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        二維液相色譜接口的改進及其在蛋白質(zhì)組學研究中的應用

        2010-01-11 02:39:30李篤信張凌怡杜一平張維冰
        色譜 2010年2期
        關(guān)鍵詞:遼寧大連利特分析儀器

        李篤信, 張凌怡, 李 彤, 杜一平, 張維冰*

        (1.華東理工大學,上海 200237;2.大連依利特分析儀器有限公司,遼寧大連 116023)

        In recent years,the com p rehensive tw o-dim ensional liquid chrom atography(2D-LC)has gained m uch attention forthe separation ofcom p lex sam p les in various fields of research[1,2].Several2D-LC system s have been developed through the past over a decade[3-5].Colum n sw itching betw een the tw o dim ensions is a delicate operation to design and imp lem ent for its functions of connections,valving,storage and enrichm ent[6].

        There are several kinds of interfaces[7,8] have been used in online2D-LC system,like samp le loop,parallel colum n and trap colum n interface.Sam p le loop interface is w idely used in2DLC system. How ever,sm all loop volum e lim its the flow rate and colum n dim ension of the first dim ension.The use of parallel colum n in the second dim ension colum ns can carry out several second dim ension separations sim ultaneously [9]. A lthough this im p roves the overall speed of the 2D-LC analysis,it requires m ore elaborate instrum entation,and causes difficulty in the data analysis[10,11].

        The trap colum n interface[12]refers to packing suitable adsorbent in the sam p le loop to collect the fractions eluted from the first dim ension colum n and storing them for periodic re-injection into the second dim ension colum n.W hile one colum n traps the com ponents at its head,the other colum n back-flushes to induce the com ponents to the second dim ension separation[6,13].

        Excep t for the various com binations of the tw o dim ensions,m ost system s em phasized the separation speed of the second d im ension[14,15]. Several kinds of the fast separation(fast gradient elution or/and short colum n)w ere used in the second dim ension in the online2D-LC system [16 -19]. How ever, the second d im ension could achieve high separation speed but lose efficiency w hen a shorter colum n w as used.In this paper,a longer trap colum n w as em p loyed to construct an interface that allow ed increased separation efficiency in the second dim ension w ithout affecting the valve sw itching frequency. The com ponents could be p re-separated through interface before entering to the second dim ension.The rat serum sam p le w as used to evaluate the perform ance of the2D-LC system.

        1 Exp e rim en t

        1.1 C hem ica ls and reagen ts

        A ll of the w ater used in the experim ent w as p repared using a Sartorius A rium 611 System (Sartorius, Germ any). Trifluoroacetic acid (99%)w as obtained from J&K Chem ical(B eijing,China).Acetic acid,sodium acetate and N aCl of analytical grade w ere purchased from Sinopharm Chem ical Reagent Co.,L td. (Shanghai,China).Acetonitrile(HPLC gradient grade) w as purchased from Xingke(Shanghai Xingke B iochem istry Co.,Ltd.(Shanghai,China).Rat serum sam p le w as kindly donated by Laboratory of System s B iology (Institutesof B iom edical Sciences,Fudan U niversity).Rat blood w as centrifuged at5 000r/m in for10m in;the upper layer w as taken as the sam p le.

        1.2 Instrum en ta tions

        For the first dim ension separation,a standard Elite230Series chrom atographic instrum ent controlled by EC2000Chem station softw are version v1.5 (EliteHPLC L td.,D alian, China)w as used.This instrum ent w as equipped w ith M odel 7725injection valve (Rheodyne,USA)w ith a hom e m ade300μL sam p le loop and abinary pum p ing system.A w eak anion exchange(WAX) colum n(AX250mm×4.6mm,5μm,B ioBasic) w as used as the first dim ension.A reversed-phase (RP)colum n(ODS-BP50mm ×4.6mm,5μm, SinoChrom)w as chosen as the second d im ension. Independent binary pump system identical to first dim ension w as used.Tw o RP colum ns(ODS-BP 35mm ×4.6mm,5μm,SinoChrom)w ere connected by10-port valve(M odel EV750-102,Rheodyne,USA)through0.01″i.d. PEEK tubing to serve as trap colum n.The UV detector w as set at the end of the second dim ension colum n.

