Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

MC LCCHNG I: PROTEIN.......................................................................................................................................2 BI 1: PHN NG BIURE.........................................................................................................................2 BI 2: KT TA THUN NGHCH BNG MUI TRUNG TNH.........................................................4 BI 3: PHNG PHP SORENSEN.........................................................................................................6 (PHNG PHP XC NH M FORMOL )..............................................................................................................6 BI 4: NH LNGPROTEIN BNG PHNG PHP KJELDAHL..................................................7 (GING TRN M HNH).....................................................................................................................................7 CHNG II: ENZYME......................................................................................................................................9 BI 1: NH HNG CA NHIT N HOT TNH CA ENZYME..........................................9 BI 2: NH HNG CA CC CHT HOT HA V KM HM N HOT TNH CA ENZYME...................................................................................................................................................10 BI 3: XC NH HOT -AMYLASE THEO PHNG PHP WOHLGEMUTH...................11 CHNG III: GLUCIDE.................................................................................................................................13 BI 1: PHN NG VI THUC TH FEHLING..................................................................................13 BI 2: PHN NG SELIWANOFF..........................................................................................................15 BI 3: XC NH NG KH BNG PHNG PHP QUANG PH K....................................17 CHNG IV: VITAMIN..................................................................................................................................19 BI 1: PHNG PHP NH TNH VITAMIN A.................................................................................19 BI 2: PHNG PHP NH TNH VITAMIN C.................................................................................20 BI 3: PHNG PHP NH LNG VITAMIN C.............................................................................21 BI 1: S NH TNG HA LIPIDE...................................................................................................23 BI 2: PHN NG X PHNG HA.....................................................................................................24 BI 3: XC NH CH S IOD CA CHT BO.................................................................................25 BI 4: XC NH CH S ACIDE CA CHT BO............................................................................27 BI 5: XC NH LIPIT TNG S........................................................................................................28 (GING TRN M HNH)...................................................................................................................................28

- 2010 -

1

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Chng I: PROTEINBi 1: PHN NG BIURENguyn tc: y l phn ng c trng ca lin kt peptide (CO-NH-) Trong mi trng kim, cc hp cht c cha t hai lin kt peptide tr ln c th phn ng vi CuSO4 to thnh phc cht mu xanh tm, tm, tm hay . Cng mu thay i ty thuc vo di mch peptide Phn ng ny thng thng cng dng nh lng protein

Nguyn liu v ha cht: Dung dch lng trng trng 1% Ure tinh th Dung dch NaOH 10% Dung dch CuSO4 1%

Cch tin hnh & kt qu:iu ch biure: cho vo ng nghim kh mt t tinh th ur, ung trn ngn la yu. Lc u ur nng chy, n khi bt u cng li th ngng un. Qu trnh ny to ra biure, acide cianuric v amoniac Dng 2 ng nghim:

ng nghimng 1 (ng biure)

Cho vo mi ng nghim 1ml dd NaOH 10% v 1-2 git CuSO4 1%Dung dch tm

ng 2 (3ml dd lng trng trng 1%)

Dung dch mu tm

PTP: (CO-NH-)+ Cu(OH)2 Ch khng cho qu nhiu CuSO4 v mu xanh ca Cu(OH)2 c to thnh Gii thch:

- 2010 -

2

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Thuc th ca phn ng mu biure l dung dch NaOH v CuSO4, i khi n cn c gi l tc cht biure Tuy nhin ch biure y khng phi l thuc th c cha biure m l v c biure v protein u cho phn ng mu ging nhau vi NaOH v CuSO4, Cng mu thay i ty thuc vo di mch peptide Phn ng so mu Biure, lin kt peptide vi Cu2+ trong MT kim, l phn ng c trng nhn bit protein

Nguyn tc cc phn ng mu khc ca protein: Phn ng xantoproteic: c trng vi cc axit amin vng thm. Cc gc amino acid Tyr,Trp,Phe trong protein tc dng vi HNO3 c to thnh mu vng v sau khi thm kim s chuyn thnh da cam Phn ng ninhydrin: c trng vi cc - axit amin. Phn ng Pholia: c trng vi cc axit amin cha lu hunh Phn ng Millon : pht hin Tyrosin. gc Tyr tc dng vi thy ngn nitrate trong HNO3 c to thnh kt ta mu nu t Phn ng Folin: pht hin Tyrosin, Tryptophan. Phn ng Sakaguchi: pht hin Arginin. Gc Arg tc dng vi dung dch kim ca -naphtol v hypobromite cho mu anh o

- 2010 -

3

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 2: KT TA THUN NGHCH BNG MUI TRUNG TNHNguyn tc: Cc mui ca kim loi kim v kim th (thng dng l (NH4)2SO4 , Na2SO4, NaCl, MgSO4) c tc dng gy kt ta thun nghch protein. Sau nu loi b nhanh cc yu t gy kt ta ,protein tr v trng thi dung dch keo bn. Cc protein khc nhau c th b kt ta v cc nng mui khc nhau,v vy c th dng mui tch ring cc protein ra khi hn hp ca chng.

