MT S BI TH NGHIM THC HNH THEO CHNG TRNH OLYMPIC

V Gio dc Trung hc B Gio dc v o to

Bi 1. Xc nh St c trong thuc vin cha St

L thuyt St l mt thnh phn thit yu ca hng cu (hemoglobin), gip vn chuyn oxi trong mu n mi phn ca c th. N cng gi vai tr sinh t trong nhiu phn ng trao i cht. Thiu st c th gy bnh thiu mu l h qu ca mc hng cu trong mu thp. Thiu st l s suy dinh dng khong cht ph bin nht trn th gii. Mt cch gim s thiu ht st l cha tr bng vin cha st. Hot cht trong thuc vin cha st l st(II) hin din trong thuc vin kho st di dng st(II) fumarat. Ngoi hp cht hu c st(II) ny, thuc vin c cha cc cht khc nh nhng tc nhn lin kt. Cu trc ca axit fumaric l:
OH O O OH

Axit fumaric St(II) v 1,10-phenanthroline to phc c mu vng cam/ [(C12H8N2)3Fe]2+. Mt quang (absorbance) ca phc ny, xc nh ti 510 nm trong dung dch m (pH=8) l mt php o hm lng st ca vin thuc. Do 1,10-phenanthroline ch lin kt vi st(II) v st(II) d b oxi ha thnh st(III), thm hidroxiamoni clorua (hydroxylammonium chloride) kh ton b st(III) thnh st(II). Mt s phn ng n gin l: 2 NH2OH + 4 Fe3+ N2O + 4 H+ + H2O + 4 Fe2+
N N

1,10-Phenanthroline

1,10 phenantroline

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Thit b v Ha cht tin hnh

THIT B HA CHT

Cn Ci, chy s; Cc 100 mL; Thit b siu m (ultrasonic bath); Bp in; Phu Hirsch cha mt lp nh cht gip lc nhanh; Bnh nh mc 250 mL v 100 mL; Pipet; Quang ph k; Phng php tin hnh

Vin thuc cha Fe (II) HCl 4M; Dung dch 1,10-phenanthroline; Dung dch hydroxylammonium chloride;

Dng cn xc nh khi lng ca vin thuc cha st chnh xc n 1 mg. Vin thuc c tn cn thn thnh bt trong mt ci v chuyn nh lng vo cc 100 mL bng mt lng nh nc ct. Thm axit clohidric (5 mL, 4 M). un nng cc cht trong cc n khong 60 oC trn bp in. Dung dch i sang mu vng. t cc vo thit b siu m (ultrasonic bath) trong t nht 5 pht. Gi cc n nh bng mp xp (styrofoam). Phu Hirsch cha mt lp nh cht gip lc nhanh (Hi-flo filter aid) c lm m v p cht trn lc, dng phu ny lc huyn ph bng cch ht. Ra cht gip lc nhanh (Hi-flo filter aid) bng lng d nc ct. Nc lc c chuyn cn thn vo bnh nh mc (250 mL) v thm nc ct, khuy lin tc iu chnh th tch cui. Dng pipet ht 10 mL dung dch ny v cho vo bnh nh mc 100 mL. Li iu chnh th tch bng nc ct ng thi khuy u. T dung dch ny, dng pipet ly 10 mL v cho vo bnh nh mc 100 mL. Sau , thm dung dch 1,10-phenanthroline (10 mL) v dung dch hidroxi

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amoni clorua (hydroxylammonium chloride) (1 mL) . K tip, iu chnh th tch dung dch bng dung dch m (pH 8). Mt quang ca dung dch ny c o bng my so mu (quang ph k) ti 510 nm so vi nc trong cuvet 1,000 cm. Hy tnh lng st trong vin thuc cha st da trn hp th mol (h s tt, e) bit ca phc st(II) phenanthroline ti 510 nm. hp th mol ca phc st(II) phenanthroline ti 510 nm bng 11100 M-1cm-1. Quan trng lai b sai lch trong mt quang khi ni vi my so mu s dng, mt h s iu chnh c ghi trn my so mu hc sinh dng trong th nghim. Mt quang quan st c cn phi nhn vi h s ny thu c mt quang ng ca dung dch phc st.

Bi 2. Xc nh cc mu v c cha bit
L thuyt C 12 mu cha bit ng trong ti bng cht do bao gm 9 dung dch cha bit, mi dung dch c ng trong ng nh git v 3 mu cht rn ng trong ba l ming rng. Tt c cc mu cha bit u c nh s vi 3 ch s. Hy kim tra cn thn cc m s mu theo danh sch cc mu v c cha bit ri vit s bo danh v tn ca mnh vo t giy. (Danh sch c km theo cc mu cha bit ca hc sinh). Mi l ng cht rn c khong 20 miligam di dng bt hoc tinh th ca mt hp cht tinh khit. Mi ng nh git cha khong 1,5ml dung dch ca mt hp cht tinh khit c ha tan trong nc ct. Nng ca cc dung dch cha bit nm trong khong t 0,05 n 0,5 M(mol/lit). Cc dung dch cha bit l nh sau: HCl NaOH Ch : H2O2 Na2CO3 H2SO4 Na2SO3 ZnCl2 BaCl2 NH4SCN K4Fe(CN)6

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(1) C 2 mu cha bit c lp li. (2) H2O kt tinh trong tinh th ngm nc c b qua trong cc cng thc cho trn. Trn bn th nghim ca hc sinh c mt hp nha ng cc dng c, mu cha bit v cc thuc th c s dng trong bi thc hnh ny. Danh sch cc dng c Dng c in cc dy Pt Hp ng pin Bn lm trng nh git Ko ct Tha cph Danh sch thuc th Thuc th KI FeCl3 S lng. 1 1 1 1 2 Dng c in cc dy Au Pin Bn mng bng nha mu en ng nh git (1 mL) S lng. 1 2 1 5

Nng . Thuc th 0.1M pp (phenolphtalein) 0.1M Dung dch tinh bt

Nng . 0.01 0.01

Mc c hi v an ton ca cc ha cht Ha cht Axit clohidric Axit sunfuric Dung dch Natri hidroxit Dung dch Hydroperoxit Dung dch Natri cacbonat Dung dch Bariclorua Dung dch Natrisunfit Dung dch Km clorua Dung dch Kali Cng thc HCl H2SO4 NaOH H2O2 Na2CO3 BaCl2 Na2SO3 ZnCl2 K4Fe(CN)6 c hi 36/37/38 35 35 22-41 36 20-25 31-36/37/38 22-34-50/53 32 an ton 26 26-30-45 26-36/37/39-45 26-39 22-26 45 26-36 26-36/37/39-45-6061 22-24/25

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hexaxyanoferat (II) Dung dch Amoni thioxyanat St (III) clorua (rn) Kali iotua (rn) Dung dch tinh bt Cht ch th Phenolphthalein Tin hnh

NH4SCN FeCl3 KI -

20/21/22-32-52/53 22-34 40

13-61 26-36/37/39-45 22-24/25 * 36/37

1. S dng bn thuc th c cp, cc phn ng gia cc mu cha bit vi nhau v thit b in phn n gin nhn bit cc mu cha bit v vit tr li ca em (di dng s vi 3 ch s - nh cch nh s mu ca cc mu cho) vo cc trng trong t phiu tr li.
Ch : Sau khi kt thc cng vic hy cho hai dy vng (Au) v Platin(Pt) v cc pin vo cc
ti nilon ban u ca chng ri li tt c dng c v ha cht (k c cc mu cha bit) vo hp nha ng v tr ban u.

2. Trong bi thc hnh ny hc sinh thc hin mt lot php th xc nh (hoc khng nh) cc mu cha bit. Hc sinh cn nm c cc phn ng ho hc lin quan n cc php th tin hnh v vit c cc phng trnh phn ng: A. Vit phng trnh in phn xy ra dng ion rt gn c ghi trng thi tn ti khng nh mt mu cha bit l dung dch cha ZnCl2. B. Vit mt phng trnh phn ng dng lm sch kt ta Zn trn b mt in cc bng cc dng c v ha cht cho trong bi ny.

