va ly trch Thc hnh Cho 1mL dung dch ADN (chun hoc mu phn tch) vo cuvette thch anh v o hp th nh sng 260nm. Mu i chng l dung dch dng pha ADN. Ch : nu hp th mu ADN phn tch ln hn 1 th cn pha long dung dch. T hp th A ca mu ADN chun v mu ADN cn phn tch tnh ra lng ADN c trong dung dch cn phn tch: Ta c: Ax = Cxl Ach = Cchl Khi l = hng s
AX C X A = C X = C ch X Ach C ch Ach

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Gio Trnh Thc Tp Sinh Ha A hay : C X = C ch X n Ach

trong : Ax: hp th ca dung dch ADN cn phn tch 260nm Ach : hp th ca dung dch ADN chun 260nm Cch : nng dung dch ADN chun (g/mL) Cx: nng dung dch ADN cn phn tch (g/mL) n : h s pha long Lu : Phng php ny n gin, nhanh, nhng c hn ch l hp th A c th b nh hng khi trong mu cha ARN v protein. V vy, thc t phng php ny thng c s dng nh lng mu ADN tinh khit. 7.4.1.2. nh lng ADN theo phng php Dise Nguyn tc: Khi un nng dung dch ADN vi thuc th diphenylamine to thnh hp cht c mu xanh hp th bc sng cc i 595nm. Ho cht - Dung dch ADN chun 500g/mL - Dung dch ADN cn phn tch - HClO4 0,5N (hoc TCA 5%) - Thuc th diphenylamine Tin hnh a. Xy dng ng chun ADN Pha dy dung dch ADN c nng t 0 - 500 (g/mL) t dung dch ADN chun 500 (g/mL). Sau cho cc ha cht theo bng sau: ng nghim Nng ADN (g/mL) Th tch dung dch ADN gc (L) Th tch dd m (mL) Th tch dd HClO4(mL) 1 0 0 400 1,6 2 50 40 360 1,6 3 100 80 320 1,6 4 200 160 240 1,6 5 300 240 160 1,6 6 400 320 80 1,6 7 500 400 0 1,6

y kn v lc u cc ng nghim, em un cch thy ang si trong 30 pht. Sau lm ngui dung dch ri thm vo mi ng nghim 4 mL thuc th diphenylamine. Tip tc un cch thy ang si 20 pht. ngui v o hp th bc sng 595nm. V th tng quan gia hp th v nng ADN. b. Chun b mu Tin hnh tng t nh cc ng ADN chun, nhng thay vo l dung dch ADN cn phn tch. em o hp th bc sng 595nm T kt qu hp th, i chiu vi th chun ADN tnh ra lng ADN trong mu. 64 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

7.4.2. nh lng ARN 7.4.2.1. nh lng ARN bng cch o hp th nh sng bc sng 260nm Nguyn tc: tng t nh phng php nh lng ADN Ha cht - Dung dch ARN chun 40g/mL - Dung dch ARN va ly trch Tin hnh Cho 1mL dung dch ARN (chun hoc mu phn tch) vo cuvette thch anh v o hp th nh sng 260nm. Mu i chng l dung dch dng pha ARN. Ch : nu hp th mu ARN phn tch ln hn 1 th cn pha lang dung dch. T hp th A ca mu ARN chun v mu ARN cn phn tch tnh ra lng ARN c trong dung dch cn phn tch: Ta c: Ax = Cxl Ach = Cchl Khi l = hng s hay : C X = C ch
AX n Ach AX C X A = C X = C ch X Ach C ch Ach

trong : Ax: hp th ca dung dch ARN cn phn tch 260nm Ach : hp th ca dung dch ARN chun 260nm Cch : nng dung dch ARN chun (g/mL) Cx: nng dung dch ARN cn phn tch (g/mL) n : h s pha lang Phng php ny c nhng u v nhc im nh i vi phng php nh lng ADN. 7.4.2.2. nh lng ARN theo phng php Meibaum Nguyn tc: Khi un nng dung dch ARN vi thuc th orcinol s to thnh hp cht c mu xanh l cy hp th bc sng cc i 670nm. Cng mu t l vi hm lng ARN trong dung dch. Ho cht - Dung dch ARN chun 500g/mL - Dung dch ARN cn phn tch - Dung dch H2SO4 10% - Thuc th orcinol Tin hnh a. Xy dng ng chun ARN Pha dy dung dch ARN c nng t 0 500 (g/mL) t dung dch ARN chun 500 (g/mL). Sau cho cc ha cht theo bng sau: 65
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Gio Trnh Thc Tp Sinh Ha

ng nghim Nng ARN (g/mL) Th tch dd ARN gc (L) Th tch dd m (mL) DD H2SO4 10% (mL)