Nguyn liu v ha cht: Dung dch lng trng trng khng pha long. Dung dch (NH4)2SO4 bo ha . Tinh th NaCl. Nc ct.

Cch tin hnh & kt qu:1) Cho vo ng nghim 3ml lng trng trng + 3ml dd (NH4)2SO4 bo ho, lc u n khidung dch bn bo ho v xut hin kt ta globulin. 5 pht, lc b kt tu ( thm t giy lc bng dd (NH4)2SO4) vo mt ng nghim khc . Cho vo dch lc 3g tinh th (NH4)2SO4 (nu dch lc l 4ml) cho n khi dung dch bo ho, lc, albumin kt ta,lc, thu kt ta Cho ring kt ta globulin v albumin vo 2 ng nghim tng ng, thm vo 2 ng t 23ml nc ct, lc u. Kt qu: ng 1: tc l ng cha kt ta globulin ,sau khi cho nc ct vo th kt ta tan t t trong nc ct . Do Globunlin vn dng kt ta , l do Globulin b bin tnh ,khng quay v trng thi dung dch keo ngay c ( hin tng kt ta khng thun nghch ). ng 2 : tc l ng cha kt ta albumin,sau khi cho nc ct vo th kt ta tan ngay trong nc ct. Do Albumin tr v trng thi dung dch keo nh ban u, kt ta tan hon ton (hin tng kt ta thun nghch). 2)Cho vo ng nghim 3ml lng trng trng , cho tip NaCl vo ,dng a thu tinh khuy cho n khi dung dch bo ho. yn 5 pht th thy xut hin kt ta a . Lc kt ta sau acid ho phn dch lc bng cch thm vo 1 git CH3COOH 1% ,th thy xut hin kt ta b. Kt qu: Kt ta a l Globulin. Kt ta b l Albumin.

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4

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Gii thch : Mi loi protein kt ta cc nng khc nhau .Khi cho NaCl vo ,u tin Globulin kt ta trc .Sau lc ht kt ta ,cho vo dch lc CH3COOH 1%, pH gim lm xut hin kt ta Albumin.

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5

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 3: PHNG PHP SORENSEN (Phng php xc nh m formol)Nguyn tc: Cc amino acide c th phn ng vi formol trung tnh to thnh dn xut metylenic.Kt qu l nhm amin mt tnh cht c bn ca n ,ngc li nhm cacboxyl trong amino acide tn ti dng metylen t do c th chun vi NaOH . Phng php c ng dng trong trng hp nhm cacboxyl v nhm amin t do bng nhau.

Nguyn liu v ha cht: khi dng). Dung dch formol (pha formol : nc ct t l 1:1 v ch chun b trc Nc mm,nc ct. Dung dch NaOH 0,1N. Thuc th Phenolphtalein 3%.

Cch tin hnh & kt qu:Ly 2 bnh nn v nh du bnh 1 v bnh 2 Bnh 1 : Dng lm bnh kim tra. Cho vo 50ml dd formol v thm vo 3 git phenolphthalein 3%.Sau dng dd NaOH 0,1N hiu chnh mu trong bnh cho n khi ch cn mu hng nht. Ta c formon trung ha Bnh 2 : Dng lm bnh th nghim. Cho vo 10ml dch mu ( c pha long t 5 n 20 ln ), thm 10ml dd formol trung ho v 3 git phenolphthalein 3%, lc u cho phn ng xy ra hon ton. Sau chun bng dd NaOH 0,1N cho n khi dd trong bnh 2 c mu hng (m hn mu bnh 1) Thc hin 3 th tht v 3 th khng(thay dd m bng nc ct), ly tr s trung bnh:

Th khng Ln 1 Ln 2 Ln 3 Trung bnh0,4 0,5 0,4 V1= 0,45ml = 0,00045 l

Th tht10,6 10,7 10,5 V2= 10,6ml = 0,0106 l

Lng m formol c trong 1l nguyn liu : M(g/l) = 1,4 ( V2 V1 )/a Trong : V1 l th tch NaOH 0,1N trung bnh ca 3 ln th khng V2 l th tch NaOH 0,1N trung bnh ca 3 ln th tht a l lng mu (ml) ng vi 10ml dch mu pha long x ln (a= 1 ml = 0,001 l). M(g/l) = 1,4 (0,0 106 0,00045 )/0.001 = 14,21