Bi 3. Xc nh cacbonat v hiro photphat trong mt mu lm cht mi L thuyt

Na2CO3, CaCO3 v Na2HPO4 l cc thnh phn chnh ca cc bt mi. Trong bi th nghim ny, phi xc nh cc ion cacbonat v hiro photphat trong mt mu mi bng hai chun axit-baz.
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u tin, thm mt lng chnh xc axit clohiric (c ly vi lng d) vo mu th. Phn ng xy ra, hiro photphat chuyn thnh H3PO4, cn cc ion cabonat chuyn thnh CO2 sau thot ra ht khi b un si. Cc ion canxi c ban u trong mu chuyn vo dung dch. V cc ion ny c th nh hng n kt qu phn tch nn chng c kt ta trong CaC2O4 v lc b trc khi chun . Tip n, axit photphoric va to thnh c chun bng dung dch NaOH c nng chnh xc vi hai cht ch th khc nhau l: Bromcrezon xanh (Bromocresol Green, BCG) v Thymolphthalein (TP). Bc th nht ca chun ny l: H3PO4 (v lng d HCl) c chun ti ion H 2PO4-, im kt thc bc chun ny c mi trng hi axit (pH khong ~4.5). im ny lm cho BCG chuyn t mu vng sang mu xanh. Bc th hai ca chun ny: tip tc bc trn cho ti khi to ra HPO42-. im kt thc bc hai ny xy ra khi TP khng mu chuyn sang mu xanh (mi trng c tnh kim, pH vo khong 10). Lng ion CO32- trong mu c tm ra khi da vo lng khc nhau gia: a) b) Lng cht chun ng vi lng ban u ca HCl ( dng ha tan mu) Lng cng ca cht chun ng vi im kt thc chun th hai (ch th TP). Lng ion HPO42- c tm ra khi da vo lng khc nhau gia lng cht chun dng t ti hai im kt thc chun (ch th TP v BCG). Thit b v Ha cht tin hnh
THIT B HA CHT

Cn Phu lc; Cc 100 mL; ng ong; Bp in; Bnh hnh nn (Erlenmeyer);

Bt mi cha Na2CO3, CaCO3 v Na2HPO4 HCl 1M; Nc ct Dung dch K2C2O4 15%; Dung dch NaOH;

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 Bnh nh mc 100 mL; Pipet;

Dung dch Thymolphthalein (TP) Dung dch BromoCresol Green (BCG)

Qui trnh tin hnh

Bc 1. Ha tan mu v ui CO2 Thm ng 10,00 mL dung dch HCl nng khong 1 mol/L (xem tr s chnh xc ny c ghi trn nhn ca l) vo mu bt mi c trong cc c y bng mt knh ng h thy tinh (mi thao tc phi chnh xc: ly dung dch bng mt pipet! Khng c b np y cc ra trnh tht thot ha cht! ). Sau giai on thot kh mnh kt thc, dng bp in cn thn un nng dung dch trong cc (vn phi y cc bng mt knh ng h) ti khi ht kh thot ra. Tip n, un cn thn dung dch cn li trong cc cho si trong khong 2-3 pht. Bc 2. Kt ta canxi Nhc cc khi bp in. Dng nc ct ra phn hi nc ngng t mt knh ng h cho chy vo cc. Dng ng ong ly 1-2 mL dung dch K2C2O4 15%. Cho t t lng ny theo thnh vo cc (mt khong 10 n 20 pht) cho ti lc kt ta hon ton. Dng khong thi gian ch i ny xc nh nng chnh xc ca dung dch NaOH (theo phng php di y). Bc 3. Xc nh nng chnh xc ca dung dch NaOH Dng mt pipet ly 10,00 mL dung dch HCl ri cho vo bnh nh mc 100 mL, thm nc ct cho n vch, lc bnh trn u. Rt dung dch NaOH y buret. Dng pipet ly 10,00 mL dung dch HCl trong bnh nh mc cho vo mt bnh hnh nn (Erlenmeyer). Thm vo erlenmeyer ny 1-2 git dung dch Thymolphthalein ri chun bng dung dch NaOH ti khi mu xanh xut hin trn vng xoy ca dung dch v bn ch trong khong 5 - 10 giy. Ti y v phn sau: Hy lp s chun mc cn thit. Cn lu rng tr s th tch dung dch cn dng nhiu nht v t nht ch cch nhau c 0,10 mL. S liu th tch dung dch bo co c chnh xc ti 0,01 mL.

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3.1a Hy in y vo bng trong Phiu tr li. 3.1b Hy tnh nng ca dung dch NaOH (theo mol/L). Bc 4. Lc b canxi oxalat Sau khi kt ta c hu ht CaC 2O4, dng phu lc dung dch vo bnh nh mc 100 mL. Nc lc ny hi b c do c mt lng nh canxi oxalat nhng khng gy nh hng ti vic chun . Dng nc ct ra sch kt ta ri b giy lc c kt ta vo thng ng rc. Thm nc ct vo bnh ng nc lc cho ti vch v lc u. Bc 5. Chun mu dng Bromocresol Green Dng pipet ly 10,00 mL nc lc thu c sau bc 4 cho vo mt Erlenmeyer ri thm tip vo 3 git dung dch BCG. Hy chun b mt Erlenmeyer khc c dung dch i chng gm 3 git dung dch NaH2PO4 15%, 3 git dung dch BCG v 15-20 mL nc ct. Dng dung dch NaOH chun dung dch nc lc ti khi mu ca dung dch ny trng vi mu ca dung dch i chng th dng. 3.2 Hy in y vo bng trong Phiu tr li. Bc 6. Chun mu dng Thymolphthalein Dng pipet ly 10,00 mL nc lc thu c sau bc 4 trn cho vo mt Erlenmeyer ri thm tip vo 2 git dung dch TP. Dng dung dch NaOH chun dung dch nc lc ti khi mu xanh xut hin v bn trong khong 5 10 giy th dng. 3.3 Hy in y vo bng trong Phiu tr li. Bc 7. Cc tnh ton 3.4. Hy tnh khi lng ca CO32- trong mu dng. 3.5. Hy tnh khi lng ca HPO42- trong mu . Bc 8. Cc cu hi thm cho bi th nghim ny Hy tr li cc cu hi sau y vo Phiu tr li. 3.6a. Hy ch ra mt phn ng (vit phng trnh) lm cn tr s phn tch mu khi c mt Ca2+.

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3.6b. Danh mc cc li c th phm phi cc bc khc nhau c nu ra trong bng ca Phiu tr li. Hy ch ra trong s nhng li no dn n sai s khi xc nh hm lng ca CO32- v/hoc HPO42-. Hy dng cc k hiu sau y: 0 nu khng c sai s nh c d on, + hoc nu c sai lch nhiu hn (sai s dng) hoc sai lch t hn (sai s m) so vi thc t.

Bi 4. Chun complexon; V d ca s xc nh ion kim loi dng php o complexon.

L thuyt Nng ion Ni2+ c th c xc nh bng s to phc vi EDTA (etylendiamin tetraaxetat). EDTA l mt ligand nhiu rng to phc 1: 1 vi ion Ni 2+. Cht ch th l murexide cng c th to phc vi ion Ni2+ nhng phc ny khng bn bng EDTA. Mc ch ca th nghim ny l xc nh lng nc kt tinh trong niken sunfat. Ha cht cn thit: Niken sunfat (300 mg) Cht ch th murexide Ammoni clorua (3 g) Ammoniac m c (20 mL) Dng c cn thit: Cn Cc ong 100 mL Bnh tam gic B thit b chun Tin hnh Cn chnh xc khong 300 mg niken sunfat v ha tan vo nc. Dng
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M an ton: R 20/21/22, 42/43, 45, 46 S 26, 27, 28, 36/37/39, 45 R 22 S 36 R 22, 36 S 22 R - S 22, 24/25

Dung dch EDTA tiu chun

cc ong 100 mL. iu ch dung dch m bng cch ha tan 2,7 g ammoni clorua v 17,5 mL ammoniac m c trong 50 mL nc. y dung dch EDTA tiu chun 0,01 M vo mt buret. Dng pipet ly 10,00 mL dung dch niken sunfat cho vo cc hnh nn 200 mL v pha long vi khong 90 mL nc. Va thm va khuy u 10 mL dung dch m vo cc hnh nn. Thm mt t cht ch th murexide rn v m bo tan ht. Chun vi dung dch EDTA n khi i mu t vng sang tm. Khi mu i chm, thm mt t ammoniac m c lc cui chun . Th nghim ny cn c thc hin hai ln. Ghi li cc s liu sau: 1. Lng dung dch EDTA theo mL. Cng ghi li chnh xc chun ca dung dch. 2. Khi lng niken sunfat.xH2O. 3. Tnh nng Ni2+ trong dung dch. 4. Tnh s mol nc kt tinh trong mt mol niken sunfat.

