《仪器分析和物理化学》课程教学资源（毛细管电泳实验文献）Performance of a physically adsorbed high-molecular-mass polyethyleneimine layer as coating for the separation of basic proteins and peptides by capillary electrophoresis.pdf_大学文库

JOURNAL OF CHROMATOGRAPHY A ELSEVIER Journal of Chromatography A.708(1995)356-361 Short communication Performance of a physically adsorbed high-molecular-mass polyethyleneimine layer as coating for the separation of basic proteins and peptides by capillary electrophoresis F.Bedia Erim',Alejandro Cifuentes,Hans Poppe,Johan C.Kraak* Laboratory for Analytical Chemistry.University of Amsterdam.Nieuwe Achtergracht 166.1018 WV Amsterdam,Netherlands First received 17 November 1994:revised manuscript received 27 March 1995;accepted 27 March 1995 Abstract A simple method for the preparation of a polyethyleneimine(PEI)coating on the iner suface of fused-silica capillaries for capillary electrophoresis(CE)is reported.The PEI layer can be coated on the silica surface by just flushing the capillary with a solution containing high-molecular-mass PEI.The physically adsorbed layer appears to be very stable and can be used in a pH range of 3-11.In comparison to described methods to fabricate an immobilized PEI layer,the proposed method docs not require an immobilization step,is simple and the preparation time of th coating is less than two hours.Good and peptides were obtained on the same PEL-coated epillry ellson erent basic proteins efficiencies ranging from 300 000-500 000 plates per meter were normally found. 1.Introduction Different methods have been developed in CE to diminish interaction between proteins and the In CE analysis of proteins,interaction of the silica surface (see Refs.[1-4]for exhaustive and biopolymers with the capillary wall seems to be up-to-date reviews).The most successful meth- the main reason for the loss in efficiency com ods are the ones whereby the silanol groups are pared to that predicted by theory.Furthermore, shielded by a polymer layer [5-14].The use of protein adsorption on the internal surface of the such polymeric coatings has led to high efficien- capillary can cause poor reproducibility of migra cies and reproducible protein separations. tion time and low protein recovery.The ad- The PEI coating as developed by Towns and sorption is believed to be due to the electrostatic Regnier [8].and adapted by others [10,12.15] interactions between positively charged residues seems to be a very interesting solution to this of the protein and the negatively charged silanol problem.This coating is different from most groups which are intrinsic to the fused-silica polymer layers pecause the surface bears surface. positive charge.The PEI coating is particularly suited for the separation of basic proteins since at acidic pH the surface and proteins have the Pre nent of Chemistr Technical Uni- same positive charge.Under these conditions the proteins are repelled from the surface in analogy 0021-9673/95/$09.50 1995 Elsevier Science B.V.All rights reserved SSD10021-9673(95)00394-0

ELSEVIER Journal of Chromatography A, 708 (1995) 356-361 JOURNAL OF CHROMATOGRAPHY A Short communication Performance of a physically adsorbed high-molecular-mass polyethyleneimine layer as coating for the separation of basic proteins and peptides by capillary electrophoresis F. Bedia Erim 1, Alejandro Cifuentes, Hans Poppe, Johan C. Kraak* Laboratory for Analytical Chemistry, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, Netherlands First received 17 November 1994; revised manuscript received 27 March 1995; accepted 27 March 1995 Abstract A simple method for the preparation of a polyethyleneimine (PEI) coating on the inner surface of fused-silica capillaries for capillary electrophoresis (CE) is reported. The PEI layer can be coated on the silica surface by just flushing the capillary with a solution containing high-molecular-mass PEI. The physically adsorbed layer appears to be very stable and can be used in a pH range of 3-11. In comparison to described methods to fabricate an immobilized PEI layer, the proposed method does not require an immobilization step, is simple and the preparation time of the coating is less than two hours. Good reproducibilities of migration times of basic proteins and peptides were obtained on the same PEI-coated capillary as well as on different PEI-coated capillaries. For basic proteins efficiencies ranging from 300 000-500 000 plates per meter were normally found. I. Introduction In CE analysis of proteins, interaction of the biopolymers with the capillary wall seems to be the main reason for the loss in efficiency compared to that predicted by theory. Furthermore, protein adsorption on the internal surface of the capillary can cause poor reproducibility of migration time and low protein recovery. The adsorption is believed to be due to the electrostatic interactions between positively charged residues of the protein and the negatively charged silanol groups which are intrinsic to the fused-silica surface. * Corresponding author. l Present address: Department of Chemistry, Technical University of Istanbul, Maslak 80626, Istanbul, Turkey. 