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6

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 4: NH LNGPROTEIN BNG PHNG PHP KJELDAHL (Ging trn m hnh)Nguyn tc: Di tc dng ca H2SO4 c nhit co ,cc hp cht hu c c cha nit b phn hy v b oxy ha n CO2 v H2O ,cn nit chuyn thnh ammoniac v tip tc kt hp vi H2SO4 to thnh mui amoni sulphate . Qu trnh c tin hnh theo cc bc sau : V c ha mu : R-CHNH2 COOH + H2SO4 CO2 + H2O + (NH4)2SO4 Ct m : (NH4)2SO4 + 2 NaOH Na2SO4 + 2NH3 + 2H2O Sau lng NH3 c hi nc li cun bng mt dng c l my Parnas Wargner (my chng ct m )v c dn n mt bnh tam gic c cha mt lng thi H2SO4.T y cho php chng ta xc nh c lng NH3 phng thch ra ,c ngha l xc nh c lng m c trong mu nguyn liu : NH3 + H2SO4 (NH4)2SO4 + H2SO4d Chun H2SO4 d bnh hng bng dung dch NaOH 0,01N

Nguyn liu v ha cht: Vt phm c cha Nit ( nc mm ,u h ,cc loi thc phm ) H2SO4m c. K2SO4 tinh khit. CuSO4 tinh khit. Se. NaOH 40%. H2SO4 0,1N : 2,8ml H2SO4 m c (d=1,84/lt). NaOH 0,1N : 4g NaOH tinh th/lt. Phenolphtalein 1% trong cn . HCl long . (Nn dng ng chun chun b H2SO4 v NaOH 0,1N)

Cch tin hnh & kt qu: u tin ht 1ml (i vi nguyn liu lng ) hay cn chnh xc 1g (i vi nguyn liu kh nghin nhuyn ) cho vo bnh Kjendanl ,cho thm 10ml H2SO4 m c (d=1,84). tng qu trnh v c ha (t chy) cn cho thm cht xc tc.Tt nht l dng 0,5g hn hp K2SO4 : CuSO4 : Se (100:10 :1). C th dng mt mnh Se kim loi 0,05g hoc dng hn hp CuSO4 v K2SO4 hoc acid perchloric. Hn hp xc tc - 2010 7

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

c tc dng lm tng nhit si,do lm tng vn tc ca qu tnh phn ng. Sau khi thm cc cht xc tc un nh hp cho n khi dunh dch hon ton mt mu. Ch un mnh khi hn hp hon ton chuyn sang dch lng. Trong qu trnh un thnh thong lc nh,trng kho lo sau cho khng cn mt vt en no ca mu nguyn liu th nghim cha b phn hy st li trn thnh bnh .un cho ti khi dung dch hon ton trng. Sau chuyn ton b dung dch sau khi v c ha xong bnh Kjendahl vo bnh nh mc 100ml ,thm nc ct cho n ngn chia,lc u .Dng c ct trc khi s dng phi ra sch : ly vo bnh tam gic 10ml H2SO4 0,1N ,thm vi git ch th phenolphthalein v lp vo my ct nh m hnh . un si nc trong bnh cu to hi nc ,m nc v ng sinh hn .Sau qua phu cho vo bnh ct 10ml dd th nghim t bnh nh mc v tip 8-10ml dung dch NaOH 40% .Trng phu bng mt lng nh nc ct ,ri ng kha .Hi nc t bnh cu sc qua bnh phn ng v ko theo NH3 sang bnh hp ph . Qu trnh ct kt thc sau 15 pht .nh phn lng H2SO4 d bng dung dch NaOH 0,1N. Sau khi ly bnh hp ph t bnh nc ct vo ,ng kha,ng thi m kha bnh ra .Dung dch chuyn t bnh ct sang bnh ra ,tho b dung dch bn i .Sau khi th nghim xong phi ra sch my ct vi lng 1 ln bng HCl long v nhiu ln bng nc ct . Tnh kt qu : Hm lng nit tnh theo cng thc sau : (a b).0,0014.10000 .100 X = 10.V X: hm lng nit (g/l) a: s mol H2SO4 0,1N em hp th NH3 b: s mol NaOH 0,1N tiu xchun tiu tn cho chun s V: s ml phm vt em v c ha 0,0014: lng g nit ng vi 1ml H2SO4 0,1N

- 2010 -

8

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Chng II: ENZYMEBi 1: NH HNG CA NHIT N HOT TNH CA ENZYMENguyn tc: Nhit c nh hng quan trng n tc phn ng ca enzyme. S tng tc phn ng ca enzyme bng cch nn nhit ch c hiu qu trong mt khong nhit tng i hp Nhit ti thch ca phn ln cc enzyme l trong khong 40-600C. nhit trn 800C hu ht cc enzyme b bin tnh khng thun nghch