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Bi 5. Phn tch nh tnh cc hp cht hu c

th nghim ny bn phi nhn bit 7 cht rn cha bit ghi trong danh sch cc cht trang 7, chng l cc thuc ph bin trong cuc sng hng ngy v l cc tc nhn hu ch trong ha hu c. t iu ny, phi tin hnh cc phn ng ho hc theo qui trnh sau v phn tch kt qu thu c. Cc l dn nhn cht cha bit nh: L U-1, L U-2, L U-3, L U-4, L U-5, L U-6, L U-7 Phn ng th 1: Th tnh tan Lc ng nghim vi CH3CN, 1M HCl, nc v 1M NaOH. Phn ng th 2: th vi 2,4-DNPH Ha tan mt cht cha bit vi 95% EtOH v th vi dung dch ca 2,4dinitrophenylhydrazin trong axit sunfuric c v 95% ethanol (2,4-DNPH). Phn ng th 3: th vi CAN Trn dung dch Xeri(IV) ammoni nitrat trong HNO3 long (k hiu nhn l CAN) vi CH3CN c hn hp. Cho cht cha bit vo dung dch hn hp. Nu c s i mu dung dch, th dung dch ny c th cha ancol, phenol hoc andehit. Phn ng th 4: Php th Bai (Baeyer) Trong ng nghim, ha tan cht cha bit vi CH 3CN. Va lc va cho t t vo dung dch th 5 git dung dch 0.5% KMnO4. Phn ng th 5: Th pH Trong ng nghim, ho tan cht cha bit vi 2 ml 95% EtOH. Dng giy pH o pH ca dung dch. Phn ng th 6: th vi st (III) clorua Ly dung dch thu c t Phn ng th 5 v cho 5 git dung dch 2.5% FeCl3.

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Thit b v Ha cht tin hnh

THIT B HA CHT

Cn Phu lc; Cc 100 mL; ng nghim; Tha;

7 cht cha bit HCl 1M;

NaOH 1M;

Nc ct Dung dch KMnO4; CH3CN; Dung dch CAN 2,4 DNPH

Dung dch FeCl3 2,5% C2H5OH Giy pH

Qui trnh tin hnh Cc li khuyn hu ch a) Trng lng ca tha (spatula) ly y cht khong 15~20 mg. b) Lau k tha bng giy lau sau khi dng ly cht. c) Sau khi cho bt k tc nhn no miu t di y vo dung dch ca mu cha bit, phi trn k v quan st thn trng hn hp thu c. d) nhn im ti a, phi tin hnh tt c cc phn ng th v ghi vo bng. Phn ng th 1: Th tnh tan Cho vo ng nghim mt tha y cht (15~20 mg) cha bit v1 ml of CH3CN. Lc ng nghim v ghi li tnh tan. Lp li th nghim vi 1M HCl, nc v1M NaOH. Phn ng th 2: th vi 2,4-DNPH Cho khong 15~20 mg mt cht cha bit vo ng nghim v ha tan vi 2 ml 95% EtOH (i vi cc cht tan c trong nc, th ly khong15~20 mg ho

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vo trong 1 ml nc). Cho vo 5 git dung dch ca 2,4-dinitrophenylhydrazin trong axit sunfuric c v 95% ethanol (k hiu nhn l 2,4-DNPH). Phn ng th 3: th viCAN Trn 3 ml dung dch Xeri(IV) ammoni nitrat trong HNO3 long (k hiu nhn l CAN) vi 3 ml CH3CN trong ng nghim. ng nghim khc cho khong 15~20 mg cht cha bit vo 1 ml dung dch hn hp. (i vi cht tan trong nc, th u tin ho khong 15~20 mg mu trong 1 ml nc, v sau cho thm 1 ml thuc th CAN. Nu c s i mu dung dch, th dung dch ny c th cha ancol, phenol hoc andehit. Phn ng th 4: Php th Bai (Baeyer) Trong ng nghim, ha tan khong 15~20 mg cht cha bit vi 2 ml CH3CN (i vi cht tan c trong nc, th ho khong 15~20 mg cht vi 1 ml nc). Va lc va cho t t vo dung dch th 5 git dung dch 0.5% KMnO4. Phn ng th 5: Th pH Trong ng nghim, ho khong 15~20 mg cht cha bit vi 2 ml 95% C2H5OH (i vi cht tan c trong nc, th ho khong 15~20 mg cht vi 1 ml nc. Dng giy pH o pH ca dung dch Phn ng th 6: th vi st (III) clorua Ly dung dch thu c t Phn ng th 5 v cho 5 git dung dch 2.5% FeCl3. Ghi kt qu 1. Ghi cc kt qu th vo t Phiu Tr li. Vit O nu tan, cn X nu khng tan i vi phn ng th tnh tan. Vit (+) i vi phn ng dng tnh, cn () cho phn ng m tnh i vi cc phn ng th 2 ~ 4 v 6. Vit a, b v n tng ng vi dung dch c tnh axit, baz hoc trung tnh, cn pH vi phn ng th 5. 2. Da trn kt qu th, hy cho bit cu to ph hp ca cc hp cht cha bit, suy t cc cht cho trong danh sch. Vit cht ny vo thch hp. Cc hp cht cha bit c th l

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COOH OH NH2 (A) HO OCH3 (E) HO OCH3 (F) HO HO

OH NH2 HCl

OH (G)

H CH3 O CH3 COOH H3C (K) OH CH3 (M) H3CO N (Q) HO N HCl

H N HO (T) O

CH3 HO

CHO

OH OCH3

OCH3 (V)

O (W)

Bi 6. Sc k trao i ion cc aminoaxit

Trao di ion l mt phng php phn tch v iu ch quan trng cho php phn tch cc cht mang in. S tng tc gia cc nhm ion ca cht vi cc gc gn trn nha l c s ca phng php ny. bi ny, phi phn tch mt hn hp cc aminoaxit, tip theo th nh tnh tng loi aminoaxit c tch ra t ct bng phn ng mu c trng. Do th sinh phi sp hng o ph nn chng ti ngh bn hy bt u vi bi thc hnh s 1.
O N N H NH2 OH HS NH2 O OH HN NH2 NH NH2 O OH

His

Cys

Arg

Cho mt hn hp gm ba aminoaxit: histidin, cystein v arginin (xem cu trc trn). Polistyren lin kt cho bi gc sunfat l nha trao i cation (xem s

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di y). Trc khi th nghim ct sc k trao i ion c nhi sn v cn bng vi Dung dch ra gii 1 (pH 4,9). Qui trnh tin hnh Tin hnh sc k. Bc 1. a dung dch cc aminoaxit ln ct sc k. u tin, m kha cho dung mi trong ct chy xung bnh tam gic (Erlenmeyer) c ghi Cht thi sao cho dung mi vn cn nm trn b mt cht nhi v trnh khng cho n b kh. ng kha li v thn trng dng mt syranh cho dung dch phn tch ln ct. M kha v cho dung dch ny ngm vo cht nhi (x dung mi vo bnh Cht thi). ng kha ct v cn thn m (nh) t t kp ng cho chy vo khong 1 mL Dung dch ra gii 1 (ng vi ~ 1 cm ca cht lng trn ct). Dng hai tay ni cht u ni c nhm trong (nhm ci) u ct vo u ni c nhm ngoi (nhm c) u ng dn dung dch ra gii 1 (xem k vic ni cht u thy tinh vi ct). B bnh Cht thi ra v thay vo cc ng nghim trn gi. M t t kp ng v m kha dung dch ra gii chy xung qua ct. Bt u qu trnh ra gii (lun m kha ct khi bt u ra gii v ng kha li khi ngng ra). Thu gom cc phn on vo ng nghim, ly khong 2,5 mL (xem mi tn s ). Nu thy cn th dng bt d nh du. Sau khi gom c t 4 n 8 ng, ngng ra gii v sau phn tch nh tnh cc mu (phn on) thu c.

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ng kp ng u ni nhm ngoi u ni nhm trong lp dung mi nha trao i ion Kha ct dung dch ra gii

nh tnh cc mu thu c nh tnh cc aminoaxit da trn phn ng ca nhm -amino vi natri 2,4,6trinitrobenzen sunfonat (TNBS):

NH2 HOOC R

+ O Na O S O

NO2 HOOC NO2 R NO2

O 2N NH NO2 O 2N

NaHSO3

nh tnh c thc hin trong cc l ca tm nha polistyren, mi l tng ng vi mi ng nghim xc nh. Trc khi th, u tin hy trn 1 mL dung dch TNBS vi 10 mL dung dch m cacbonat v sau cho 0,1 ml hn hp thu c vo mt na cc l trn tm nha (t A1 n H5). Tip theo, cho 0,1 mL ca phn on cn phn tch vo l. Bt u th vi A1, v tip tc vi B1, C1, v.v. (di chuyn t trn xung v t tri sang phi). Nu aminoaxit c mt