        2 Resu lts and d iscuss ion

        2.1 P rincip a l of the in te rface

        In order to increase the speed of the second dim ensional separation,fast separation of second dim ension is often used in the2D-LC system s.A lthough it increases the valve sw itching the frequency,it also causes colum n efficiency loss. In our w ork,to acquire the enhanced colum n efficiency and m aintain the separation speed,longer trap colum ns w ereem p loyed and w erealw ays flushed forw ard w hatever in the loading status or elution status. The trap colum ns had tw o functions:one w as to trap the elution;the other w as to p re-separate the com ponents retained on the colum n.The p rincip les of im p roved trap colum n interface are show n in Fig.1.

        F ig.1 Princip les of the in te rface in2D-LC system

        Colum n1and2,served as thetrap colum ns and had the function of p re-separation.A t position1,colum n1w as connected to the first dim ension.The fractions from the p revious dim ension w ere cap tured by colum n1and retain at the head of the colum n.Solvent of the first dim ension w as drained to the w aste directly.W hen changed to position2,the colum n1w as connected to the second dim ension directly.Increased organic solvent gradient from the pum p system eluted the colum n1and second d im ension colum n in series. Com ponent enriched at the head of colum n1in position1got retention at colum n1and the second d im ension colum n,and w ere eluted by the organic solvent gradient gradually.A t position2, colum n1perform ed the function of p re-separation.Sim ultaneously,colum n2p lays the role of trap colum n.W hen the separation of the second dim ension com p leted,the valve w as sw itched to the position1,and colum n2w as eluted by the pum p system and the circulation p rocess started. The p re-separation w as carried out through the longer trap colum n before com ponents arrived the second d im ension.The sw itching frequency of the valve can increased the separation efficiency of the second dim ension.

        F ig.2 O ne dim ensiona l sepa ra tions of a ra t se rum sam p le(a.IEX sepa ra tion;b.RP sep a ra tion)Conditions:a.B ioBasic AX colum n,250mm ×4.6mm,5 μm;m obile phases,(A)20mm ol/L Tris-HCl(pH7.5)and (B)20mm ol/L Tris-HCl,3m ol/L NaCl; flow rate,0.2 mL/m in;gradient elution,0-60m in,0%B-60%B;detection w avelength,280nm;b.SinoChrom ODS-BP colum n,250mm ×4.6mm,5μm;m obile phases,(A)w ater containing0.1% TFA and(B)acetonitrile containing0.1%TFA;flow rate,1 mL/m in;gradient elution,0-8m in,30%B-40%B;8-40 m in,40%B-60%B;detection w avelength,280nm.

        2.2 C h rom a tograp h ic cond itions

        Before the2D-LC analysis,the rat serum w as investigated by the one d im ensional m ode. The WAX colum n and the RP colum n w ere used and conditioned to get the op tim ized chrom atographic param eters. The chrom atogram s of the one dim ensional separation are show n in Fig.2.In Fig. 2a,only a few peaks are recognized in the ion exchange chrom atography(IEX)separation m ode, w hich did not give a pow erful resolution. The sam e situation exists in the RP separation in Fig. 2b.How ever,the one dim ensional separation w as restricted by its low er capacity.

        2.3 2D-LC sep a ra tion of ra t se rum sam p le

        W hen the35mm colum n w asused as the trap colum n to perform the p re-separation function, the separation pow er of the second d im ension, w hich w as50mm long,w ould theoretically increase by70%.B ecause of the volum e of the m ixer and the trap colum n,w hich generated a delay tim e,the t im e of m obile phase of the initial gradient reaching the separation colum n w aspostponed by2.5m in.As aresult, the valve w as sw itched w hen the gradient had passed2.5m in and data acquisition started at the sam e tim e.The m odulation tim e of the second dim ension w as5 m in. The chrom atogram s of rat serum sam p les separatedon this2D-LC system areshow n in Fig.3.

        F ig.3 2D-LC chrom a togram s of aqueous ex tract of ra t se rum sam p lesConditions:first dim ension,B ioBasic AX colum n,250mm × 4.6mm,5μm;m obile phases,(A)20mm ol/L Tris-HCl(pH 7.5)and(B)3m ol/L NaCl,20mm ol/L Tris-HCl;flow rate: 0.2mL/m in;gradient elution,0-60m in,0%B-60%B;Second dim ension,SinoChrom ODS-BP colum n,50mm ×4.6 mm,5μm;m obile phases,(A)w ater containing0.1%TFA and (B) acetonitrile containing0.1%TFA; flow rate, 1 mL/m in;gradient elution,0-2.5m in,30%B-60%B;2.5-5 m in,0%B-30%B;detection w avelength,280nm.