Cch tin hnh & kt qu:Chun b 4 ng nghim, cho vo mi ng 2ml dd tinh bt 1% ng 1 : gia nhit 85-900C ng 2 : cho vo nc ng 3 : t m 45-500C ng 4 : nhit phng Gi cc ng trong 15 pht dd trong ng t n cc nhit tng ng Tip tc cho vo mi ng 0,5ml dd amylase cng t cc nhit trn v t cc ng nghim cc nhit c Sau 1, 2, 4, 6, 8 v 12 pht ly t mi ng 1 git, nh vo cc git thuc th Lugol chun b sn trn bn knh. Quan st mu to thnh

ng 1 1 pht 2 pht 4 pht 6 pht 8 pht 12 phtXanh en Xanh en Xanh en Xanh en Xanh en Xanh en

ng 2Tm en en nht Nu en Nu en Nu t Nu en

ng 3en Tm en Nu nht Nu nht nht Nu nht hn Nu rt nht

ng 4en m Nu t Nu t nht Nu t nht Nu t nht hn Mu ca lugol

Qua bng trn ta thy : trong ng 1 v ng 2 th mu dd tinh bt thay i khng ng k, trong ng 3 v ng 4 mu ca dd tinh bt thay i nh enzyme thy phn Gii thch : Nhit c nh hng quan trng n tc phn ng ca enzyme. Nhit ti thch ca phn ng enzyme nm trong phm vi 40-500C v c th bin i ph thuc vo nhiu yu t nh sch ca enzyme, thi gian phn ngNhit m enzyme b mt hon ton hot tnh xc tc gi l nhit ti hn, thng vo khong trn 70 0C. nhit ti

- 2010 -

9

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

hn, enzyme b bin tnh, t khi c kh nng c hi phc li hot . Ngc li, di 00C, hot enzyme tuy b gim nhng li c th tng ln khi a v nhit bnh thng

Bi 2: NH HNG CA CC CHT HOT HA V KM HM N HOT TNH CA ENZYMENguyn tc:Cht hot ha c kh nng lm tng cng tc dng ca enzyme. Cht km hm l gim i lc ca enzyme vi c cht hay lm enyme mt kh nng kt hp vi c cht

Cch tin hnh & kt qu:Chun b 3 ng nghim:

ng nghimng 1 1ml nc ct

Sau cho vo mi ng 1ml nc bt v 1ml dd tinh bt, lc u. Sau vi pht, thm vo mi ng vi git thuc th Lugol, lc uMu vng hi m

ng 2

1ml dd NaCl 1%

Mu vng nht hn

ng 3

1ml dd CuSO4

Mu xanh en

Kt qu: Ta thy enzyme ha tan tt trong nc, trong dung dch mui long nhng khng tan trong dung mi khng phn cc

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10

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 3: XC NH HOT -AMYLASE THEO PHNG PHP WOHLGEMUTHNguyn tc: Cc amylase l cc enzyme thy phn tinh bt, c 2 loi l -amylase v amylase. S tc dng ca hn hp -amylase v -amylase trn tinh bt cho ta hn hp cng lc cng nhiu glucose, maltose v dextrin n gin xc nh hot ca enzyme, ta xc nh nng enzyme thp nht c kh nng gy thy phn hon ton tinh bt cho cc sn phm khng i mu dd iode. l nguyn tc ca phng php Wohlgemouth n v Wohlgemouth l lng enzyme cn thit thy phn hon ton 1mg tinh bt 37oC sau 30 pht c Cl- lm cht hot ha

Nguyn liu v ha cht: Dung dch amylase (dd nc bt pha long 10 ln) => nc bt ca bn n Dung dch tnh bt 0,1% Dung dch NaCl 0,9% Dung dch Iode 0,02N Nc ct

Cch tin hnh & kt qu:Chun b 10 ng nghim, nh s t 1-10,cho vo mi ng 1ml dd NaCl 0,9%. Trong ng nghim 1 cho vo 1ml dch enzyme, lc u. Sau ly 1ml t ng nghim 1 cho vo ng nghim 2, lc k v lp li cho ti ng nghim 10 th ht 1ml v b i. Trong mi ng nghim cho vo 2ml dd tinh bt 0,1%, lc u v vo t m n nhit 37 oC trong 30pht. Khi thi gian ht, lm ngui cc ng n nhit phng v cho vo mi ng 2 git dd Iode 0,02N v lc u

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11

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Kt qu: Ta thy 6 ng nghim u thy phn hon ton. ng 7 phn hy c , cn 3 ng cn li hon ton khng b phn hy Hot enzyme: X= 2n *2 (trong n l s th t ng nghim c nng enzyme thp nht v c thy phn hon ton)