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trong phn on phn tch th mu vng m s xut hin trong l trong khong 3 pht. Ly mu trong l u lm chun i chiu. nh gi ng mu, bn nn tm nha ln t giy trng. Lu : Dng pipet my ly tt c cc cht lng m c th tch 0,1 mL. Bn nn dng mt u ht nha cho tt c cc phn on c mt cht (nh). 6.1a nh du m t s lc cng mu (nh tnh) trn tm nha (c l ) vo Phiu Tr li. Dng cc k hiu sau: (-)- khng mu, 1-mu yu, 2- mu va phi v 3- mu mnh. Tip tc nh du s m t ny trong qu trnh sc k. Tip tc ra gii thu cc phn on v phn tch chng cho n khi bn nhn c t nht 2 l c mu nh l A1 , iu ny ch ra rng aminoaxit th nht hon ton ra ht khi ct (kt thc nh (peak) th nht). Tin hnh sc k. Bc 2. Ngay sau khi kt thc thu gom nh (peak) th nht, bn phi thay Dung dch ra gii th 2. lm iu ny, hy ng kha ct, ng (vn cht) kp ng dn (Quan trng !), tho ng dn ang ni vi chai ng Dung dch ra gii th 1 v ni n vi chai ng Dung dch ra gii th 2. Gi cht u ni nhm u ct. 6.1b. Khi cc Dung dch ra gii c thay i, hy nh du bng cch v cc ng thng nm gia cc l tng ng tm nha. Tip tc ra gii, thu cc phn on v phn tch nh tnh chng nh miu t trn. Tin hnh sc k. Bc 3. Ngay sau khi kt thc thu gom nh (peak) th 2, bn phi thay Dung dch ra gii th 3 nh miu t bc 2. Tip tc sc k cho n khi aminoaxit th 3 hon ton ra khi ct. Dng qu trnh sc k bng cch ng kha ct v vn cht kp ng. Da vo kt qu phn tch nh tnh, hy chn nhng phn on c cha cc aminoaxit.

235

6.1.c Hy in vo Phiu Tr li nhn ghi (s th t) ca cc l ng vi cc phn on chn trn. 6.2 Gp li cc phn on c cng mt nh v dng ng ong o th tch ca tng phn on gp. Bo co th tch ca cc phn on gp ngoi tr lng dng cho phn tch nh tnh. Ghi cc kt qu thu c vo Phiu Tr li. Rt cc phn on gp vo l thy tinh nu c ghi nhn Peak 1, Peak 2 Peak 3. Chun b cc mu phn tch nh lng trn my quang ph nh m t di y. Khi kt thc bi thi thc hnh, hy nt cc l v chng trn bn. Cc phn on gom sau s c nhn vin phng th nghim phn tch kim tra li. Phn tch quang ph i vi mi mu, bn cn phi a 2 cuvet cho ngi o mu. Chun b mu nh sau. Quan trng! Khi bo qun, lun cuvet trong hp! Tt c cc cuvet c 2 mt hng v 2 mt trn nm thng ng dng o. Khi dng cuvet, khng c chm vo mt dng o, nu khng bn s thu c gi tr mt quang sai. Php th s 1(nh 1). Nng cystein c xc nh bng phn ng Ellman:
NO2 O O
-

O O

+ NH3

O-

O SO-

+
SH

OH

H3N

S
-

S NO2

+
NO2

-H 2O

NO2

236

ng nghim A1 (ng i chiu). Cho 0,1 mL Dung dch ra gii 1 ly t ng nha nh vo mt ng nghim v cho thm vo 2.9 mL tc nhn Ellmann. ng nghim B1 (ng mu phn tch). Cho 0,1 ml dung dch phn tch vo mt ng nghim v cho thm vo 2.9 mL tc nhn Ellmann. Trn u cc ng nghim v chuyn mi hn hp sang cc cuvet tng ng c ghi A1 (cho mu i chiu) v B1 (cho mu phn tch). Mu th s 2 (nh 2). Xc nh nng histidin da trn kh nng ca gc imidazol phn ng vi cc hp cht diazo (phn ng Pauli). ng nghim A2 (ng i chiu). Cho 2,8 mL dung dch m Tris-HCl vo mt ng nghim, cho thm vo 0,1 mL Dung dch ra gii 2 ly t ng nha nh v 0,1 mL tc nhn Pauli. ng nghim B2 (ng mu phn tch). Cho 2,8 mL dung dch m Tris-HCl vo mt ng nghim, tip theo cho thm 0,1 mL dung dch cn phn tch v 0,1 mL tc nhn Pauli. Trn u cc ng nghim v chuyn mi hn hp sang cc cuvet tng ng c ghi A2 (cho mu i chiu) v B2 (cho mu phn tch). Mu th s 3 (nh 3). Xc nh nng ca arginin da trn kh nng ca gc guanidin phn ng vi mt s phenol trong iu kin kim v cht oxi ha (phn ng Sakaguchi). ng nghim A3 (ng i chiu). Cho 0,1 mL Dung dch ra gii 3 vo mt ng nghim, tip theo cho thm 1,5 mL dung dch NaOH 10%, 1 mL dung dch 8hydroxiquinolin v 0,5 mL dung dch natri hypobromua. ng nghim B3 (ng mu phn tch). Cho 0,1 mL dung dch phn tch vo mt ng nghim, tip theo cho thm 1,5 mL dung dch NaOH 10%, 1 mL dung dch 8-hydroxiquinolin v 0,5 mL dung dch natri hypobromua. Lc mnh cc ng nghim trong 2 pht ( Quan trng!) v quan st s to thnh mu vng cam. Cho 0,2 mL dung dch ur 8 M vo mi ng, trn k v ly

237

khong 3 mL ca mi hn hp cho vo cc cuvet tng ng ghi A3 (cho mu i chiu) v B3 (cho mu phn tch). Tt c cc hn hp cn phi phn tch bng quang ph khng c sm hn 10 pht v khng c mun hn 2 gi sau khi chun b xong mu. a 6 cuvet cho nhn vin o mu. Trong trng hp phi sp hng ch, hy ngh nhn vin o mu ghi m s th sinh ca bn ln bng nh du sp hng. Bn s c gi khi n lt mnh. Trong thi gian ny bn c th bt u lm bi th hnh s 2. Trong trng hp cc mu ca bn khng kp kho st trong khong thi gian ph hp nu trn (hon ton khng th xy ra), bn phi chun b li mu mi. Nhn bn in ph cc mu ca bn v kim tra li. K nhn vo bn ph v xin ch k ca nhn vin o mu. 6.3 Hy xc nh hp ph cc bc sng tng ng v tnh hm lng (theo mg) ca mi aminoaxit trong hn hp ca bn. di quang l 1,0 cm. in vo Phiu Tr li, nh rng 1 mol ca mi aminoaxit cho 1 mol sn phm tng mg. D liu i chiu: Gi tr ca cc h s tt: 410 nm nm Sn phm ca phn ng Sakaguchi: 7700 M-1 cm-1 500 nm 6.4 V 3 cu trc cng hng ca cc gc phn t tham gia vo s to thnh hn hp mu trong phn ng Ellmann Khi lng phn t ca Cystein 121 g/mol Arginin 174 g/mol Sn phm ca phn ng Ellmann: 13600 M-1 cm-1 aminoaxit: Sn phm ca phn ng Pauli: 6400 M-1 cm-1 470 Histidin 155 g/mol

238

Bi 7. Phn tch nh lng Axit Ascorbic trong vin Vitamin C

L thuyt Thnh phn chnh trong vitamin C thng mi l axit ascorbic (H 2C6H6O27, FW = 176,12). Axit ascorbic va l mt axit, va l mt cht kh, do , c chun axit-baz v chun oxi ha kh u c th s dng xc nh lng axit ascorbic trong nhng vin vitamin C thng mi. Vitamin C l 1 cht chng oxy ha cn thit i vi dinh dng ca con ngi. Thiu vitamin C c th dn n bnh scurvy (scobat) c trng khin cho xng v rng khng bnh thng v mt s bnh khc. Vitamin C l tn thng gi ca axit L-ascorbic (AsA), c danh php quc t l 2-oxo-L-threo-hexono-1,4-lactone-2,3-enediol, CTPT C6H6O6 (M= 176,1g/mol), CTCT nh sau:

Trong cng thc cu to ca ascorbic ta nhn thy c C 4 v C5 l 2 cacbon bt i xng, v vy ascorbic c 4 ng phn quang hc l axit Lascorbic, axit izo L-ascorbic, axit D-ascorbic v axit izo D-ascorbic. Trong s cc ng phn ny ch c axit L-ascorbic v izo L-ascorbic l c tc dng cha bnh cn 2 ng phn cn li l cc khng vitamin, tc l c ch tc dng ca vitamin. Trong t nhin ch tn ti dng axit L-ascorbic, cc ng phn cn li c to ra theo con ng tng hp. Axit ascorbic tn ti dng tinh th hoc bt kt tinh trng hoc hi ng vng, khng mi, c v chua, tan nhiu trong nc (300g/lt), t tan hn trong ru v khng tan trong chloroform, benzene hay cc dung mi h c khng phn cc. Axit ascorbic rt d b phn hy di tc dng ca nh sng v nhit . Dung dchaxit ascorbic khng bn, rt d b oxy ha di tc dng ca oxy