1 0 0 400 1,6

2 50 40 360 1,6

3 100 80 320 1,6

4 200 160 240 1,6

5 300 240 160 1,6

6 400 320 80 1,6

7 500 400 0 1,6

y kn v lc u cc ng nghim, em un cch thy ang si trong 30 pht. Sau lm ngui dung dch ri thm vo mi ng nghim 2 mL thuc th orcinol. Lc u v tip tc un cch thy ang si 20 pht. ngui v o hp th bc sng 670nm. V th tng quan gia hp th v nng ARN. b. Chun b mu Tin hnh tng t nh cc ng ARN chun, nhng thay vo l dung dch ARN cn phn tch. em o hp th bc sng 670nm. T kt qu hp th o c, i chiu vi th chun ARN tnh ra lng ARN trong mu.

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CHNG 8. PH CHNG

8.1. XC NH ACID BO BNG SC K KH Sc k kh l mt phng php nhanh v chnh xc dng nh tnh v nh lng nhiu hp cht da trn cc cht chun. Sau y xin gii thiu mt phng php xc nh acid bo bng sc k kh. Dng c v ha cht - My sc k kh - ng bm mu (syringe) - Bp un cch thy - Bnh tam gic 100ml - Bnh chit 250ml - Phu lc - Giy lc - My c quay chn khng - KOH 0,5N - Boron trifluoride methanol - Hexan - Na2SO4

Tin hnh Methyl ha mu Cn 100g du thc vt trong bnh tam gic 100 mL c np y (v d nh du u phng c trch bng my Soxhlet). Tip tc cho thm 5 mL KOH 0,5N ri un cch thy 800C trong 5 pht. Thm vo 5ml hn hp boron trifluoride methanol v tip tc un cch thy 800C trong 5 pht. Cho thm 5 mL n-hexane v tip tc un cch thy 800C trong 1 pht. Thm vo bnh tam gic 50 mL hexane s thu c mt hn hp phn lp. Dng bnh chit tch hai dung dch trong hn hp. Lp di l dung dch mui bo ha. Lp trn l dung dch cha acid bo trong hexane s c dehydrate ha bng cch thm vo mt t Na2SO4 ri lc bng giy lc. Dung dch lc thu c s c em c quay chn khng ri thm vo 5 mL hexane thu c dung dch methyl ha sn sng dng cho sc k kh. Phn tch bng sc k kh Sc k kh c chun b trong iu kin sau: - Ct mao qun PEG-20M (kch thc 30m x 0,25mm). - Detector: FID (Flame Ionization Detector). - Nhit khi u (initial temperature): 1450C. - Thi gian khi u (initial time): 5 pht. - Nhit lc bm mu (injection temperature): 2100C. - Chng trnh gia nhit (program rate): 40C/ pht. - Nhit ca detector (detector temperature): 2100C. - Nhit cui cng (final temperature): 2100C. - Thi gian phn tch (final time): 40 pht. - Kh mang (carrier gas): He. 67 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

- Cc thng s cung cp kh np vo my sc k kh: + Ngun kh He: 0,5 Mpa. + Khng kh: 0,5 kg/ cm2. + H2: 0,6 kg/ cm2. + Carrier (P1): 1 kg/ cm2. + Carrier (P2): 1,8 kg/ cm2. - Sau khi kim tra cc thng s trn my hon chnh c th bm mu chun vo phn tch. Sau khi phn tch mu chun th bm tip mu phn tch. Th tch mu bm vo c methyl ha trong hexan (injection volumn) l 1L. So snh thi gian lu mu (retention time) ca mu phn tch v mu chun bi cc nh (peak) ca ng biu din suy ra loi acid bo tng ng. Lng acid bo ca mu phn tch c xc nh bng cch so snh din tch cc nh ca ng biu din mu chun vi mu phn tch c thi gian lu tng ng nhau c cho bi my sc k kh sau khi phn tch. Nng ca mt acid bo Cn no c th c tnh nh sau:

Sn x Cs Cn = Ss
Trong : - Cn l nng cht cn tm - Sn l din tch ca ng biu din mu phn tch n - Cs l nng ca cht chun - Ss l din tch ca ng biu din mu chun 8.2. XC NH VITAMIN BNG PHNG PHP SC K LNG CAO P (HPLC) Cc thit b phn tch ngy cng c ph bin v thay dn cc phng php phn tch c. Ni dung ca phn ny nhm a ra cho ngi hc cc phng php phn tch vitamin c bn bng my sc k lng cao p m phng th nghim chuyn su c trang b. Cc phng php phn tch sau da trn iu kin c bn ca my sc k lng ca nh sn xut Shimadzu v Hitachi. 8.2.1. Phn tch vitamin A v vitamin D Vitamin A v vitamin D l vitamin thuc nhm tan trong cht bo. Bi thc hnh s dng vitamin A acetate v vitamin D3. Dung mi dng pha hai vitamin ny l acetonitrile v methanol theo t l 75:25 (v/v). - Chun b mu: Dng hai ng nghim nh c np c 2 mL. Cn ln lt 1g vitamin A v 1g vitamin D cho vo mi ng nghim. Tip tc cho 2 mL dung dch acetonitril v methanol vo mi ng nghim ha tan vitamin, y np ng nghim v trn u bng my lc. Mu sn sng cho phn tch. - Chun b my sc k: C th dng ct sc k Inertsil ODS-80A 5M (250 x 4,6mm). Ct sc k c ra bng 70% MeOH vi bm B (pump B) ti thiu l 1 gi. 68
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Gio Trnh Thc Tp Sinh Ha

Dung mi cho pha di ng l acetonitrile : methanol theo t l 75:25 (v/v), tc bm 1 mL/ pht (pump A). Nhit ct sc k 40C (Column oven). Detector: UV 280 nm. - Tin hnh phn tch: Mu cha vitamin s c bm vo my vi th tch l 1L. Vitamin A v vitamin D c phn tch trong cng mt iu kin nu trn vi hai mu c phn tch ring l. Vi iu kin phn tch nu trn th vitamin A acetate s cho thi gian lu (retention time) khong 7,2 pht v vitamin D3 s cho thi gian lu khong 2,4 pht. 8.2.2. Phn tch vitamin E Vitamin E l vitamin thuc nhm tan trong cht bo. Vitamin E c th c trch t du thc vt nh du u phng. - Chun b mu: Cn 2 g du u phng c trch bng phng php Soxhlet. Sau cho vo ln lt 0,5 mL dung dch mui NaCl 1%, 10 mL pyrogallolethanol 3% v 1 mL dung dch KOH 60% ri un 70C trong 30 pht sau lm ngui nhit phng th nghim. Tip tc cho 22,5 mL dung dch mui NaCl 1%, v 15 mL dung dch ethyl acetate-hexane ri trn mu u bng my lc trong 5 pht. Mu sau s c em ly tm 2000 rpm trong 5 pht. Sau khi ly tm s thu c vitamin E trong dung mi phn bn trn ng nghim, phn bn di cha nc. Rt ly phn bn trn ca ng nghim v tip tc cho vo 15 mL dung dch ethyl acetatehexane ri trn mu u bng my lc trong 5 pht. Mu sau s c em ly tm 2000 rpm trong 5 pht. Qu trnh ny c th lp li trong 2-3 ln thu c mu tt sn sng dng cho phn tch. - Tin hnh phn tch: Mu sau khi c trch s c lm kh bng kh nit ri ha tan bng 50 mL hexane s dng cho phn tch bng HPLC. My sc k lng cao p s c lp vi ct sc k Finepak SIL 5m (250 x 4.6 mm) v m nhit 400C. Trc khi phn tch, ct sc k c ra bng hexane vi tc bm t 1,2 1,5 mL/ pht (pump A). Tin trnh phn tch c thc hin vi pha di ng cha acid acetic : 2-propanol : n-hexan (5 : 6 : 1000 v/ v/ v) vi tc 1,5 mL/ pht (pump B). Detector: Fluorescent detector (excitation 298 nm, emision 325 nm). Mu cha vitamin E s c bm vo my vi th tch l 1L phn tch. Mu vitamin E chun cng c tin hnh tng t so snh vi mu phn tch. 8.2.3. Phn tch vitamin K Vitamin K l vitamin thuc nhm tan trong cht bo. y gii thiu phng php phn tch vitamin K vi mu chun l vitamin K1. - Chun b mu: Cn 1 mg vitamin K1 pha trong 2 mL CH3CN. Ha tan mu v trn u bng my lc. Mu sn sng dng cho phn tch. - Tin hnh phn tch: My sc k lng c lp vi ct nhi COSMOSIL/ COSMOGEL 5m (250 x 4,6mm). Ct sc k c ra trc vi CH3CN. Pha di ng c tin hnh vi CH3CN, tc bm 1,5 mL/ 1 pht. Ct sc k c m nhit 40C. Detector: UV 254 nm. Th tch mu bm l 20 L. Quan st v ghi nhn thi gian lu mu. 8.2.4. Phn tch acid nicotinic (vitamin B3) Acid nicotinic l vitamin thuc nhm B tan trong nc. Vic phn tch c th tin hnh bng sc k lng cao p. 69 http://www.ebook.edu.vn