X= 26 *2=128 Gii thch: Khi chng ta pha long nc bt 10 ln v dch enzyme trong nc bt li c pha long n 10 ln tip theo (qua 10 ng nghim) th n ng nghim 7, lng tinh bt cn rt t nn ch thy phn c . n 3 ng nghim cui (ng nghim 8,9,10), lng enzyme khng cn nn khng th thy phn c tinh bt Vai tr ca NaCl trong th nghim: Cl- trong NaCl lm cht hot ha enzyme

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12

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Chng III: GLUCIDEBi 1: PHN NG VI THUC TH FEHLINGNguyn tc: Do c cha chc aldehyde hoc ceton cho nn cc monosaccharide c tnh kh v c gi l ng kh. Nu 2 monosaccharid kt hp vi nhau bng 2 hydroxyl glucoside th disacccharide to thnh b mt tnh kh. Cn nu nhm OH glucoside ca nhm ny lin kt vi nhm OH alcol ca monosaccharid kia th disaccharid to thnh vn cn tnh kh Khi un ng kh vi dd thuc th Fehling th kt ta ca Cu2O hnh thnh ( do ng kh kh Cu(OH)2 c trong Fehling thnh CuO2)

Nguyn liu v ha cht: Dung dch Glucose 1% Dung dch Mantose 1% Dung dch Saccharide 1% Thuc th Fehling A (CuSO4) Thuc th Fehling B (Seignet + NaOH)

Cch tin hnh & kt qu:Ly 3 ng nghim, nh s t 1-3 nh sau : ng 1 : cho 2ml glucose 1% + 1ml Fehling A + 1ml Fehling B + un trong 5 pht - Kt qu: c kt ta gch ti ( Cu2O ) - PTP:

ng 2 : cho 2ml maltose 1% + 1ml Fehling A + 1ml Fehling B + un trong 5 pht - Kt qu: c kt ta gch ti ( Cu2O ) - PTP:

ng 3 : cho 2ml saccharide 1% + 1ml Fehling A + 1ml Fehling B + un trong 5 pht - Kt qu: khng hin tng - PTP: - 2010 13

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5 Gii thch: Thuc th Fehling l hn hp 2dd : dd CuSO4 v dd mui Seignet vi NaOH. Khi trn 2 dd trn vi nhau th xy ra phn ng : CuSO4 + 2 NaOH Cu(OH)2 + Na2SO4 Sau dd CuSO4 tc dng vi mui seignet to mui phc ha tan, dd c mu xanh thm Mui phc trn l hp cht khng bn Trong mi trng kim, cc mono v 1 s dixacarit kh Cu2+ di dng alcolat ng thnh Cu2+, chc aldehit b oxi ha thnh axit hoc mui tng ng Do glucose v maltose c tnh kh nn khi un vi dd thuc th Fehling th kt ta ca Cu2O hnh thnh ( do ng kh Cu(OH )2 c trong Fehling thnh CuO2 )

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14

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 2: PHN NG SELIWANOFFNguyn tc: Khi trn dd acid ascorbic vi dd xanh metylen th dd s mt mu. Phn ng xy ra do acid ascorbic b oxi ha thnh acid dehidro ascorbic v xanh metylen tr thnh dng khng mu

Nguyn liu v ha cht: Dung dch fructose 1% Dung dch glutose 1% Dung dch NaCl 1% Thuc th Seliwanoff

Cch tin hnh & kt qu:Cho vo mi ng nghim 2ml thuc th Seliwanoff v thm vo : ng 1 : 2ml dd fructose 1% ng 2 : 2ml dd glucose 1% ng 3 : 2ml nc ct

Tt c un cch thy trong 5 phtng 1

Kt quC mu anh o ti

ng 2

Mu vng nht

ng 3

Khng hin tng

Gii thch : Phn ng seliwanoff c trng i vi cc fructose v cc cetohexose nhng khng nhy i vi cc aldohexoz

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15

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

- 2010 -

16

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 3: XC NH NG KH BNG PHNG PHP QUANG PH KNguyn tc: Cc loi ng kh c kh nng kh Ferricyanur (Fe3+) thnh Ferrocyanur (Fe2+) ion ny c mu xanh m (xanh prusse). Do ta c th dng phng php so mu nh lng ng Chun b mu: 1g nguyn liu thm + H2SO4 10% (ngp mu), un cch thy khong 1h, ly ra cho NaOH vo trung ha n pH=7, lc nu c cn ta c dung dch mu Ly 1ml dd mu pha long thnh 30-100ml dd dng xc nh ng kh

Chun b mu: 1g nguyn liu thm + H2SO4 10% (ngp mu), un cch thy khong 1h, ly ra cho NaOH vo trung ha n pH=7, lc nu c cn ta c dung dch mu Ly 1ml dd mu pha long thnh 30-100ml dd dng xc nh ng kh