239

khng kh, c bit l khi c mt mt s kim loi nng: Fe, Cu V vy cn phi bo qun vitamin C trong bng ti v nhit thp. Ha tnh ca vitamin C l ha tnh ca nhm chc lacton, ca cc nhm hydroxyl, ca lin kt i, song quan trng nht l nhm chc endiol. Chnh nhm ny quyt nh tnh axit v tnh kh ca axit ascorbic. Nguyn tc phng php 1. Phng php axit baz Trong dung mi nc, axit ascorbic l axit phn ly hai nc vi cc gi tr pKa ln lt bng 4,2 v 11,6 tng ng vi s phn ly H + ca nhm OH nh vo C3 v C2 . Axit ascorbic d dng phn ng vi cc dung dch kim to mui.
CH2OH H H HO OH O O OH

NaOH

HOH2C

(CHOH)3

C O

COONa

H2O

nh lng axit ascorbic c th dng phn ng chun nc 1 vi NaOH, ch th phenolphatalein. 1. Phng php oxi ha kh: Axit L- ascorbic b oxi ha thnh axit L- dehydroascorbic theo bn phn ng oxiha sau y ( E0= 0,127V pH=5)
CH2OH H H HO OH O H O OH H O O CH2OH OH O O

2H+

2e-

Axit ascorbic

Axit dehidroascorbic

Qu trnh oxy ha ascorbic xy ra hai mc khc nhau:

240

- S oxy ha thun nghch vitamin C thnh axit dehydroascorbic: tnh cht ny v cng quan trng i vi tc dng sinh hc ca axit ascorbic l tham gia xc tc cc qu trnh oxy ha kh xy ra trong c th. - S oxy ha bt thun nghch bin vitamin C thnh cc sn phm khc khng c hot tnh v bin mu. Phn ng ny tng nhanh theo pH v nhit ca dung dch. Cc cht oxy ha thng dng oxi ha axit ascorbic l: brom, iot, thuc th Fehling, dung dch KMnO4, dung dch AgNO3, 2,6diclorophenolindophenol. Phng php chun c tin hnh bng cch nh t t dung dch thuc th t buret vo dung dch c cha axit ascorbic trong mi trng thch hp. im tng ng c nhn nh s chuyn mu ca dung dch khi c cht ch th thch hp. Phng php ny c th p dng xc nh trc tip vitamin C trong cc mu thc phm. Trong cc i tng khc nh rau qu, thc phm, nc gii kht c thnh phn tng i phc tp, cha nhiu cht kh khc nhau, dung dch c v c mu, gy kh khn trong vic xc nh im cui ca qu trnh chun . Trong th nghim ny hm lng vitmain C trong vin nn c xc nh bng phng php chun axit- baz hoc chun oxi ha kh. Axit ascorbic c xc nh da trn phn ng oxi ha n bng iot (trong KI d) theo phng php chun trc tip vi cht ch th h tinh bt. Th nghim ny gm hai phn, phn u dng chun axit-baz xc nh lng axit ascorbic trong mt vin vitamin C. Phn th hai dng chun oxi ha kh thc hin xc nh tng t. S nh gi c da trn s chnh xc ca mi php chun . Tnh 30% cho chun axit-baz, tnh 60% cho chun oxi ha kh v 10% cho s so snh hai phng php. KIM TRA THUC TH V THIT B TRC KHI TH NGHIM Thuc th Dung dch NaOH (trn nhn c ghi nng ) Thit b ng ong 10 mL
241

x1

100 mL Dung dch Thiosunfat (Na2S2O3) Cc thy tinh 100 mL (trn nhn c ghi nng ) Dung dch Iod (0.01 M) 250 mL Cht ch th Dung dch Phenonphtalein Dung dch metyl Dung dch h tinh bt Bnh Erlenmeyer 125 mL 250 mL

x1 x2 x2 x4 x2

Giy lc x 10 Giy cn x 10 Mold and Pastel 1 b Buret (1 rack) x2 Buret Brush x1 Bnh nh mc, 100 mL x1 Spatula x1 Phu x 1 Pipet (20 mL) / Bm an ton Pipet Pasteur (ng nh git) x 6 Bn chi x1

1 b

Tin hnh: Ha tan vin vitamin C trong nc, lc nu cn thit. Th tch cui cng ca dung dch nn l 100 mL. Chun b cc dung dch: * Dung dch vitamin C chun: Cn chnh xc lng c 0,1 gam axit ascorbic trn cn phn tch v chuyn nh lng vo bnh nh mc dung tch 250 ml. Thm khong 2 gam axit oxalic vo bnh nh mc, thm nh mc n 2/3 th tch bnh v lc u cho cht rn tan ht sau nh mc n vch mc bng nc ct. Nt kn bnh trnh s oxi ha ca oxi khng kh. Dung dch ny c dng chun dung dch iot. * Dung dch it:

242

Ha tan 5 g KI v 0,268 g KIO 3 trong 200 ml nc ct, thm 30 ml axit sunfuric 3 M v chuyn vo bnh nh mc 500 ml, nh mc n vch mc, ta c dung dch KI3 Phn 1: Chun axit-baz. 1-1 Dng pipet pipet 10 mL ht dung dch trn cho vo mt bnh Erlenmeyer. Chn cht ch th thch hp thc hin s chun . 1-2 Lp li 3 ln bc th 2. Phn 2: Chun oxi ha kh 2-1 S dng dung dch thiosunfat chun xc nh nng dung dch iod cho. 2-1-1 Dng pipet 20 mL a dung dch iodin vo bnh Erlenmeyer, ri chun bng cch s dng dung dch Na 2S2O3 chun. Dng tinh bt lm cht ch th. 2-1-2 Lp li 3 ln bc th 4. 2-2 Xc nh lng axit ascorbic. 2-2-1 chun vi dung dch iod. 2-2-2 Bng d liu 3 3-1 Chun axit - baz
Chun ln 1 Chun ln 2 Chun ln 3 Dung dch Vitamin C Dung dch Vitamin C Dung dch Vitamin C mL; Dung dch NaOH dng mL; Dung dch NaOH dng mL; Dung dch NaOH dng mL mL mL

Dng Pipet 10 mL a dung dch t

bc 1 vo bnh Erlenmeyer. Thm vo vi git tinh bt lm cht ch th v Lp li 3 ln bc th 6.

3-2 3-2-1

Chun oxi ha kh Xc nh nng iot

Chun ln 1 Chun ln 2 Chun ln 3 Dung dch Iod Dung dch Iod Dung dch Iod mL; Dung dch Na2S2O3 dng mL; Dung dch Na2S2O3 dng mL; Dung dch Na2S2O3 dng mL mL mL

3-2-2 Xc nh axit ascorbic

243

Chun ln 1 Chun ln 2 Chun ln 3

Dung dch Vitamin C Dung dch Vitamin C Dung dch Vitamin C

mL; Dung dch Iod dng mL; Dung dch Iod dng mL; Dung dch Iod dng

mL. mL. mL.

Cu hi 7-1 7-2 Gi s axit ascorbic l mt n axit, dng d liu t chun axit-baz tnh lng axit ascorbic trong c vin vitamin C. Phn ng ca I2 vi Na2S2O3 nh sau: 2 S2O32- + I2 S4O62- + 2ITnh nng dung dch iod. 7-3 Phn ng ca axit ascorbic vi I2 l: H2C6H6O6 + I2 C6H6O6 + 2 I- + 2H+ Tnh lng axit ascorbic trong c vin vitamin C 7-4 So snh u im v khuyt im ca hai phng php chun .

