cweraCcpge,A0c3R CHROM.22 580 Polyethyleneimine-bonded phases in the separation of proteins by capillary electrophoresis JOHN K.TOWNS and FRED E.REGNIER Department of Chemistry,Purdue University.West Lafayette.IN 47907(U.S.A.) ABSTRACT ules.Polyethylene mine is adsorbed to the inner an of f nd the adso rbed coating cross-linked into a stable layer.Ca etveneimine-coated silica gave u nique separations owing to the reversal of electro-osmotic flow caused by the positively charged coating.The resulting coatin as stable from pH 2-12 and could be used over a wide pH range without substantial change in electro-osmotic fow.High-molecular-weight polymers were needed to give thick coatings which mask silanol groups on the wall.Proteins were resolved quickly and efficiently with good recovery using capillaries of 50 cm in length. INTRODUCTION Ad is a problem in capil ry 2 s par adsorption dir ticalartytnueinthecaseo 0 of ba recovery and ze the separation solutes,adsorption t be kept capilla owsanoth 1a1e which rd the athod ation of eit in this double la otic flow.The rate of this flow is Reaiedi.harEgh6esiyandiacTeaseswihtheionizationofsurfaceanolsfrompH 3 to 9.The net velocity of a charged solute in a fused silica capillary is the sum of the rates of both convective and electrophoretic transport.Ideally,the convective component of transport should remain constant while the electrophoretic component is being varied with pH to optimize selectivity in a separation.This would only be possible in capillarics with no charge or constant charge at the surface. hnooipm1hamoAmon0tsgyygeiyPeeigorpic2n3eS 021-9673/90/503.50 1990 Elsevier Science Publishers B.V
Journal of Chromatography, 516 (1990) 69-18 Elsevier Science Publishers B.V., Amsterdam CHROM. 22 580 Polyethyleneimine-bonded phases in the separation of proteins by capillary electrophoresis JOHN K. TOWNS and FRED E. REGNIER* Departmeni of Chemistry, Purdue University, West Lafayette, IN 47907 (U.S.A.) ABSTRACT A hydrophilic, positively charged, durable coating has been developed for capillary electrophoresis of macromolecules. Polyethyleneimine is adsorbed to the inner wall of fused silica capillaries and the adsorbed coating cross-linked into a stable layer. Capillaries of polyethyleneimine-coated silica gave unique separations owing to the reversal of electro-osmotic flow caused by the positively charged coating. The resulting coating was stable from pH 2-12 and could be used over a wide pH range without substantial change in electro-osmotic flow. High-molecular-weight polymers were needed to give thick coatings which mask silanol groups on the wall. Proteins were resolved quickly and efficiently with good recovery using capillaries of 50 cm in length. INTRODUCTION Adsorption of positively charged species onto the walls of fused-silica capillaries is a problem in capillary zone electrophoresis. This is particularly true in the case of basic proteins and peptides where adsorption diminishes solute recovery and resolution. Therefore, in order to optimize the separation of basic solutes, adsorption to the capillary wall must be kept to a minimum. Electra-osmotic flow is another issue that must be considered in fused-silica capillaries. Ionization of surface silanols generates an electrical double layer which migrates toward the cathode when an electric potential is applied to the capillary. Migration of positive ions in this double layer pulls the solution through the capillary, producing the phenomenon known as electro-osmotic flow. The rate of this flow is related to charge density and increases with the ionization of surface silanols from pH 3 to 9. The net velocity of a charged solute in a fused silica capillary is the sum of the rates of both convective and electrophoretic transport. Ideally, the convective component of transport should remain constant while the electrophoretic component is being varied with pH to optimize selectivity in a separation. This would only be possible in capillaries with no charge or constant charge at the surface. To this end, modification of capillaries by either masking or deactivating surface silanol groups has been performed by physically coating capillary walls with 0021-9673/90/%03.50 0 1990 Elsevier Science Publishers B.V. *