Cch tin hnh & kt qu:Thc hin 9 ng nghim theo mu sau: S ng ml glucose 0,02mg/ml ml nc ct ml ng nh nng Nng trong mi ng chun (g/ml) 1 0 1 0 2 0 1 0 3 0,05 0,95 1 4 0,1 0,9 2 5 0,2 0,8 4 6 0,4 0,6 8 7 0,6 0,4 12 8 1 17,994 9 2 20,337

Cho vo mi ng 1ml thuc th A (Na2CO3 + KCN/H2Oct) v 1ml thuc th B(K3Fe(CN)6/ H2Oct), lc u, un si cch thy 15 pht Lm lnh, thm 5ml thuc th C (Fe[NH4(SO4)2] + Lauryl sulfat/H2SO4), lc u, yn 15pht o Mode Absorbance vi di sng 690nm

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17

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

th theo nng ng v hp thu1.6 1.4 1.2

hp thu

1 0.8 0.6 0.4 0.2 0 0 5 10 15 20 25

Nng

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18

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Chng IV: VITAMINBi 1: PHNG PHP NH TNH VITAMIN ANguyn tc:Di tc dng ca H2SO4 m c , Vitamin A b mt nc v to thnh sn phm ngng t c mu khng bn .Sau bin i nhanh thnh mu c trng ca liporom (nu en).

Cch tin hnh & kt qu: Nh 1 git du c ha tan trong 25 git Cloroform, sau cho 1 git H2SO4 vo ri lc u Do khi tc dng vi H2SO4, vitamin A trong du c b mt nc to thnh sn phm ngng t c mu hng khng bn, sau bin i nhanh thnh mu nu en

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19

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 2: PHNG PHP NH TNH VITAMIN CNguyn tc:Khi trn dd acid ascorbic vi dd xanh metylen th dd s mt mu .Phn ng xy ra do acid ascorbic b oxy ho thnh acid dehydro ascorbic v xanh metylen tr thnh dng khng mu

Nguyn liu v ha cht: Dd Vitamin C. Xanh metylen 0,01%, Dd NaOH 5%.

Cch tin hnh & kt qu:Chun b 2 ng nghim: ng 1: 1ml dd Vitamin C Thm vo 2 git xanh T mu xanh trong sut metylen, lc u ng 2: 1ml nc ct Thm vo 3 git NaOH 5%, Vn gi nguyn mu trong lc u sut Gii thch: Do Vitamin C lm mt mu dd xanh metylen

- 2010 -

20

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 3: PHNG PHP NH LNG VITAMIN CNguyn tc: xc nh hm lng Vitamin C (Acid ascorbic) trong nguyn liu bng phng php chun iod ngi ta cho tt c acid ascorbic b oxy ho bi iod , phn iod cn tha s cho mu xanh vi dung dch tinh bt . iu ni ln phn ng kt thc

Nguyn liu v ha cht: dd HCl 5%. dd I2 0,01N. dd tinh bt 1% . Chanh tri ,bi ,cam.

Cch tin hnh & kt qu: Ly 10g tht tri chanh v 20ml HCl 5% vo ci s gi ,nghin nh cho n khi dung dch c dng ng th, dng nc ct chuyn ton b dch chit ng th vo trong bnh nh mc 100ml, thm nc ct n vch 100ml. Lc u cho lng axit ascorbic c trong nguyn liu c ho tan hon ton, lc cn vo trong bnh tam gic 250ml, ta c dung dch mu hay dd nguyn liu Ht 20ml dd nguyn liu c lc cn vo trong bnh nn, thm5-10 git h tinh bt 1% Dng I2 0,01N chun cho n khi dung dch bt u xut hin mu xanh lam nht l c Lp li chun 3 ln, ly kt qu trung bnh ta c V(ml) dd I 2 0,01N dng chun Vitamin C

Chun bng dd I2 0,01N Ln 1 Ln 2 Ln 3 Trung bnh Trong : 0,5 0,5 0,6 V= 0,533ml V .V1 .0,00088 .100 V2 .m

Hm lng Vitamin C : X(%) =

m : lng mu th nghim (5gr) 0,00088 : s gram acid ascorbic ng vi 1ml dd I2 0,01N. V1 : th tch dd mu (50ml). V2 : th tch dch mu ly xc nh ( 20ml). V: s ml Iode 0,01N dng chun

X(%) =

0,533.50.0,00088 .100 = 0,023452 % 20.5 Phn ng oxy ho acide ascorbic : - 2010 21

Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

It tng i khng tan trong nc, nhng iu ny c th ci thin bng cch pha trn it vi ioua v hnh thnh triioua: I2 + I - I3 Triioua oxy ha vitamin C to acid dehydroascorbic: C6H8O6 + I3 + H2O C6H6O6 + 3I - + 2H +

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Chng V: LIPIDEBi 1: S NH TNG HA LIPIDENguyn tc: S nh tng ho lipide trong c th l giai on u ca s hp th chung. gi s cn bng gia mi trng phn ho ( H2O ) vi pha phn tn (lipide) th phi cn c cht gy nh tng ( natri carbonat , protein, x phng , mui mt,.)