Bi 8. Tng hp v phn tch Aspirin

L thuyt Aspirin, acid acetylsalicylic l mt axit hu c c cha c este hu c. N c s dng rng ri trong y hc nh mt loi thuc gim au v nh mt loi thuc gim st. N thng c iu ch bng phn ng ca axit salixylic vi anhydrit axetic theo phn ng sau:

axit Salixylic

anhydrit axetic

axit axetyl salixylic

axit axetic

Mt lng axit axetylsalixylic c th c xc nh bi s chun vi mt baz mnh nh Natri hyroxit CH3COO-C6H4COOH(aq) + OH-(aq) CH3COOC6H4COO-(aq) + H2O(l)

244

Tuy nhin, axit axetylsalixylic cng l mt este nn d dng b thy phn khi chun vi mt baz mnh, do trong mi trng kim n b phn hy dn n sai st trong s phn tch. Nh vy, khi p dng phng php chun th tt c axit c mt trong dung dch s thy phn hon ton trong NaOH d. Mt mol axit trong aspirin phn ng va vi mt mol NaOH, mt mol este trong aspirin phn ng va vi mt mol NaOH. Nh vy s mol NaOH phn ng s gp i s mol aspirin, sau lng NaOH tha s c chun vi dung dch axit chun. Trong th nghim ny, axit acetylsalicylic s c chun b, tng lng acid c mt s c xc nh bng cch s dng mt phng php chun li. Ha cht v Thuc th axit Salixylic HOC6H4COOH anhydrit axetic (CH3CO)2 axit photphoric H3PO4 v axit sunfuric m c Etanol C2H5OH Natri hyroxit NaOH 0,50 mol.L1. axit clohyric HCl 0,30 mol.L1. Cht ch th phenolphtalein. Cht CH3CO2C6H4C CH3C2O3CH3 O2H H3PO4 H2SO4 C2H5OH NaOH(aq) HCl(aq) Trng thi Cht rn Cht lng m c m c Cht lng 0.50 molL0.30 molLK hiu R 22 36 37 38 41 61 10 20 22 34 23 24 25 35 36 37 38 23 49 24 25 35 36 37 38 11 49 20 21 22 36 37 38 35 40 23 25 34 38 K hiu S 22 26 36 37 39 26 36 37 39 45 23 30 36 37 39 45 23 30 36 37 39 45 7 16 24 25 36 37 39 26 45 37 39 45 26 36 37 39 45

Thit b v thy tinh cc, 100 ml, Bnh tam gic (Erlenmeyer) 250 mL
245

phu Buchner giy lc

 bnh lc chn khng Pipet, 5 ml v 10 ml Xi lanh c chia , 50 ml buret, 50 ml Thanh khuy Ming knh ng h A. Tng hp Aspirin, axit axetylsalixylic 1. Cn chnh xc 3,00 gam axit salixylic trong bnh tam gic nh mc 100 mL 2. Thm 6,00 mL anhyrit axetic v 4 8 git axit photphoric vo bnh v khuy, trn k 3. un nng dung dch n khong 80 100oC bng cch t bnh trong nc nng khong 15 pht. 4. Thm tng git 2,0 ml nc lnh cho n khi anhydrit axetic phn hy hon ton v sau thm 40 mL nc v lm mt dung dch trong bnh nc . Nu tinh th khng xut hin, dng thanh khuy c vo thnh bnh to ra kt tinh. 5. Dng giy lc s dng lc. Lc cht rn bng cch lc ht thng qua phu Buchner v ra cc tinh th vi vi ml nc lnh vo khong 5oC. 6. kt tinh li, bng cch ha tan cc tinh th vo cc v thm 10 ml etanol, sau thm 25 ml nc m. 7. y cc bi ming knh ng h v khi s kt tinh bt u th t cc trong bnh nc hon tt s kt tinh li. 8. p dng ht lc nh m t trong bc 5. 9. t cc sn phm vo giy lc vi ming knh ng h v sy kh 100 C trong khong 1 gi ri xem xt sn phm. 10. Xc nh im nng chy (135 C) xc minh tinh khit. B. Xc nh lng axit acetylsalicylic ng mao dn im nng chy Nhit k, 110 C Nhit nng chy b my Bnh ra

246

1. Ha tan 0,5 g aspirin vo 15 ml etanol trong mt bnh tam gic 250 ml. 2. Thm 20 mL dung dch NaOH 0.50 molL-1. 3. tng tc phn ng thy phn, un nng cc mu trong mt cc nc khong 15 pht sau khi b sung hai hoc ba chip si vo bnh xoy trn bnh thnh thong. Ch : Trnh un si, bi v cc mu c th b phn hy. 4. Lm lnh mu n nhit phng v thm 2- 4 git ch th phenolphtalein vo bnh. Mu sc ca dung dch l mu hng nht. Nu dung dch khng mu th thm 5 ml dung dch NaOH 0,50 mol. L-1 ri lp li cc bc 3 v 4. 5. Ghi li tng khi lng dung dch NaOH 0,50 mol.L-1 c thm vo. 6. Chun baz tha trong dung dch bng dung dch HCl 0,30 mol.L-1 cho n khi mu hng bin mt v dung dch tr nn c. 7. Ghi li khi lng dung dch HCl 0,30 mol.L-1 c thm vo. 8. Lp li cc bc ca s chun hai ln na bng cch s dng hai mu mi. X l d liu 1. Tnh hm lng ca Aspirin chun b:

Axit salixylic

Anhyrit axetic
3,00 13812 ,

Axit axetylsalixylic

Axit axetic

Khi lng aspirin theo l thuyt: n(salicylic acid) = = 0,0217 mol, n(aspirin) = 0,0217 mol

Khi lng aspirin = 0.0217180,2 = 3.906 g Thc nghim: Khi lng sn phm kh (aspirin) thu c theo thc nghim = 3,03 g
247

n(aspirin) hay 3,03/180,2 = 0,01682 mol Hm lng ca aspirin =

3,03 100% = 3,91

77%

Hm lng thp do tan ca aspirin trong nc lnh 2. Tnh lng axit axetylsalixylic c trong mu asppirin n(axit axetylsalixylic) theo l thuyt= 40 ml NaOH 0,5 M cha 20 mmol Chun 1,00 g mu vi HCl 0.30 M, thc nghim dng trung bnh 27.0 ml 27 ml HCl 0,3M cha 8,10 mmol n(NaOH dng phn ng vi axit axetylsalixylic) = 20.0 8.10 = 11.9 mmol 1,0 mol axit axetylsalixylic phn ng vi 2,0 mol NaOH nn n (axit axetylsalixylic) =
11,9 = 2 1,00g 180,0g/m ol

= 5.55 mmol

5.95 mmol

loi b axit axetic to ra sau phn ng, qu trnh kt tinh li (ti kt tinh) c lp i lp li v ra sch mu bng nc d. Do lng mu gim t 1,50 g n 1,05 g. Trong s chun ti kt tinh 1,00 g mu vi HCl 0.30 M, thc nghim dng trung bnh 28.9 ml. 28,9 ml HCl 0,30M cha 8,67 mmol n(NaOH dng phn ng vi axit axetylsalixylic) = 20,0 8,67 = 11,3 mmol 1,0 mol axit axetylsalixylic phn ng vi 2,0 mol NaOH nn n (axit axetylsalixylic) =
11,3 2

= 5,67 mmol

3. Tnh tinh khit ca aspirin v biu th bng t l phn trm khi lng im nng chy ca mu l 132oC ch ra rng mu khng tinh khit. Khi lng axit axetylsalixylic tm thy trong (b) nhiu hn so vi l thuyt ch ra rng phng php tnh cho thy khng ch axit axetylsalixylic m cn axit salixylic v sn phm ph axit axetic cng khng phn ng. V vy n s
248

nh hng n tinh khit ca mu aspirin.

Bi 9. Thy phn N-axetyl-alanin bng enzim; v d ca qu trnh ha hc thn thin vi mi trng

L thuyt: Phn ng sinh hc c xc tc bi enzim. Nhiu enzim biu l kh nng chn lc cao v thng c th xc tc chn lc cho mt ng phn i quang ca hn hp trit quang. Trong ha hc hin i enzim c dng cho nhiu qu trnh tin hnh trong ng nghim, c bit trong s tng hp cc ng phn quang hc tinh khit. Th nghim ny xem xt s thy phn N-axetyl-alanin vi enzim axylaza I (acylase I). S phn ng c nu di y:

Hn hp raxemic N-axetyl-alanin ng vi ninhidrin nh di y.

N-axetyl-alanin

alanin

C th theo di din tin phn ng da trn s to thnh alanin nh phn

ninhidrin

indan-1,2,3-trion

alanin Ha cht cn thit: N-axetyl-alanin raxemic (265 mg) Acylase I (10 mg)

anion mu tm lmax = 592 nm M an ton: R-SR-S249

Liti hidroxit (85 mg) 36/37/39 Dimetyl sunfoxit (khang 70 mL) m liti axetat pH 5,2 Tin hnh:

R 23-34 S 45-26-27-36/37/39

Dung dch ninhidrin (Sigma N1632) (2 mL) R 11-20/21/22-34 S 16-26-27R 36/37/38 S 26-36-23 R 20/21/22-63 S 22-36