J.K.TOWNS,F.E.REGNIER ads inish y do not solv the very o teins and cause ectro-osmotic flow to vary widely with pH.Coating stability is also a problem. Hydrop ilic organosilanes can erode from columns in a matter of days* This paper focuses on the preparation of fused-silica capillaries with a positively charged polymer coating.The function of this bonded phase is to enhance the separation and recovery of positively charged species,to stabilize electro-osmotic flow across the pH range from 5 to 11 and to extend the useful life of capillaries. EXPERIMENTAL Chemicals Polyethyleneimine (PED)6(average rel.mol.mass,M,=600)was purchased from Polysci nces (Warrington.PA..U.S.A.).PEI 18 and 200 were gifts from Dow Chemical (Midland.MI,U.S.A.).Ethyler eycol diglycidyl ether (EDGE)and lamine(TEA)we has sed from Aldrich(Milw .WI,U.S.A.).Allothe were commercially obta ed reagent grade f available e purest grade Lysozyme(egg white),cytochrome(horse heart),chymotrypsinogenA(bovine pancreas),ribonuclease A (bovine pancreas),and myoglobin (horse heart)were purchased from Sigma(St.Louis,MO,U.S.A.).The neutral markers,mesityl oxide and dextran blue (M 2000000).were purchased from Aldrich. instrumentation Capillary electrophoresis was performed on an instrument based on an in-house design.All high-voltage components of the system were contained in a Lucite cabinet fitted with a safety interlock that would interrupt the line voltage to the transformer in the power su 60/EI(Spel upply when the cabinet door was opned.A Speliman Model FHR3P n High Volta Electronics,Plair NY.U.S.A.)po ver supply was used to apply the electri cld a Th nnect 2-gauge plati ele des ml pply eser ng with the ary U.s. capill aries (Polymi )of 50 d73 .200 the tot ength varying between 50 an cm and ti separa cm.On-line on was perlorme variable wave ength n UV ab E,U S.A).be tionw ore at 20 for the proteins and peptides and 25 nm for the me sityl oxide The signal from th detector was fed to a Linear 2000(Linear,Reno,NV,U.S.A.)strip chart recorder Electrophoresis Protein solutions of a concentration of 1-5mg/ml were injected into the capillary by syphoning for a fixed time(1-3 s)at a fixed height(5-8 cm).Mesityl oxide was used as the neutral marker along with dextran blue to determine any sizing differences in the coating.Several buffer solutions were used over the pH range of 3 to 11:0.01 Macetate at pH 3 and 5,0.01 M hydroxylamine-HCI at pH 7,0.01 M diaminopropane at pH 9 and 11.Salt was added to each buffer to give comparable ionic strengths and
70 J. K. TOWNS, F. E. REGNIER methylcellulose1T2 and by silane derivatizatiotr-‘, respectively. Although these coatings reduce both adsorption and electro-osmotic flow, they do not solve these problems. Residual silanols still diminish the recovery of basic proteins and cause electro-osmotic flow to vary widely with pH. Coating stability is also a problem. Hydrophilic organosilanes can erode from columns in a matter of days4. This paper focuses on the preparation of fused-silica capillaries with a positively charged polymer coating. The function of this bonded phase is to enhance the separation and recovery of positively charged species, to stabilize electro-osmotic flow across the pH range from 5 to 11 and to extend the useful life of capillaries. EXPERIMENTAL Chemicals Polyethyleneimine (PEI) 6 (average rel. mol. mass, M, = 600) was purchased from Polysciences (Warrington, PA., U.S.A.). PEI 18 and 200 were gifts from Dow Chemical (Midland, MI, U.S.A.). Ethyleneglycol diglycidyl ether (EDGE) and triethylamine (TEA) were purchased from Aldrich (Milwaukee, WI, U.S.A.). All other reagents were commercially obtained, reagent grade if available or the purest grade obtainable if not. Lysozyme (egg white), cytochrome c (horse heart), chymotrypsinogen A (bovine pancreas), ribonuclease A (bovine pancreas), and myoglobin (horse heart) were purchased from Sigma (St. Louis, MO, U.S.A.). The neutral markers, mesityl oxide and dextran blue (Mr 2 000 000), were purchased from Aldrich. Instrumentation Capillary electrophoresis was performed on an instrument based on an in-house design. All high-voltage components of the system were contained in a Lucite cabinet fitted with a safety interlock that would interrupt the line voltage to the transformer in the power supply when the cabinet door was opened. A Spellman Model FHR 30P 60/EI (Spellman High Voltage Electronics, Plainview, NY, U.S.A.) power supply was used to apply the electric field across the capillary. The power supply output was connected to 22-gauge platinum-wire electrodes immersed in 3-ml buffer reservoirs along with the capillary ends. Polyimine-coated fused-silica capillaries (Polymicro