Gio Trnh Thc Tp Sinh Ha

- Chun b mu: Cn 1g acid nicotinic ri pha trong 2 mL methanol 70% trn mu u bng my lc dng cho phn tch. - Tin hnh phn tch: My sc k lng c lp vi ct Inertsil ODS-80A 5M (250 x 4,6mm). Ct sc k c ra trc vi methnol. Pha di ng c tin hnh vi methanol 70%, tc bm 1 mL/ 1 pht. Ct sc k c m nhit 40C. Detector: UV 210 nm. Th tch mu bm l 1L. Quan st v ghi nhn thi gian lu mu. Nu n nh iu kin phn tch nh trn th thi gian lu mu s vo khong 3,7 pht. 8.3. PHNG PHP XC NH M m l lng nc t do c trong thc phm. y l ch tiu quan trng trong vic phn tch xc nh gi tr dinh dng v cht lng thc phm Phng php thng dng nht xc nh m thc phm dng rn l sy kh. Nguyn tc: Dng sc nng lm bay ht hi nc trong mu phn tch. Cn trng lng mu trc v sau khi sy kh, t suy ra phn trm nc c mt trong mu phn tch. Dng c thit b: - T sy iu chnh c nhit - Cn phn tch chnh xc n 0,0001g - Bnh ht m, pha di cht ht m ( H2SO4, silicagel,...) - Cc cn s hoc a nhm Tin hnh: Chun b mu: Mu c nghin vi kch c khang 0,75 mm (hoc 2mm i vi mu phn tch nhy vi nhit. Ly cc cc cn hoc a nhm v mt a thy tinh dp u em sy 100oC105oC cho n trng lng khng i. ngui trong bnh ht m v cn chnh xc n 0,001g Cho khang 2 gam mu phn tch vo cc cn. Cn tt c vi chnh xc nh trn. Dng que thy tinh dn u thnh lp mng. Cho tt c vo t sy 100oC-105oC, sy kh n trng lng khng i, thng thng khang 6 gi. Trong thi gian sy, c sau 1 gi li dng a thy tinh nghin nh cc phn vn cc, sau dn u v tip tc sy. Sy xong, em lm ngui bnh ht m (25-30 pht) v em cn phn tch vi chnh xc 0,0001 g Cho li mu vo t sy 100oC-105oC trong 30 pht, ly ra ngui bnh ht m v cn nh trn n trng lng khng i. Kt qu gia 2 ln cn lin tip khng c cch qu 0,5 mg cho mi gam cht th. Tnh kt qu: X=
(G1 G2 ) x100 G1 G

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trong : - G : trng lng ca cc cn v a thy tinh - G1: trng lng ca cc cn, a thy tinh v trng lng mu phn tch trc khi cn - G2: trng lng ca cc cn, a thy tinh v trng lng mu phn tch sau khi sy n trng lng khng i Ch : - Mt mu phn tch phi c lp li t nht 2 ln, kt qa cui cng l trung bnh cng ca kt qu hai ln xc nh song song. Sai lch gia kt qu hai ln xc nh song song khng c ln hn 0,5%. - Phng php sy kh thng a n kt qu khng chnh xc cho mu phn tch c cha tinh du, cn, acid bay hi, urea hoc thc phm c cha nhiu ng, m. 8.4. PHNG PHP XC NH HM LNG TRO Tro l thnh phn cn li ca thc phm sau khi nung chy hon ton ht cc cht hu c. Tro thc cht l cc loi mui khong, do mu phn tch c ln cc cht bn (t, ct) cn c loi tr trc khi em nung. 8.4.1. Hm lng tro tan phn Nguyn tc: Dng sc nng 550oC-600oC nung chy hon ton cc cht hu c. Phn cn li em cn v tnh ra phn trm tro c trong mu phn tch. Dng c thit b: - L nung iu chnh c nhit n 550oC-600oC - Cn phn tch chnh xc n 0,0001g - Bnh ht m, pha di cht ht m ( H2SO4, silicagel,...) - Chn nung bng s hoc kim loi kn, bch kim - Nc oxy gi (H2O2) 30% hoc HNO3 m c Tin hnh: Nung chn s ra sch l nung ti 550oC-600oC n trng lng khng i. ngui bnh ht m v cn chnh xc n 0,0001g Cho khong 2 gam mu phn tch vo chn s. Cn tt c vi chnh xc nh trn. Cho tt c vo l nung sy n 600oC. Nung cho n khi tro trng, ngha l ht cc cht hu c, thng thng khang 6-7 gi ty loi mu. Trng hp tro cn en, ly ra ngui, cho thm vi git H2O2 30% hoc HNO3 m c v nung li cho n khi tro trng. ngui bnh ht m v cn n chnh xc nh trn. Tip tc nung thm nhit trn trong 30 pht ri ngui trong bnh ht m v cn cho ti trng lng khng i. Kt qu gia 2 ln nung lin tip khng c cch qu 0,5 mg cho mi gram cht th. 71
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Gio Trnh Thc Tp Sinh Ha