Nguyn liu v ha cht: Du lc ( du u phng ). Gelatin 2%. R ng

Cch tin hnh & kt qu: Chun b 2 ng nghim sch ,, lau kh , v tin hnh nh du ng nghim thnh ng 1 v ng 2. Tip theo cho vo c 2 ng 0,5ml du lc . Sau cho vo mi ng : ng 1 : 0,5ml gelatin 2% . ng 2 : 0,5ml nc mt r ng . Lc mnh c 2 ng . * Kt qu :

Kt qu ng 1To thnh 2 lp ,lp du ni ln trn.(l nhng ht du khng nhp vo nhau ni ln trn). Chuyn thnh trng c , mt thi gian s phn lp .

Gii thchBn cht l protein nh tng mnh hn r ng

ng 2

Cho mt r ng vo th do trong r ng do c mui mt l cht gy nh tng nn xy ra hin tng nh tng nhng yu hn ng 1

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 2: PHN NG X PHNG HANguyn tc: Ch s savon ha l lng mg KOH cn trung ha acide bo t do cng nh lin kt c trong 1g cht bo Di tc dng ca kim, glyxerit b thy phn to thnh glyxerin v x phng ( mui Natri hoc Kali ca acid bo )

Cch tin hnh & kt qu: Cho vo cc s 0,5ml du lc + 10 ml KOH 30% Khy u v un cch thy cho n khi qunh li th ta c x phng, nu cho nc vo lc ln s thy xut hin bt x phng Gii thch: Di tc dng ca enzyme, axit hoc kim, un nng, m b thy phn to thnh glixerin v axit bo hoc mui ca axit bo bc cao gi l x phng Phn ng nh sau :

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 3: XC NH CH S IOD CA CHT BONguyn tc: Ch s iod ca cht bo l s gram iod kt hp vi 100gr cht bo Xc nh ch s iod l da trn kh nng ca iod kt hp c vi acide bo ch nhng lin kt i, v mi ni i ca lipit s cho phn ng cng vi 2 nguyn t ca nhm halogen. Do , nu ta cho cht bo tc dng vi mt lng tha iod v xc nh lng tha y gip ta suy ra ch s iod Nh vy ch s iod xc nh tng qut cc acid bo khng no trong cht bo

Nguyn liu v ha cht: Du n Cn 96o Dd I2 0,1N Dd Na2S2O3 0,1N dng chun Dd tinh bt 1%

Cch tin hnh & kt qu: Ly 6 bnh tam gic: Bnh 1,2,3 (mu trng):1ml nc ct Bnh 4,5,6 (mu nguyn liu): 12g du n Thm vo mi bnh 10ml cn 96o lc u, thm 10ml I2 0,1N. Sau dng giy bt kn ming bnh tam gic trnh bay hi I2

1ml H2O ctBnh (1) (2) (3) (4)

2g Du n(5) (6)

yn trong ti t 10 15 pht, ri sau chun bng Na2S2O3 0,1N n khi dung dch c mu vng nht Na2S2O3 0,1N dng chun (ml) 9,7 9,7 9,8 9,7 9,6 9,7

Thm vo mi bnh 1ml dung dch tinh bt 1%, v tip tc chun n khi mt mu xanh Na2S2O3 0,1N dng chun (ml) 0,4 0,3 0,4 0,5 0,3 0,4

Tnh ch s iod: a: s ml Na2S2O3 0,1N dng chun mu trng 9,7 + 9,7 + 9,8 0,4 + 0,3 + 0,4 + = 10,1 (ml) a = 3 3 b: s ml Na2S2O3 0,1N dng chun mu nguyn liu

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Thc hnh Ha Sinh 9,7 + 9,6 + 9,6 0,5 + 0,3 + 0,4 + = 10,06667 (ml) b = 3 3 m: trng lng mu tnh bng gram 0,0127: s gram iod ng vi 1ml Na2S2O3 0,1N

Lp K14S1_Nhm 2_t 5

Ch s I2 =

( a b). 0,0 2 .1 0 17 0 (10,1 10.06667 ).0,0127 .100 = = 0.020955 m 2 Gii thch : Ch s iod cho ta bit mc cha no ca cc acid bo c trong thnh phn ca cht bo Da vo phng php tha tr, tc l cho 1 lng d iod phn ng cng vi du bo, ri i chun iod cn li nh vo Na2S2O3, suy ra lng iod cng vo du bo. Ch s iod cng cao chng t cht bo cng lng v kh nng b oxi ha cng nhanh

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 4: XC NH CH S ACIDE CA CHT BONguyn tc: Ch s acide ca cht bo l s milligram KOH cn thit trung ho ht nhng acid bo t do cha trong 1g cht bo. Dng KOH 0,1N trung ho cc acide bo t do trong nguyn liu vi phenolphtlein lm cht ch th mu .