0021-9673/95/$09.50 © 1995 Elsevier Science B.V. Allrights SSDI 0021-9673(95)00394-0 Different methods have been developed in CE to diminish interaction between proteins and the silica surface (see Refs. [1-4] for exhaustive and up-to-date reviews). The most successful methods are the ones whereby the silanol groups are shielded by a polymer layer [5-14]. The use of such polymeric coatings has led to high efficiencies and reproducible protein separations. The PEI coating as developed by Towns and Regnier [8], and adapted by others [10,12,15], seems to be a very interesting solution to this problem. This coating is different from most polymer layers because the surface bears a positive charge. The PEI coating is particularly suited for the separation of basic proteins since at acidic pH the surface and proteins have the same positive charge. Under these conditions the proteins are repelled from the surface in analogy reserved

F.B.Erim et al.J.Chromatogr.A 708 (1995)356-361 51 to what has been described by Lauer and trypsinogen and a-chymotrypsinogen (bovine MeManigill [16].The PEI coating has the addi tional advantage that the separation of basic C(MO.USA) as receive the move to the ano i.e. SA 6.101.10 ng hig s PEI (M, ch 认a en AG(E it the were dissolved at the con ther CE stud ed that PEI sticks rations indicated (ranging from 0.2 to 2 mg irreversibly on the silica surface, even under ml)in water.previously purified by passage harsh conditions such as flushing the capillary through a PSC filter assembly (Barnstead,Bos with strong basic and acidic solutions.It is well ton,MA,USA).The samples were stored at known that polymers strongly adsorb onto silica 20C and warmed to room temperature before surfaces and their adsorption has been applied to molecula mask the silanol groups on the surface of fused 10 was from silica capillaries [11,17].Since the preparation of erland) an immobilized Me nc acid bonded PE co Darm Ge lab nous an ning (as r mos mica PEI I ris bvdro well.T (3-cvclohexylam thyl]meth 0-1-p nesulfonic acid)and focused on the r roducibility,the efficiency and MES (all from Aldrich,Axel.Netherlands)were the long-term stability of the coating,using some used in the various running buffers.These buf. basic proteins and peptides as test compounds. fers were used in a 50 mM concentration and at pH values of 3(chloro-acetic),5.5(acetic,ME succinic,malonic). and ES),8 and 8 2.Materials and methods (Tricine) 9-11 (CAPS).The bufte rs were at 4C and warmed to room temperature berore 2.1.Instrumentation use. rations 2.3.Coating procedure (Lauer-Labs E Nethe ini system with temperature controer Before coating the capillary with PEI the a Linear M-200 variable-wavelength UV-Vis external polyimide coating was burned-off over a detector (Linear Ins.Corp.,Reno,NV.USA) length of 5 mm in order to make the detection operated at 214 nm.Fused-silica capillaries window The fused-silica capillary was frs (Polymicro Technologies Inc 、Phoenix.AZ etched by fush ning the capillary with a solution o USA)with 75 um I.D.and 360 um O.D.were 1 M sodium hydroxid min at use and effective (from the injec ter for l me pressure are indo the the capillarie the nd the ed at t si The injection wa using contro olut left in the capillary one ho pres ure the solution essed out of the 2.2.Samples and chemicals was rinsed with water for 15 min and with running buffer for 15 min.A washing step of 1 Lysozyme (chicken egg white),cytochrome c min with buffer was used etween injectio (horse heart).ribonuclease A (bovine pancreas). This procedure can be carried out by using the