Tnh kt qa: Hm lng tro ton phn: X= trong : - G: trng lng ca chn s - G1: trng lng ca chn s v trng lng mu phn tch trc khi cn - G2: trng lng ca chn s v trng lng tro trng sau khi nung n trng lng khng i 8.4.2. Xc nh hm lng tro ha tan v khng ha tan trong nc Tin hnh nung mu phn tch nh mc 9.2.1., sau ha tan tro ton phn vo nc ct si. Lc qua giy lc khng tro v hng dch lc vo mt chn s nung, ngui v cn sn. Ra li phn tro khng tan, giy lc v phu bng nc ct si nhiu ln. Dch lc cho ht vo chn s em bc hi nc 100oC, sau em nung n tro trng 550oC-600oC trong 30 pht. ngui trong bnh ht m v cn chnh xc n 0,0001g Tnh kt qu Hm lng tro tan trong nc: X2 = trong : - G * : trng lng ca chn s
* : trng lng ca chn v trng lng tro tan trong nc - G2
* (G2

(G2 G ) x100 G1 G

G * ) x100 P

- P: trng lng mu phn tch T kt qu trn ta c th suy ra hm lng tro khng tan trong nc: X3 = X1-X2 trong : X1 : hm lng tro ton phn X2 : hm lng tro tan trong nc

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TI LIU THAM KHO


- Tsumura T. et al. 1993. Rapid enzymatic assay for ascorbic acid in various foods using peroxidase. Journal of Food Science. 58(3): 619-622. - Phng th nghim ha hc thc phm, Trng i Hc Nihon, Nht bn. 2005. Phng php th nghim sinh ha thc phm (nguyn bn ting Nht). - Becker J.M., Caldwell G. A., Zachgo E.A. 1996. Biotechnology A laboratory Course. Academic Press. - nh H, ng Th Hoi An, Nguyn Th Ho, Phm Th Mai, Trn Thanh Lan Phng, Th Thanh Thy v L Xun Trng (i Hc Y Dc Thnh Ph H Ch Minh). 2003. Ha sinh y hc. Nh Xut Bn Y Hc. - Hames B. D. and Hooper N.M. 2000. Instant notes biochemistry (second edition). BIOS Scientific Publishers Limited. - Nguyn c Lng. 2003. Th nghim cng ngh sinh hc (tp 1). NXB i Hc Quc Gia thnh ph H Ch Minh. - Nguyn Vn Mi. 2001. Thc hnh ha sinh hc. Nh Xut Bn i Hc Quc Gia H Ni. - Price Nicholas C. and Stevens Lewis. 1999. Fundamentals of enzymology (third edition). Oxford University Press. - Phm Th Trn Chu, Nguyn Th Hin v Phng Gia Tng. 1997. Thc hnh ho sinh hc. Nh xut bn gio dc. - Phm Thu Cc. 2001. Gio trnh sinh ho phn I. T sch i Hc Cn Th. - Phm Thu Cc. 2002. Gio trnh sinh ho phn II. T sch i Hc Cn Th. - Schopfer Mohr. 1995. Plant physiology. Springer. - T Sinh Ho (B mn Cng Ngh Thc Phm, Khoa Nng Nghip, Trng i Hc Cn Th). 2003. Gio trnh thc tp sinh ho. Ti liu lu hnh ni b. - Zubai Geoffrey. 1998. Biochemistry (fourth edition). WCB McGraw-Hill.

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