Nguyn liu v ha cht: Du n tinh luyn , Cn tuyt i 980 . Dd KOH 0,1N chun . Ch th phenolphthalein 1%

Cch tin hnh & kt qu: Trong bnh tam gic 250ml, cn khong 5g du n v thm vo 50ml cn 980 ho tan du n .Sau em i un nh 600C Chun nhanh vi dd KOH 0,1N khi dd trong bnh tam gic cn hi m , thm 3 git ch th phenolphthalein, chun n khi dd trong bnh tam gic c mu hng nht bn trong 30 giy Lm lp li th nghim 3 ln ly th tch trung bnh

Chun bng dd KOH 0,1N Ln 1 Ln 2 Ln 3 Trung bnh34,5 35 34 V= 34,5ml

Ta c: 5,61 l s mg KOH ng vi 1ml KOH 0,1N V : s ml KOH 0,1N dng chun . m : trng lng cht bo cn (5000mg) 5,61.V 5,61.34,5 = = 0,38709 Ch s Acid = m 5000 Gii thch : Ch s acid: l s mg KOH cn thit trung ha cc acid bo t do c trong 1g cht bo. Ch s acid cho bit c cht lng cht bo, ch s acid cng cao chng t cht bo khng ti, b phn hy v b oxi ha mt phn

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Bi 5: XC NH LIPIT TNG S (Ging trn m hnh)Nguyn tc: Dng dung mi k nc trch li hon ton lipide t nguyn liu c nghin nh. Mt s thnh phn ha tan trong cht bo cng c trch li theo, bao gm sc t, cc vitamin tan trong cht bo, cc cht mi tuy nhin hm lng ca chng thp. do c ln tp cht, phn trch li c gi l lipide C 2 phng php xc nh : Phng php xc nh trc tip : chit xut lipide ra khi nguyn liu v cn trc tip Phng php xc nh gin tip : chit xut lipide ra khi nguyn liu v cn li nguyn liu

Nguyn liu v ha cht: Dung mi chit xut lipide thng dng l ether etylic Nguyn liu c nghin nh ,sy kh n khi lng khng i.

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

Thit b Soxhlet :

Cch tin hnh & kt qu: Chun b ti bng giy lc ng nguyn liu hoc dng ng hnh tr ng mu c sn ( ti giy lc c ct hnh ch nht, chiu di gp 2,5 ln chiu rng, gp thnh ti tr c ng knh b hn tr chit). Ti c sy kh n khi lng khng i v c cn trn cn phn tch ( nu xc nh theo phng php gin tip ) Cn chnh xc 2-5 g ri cho mu vo ti giy, gp kn mp ti, t ti c mu phn tch vo tr chit Phng php xc nh trc tip + + + + Trc khi chit, bnh cu c sy kh n kh lng khng i t bnh cu ln ni cch thy v cho ete vo th tch bnh Cho ti nguyn liu vo tr chit Lp tr chit vo bnh cu

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Thc hnh Ha Sinh

Lp K14S1_Nhm 2_t 5

++ + +

Cho dung mi vo tr chit n ngp ti nguyn liu. Mc dung mi n phn trn ng xifon tr chit Lp ng sinh hn, ngm nguyn liu trong dung mi mt vi gi t my Soxhlet vo ni cch thy sao cho s ln dung mi rt t tr chit xung bnh cu khong 10-15 ln trong 1 gi Th lipide chit ht cha bng cch ly mt vi git ete t u cui tr chit cho ln a knh ng h sch, cho bay hi ht ete, nu khng c lipide trn a knh, xem nh lipide c chit hon ton Khi chit xong, ly bnh cu ra, lp ng sinh hn vo v ct ly ete Sy bnh cu c cha lipide ( 60-700C trong 30 pht ) n khi lng khng i ri em cn

+

+

Tnh kt qu

Hm lng lipide c trong 100g mu nguyn liu: X : hm lng lipide tnh theo % a : khi lng bnh v lipide (g) b : khi lng bnh (g) c : khi lng mu tch lipide (g) Phng php xc nh gin tip : (a b).100 X = c + Sau khi kt thc th nghim nh trn, ly ti mu nguyn liu ra khi bnh chit, cho bay hi ht dung mi, sy kh n trng lng khng i

Tnh kt qu

Hm lng lipide c trong 100g mu nguyn liu : X = (a b).100 c X : hm lng lipide tnh theo % a : khi lng ti mu nguyn liu trc khi chit (g) b : khi lng ti mu nguyn liu sau khi chit (g) c : khi lng nguyn liu ly xc nh cc ch s ca cht bo

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