Technologies, Phoenix, AZ, U.S.A.) of 50 and 75 pm I.D., 200 ,um O.D. were used with the total length varying between 50 and 100 cm and the separation length from 35 to 85 cm. On-line detection was performed with a variable-wavelength UV absorbance detector (Model V4; Isco, Lincoln, NE, U.S.A.). Detection was monitored at 200 nm for the proteins and peptides and 254 nm for the mesityl oxide. The signal from the detector was fed to a Linear 2000 (Linear, Reno, NV, U.S.A.) strip chart recorder. Electrophoresis Protein solutions of a concentration of l-5 mg/ml were injected into the capillary by syphoning for a fixed time (l-3 s) at a fixed height ( 5-8 cm). Mesityl oxide was used as the neutral marker along with dextran blue to determine any sizing differences in the coating. Several buffer solutions were used over the pH range of 3 to 11: 0.01 M acetate at pH 3 and 5, 0.01 M hydroxylamineHC1 at pH 7, 0.01 M diaminopropane at pH 9 and Il. Salt was added to each buffer to give comparable ionic strengths and
CE OF PROTEINS 71 currents.During electrophoresis,current through the capillary never exceeded 50 uA with all analyses being run at ambient temperature.No temperature control of the capillary was employed.Between analyses,the capillary was flushed with 1%TFA in propanol,deionized water and the separation buffer for 1 min. of was treated with 1.0 M NaOH for 15 min.followed by with dei er Re evaporated fr by connecting the ca a gas chro e sure of 400 kPa.The ethanolic solution of polyeth through the capillary by syringe and allowed to sit for 8 e was the pulle solution was removed from the capillary by pushing nitrogen through the capillary. Nitrogen flow was continued through the capillary for 2 h to evaporate the methanol. In the case of PEI 200,which has water added to reduce the viscosity,the capillary was heated at 80C for 4 h to remove the 33%water.Next,a 70%solution of EDGE in TEA was pulled into the capillary and allowed to sit for I h.This solution was then pushed out with nitrogen.Nitrogen flow through the capillary was continued at 400 kPa for 3 h.The capillary was then heated at 80'C for 30 min. Picric acid assay A 2 m x 100 um I.D.PEI-coated capillary was washed with methylene chloride for 30 min followed by 0.2 M picric acid in methvlene e chloride Inb ric ac was removed with an additional methylene chloride wash.Bo with 5%(v/v)TEA in 1 me ng wit olution until th ide into a didiedto0.1mlandthctriethA ng om the capillary wa colorless.The eluent was then ne picrate assayed ctrophotometrically.The molar extinction coefficient of TEA picrate in methylene &hioi8e1450a38m” RESULTS AND DISCUSSION surface derivatization The synthetic route(Fig.1)used to prepare positively charged capillaries in these atudies was derived from techniques used in liquid chromatography to prepar silica-based anion-exchange packing materials.It has been sho that PEI is strongly adsorbed from organic solvents onto a silica surface where it may ently he cross-linked with a multifunctional oxirane This cross-linked layer is held in place by electrostatic adsorption at cannot beeluted fr ith is a funet tion of both EDGE bridg th hydrophilic a polya an intr mec n ermole purp 0 slink coa nto place as wn in Fig 1. In contrast intramolecula reactions polyamine molecu e or more generally derivatize the polyamine with free oxiranes.These oxiranes are subsequently
CE OF PROTEINS 71 currents. During electrophoresis, current through the capillary never exceeded 50 PA with all analyses being run at ambient temperature. No temperature control of the capillary was employed. Between analyses, the capillary was flushed with 1% TFA in propanol, deionized water and the separation buffer for 1 min. Capillary coating Capillaries were first treated with 1 .O A4 NaOH for 15 min. followed by 15 min. of washing with deionized water. Residual water was evaporated from the capillaries by connecting the capillaries to a gas chromatography oven at 80°C for 1 h under a nitrogen pressure of 400 kPa. The methanolic solution of polyethyleneimine was then pulled through the capillary by syringe and allowed to sit for 8 h. The methanol-PEI solution was removed from the capillary by pushing nitrogen through the capillary. Nitrogen flow was continued through the capillary for 2 h to evaporate the methanol. In the case of PEI 200, which has water added to reduce the viscosity, the capillary was heated at 80°C for 4 h to remove the 33% water. Next, a 70% solution of EDGE in