F.B. Erim et al. / J. Chromatogr. A 708 (1995) 356-361 357 to what has been described by Lauer and McManigill [16]. The PE! coating has the additional advantage that the separation of basic compounds is fast, because the basic analytes move to the anode, i.e. in the same direction as the electroosmotic flow. When testing a high-molecular-mass PEI (M r 6.10~-1.106) as additive to the buffer in another CE study, we discovered that PEI sticks irreversibly on the silica surface, even under harsh conditions such as flushing the capillary with strong basic and acidic solutions. It is well known that polymers strongly adsorb onto silica surfaces and their adsorption has been applied to mask the silanol groups on the surface of fusedsilica capillaries [11,17]. Since the preparation of an immobilized bonded PEI coating is rather laborious and time-consuming (as for most polymer coatings), we found it worthwhile to investigate whether a physically adhered PEI layer can be used as coating as well. The study was focused on the reproducibility, the efficiency and the long-term stability of the coating, using some basic proteins and peptides as test compounds. 2. Materials and methods 2.1. Instrumentation trypsinogen and t~-chymotrypsinogen (bovine pancreas) were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and used as received. All the short peptides were from Nutritional Biochemicals Corporation (Cleveland, OH, USA) and the long ones from Bachem Feinchemikalien AG (Bubendorf, Switzerland). Proteins and peptides were dissolved at the concentrations indicated (ranging from 0.2 to 2 mg/ ml) in water, previously purified by passage through a PSC filter assembly (Barnstead, Boston, MA, USA). The samples were stored at -20°C and warmed to room temperature before use. Polyethyleneimine (PEI, molecular mass range 6. 105-1 • 106) was from Fluka (Fluka AG, Buchs, Switzerland). Acetic acid and formic acid (E. Merck, Darmstadt, Germany), succinic acid (BDH Laboratory Chemicals Division, Poole, UK), chloro-acetic acid, malonic acid, Tricine (N-tris[hydroxymethyl]methyl-glycine), CAPS (3-cyclohexylamino-l-propanesulfonic acid) and MES (all from Aldrich, Axel, Netherlands) were used in tbe various running buffers. These buffers were used in a 50 mM concentration and at pH values of 3 (chloro-acetic), 5.5 (acetic, MES, succinic, malonic), 7 and 7.5 (MES), 8 and 8.5 (Tricine), 9-11 (CAPS). The buffers were stored at 4°C and warmed to room temperature before use. Separations were carried out using a Prince (Lauer-Labs, Emmen, Netherlands) injection system with temperature controller, connected to a Linear M-200 variable-wavelength UV-Vis detector (Linear Ins. Corp., Reno, NV, USA) operated at 214 nm. Fused-silica capillaries (Polymicro Technologies Inc., Phoenix, AZ, USA) with 75 /zm I.D. and 360/zm O.D. were used; the total and effective (from the injection point to the detector) lengths of the capillaries are indicated in the figures. The injection was carried out at the cathodic side using controlled pressure for a fixed time. 2.2. Samples and chemicals Lysozyme (chicken egg white), cytochrome c (horse heart), ribonuclease A (bovine pancreas), 2.3. Coating procedure Before coating the capillary with PEI the external polyimide coating was burned-off over a length of 5 mm in order to make the detection window. The fused-silica capillary was first etched by flushing the capillary with a solution of 1 M sodium hydroxide for 30 min at 1 bar and with water for 15 rain at the same pressure. Then the capillary was flushed with a solution of PEI in water at 1.5 bar for 10 min and the PEI solution left in the capillary for one hour. Next the polymer solution was pressed out of the capillary with air at 1.5 bar. Finally the capillary was rinsed with water for 15 min and with running buffer for 15 min. A washing step of 1 min with buffer was used between injections. This procedure can be carried out by using the

358 F.B.Erim et al.J.Chromatogr.A 708(1995)356-361 Prince instrument in automatic mode,which ueo (108 m*2/sV) allows to coat each capillary unattendedly over- night. .1%PE %PE图 3.Results and discussion 10%E and the surface of the fused-silic a PEI layer on the capillary wall and thus masks the underlying silanol groups from unwanted interactions with biopolymers.Moreover,the adsorbed PEI layer has a positive charge over a wide pH range,which results in an electroosmot ic flow towards the anode.This anodic electro phoretic flov favours the separatio substances proteins DH 3.1.Effect of the pel concentration in the coating solution Fig.1.Plots of electroo c flow versus pH on capillaries oated wth1.1.5and1()PEI solutions in water. employe In order to determine whether the PEI con- centration in the coating solution has an effec on the nature of the dynamically generated PEI flow(EOF)was found.A similar behaviour has n ot erved by other hors pH on PE In orde in tha the eff er the H g 3-10.4 pHi an Fig.1 shows the the h As can be s n the i0 adding diffe nt a flow is always towards the anode over the in- keep the current the same (ionic strength is then vestigated pH range.This behaviour indicates also approximately constant).Some EOF values that the basic PEI molecules are strongly ad- changed slightly;however,the same trend as sorbed on the silica surface and that the residual shown in Fig.1 was observed. amine groups of the PEl create a positively charged surface. 