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Nhm 4: Nguyn Th Th Thanh Tuyn Ung Quang Nht Trn Th Hng Tm

BI TH NGHIM S 1 NH LNG AMINO ACID BNG NINHYDRIN


I. NGUYN TC Nguyn tc Di tc dng ca ninhydrin, acid amin oxi ho b gim bt mt nguyn t C, n tr thnh dng aldehyde ri n ammoniac v carbonic, acid amin.Nh vy cc acid amin c khi lng phn t nh hn v amoniac to thnh s oxi ho ninhydrin to nn phc hp c mu xanh tm, cng mu tng ng vi gi tr nit amin ca acid amin. Phn ng ca acid amin vi ninhydrin l c s nh lng cc hp cht theo phng php kh k bng cch tnh s lng CO2 hoc NH3 bay ra, cn phng php so mu li o cng mu, n ph thuc nng phc hp ca ninhydrin vi NH3. Phn ng mu ninhydrin cng xy ra vi acid amin mch bn ca peptide v protein cng nh vi mui amoni, glucozamin v ammoniac. nh lng chnh xc acid amin, dung dch nghin cu phi khng c hp cht trn. Phn ng ca acid amin vi ninhydrin c s dng nhiu nh tnh v nh lng acid amin, c bit l phng php sc k trn giy v sc k trn ct nha trao i ln nh my phn tch acid amin t ng do Spackman, Stein v Moose xng. Phn ng ny rt nhy, c th

cho php ta xc nh c cc acid amin c nng rt nh (vi microgam).

II.

THC HNH

2.1 Ha cht Dung dch mu cha acid amin c khong 3-5g nit trong 1ml Dung dch ninhydrin: ho tan 2g ninhydrin trong 50ml dung dch etanol 50%. Dung dch m citrate 0,4M; pH = 5. Dung dch etanol 50%. Dung dch chun leucin

2.2 Dng c th nghim Bnh nh mc 10ml. Pipet 1ml, 5ml. ng nghim 10ml Ni . My so mu 2.3 Cch tin hnh Ly 0,1ml dung dch nc mm cho vo bnh nh mc 100ml,thm nc ct vo cho n 100ml ta c dung dch acid amin cn nh lng. Ly 1ml dung dch acid amin pha long trn cho vo ng nghim. Cho thm 1,5ml dung dch m citrate 0,4M v 2ml dung dch ninhydrin. Lc u ng nghim v t vo ni nhit un si 30 pht. Ly ng nghim trong ni nhit ra v vo bnh nh mc 10ml.Cho thm etanol 50% n 10ml. o gi tr hp thu OD 570nm. Chnh my v vi ng kim tra, ng c cc ha cht nh ng ban u nhng thay dd acid amin bng nc ct cng th tch. Gi tr hp th nhn c tng ng vi hp th ca dung dch chun leucin bit nng (cng mu trong phn ng ninhydrin tng ng vi cng mu trong dung dch chun leucin). ng chun leucin: Ly 5 ng nghim, t ln gi.V ln lt cho vo tng ng nghim:

ng 1 2 3 4 5

Dung dch leucin (g) 20 40 60 80 100

Lm phn ng vi cc ha cht trn v o hp th

III. KT QU Thi gian kho st : 10 pht ng Nc 1 Mu sc OD ++++ 0,112 + 0 ++ 0,033 2 +++ 0,075

Mu

Dung dch leucin 3 4 5

+++++ ++++++ +++++++ 0,143 0,177 0,209

OD
0.25 0.2 0.15 0.1 0.05 0 0 -0.05 20 40

ng chun
y = 0.0022x - 0.0042 R = 0.9871

ng chun Linear (ng chun)

60

80

100

120

Nng leucin ( g)

IV. BIN LUN KT QU Cc axit amin khi phn ng vi ninhydrin s b dezamin ha, oxy ha v decacboxy ha to NH3, CO2, v aldehyt tng ng.

Phn ng ninhydrin l phn ng mu, dng nh tnh v nh lng axit amin, imino axit v amin. Khi un nng ninhydrin trong mi trng kim vi cht c nhm chc amin bc 1 s thu c sn phm c mu tm xanh c maximum hp th 570 nm. Mt quang ca sn phm thu c tuyn tnh ph thuc vo s lng nhm amin t do. Da vo tnh cht ny ngi ta c th nh tnh c axit amin.

i vi ng chun: theo th t ng, nng dung dch leucin tng dn, lng phc hp sinh ra t phn ng cng tng nn mu xanh tm cng m dn theo th t ng, lng OD cng tng dn.ng nc ch c mu vng ca ninhydrin, khng c mu xanh v trong ng khng c acid amin to ra phc mu xanh tm. Mu: c pha long vi lng thch hp nn thuc ng chun y = 0.0022x 0.0042 vi OD = 0.112 C =53g

V. PHN M RNG Phng php sc k giy L mt phng php sc k dng giy hp th phn tch mt lng nh vt cht lng mang (cht ho tan) v ly ra khi vch cht ho tan tin gn u kia ca mt bng giy. C th dng sc k hai chiu nu dng giy vung v hai cht ho tan khc nhau. Giy c rt ra khi cht lng mang th nht, quay mt gc vung v nhng vo cht lng th hai. Ta s c bn hai chiu ca cc thnh phn git th. S ging nhau, khc nhau c th tm thy bng cch o gi tr Rf ( chuyn ng tng i) Nguyn tc ca phng php ny l da vo s phn b gia hai pha dung mi: dung mi c nh v dung mi di ng. Dung mi c nh thng l nc gi giy sc k ( trong iu kin bo ha hi nc, giy c th gi 15-20% nc tnh theo trng lng giy). Dung mi di ng thng l mt dung mi ha c bo ha nc di chuyn trn t giy theo mao dn ko theo cc cht trong dung dch. Tc di chuyn ca tng cht khng ging nhau v mi cht c c trng bi mt tr s nht nh c gi l Rf. C nhiu sc k giy khc nhau l sc k mt chiu i xung, sc k vng nm ngang v sc k hai chiu..Loi giy thng dng l Whatman s 1 v Shleicher-Schull 2044 a v b, dung mi gm cc cht nh 4 Butanol: 1 Acetic acid: 5 nc (dng cho chiu th nht: 3 phenol: 1 nc (dng cho chiu th 2). Trong : - a l khong cch dch chuyn ca cht phn tch - b l khong cch dch chuyn ca dung mi Sc k lp mng Nguyn tc ca phng php ny dc trn l thuyt ca sc k giy, ngha l cng da trn s phn b cc cht gia hai pha: cht hp ph c trn

rng trn mt phin knh to thnh mt lp mng v pha di ng l dung mi thch hp. Dung mi di chuyn lm dch chuyn cc cht trong mu th. Cc cht hp ph thng dng l Silicagel, alumin acid,Sephadex,v.v c kt hp vi thch cao (gypse) dn vo phin knh Sc k kh Nguyn tc ca phng php ny l li dng tnh cht kh bay hi ca cc acid amin nn c th s dng chng trnh nhit chuyn chng thnh cc dn xut ( thng l N-acetyl-amin). Cho tc dng acid amin vi cn amylic v HBr khan, sau cho tc dng hn hp ny vi anhydric acetic. Ct thng dng l loi Chromosorb W (60-80 mesh) c mt lp polyetylenglyco 1% (cobowax 1564 hoc 6000). Chng trnh nhit gia 1250 C v 1550 C, tc chy 60-240 ml/pht. Phng php Sorensen 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 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 v - 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

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