Ha tan 262 mg (2,0 mmol) hn hp raxemic N-axetyl-alanin trong 10 mL nc. Sau thm t t v khuy nh dung dch cha 84 mg (2,0 mmol) liti hidroxit monohidrat trong 4 mL nc. Theo di pH bng giy pH n khi t pH = 7. Thm dung dch acylase I (10 mg) trong nc ng thi khuy mnh trong 2 pht. [ iu ch dung dch enzim ny bng cch thm enzim vo 5 mL nc v lc bng lc thy tinh nh c ph vi diatomit]. K tip, thm nc vo th tch tng cng t chnh xc 20,0 mL. Hn hp phn ng c gi ti nhit 37C (chng cch thy) trong 60 pht. Dng ng tim hoc pipet nh ly chnh xc 0,25 mL dung dch enzim cho vo ng nghim ri thm 1,25 mL ninhidrin (Sigma N 1632), (1.25 mL). un nng hn hp ny trong nc si khong 20 pht, lc y s c mu tm thm. Sau khi ngui, cho hn hp ny vo dung dch m c cha dung dch liti axetat 4 M (pH = 5,2) v dimetyl sulfoxit theo t l 1 : 3 trong ng ong 250 mL. iu chnh th tch n 250 mL. Sau o s hp th bng ph k ti l = 592 nm. Dng ninhidrin trong cng dung dch m (liti axetat trong dimetyl sulfoxit) tham chiu. e purple complex = 13350 L mol1 cm1. Ghi li cc s liu sau: 1. Nng ban u ca hn hp raxemic N-axetyl-alanin. 2. Hp th ti l = 592 nm. 3. Tnh s mmol alanin to thnh trong phn ng enzim. Dng nh lut Lambert-Beer.
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4. Tnh phn trm chuyn ha. Cu hi: 9-1 9-2 9-3 9-4 Alanin to thnh c quang hot khng? N-axetyl-alanin vn khng quang hot, quang hot hn hay tinh khit v Cng cu hi nh trn khi s chuyn ha t chnh xc 50%? C th chuyn ha trn 50% hay khng? Nu thi gian cho php, c th dng phn ng sau 10, 25, 40 v 60 pht xc nh nng alanin trong mi trng hp. T , v th biu din nng alanin theo thi gian, v c lng thi gian phn ng ti u.

mt quang hc khi s chuyn ha nh hn 50%?

Ghi ch quan trng:

Bi 10 - Phn ng ngng t andol thn thin vi mi trng.

L thuyt c gng thn thin hn vi mi trng, ngi ta ngy cng ch nhiu n vic gim thiu mt lng ln dung mi dng trong cc phn ng ha hc. Trong th nghim sau, mt phn ng andol c thc hin khi khng c dung mi.

Thit b v Ha cht s dng Thit b Cc thy tnh 25 cm3 Ha cht 3,4-dimetoxibenzandehit (DMBA)

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 Bnh tam gic 100 cm3 Tha kim loi a thy tinh pH k Phu Hirsch Phu Buchner Bm ht chn khng Thit b phn tch sc k lp mng TLC n t ngoi (UV) Tin hnh

1- indanon (C9H7ON) NaOH HCl 3M C2H5OH (ethanol) (C2H5)2O (diethyl ethe) C7H14 (Heptane) CH3COOC2H5 (ethyl ethanoat)

1. Cho 3,4-dimetoxibenzandehit (DMBA 0,50 g; 3,0 mmol) v 1-indanon (0,40 g; 3,0 mmol) vo mt cc thy tinh dung tch 25 cm 3. Dng mt tha kim loi nghin nh hai cht rn v c xt cho n khi chng to ra mt cht du trong. 2. Cho NaOH (0,1 g; 2,5 mmol) vo hn hp phn ng, nu c sinh ra bt k s vn cc no th phi nghin nh v tip tc c xt cho n khi hn hp chuyn thnh cht rn. 3. yn hn hp trong 20 pht. Sau thm 4 cm 3 dung dch HCl 3M v c xt quanh cc y xung tt c sn phm bm trn thnh cc. Dng a thy tinh u bng nghin nh bt k s vn cc no xut hin. a) o v ghi li pH ca dung dch. 4. Lc chn khng qua phu Hirsch tch ly sn phm th. Trng cc vi 2 cm3 dung dch HCl 3M v dng n ra sn phm th trn phu Hirsch, tip tc ht chn khng thm 10 pht lm kh sn phm. b) Ghi li khi lng sn phm th (c th n vn cn hi t) v cho vo l dn nhn CPA.

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5. Chy sc k lp mng (TLC) vi h dung mi Et 2O:heptane (1:1) kim tra phn ng kt thc hay cha. Cho sn hai cht u pha trong etyl etanoat. Pha sn phm th vo etyl etanoat. [Lu : c th s dng tt c 3 bn mng cho, nhng ch cn np 1 bn cho vo ti nilon c kha (Ziploc bag), ghi tn v s bo danh ca mnh. y chnh l bn mng m th sinh s v vo phiu tr li.] c) Dng n t ngoi (UV) pht hin cc vt cht; ly bt ch khoanh trn v tr ca cc vt; sao chp (copy) bn mng vo phiu tr li, v sau cho bn mng vo ti nilon c kha (Ziploc bag), ghi s bo danh ca mnh. Xc nh RF v ghi li gi tr tm c. 6. Dng mt bnh tam gic 100 cm 3 c thanh khuy t kt tinh sn phm th trong h dung mi 9:1 EtOH:H2O (Lu : dng phu thy tinh lc nng, cn phi c qu trnh ny loi i mt lng nh tp cht khng tan). Dng a thy tinh u bng nghin cht vn cc nu c. ngui bnh nn cha dung dch lc n nhit phng v sau lm lnh trong chu (dng khay xp polistyren lm chu ng ) trong mt gi, sau lc qua phu Busn (Buchner) thu sn phm sch, ri ht thm 10 pht cho kh sn phm. Cho sn phm vo l dn nhn RPA v ghi s bo danh ca mnh. d) Ghi li khi lng ca sn phm tinh ch. e) V cc cu trc c th c i vi sn phm A, da trn cc thng tin cho trong phiu tr li. f) Ph 13C NMR ca A c dn ra trang sau. Cc pic do dung mi CDCl 3, c nh du sao (*). Da vo ph ny kt lun cng thc no l ng i vi A. nh du cu tr li trong phiu tr li. g) Tnh hiu sut phn trm sn phm tinh ch, da trn cng thc ng vi cu trc m bn nu trn.

Bi 11. Tng hp Benzylhydantoin

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L thuyt -Amino axit l nhng vin gch xy dng cc peptit v protein. Chng cng thng c dng lm vt liu ban u trong tng hp dc phm. Trong th nghim ny S-phenylalanine A thin nhin c chuyn theo hai bc thnh benzylhydantoin C, l cht trung gian hu ch iu ch cc dn xut c hot tnh sinh l khc nhau.
B C1 O H 2N A Kho i l ng pha n t =165,19 OH O 1) NaOCN, NaOH (aq) 2) HCl (aq), 25 oC B HN O OH NH2 C HCl (aq), 80 oC HN O NH B C2 O

Kho i l ng pha n t =208,22

Kho i l ng pha n t =190,20

Phng php tin hnh BC 1 S dng phng php phn tch sc k lp mng TLC S dng phng php chng cch ct BC 2 S dng phng php phn tch sc k lp mng TLC S dng phng php chng cch ct S dng n cc tm (UV)

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BC 1 Gi li mt lng rt nh cht ban u A phn tch sc k lp mng TLC (xem di y). Cho (S)-phenylalanin A (500 mg, 3 mmol, lng chnh xc c ghi trn nhn ca l nh) vo mt bnh cu c di, thm natri xianat (300mg; 4,6 mmol), nc (3 mL) v mt c t (stirring bar). Thm hai git dung dch natri hidroxit (1M) trong nc vo huyn ph khuy. Bnh cu c ni vi b ngng t v un nng hn hp phn ng n 80 oC bng chng cch ct va ng thi s dng khuy t. Quan trng t n nhit thch hp kp thi v khng mt qu nhiu thi gian, hy m in nung ct ngay khi bt u th nghim ny. Dng nhit k kim tra thng xuyn v cn thn nhit ca ct. Sau khi un nng hn hp phn ng ti 80 oC trong t nht 30 pht, ngui dung dch trong sut thu c v nhit phng v vo mt cc nh hnh nn. Ra bnh cu vi mt t nc. Axit ha dung dch bng cch thm tng git axit clohidric (4 M) n pH < 3 ng thi khuy t. Thm t nc vo huyn ph trng thu c d khuy. Lc kt ta trng bng cch ht, ra kt ta bng nc ly d (trn lc) ri ra hai ln vi lng nh di-isopropyl ete loi hu ht nc cn bm vo. Dn xut ca ure B c trn lc v ht trong t nht 3 pht loi b cng nhiu cng tt dung mi cn st li. Mt lng nh dn xut ca ure B to thnh c gi li phn tch sc k lp mng TLC sau ny. BC 2 Dn xut ca ure B nay c chuyn sang bnh cu c di v thm axit clohidric (4 M, 3 mL). Cho c t vo, khuy k huyn ph v un nng n 80 oC bng chng cch ct. Thu c dung dch trong sut. Sau thi gian phn ng khong 30 pht, hn hp phn ng (lc ny cng c th c cht t kt ta) c