3.2.Effect of type of buffer and pH tive du ons nt over th ang nd the PH Thi The effect of the typ of buffer on the electro th 10%PEI amine gr oups and the higher ionization of silano capillary using acetone as neutral marker four groups on the capillary wall [8l. different buffers at H 55 ie malonic acid From Fig.1 it can be seen that capillaries succinic acid,MES and acetic acid,at the same coated with 1.5 and 10%of PEI exhibit simila concentration (50 mM)were employed.It ap peared that the electroosmotic flow w s strongly dependent on the type of anion.The electro

358 F.B. Erim et al. / J. Chromatogr. A 708 (1995) 356-361 Prince instrument in automatic mode, which allows to coat each capillary unattendedly overnight. 3. Results and discussion The PEI molecule has many positive charges and interacts strongly with negatively charged silanol groups on the surface of the fused-silica capillary [8]. This irreversible adsorption creates a PEI layer on the capillary wall and thus masks the underlying silanol groups from unwanted interactions with biopolymers. Moreover, the adsorbed PEI layer has a positive charge over a wide pH range, which results in an electroosmotic flow towards the anode. This anodic electrophoretic flow favours the separation speed of substances with a positive charge such as basic proteins and peptides. 3.1. Effect of the PEI concentration in the coating solution In order to determine whether the PEI concentration in the coating solution has an effect on the nature of the dynamically generated PEI layer, the electroosmotic flow was measured as function of the pH on capillaries coated with 0.1-10% (w/v) solutions of PEI. Different buffers were used to cover the pH range of 3-10.4. Fig. l shows the effect of the pH on the electroosmotic flow. As can be seen the electroosmotic flow is always towards the anode over the investigated pH range. This behaviour indicates that the basic PEI molecules are strongly adsorbed on the silica surface and that the residual amine groups of the PEI create a positively charged surface. The electroosmotic flow is relatively constant over the pH range 3-6 and then gradually decreases at higher pH. This latter effect can be attributed to deprotonation of the amine groups and the higher ionization of silanol groups on the capillary wall [8]. From Fig. 1 it can be seen that capillaries coated with 1, 5 and 10% of PEI exhibit similar behaviour. However, in the capillary coated with 0.1% PEI a significantly larger electroosmotic /Jeo {10"8 m'2/sV) 0 -2 -3 -4 -5 I I I r I I I I 3 4 5 6 7 8 9 10 pH Fig. 1. Plots of electroosmotic flow versus pH on capillaries coated with 0.1, 1, 5 and 10% (w/v) PEI solutions in water. Acetone was employed as neutral marker. flow (EOF) was found. A similar behaviour has been observed by other authors [8]. In order to ascertain that the effects described above were indeed due to changes in the pH and degree of coating, all the experiments described above were repeated using the same buffers but adding different quantities of NaC1 in order to keep the current the same (ionic strength is then also approximately constant). Some EOF values changed slightly; however, the same trend as shown in Fig. 1 was observed. 3.2. Effect of type of buffer and pH The effect of the type of buffer on the electroosmotic flow was investigated on a 10% PEI capillary using acetone as neutral marker. Four different buffers at pH 5.5, i.e. malonic acid, succinic acid, MES and acetic acid, at the same concentration (50 mM) were employed. It appeared that the electroosmotic flow was strongly dependent on the type of anion. The electro-