        A total of16fractions of the first dim ension w ere introduced to the second dim ension. The negative peak betw een0.8m in and1.6m in attributed to the UV absorbance of the m obile phase cap tured in the trap colum n.W hen com paring w ith the traditional trap colum n interface, the elution from0-0.8m in w as additional separation pow er p rovided by the forw ard-flushed longer trap colum n.

        According to Shalliker’s report[20]that the viscous fingering(VF)phenom enon w as generated from the velocities difference of the m obile phases of the tw o dim ensions in the region they contacted directly.W hen the viscosity of injection p lug w as bigger than that of the m obile phase,VF generated at the rear of the injection p lug.O n the contrary,VF w ould be generated at the head of the injection p lug.Since the m obile phase in the first d im ension w as pure buffer,w hose viscosity w as bigger than that of m obile phase of the second d im ension,so the VF generated at around 1.6m in.M ass transfer in direct contact regions w as very slow.The intact region betw een the m obile phases of tw o dim ensions w as just like the m oving reaction boundary.So the desalting of the fraction and re-equilibrium of the colum n can be avoided.Fast gradient elution of the second dim ension can be carried out directly.

        3 C onclus ion

        A WAX-RP2D-LC system w as constructed w ith im p roved trap colum n interface. This interface greatly increases the efficiency of the second dim ension colum n w ithout affecting the separation speed.W hen35mm trapcolum ns w ere used, com paring to a50mm second d im ensional colum n, the separation efficiency w as theoretically increased by70%. Prim ary separation of the rat serum sam p le w as used to analysis and evaluate the utility of the system. The VF phenom enon w as generated attributing to viscosity of the m obile phases of the tw o dim ensions.This interface w ould be an alternative selection to the traditional trap colum n interface in2D-LC system for the separation ofcom p lex m ixture in p roteom ics. Based on the im p roved interface, the WAX-RP 2D-LC system also could be app lied in the other areas such as the research of traditional Chinese m edicines.

        [1] Dugo P,Cacciola F,Kumm T,et al.J Chrom atogr A, 2008,1184(1/2):353

        [2] Issaq H J,Chan K C,Janini G M,et al.J Chrom atogr B, 2005,817(1):35

        [3] Bushey M M,Jorgenson J W.Anal Chem,1990,62(10): 978

        [4] M urphy R E,Schure M R,Foley J P.Anal Chem,1998,70 (8):1585

        [5] Lan T,J iao F L,Tang T,et al.Chinese Journal of Chrom atography,2008,26(3):374

        [6] Guiochon G,MarchettiN,M riziq K,et al.J Chrom atogr A, 2008,1189(1/2):109

        [7] Shellie RA,Haddad P R.Anal B ioanal Chem,2006,386 (3):405

        [8] W ang Z C,Zhang Q H,Zhao Z Y,et al.Chinese Journal of Analytical Chem istry,2005,33(5):722

        [9] Tanaka N,Kim ura H,Tokuda D,et al.Anal Chem,2004, 76(5):1273.

        [10] Felinger A,Kele M,Guiochon G.J Chrom atogr A,2001, 913(1/2):23

        [11] W agner K,M iliotis T,M arko V G,et al.Anal Chem,2002, 74(4):809

        [12] M aw uenyega K G,Kaji H,Yam auchi Y,et al.J Proteom e Res,2003,2(1):23

        [13] Sw eeney A P,Shalliker R A.J Chrom atogr A,2002,968 (1/2):41

        [14] StollD R,Carr P W.J Am Chem Soc,2005,127(14): 5034

        [15] Stoll D R,Cohen J D,Carr P W.J Chrom atogr A,2006, 1122(1/2):123

        [16] Cesla P,Hajek T,Jandera P.J Chrom atogr A,2009,1216 (16):3443

        [17] Dugo P,Cacciola F,Herrero M,et al.J Sep Sci,2008,31 (19):3297

        [18] Ikegam i T,Hara T,Kim ura H,et al.J Chrom atogr A, 2006,1106(1/2):112

        [19] Im K,Park H W,Lee S,et al.J Chrom atogrA,2009,1216 (21):4606

        [20] Shalliker R A,Catchpoole H J,Dennis G R,et al.J Chrom atogr A,2007,1142(1):48

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