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ngui n nhit phng. Lc huyn ph thu c bng cch ht, ra k vi nc v sau cng ra hai ln vi lng nh di-isopropyl ete. Sn phm c li trn lc t ht trong t nht 3 pht. Sau thu ly trn giy lc v kh trong khng kh trong t nht 30 pht. Sn phm cui C, cht B v cht ban u A (xem trn) c em phn tch sc k lp mng TLC. phn tch, cc lng nh mi cht c ha tan trong lng rt t axeton tinh khit. Cc mu nh ca cc dung dch ny c cho ln bn TLC bng cch dng cc ng mao dn c cp. S phn tch c tin hnh mi ln vi hai bn TLC. Cc bn TLC c x l vi cht ra gii l dung dch axit fomic 2% trong etyl axetat. Sau khi ra gii, phn tch cc bn TLC bng n cc tm (UV-lamp). nh du r vch u (starting line), vch cui (front line) v cc im kch hot cc tm (UV-active spots) bng bt ch. Sao chp s vo trong T Bi lm. Cc tr s Rf c xc nh. Cui cng, gi bn TLC vi kt qu phn tch tt nht trong parafilm vo trong ti nha (plastic bag) dng bng dnh dn li. Sn phm cui C c chuyn vo mt l nh xc nh khi lng rng trc (khi lng c ghi trn nhn). Cn l cha sn phm v tnh hiu sut ca sn phm C. Gio vin s kim tra cht lng ca benzylhydantoin do hc sinh iu ch v xc nh im nng chy bng thit b o im nng chy t ng.

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CC CNH BO V CC NGUY HIM C TH GP V KHUYN CO V AN TON TRONG KHI LM TH NGHIM

1. Cnh bo cac nguy c c bit (K hiu R - Risk)
R 1. Gy n khi dang kh. R 2. Nguy c n khi va p, ma sat, co la hoc ngun gy chay khac. R 3. Nguy c gy n rt cao khi va p, ma sat, co la hoc ngun gy chay khac. R 4. Tao ra cac hp cht n kim loai rt nhay. R 5. un nong co th gy n. R 6. Gy n khi tip xuc hoc khng tip xuc vi khng khi. R 7. Co th gy chay. R 8. Tip xuc vi vt liu chay co th gy chay. R 9. Gy n khi trn vi cht d chay R 10. Co th chay. R 11. D chay. R 12. Rt d chay. R 13. Khi hoa long rt d chay. R 14. Phan ng manh lit vi nc. R 15. Tip xuc vi nc giai phong khi d bc chay. R 16. Gy n khi trn vi cac cht oxi hoa. R 17. T bc chay trong khng khi. R 18. Khi s dung, co th tao ra hn hp hi vi khng khi gy chay hoc n. R 19. Co th tao ra cac peoxit gy n. R 20. Nguy him khi hit vao. R 21. Nguy him khi tip xuc vi da. R 22. Nguy him nu nut vao. R 23. Ng c khi hit vao. R 24. Ng c khi tip xuc vi da. R 25. Ng c nu nut vao. R 26. Rt c khi hit vao. R 27. Rt c khi tip xuc vi da.

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R 28. Rt c nu nut vao. R 29. Tip xuc vi nc giai phong khi c. R 30. Khi s dung, co th rt d chay. R 31. Tip xuc vi axit giai phong khi c. R 32. Tip xuc vi axit giai phong khi rt c. R 33. Gy nguy him do cac tac ng tich luy. R 34. Gy bong. R 35. Gy bong nng. R 36. Gy cay mt. R 37. Kich thich h h hp. R 38. Gy mn nga da. R 39. Nguy him do cac tac ng nghim trong khng th loai bo. R 40. Co th nguy him do cac tac ng nghim trong khng th loai bo. R 41. Nguy him do gy hong mt nng. R 42. Co th gy s mui khi hit vao. R 43. Co th gy mn nga khi tip xuc vi da. R 44. Nguy c gy n nu un nong trong binh kin R 45. Co th gy ung th. R 46. Co th gy tn hai gen. R 47. Co th gy tn hai phi. R 48. Nguy him do bi tn hai keo dai.

2. Khuyn cao v an toan (K hiu S - Safety)

S 1. Nut kin binh cha. S 2. cach xa tm vi cua tre con. S 3. Gi ni thoang mat. S 4. Bao quan cach xa khu dn c. S 5. Bao quan binh cha di cac iu kin....(cht long c nha san xut a ra chi dn ring). S 6. Bao quan di cac iu kin... (khi tr c nha san xut chi dn ring). S 7. Bao quan binh cha dang ong kin. S 8. Bao quan binh cha kh rao. S 9. Bao quan binh cha ni thng gio. S 10. Bao quan binh cha cht trong binh dang t.

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S 11. Tranh tip xuc vi khng khi. S 12. Khng bao quan binh cha dang kin. S 13. Bao quan cach xa thc phm, nc ung va thc phm cho gia suc. S 14. Bao quan cach xa ..... ( cac cht ki nhau phai c nha san xut chi inh). S 15. Bao quan cach xa nhit. S 16. Bao quan cach xa ngun phat la. Cm hut thuc. S 17. Bao quan cach xa cac cht d chay. S 18. Tip xuc va m binh cha hoa cht cn thn. S 19 Khi s dung hoa cht khng n hoc ung ng thi. S 20. Khi s dung hoa cht khng hut thuc. S 21. Khng hit bui hoa cht. S 22. Khng hit khi/khoi/ hi/ khi phun sng. S 23. Tranh tip xuc hoa cht vi da. S 24. Tranh hoa cht bn vao mt. S 25. Trong trng hp bi bn vao mt, phai ra ngay vi nhiu nc va n c quan y t. S 26. Ci bo ngay ao qun bi nhim bn hoa cht. S 27. Khi bi dinh vao da, ra ngay vi mt lng nhiu.....(do nha san xut chi inh). S 28. Khng lam kh kit binh cha. S 29. Khng bao gi c cho nc vao san phm nay. S 30. Bao quan cach xa cac cht gy chay. S 31. Cn co cac bin phap phong s phong in. S 32. Tranh va p va ma sat. S 33. Cht nay va binh cha no phai c loai bo theo cach an toan thich hp. S 34. Mc qun ao bao v thich hp. S 35. eo gng tay thich hp. S 36. Trong trng hp khng u thng thoang, phai eo thit bi tr h hp. S 37. eo phng tin bao v mt/ mt. S 38. v sinh san va cac vt dung bi nhim bn hoa cht nay, cn s dung ... (do nha san xut chi inh).

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S 39. Trong trng hp chay va/ hoc n khng c hit khoi. S 40. Trong thi gian phun khoi / phun sng phai eo thit bi tr h hp thich hp. S 41. Trong trng hp chay, s dung ....(chi ro chinh xac dung loai dung cu cu hoa nao. Nu nc lam tng nguy c thi KHNG bao gi c dung nc) S 42. Nu cam thy ngi khng khoe, n c quan y t ngay (co bin chi dn) S 43. Trong trng hp tai nan hoc nu ngi khng c khoe n c quan y t ngay (co bin chi dn).

3. V d v ngha R v S
Di-isopropyl ete (Di-isopropyl ether) Cng thc C6H14O Khi lng phn t 68 oC 102,17 im nng chy Khi lng ring R11 R19 R66 R67 S9 S16 S29 S33 Rt d chy C th to peoxit d n Lm cho da kh nt nu b dy nhiu ln Ha cht dng hi gy chng mt v bun nn Gi ni thng gi Trnh tia la - Khng ht thuc Khng c vo h thng thot nc Cn c bin php chng phng tnh in HCl 0,909 Khi lng phn t 36,46 -85 oC im si 0,72 g/cm3

Axit clohidric (Hydrochloric acid) Cng thc R11 S16 S26 S45 S7 Khi lng ring Rt d chy Trnh tia la - Khng ht thuc Nu ri vo mt, ra ngay vi nhiu nc v xin tr gip y t Khi b tai nn hoc khi thy khng khe, xin tr gip y t ngay (nu c th, mang theo nhn ha cht) Cn gi bnh tht kn

R37/38 Gy nga mt, h thng h hp v da

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Metanol (Methanol) Cng thc CH4O

o

Khi lng phn t -98 C 0,79 g/cm3 im si

32,04 65 oC

im nng chy Khi lng ring R11 Rt d chy R23-25 R39/23 24/25 S7 S16 S45

Rt c khi ngi, tip xc vi da hoc ung Rt c v gy h qu nghim trng khng th hi phc khi ngi, tip xc vi da hoc ung

Cn gi bnh tht kn Trnh tia la Khng ht thuc Mang bao tay v mc y phc bo h Khi gp tai nn hoc cm thy khng khe, xin tr gip y t ngay (nu c th, mang theo nhn ha cht)

S36/37

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