F.B. Erim et al. / J. Chromatogr. A 708 (1995) 356-361 359 osmotic mobilities with malonic acid, succinic acid, MES and acetic acid were found to be -18- 10 -9, -18" 10 -9, --50" 10 .9 and -45 • 10 .9 m 2 s -l g -1, respectively. As can be seen, the divalent acids, i.e. malonic and succinic acid, have the same electroosmotic flow, but the value is much smaller than those found with the monovalent acids, i.e. acetic acid and MES. A similar behaviour was observed when NaCI was added to the buffers in order to get similar ionic strength, as done above. A strong electrostatic interaction between the divalent anions and the amine groups of PEI, which decreases the charge on the coating and thus the zeta potential, seems to take place, which may explain this effect. 3.3. Efficiency of the PEI coating for biological compounds The performance of the PEI-coated capillaries was investigated with proteins and peptides. Fig. 2 shows a typical electrophoretogram of a mixture of basic proteins on a 10% PEI-coated capillary at pH 5.5. The PEI coating appears to be very efficient for basic proteins, and plate numbers ranging from 300 000-500 000 plates/m were normally found. Similar efficiencies were found with the 0.1, 1 and 5% PEI coating solutions. Unfortunately acidic proteins interact strongly with the coating and the performance is very poor. The PEI coating appears to be also suitable for the separation of other types of samples. As an illustration, Figs. 3A and 3B show a separation of a mixture of di- and tripeptides at pH 8.2 and a separation of longer peptides at pH 9.75, respectively. As can be seen, very efficient and fast separations can be realized. 3.4. Reproducibility and long-term stability of the PEI coating The reproducibility and the long-term stability of the PEI coating was investigated by measuring the plate numbers and retention times of some basic proteins on three 10% PEI coated capillaries. The run-to-run reproducibilities of the migration times of the proteins on the three I I I I 0 4 8 12 Time (rnin) Fig. 2. Separation of five basic proteins. 10% PEl-coated capillary, total length 63.5 cm, effective length 46.5 cm, I.D. 75 ~m. Buffer: 50 mM acetate, pH 5.5. Run voltage -28.8 kV. Injection 10 mbar for 6 s. Sample: (1) trypsinogen 0.32 mg/ml, (2) a-ehymotrypsinogen 0.32 mg/ml, (3) ribonuclease A 0.72 mg/ml, (4) cytochrome C 0.32 mg/ml and (5) lysozyme 0.32 mg/ml. UV detection at 214 nm. capillaries were excellent and the relative standard deviations (%R.S.D.) in migration times ranged from 0.5 to 1.5% (n = 6). The R.S.D. values for column-to-column reproducibilities of the migration times ranged from 1.9 to 2.8% (n = 18) and the R.S.D. in the plate numbers ranged from 10 to 15%. The long-time reproducibility of the migration times of proteins was followed on one capillary for one month. During this period 70 injections of the test proteins were performed. The migration times changed about 5% and no noticeable loss in efficiency was observed

4 1 F.B. Erim et al. J. Chromatogr. A 708 (1995) 356-361 I t I I 0 2 4 6 8 Time (min) B I. I . I I I 360 0 4 8 I 12 In order to test the stability of the coating at very basic pHs we left a 50 mM CAPS buffer at pH 11 in a 5% PEI-coated capillary for 60 h. Analysis times were measured before and after this experiment using the five basic proteins as analytes at pH 5.5. After 60 h the migration times of all proteins were slightly increased, i.e. about 3%, while the efficiency was not altered. This result indicates that the dynamically generated PEI layer is more or less irreversibly attached to the silica surface. 4. Conclusions A stable high-molecular-mass PEI layer can be dynamically generated on the inside surface of fused-silica capillaries by just filling the capillary with a solution of a high-molecular-mass PEI (M r 6. 105-1 • 106) and flushing the capillary after a certain time. The preparation of the coating is very simple, reproducible and takes less than two hours. The coating is very stable in the pH range 3-11. For basic proteins efficiencies of 300 000- 500000 plates/m were obtained. The coating appears to be suitable for the separation of biological compounds such as basic proteins and peptides but cannot be recommended for acidic proteins. Acknowledgements A.C. would like to acknowledge the financial support of the European Communities (Human Capital and Mobility Programme, bursary #ERB4001GT920989). Time (min) Fig. 3. Separation of short peptides (A) and long peptides (B) on a 5% PEI-coated capillary. Total length 85 cm, effective length 50 cm, I.D. 75 /xm. Run voltage -24 kV. Injection 10 mbar for 6 s. Sample concentration ranging from 0.2 to 0.6 mg/ml. UV detection at 214 nm. (A) Buffer: 50 mM Tricine, pH 8.2; peptides: (1) GE, (2) GGG, (3) LGF, (4) AA, (5) SGG. (B) Buffer: 50 mM CAPS, pH 9.75; peptides (1) WAGGDASGE, (2) ELAGAPPEPA, (3) SYSMEHPRWG, (4) LQAAPALDKL. References [1] W.G. Kuhr, Anal. Chem., 62 (1990) 403R. [2] W.G. Kuhr and C.A. Monning, Anal. Chem., 64 (1992) 389R. [3] C.A. Monning and R.T. Kennedy, Anal. Chem., 66 (1994) 280R. [4] S.F. Yi, Capillary Electrophoresis: Principles, Practice and Applications, Elsevier, Amsterdam, 1992. [5] S. Hjert6n, J. Chromatogr., 347 (1985) 191