I /C s phng php sc k trao i ion ( Ion exchange chromatography - IEC).

. 1.Gii thiu chung - Sc k l mt h cc k thut ha hc phn tch dng tch cc cht trong mt hn hp v c tin hnh ln u bi nh thc vt hc ngi Nga Mikhail Tsvet (Mikhail Semyonovich Tsvet) vo nm 1903 khi ng ang nghin cu v chlorophyll. - Sc k trao i ion l mt phng php sc k lng rn m pha tnh l hp cht c kh nng trao i ion ( gi l cc ionit ). - Sc k trao i ion c phn loi da trn c ch ca qu trnh tch : do i lc khc nhau ca cc ion trong dung dch ( pha ng) vi cc trung tm trao i ion ( nhm cha ion) trn cht rn ( pha tnh ). - y l phng php c s dng nhiu trong cc phng th nghim Ha sinh.Theo nhng thng k mi nht, IEC l k thut tch protein c s dng nhiu nht trong cc quy trnh tinh ch (c th bao gm nhiu bc v nhiu k thut khc nhau): IEC: 75%, sc k i lc (AC): 60%, sc k lc gel (GF): 50% cc trng hp. 2.C ch ca phng php.

- Trc tin, cht cn phn tch s gn thun nghch vi cc cht trao i bng tng tc ion gia cc nhm mang in tch tri du. - Sau , cc ion trao i c chit rt ring bit bng phng php ra gii hoc ra y.C th tch chit bng cch thay i pH (t dng hn) khin cc nhm tng tc trn cht phn tch b mt in tch .

S phn tch cc ion tch in ( hnh vung) trong mi trng trao i anion ( hnh trn )

Nguyn tc : - C s ca IEC l s cnh tranh cc nhm tch in tri du trn cht trao i gia cc ion ca cht cn tch vi cc ion khc. - Khi nng ion cnh tranh thp, cc ion cn tch s lin kt vi cht trao i, Khi nng ion cnh tranh cao, cc ion cn tch s ri ra. u im : - C phn gii cao - a dng (cc loi nha trao i khc nhau, nhiu cch kt hp gia m v pH).

- Nguyn tc tch trc tip (cn c trn khc bit in tch). - D dng tin hnh. 3. Cc loi nha trao i ion 3.1. KN Ionit l nhng i phn t acid hoc base khng tan trong nc hoc dung mi c cha trn mng li nhng ion linh ng,c kh nng trao i theo ng lng v thun nghch vi cc ion cng du trong dung dch cht in ly khi tip xc. 3.2. Phn loi Da vo du in tch ca cc ion trao i, ngi ta phn chia thnh 2 loi : - Cationit ( RH, RNa, R2Mg, R2Ca ): ion c nh ca mng li mang in tch m, thng l nhm acid. Ion linh ng mang in tch dng. Nha c kh nng trao i anion. VD : 2R-Na + CaCl2 R2-Ca++ + 2NaCl - Anionit ( ROH, RCl, R2CO3, R2SO4 ) : ion c nh l nhm base mang in tch dng. Ion linh ng tch in m. Nha c kh nng trao i cathion. VD : 2RCl + NaSO4 R2SO4 + 2NaCl - Ionit lng tnh: c th trao i c ion mang in tch dng ln ion mang in tch m. Da vo cc nhm c cng bn cht : - Ionit v c thin nhin v tng hp. - Ionit hu c thin nhin v tng hp. - Ionit c bit : c nhm to phc , ionit lng tnh , ionit redox, ionit lng, mng trao i ion.

Mt s nhm trao i ion trn nha.

4.Chit tch cc ion ca cht cn phn tch khi ct sc k. Phng php c s dng nhiu nht l phng php ra gii : - Cho V(ml) dung dch cha hn hp cc cu t (v d, hn hp hai cu t A v B, trong A c i lc vi ct nh hn B) chy qua ct. Cc cu t A, B cha trong Vml trc ht s b gi li phn trn ca ct. - Sau cho dung dch ra (thng l dung mi ho tan cc cu t) chy qua ct. Lc cc cu t b gi phn trn ca ct s b dung mi ra v a dn xung pha di. Cu t A c i lc vi ct nh hn B nn chuyn ng xung pha di nhanh hn B. Nu ct di v ch chy ca dung dch ra thch hp th sau mt thi gian cho chy dung dch ra, cc cu t tch ra thnh tng vng. Cc vng ny s tun t thot ra khi ct, mi vng li c cch nhau bng mt phn dung mi. Hiu qu tch bng phng php Trong phng php ra gii, ngi ta gradient elution cng hay dng nhng dung dch cha mt cu t c i lc vi ct nhng phi nh hn i lc ca cc cu t cn tch vi ct. S dng mt dung mi n gin c thnh phn khng i: isocratic S dng hai hay nhiu hn cc h dung mi c phn cc (polarity) khc nhau nhiu: gradient elution=> tng cht lng ca qu trnh tch (improve seperation efficiency) Hnh bn di biu din ng cong thot ca qu trnh ra gii.

5. Cch chun b v ti sinh ct - Cn ra, lm trng (mt s nha c bn dng c trng sn c th dng ngay), v x l nha thnh dng cn thit trc khi dng. Cc IEC c th ti s dng nhiu ln, sau mi ln s dng nht thit phi ti sinh (hi nguyn) nha. - Ra v lm trng trn nha vi khong 10 th tch m, lng, gn b cc ht l lng( m bo tc chy n nh).

II/ Cch tin hnh phn tch hm lng Nitrate v Nitrite bng phng php sc k trao i ion
1. Chun b mu.

- Ly 10g mu nghin nh, thm 50ml nc 60oC . - Trn u v lng trong 10 pht. - Chuyn sang bnh nh mc 200ml. Thm 50ml acetonitrile v o nh. - nhit phng. Thm nc tinh khit ti mc, lc u. - Lc qua giy lc. Sau cho dung dch ha tan qua mt mng lc, kch thc l khong 0.45m .

Acetonitrile

2.iu kin tin hnh : Ct sc k : Waters IC-PacTM Anion HC 150 x 4.6 mm column Pha ng : m Lithium borate gluconate , gradient tch (ra gii ) : acetonitrile/water (pH~6.5) , mu rau phn tch. Tc dng : 1.0 mL/min. Detector quang : UV

 Bc sng : 205 nm Dung tch mu : 40 L 3.H thng thit b: - Bnh cha dung mi pha ng - B phn kh kh - Bm cao p -B phn tim mu ( bng tay hay Autosample) -Ct sc k ( Pha tnh ) ( ngai mi trng hay trong b iu nhit ) - Detector ( nhn tn hiu ) -H thng my tnh gn phn mm nhn,tn hiu v s l d liu v iu khin h thng HPLC. - In d liu . 4. Kt qu :

- Da vo thi gian lu ca Nitrate v Nitrite dung dch chun th hin pic sc k u tin, ta c th xc nh c nh Nitrate/Nitrite trong mu cn phn tch trong tp hp cc cht m sc k trao i ion tch c pic 2 v 3.

- AU : hp th ( Absorbance Unit). 5. Kt lun phng php : Hiu qu, chn lc cao, ng dng rng ri Th tich mu nh C th khng ph hy mu (nondestructive of sample) nh lng d dng p dng c vi cc mu khng bay hi v khng bn nhit p dng c cho cc ion v c Gii hn phn tch ca phng php khng cao (ch xc nh c 0,1 mg NO2-/ lit ).