5.3. PHNG PHP Sc k lng

Sc k lng l phng php tch sc k cc cht da trn s phn b khc nhau ca chng gia hai pha khng trn ln, trong pha ng l mt cht lng chy qua pha tnh cha trong ct.

Sc k lng c tin hnh ch yu da trn c ch hp ph, phn b khi lng, trao i ion, loi tr theo kch thc hoc tng tc ho hc lp th.

Thit b

Thit b bao gm mt h thng bm, b phn tim mu, ct sc k (b phn iu khin nhit c th c s dng nu cn thit), detector v mt h thng thu d liu (hay mt my tch phn hoc mt my ghi th). Pha ng c cung cp t mt hoc vi bnh cha v chy qua ct, thng thng vi tc khng i v sau chy qua detector.

H thng bm

H thng bm trong sc k lng phi gi cho pha ng lun chy vi mt lu lng khng i. Nhng bin i p sut s c gim thiu, v d cho dung mi chy qua mt thit b gim xung. ng dn v h thng ni phi l loi chu c p sut sinh ra do h thng bm. Cc bm c th c lp vi thit b loi b bt kh.

H thng iu khin bng b vi x l c kh nng cung cp pha ng hoc hng nh (ra gii ng dng) hoc thay i t l thnh phn (ra gii gradient) theo mt chng trnh xc nh. Trong trng hp ra gii gradient, h thng bm ly cc dung mi t mt vi bnh cha v cc dung mi c th c trn ln p sut thp hoc p sut cao.

B phn tim mu

Dung dch mu th c a vo dng pha ng hoc vo v tr gn u hoc u ct nh mt b phn tim mu c kh nng hot ng p sut cao. C th dng vng cha mu th, c th tch c nh hoc thit b c th tch thay i, c th vn hnh bng tay hoc t ng. Khi tim mu bng tay c th gy ra sai s do th tch tim vo vng cha mu khng 񠠠

Pha tnh

C nhiu loi pha tnh c th c s dng trong sc k lng, bao gm:

-          Silica (silic dioxyd), nhm oxyd hoc than hot tnh dng xp thng c dng trong sc k pha thun m qu trnh phn tch da trn s khc nhau v kh nng hp ph hoc (v) phn b khi lng,

-          Nha hoc polymer c cha cc nhm chc acid hoc base, s dng trong sc k trao i ion m trong s chia tch c thc hin da trn s cnh tranh gia cc ion cn tch v cc ion trong pha ng,

-          Silica xp hoc polymer, s dng trong sc k ry phn t, s chia tch da trn s khc nhau v kch thc phn t, tng ng vi s loi tr khng gian .

-          Rt nhiu cht mang bin i ho hc c ch to t polymer, silicagel hoc than graphit xp c dng trong sc k lng pha o m s chia tch v nguyn tc c bn da trn s phn b phn t cc cht gia pha ng v pha tnh.

-          Pha tnh loi bin i ho hc c bit, v d dn xut ca cellulose hoc amylose, protein hoc peptid, cyclodextrin vv... dng tch cc ng phn i quang (sc k i quang).

Phn ln s chia tch da trn c ch phn b, s dng silica bin i ho hc lm pha tnh v cc dung mi phn cc lm pha ng. B mt ca cht mang, v d nh cc nhm silanol ca silica c phn ng vi cc thuc th silan khc nhau to thnh cc dn xut silyl c lin kt cng ha tr, che ph mt s lng khc nhau cc v tr hot ng trn b mt cht mang. Bn cht ca cc pha lin kt l tham s quan trng xc nh cc tnh cht tch ca h sc k.

Cc pha lin kt dng ph bin l:

Octyl = Si (CH2)7CH3 C8

Octadecyl = Si (CH2)17CH3 C18

Phenyl = Si(CH2)n (C6H5) C6H5

Cyanopropyl = Si(CH2)3CN CN

Aminopropyl = Si (CH2)3NH2 NH2

Diol = Si-(CH2)3-OCH(OH)-CH2-OH

Tr khi c tiu chun ring ca nh sn xut, thng thng cc ct sc k pha o da trn silaca c coi l n nh i vi pha ng c pH t 2,0 ti 8,0. Ct cha than graphit xp hoc cc ht vt liu polymer nh styren - divinylbenzen copolymer n nh mt khong pH rng hn.

Phn tch s dng sc k pha thun vi pha tnh l silica khng b bin i, than graphit xp hoc silica bin i ha hc lm cho phn cc (v d cyanopropyl hoc diol) v pha ng khng phn cc c s dng trong mt s trng hp.

i vi s tch nhm mc ch phn tch, kch thc ht ca pha tnh ph bin nht t 3 μm ti 10 μm. Cc ht c th hnh cu hoc khng c hnh dng nht nh, c xp khc nhau v din tch b mt c hiu. Nhng tham s ny cu thnh biu hin sc k ca tng pha tnh c th. Trong trng hp pha o, cc yu t b sung nh bn cht ca pha tnh, mc lin kt, v d nh di mch carbon lin kt, hoc cc nhm hot ng b mt ca pha tnh c c che ph ht hay khng. S ko ui pic, c bit ca cc cht base, c th xy ra khi c mt cc nhm silanol b mt ca silica.

Ct c lm bng thp khng g tr khi c ch dn khc trong chuyn lun ring, c chiu di v ng knh trong (f) khc nhau c s dng cho phn tch sc k. Ct vi ng knh trong nh hn 2 mm thng c coi l vi ct. Nhit ca pha ng v ct phi c gi n nh trong sut thi gian phn tch. Phn ln qu trnh tch c thc hin nhit phng, nhng ct c th c lm nng nhm thu c hiu qu cao hn. Tuy nhin, nhit ct cng khng c php vt qu 60 0C v kh nng phn hu ca pha tnh hoc s thay i thnh phn ca pha ng c th xy ra.

Pha ng

i vi sc k pha thun, thng s dng dung mi t phn cc. S c mt ca nc trong pha ng phi c hn ch v kim tra cht ch nhm thu c kt qu ti lp li. i vi sc k lng pha o, s dng pha ng cha nc, c hoc khng c dung mi hu c.

Cc thnh phn ca pha ng thng c lc nhm loi b cc tiu phn ln hn 0,45 μm. Pha ng cha nhiu thnh phn c chun b bng cch ong cc th tch qui nh (tr khi c ch nh v khi lng) ca cc thnh phn ring l ri sau trn ln vi nhau. Ngoi ra, dung mi cng c th c cp qua cc bm ring l, iu khin bng cc van chia t l thun, c th trn ln theo cc t l mong mun. Dung mi thng c loi kh trc khi bm bng cch sc kh heli, lc siu m hoc s dng h thng lc mng lc/chn khng trc tuyn nhm trnh s to bt kh trong cc o ca detector.

Dung mi dng chun b pha ng thng khng c cha cc cht lm n nh v phi trong sut (khng hp th quang) vng bc sng pht hin, nu nh s dng detector t ngoi. Dung mi v nhng thnh phn khc c dng phi c cht lng ph hp. Khi cn iu chnh pH ch thc hin vi thnh phn nc ca pha ng m khng iu chnh vi hn hp. Nu s dng dung dch m, cn phi ra h thng bng hn hp nc v dung mi hu c (5% tt/tt) nhm ngn chn s kt tinh mui sau khi kt thc qu trnh sc k.

Pha ng c th cha nhng thnh phn khc, v d mt ion tri du trong sc k to cp ion hoc mt cht chn lc i quang trong trng hp sc k s dng pha tnh khng chn lc i quang.

Detector

Detector hp th t ngoi/kh kin gm c detector chui diod l c s dng ph bin nht. Detector hunh quang, detector khc x vi sai, detector in ho, detector khi ph, detector tn x nh sng bay hi, detector phng x hoc cc loi detector c bit khc cng c th c s dng.

Cc i lng c trng cho qu trnh sc k: Xem Ph lc 5.8.

 

 

 

Phng php tin hnh

Lm cn bng ct vi pha ng v tc dng theo qui nh, nhit phng hoc nhit qui nh trong chuyn lun ring, cho n khi ng nn n nh. Chun b cc dung dich chun v dung dch th theo yu cu. Cc dung dch phi khng c c cc tiu phn rn.

Hiu nng

Qui nh v s ph hp ca h thng c m t trong Ph lc 5.8. Cc k thut tch sc k. Xc nh v iu chnh cc thng s ca h thng sc k c th c thc hin nhm p ng cc yu cu v s ph hp ca h thng cng c trnh by trong Ph lc ny.

Thnh phn v tc dng ca pha ng c qui nh trong chuyn lun ring. Pha ng l hn hp dung mi c ui kh bng bm chn khng hoc bng mt thit b ui kh khc ph hp nhng khng nh hng n thnh phn ca hn hp.

Trong qu trnh nh lng, khi trong chuyn lun ring khng qui nh dng chun ni, nn s dng b phn tim mu c th tch c nh. Trong mt s trng hp ngoi l, khi trong chuyn lun ch dn tnh theo chiu cao pc th khng cn quan tm n h s i xng.

Ct sc k thng c lm bng thp khng g c kch thc (chiu di x ng knh trong) c qui nh trong chuyn lun ring. Trong chuyn lun ring, khi pha tnh c k hiu bng mt ch ci th tra cu phn Nguyn vt liu nu di y. ng knh danh ngha ca ht pha tnh c trong ngoc n ngay sau k hiu ch ci c th. Nu khng c ch dn khc trong chuyn lun ring, qu trnh sc k c tin hnh iu kin nhit khng i trong mi trng phng th nghim. Khi s dng cc pha ng c pH cao vi ct c bn cht l silica nn s dng mt tin ct trc ct phn tch.

Tr khi c cc ch dn khc trong chuyn lun ring, h thng detector gm mt detector o quang gn vi mt cc o c th tch nh (khong 10 ml l ph hp). Phi t bc sng theo ch dn trong chuyn lun ring.

Khi dng mt my sc k c thit k ring bit c th phi thay i cc iu kin sc k ghi trong chuyn lun ring. Trong trng hp ny, ngi phn tch cn m bo rng nhng thay i cho kt qu tng ng.

Th tch tim

Khi th tch tim khng qui nh trong chuyn lun ring, nn chn mt th tch tim ph hp p dng. Chn th tch tim ph thuc vo p ng ca php phn tch, detector s dng, hiu lc ct v ton b hiu nng ca h thng sc k. Khi khng c ch dn, thng dng th tch tim l 20 ml, tuy nhin cn kim tra s thch hp trong iu kin c th.

Pc th cp

C th cn cht i chiu xc nh pc th cp. Pc th cp l mt pc c trn sc k nhng khng phi l pc chnh hay pc ca chun ni hoc pc ca dung mi hay ca cc thuc th to dn xut.

Nguyn vt liu

Cc dung mi v thuc th dng pha cc dung dch s dng trong phn tch phi c cht lng thch hp cho sc k lng.

Khi chuyn lun ring quy nh pha tnh c gn vi mt ch ci (A hoc B hoc C) l mun ni ti cc pha tnh nh m t di y:

Pha tnh A, ht silica

Pha tnh B, ht silica c bin i ha hc, gn vi nhm octylsilyl (C8)

Pha tnh C, ht silica c bin i ha hc, gn vi nhm octadecylsilyl (C18)