TEA was pulled into the capillary and allowed to sit for 1 h. This solution was then pushed out with nitrogen. Nitrogen flow through the capillary was continued at 400 kPa for 3 h. The capillary was then heated at 80°C for 30 min. Picric acid assay A 2 m x 100 pm I.D. PEI-coated capillary was washed with methylene chloride for 30 min followed by 0.2 M picric acid in methylene chloride. Unbound picric acid was removed with an additional methylene chloride wash. Bound picric acid was then released with 5% (v/v) TEA in methylene chloride into a l-mm cuvette. Washing with the amine solution was continued until the solution eluting from the capillary was colorless. The eluent was then diluted to 0.1 ml and the triethylamine picrate assayed spectrophotometrically. The molar extinction coefficient of TEA picrate in methylene chloride is 14500 at 358 nm. RESULTS AND DISCUSSION Surface derivatization The synthetic route (Fig. 1) used to prepare positively charged capillaries in these studies was derived from techniques used in liquid chromatography to prepare silica-based anion-exchange packing materials. It has been shown6-8 that PEI is strongly adsorbed from organic solvents onto a silica surface where it may subsequently be cross-linked with a multifunctional oxirane into a continuous film. This cross-linked layer is held in place by electrostatic adsorption at many sites and cannot be eluted from the silica surface with solvents used in chromatography. Coating thickness is a function of both M, and concentration of PEI in the coating solution*. EDGE was chosen as the cross-linking agent because it was of sufficient length to bridge between adjacent adsorbed PEI molecules and would contribute to the hydrophilicity of the coating. EDGE can react with a polyamine by either an intra- or intermolecular mechanism. The intermolecular reaction serves the purpose of crosslinking the coating into place as shown in Fig 1. In contrast, intramolecular reactions (not shown) can either cross-link within a polyamine molecule or more generally derivatize the polyamine with free oxiranes. These oxiranes are subsequently
2 J.K.TOWNS,F.E.REGNIER 81-0e {CH2CH2NH)m-CH2CH2N)n-CH2CH2NH2o -o0影 PEI-200 (5 in MeoH) CH2 where RI-H or fCH2CH2NYx 1-o R1 PHASE I 0 CH2CH-CH2--(CH2)2-0-CH2CH-CH2 EDGE (70%IN TEA) 8 OH 【81、09®N-GH2C2H22 where R2 =-(CH2)3-0-CH2CH-CH2-PEI si-oe@NH2 CH2 OH -(CH2)3-0-CH2CHCH2-OH PHASE II coating further hydrolyzed to produce a diol-rich coating.Polyethyleneimines are known to be branched polymers with a primary:secondary:tertiary amino group ratio of ap- proximately 1:2:1(ref.8).Since the tertiary amino groups are sterically hindered and non-reactive,the primary and secondary amino groups both dominate the electrostatic interaction of the coating with the capillary wall and react with the EDGE
12 J. K. TOWNS, F. E. REGNIER ~ _~7 PEI-200 (52 in MeOH) where RI - H or fCH2CH2&, I 0 I I CH2 0 I I CH2 si- oeerai2 I 0 I CH2 PHASE I 0 0 :, CH2 P” OH I I si- 0eeNH - CH2cH-CH2-0-CH2- R2 I CH2 0 I P2 where R2 Si- 08 eMi2 I 0 CH2 I CH2 Si-Oee -RI I P n ? z2 si- oeem~ I I OH = -(CH2)2-O-CH2;H-CH2- PEI or OH -(CH2)3-O-CH2;HcH2- OH PHASE II Fig. 1. Synthetic route to an adsorbed PEI-bonded phase. The coating process is two step: (1) adsorption of PEI 200 onto the fused-silica capillary, and (2) cross-linking of the PEI 200 polymer in order to stabilize the coating further. hydrolyzed to produce a diol-rich coating *. Polyethyleneimines are known to be branched polymers with a primary:secondary:tertiary amino group ratio of approximately 1:2: 1 (ref. 8). Since the tertiary amino groups are sterically hindered and non-reactive, the primary and secondary amino groups both dominate the electrostatic interaction of the coating with the capillary wall and react with the EDGE
CE OF PROTEINS 0.0 日+0 P图18 2.0 4.0 98 0P阳200 6.0 8.0 2040608010120 PH Infuence of polymer size on electro-omotie fow The inf e mole range from 2.There etween the electro-osmotic flow-rate,direction of flow and polymer size.For low-molecular-mass polymer(PEI 6),the electro-osmotic flow moves from the negative to the positive electrode at low pH,indicating a positively charged wall.However,as the pH is increased,there is a sharp reversal in direction of the flow between pH 7.9 and 8.0. Flow reversal is the result of a switch in the net charge on the wall from positive to negative within that very small pH range.This is unusual in that the change occurs abruptly over a small pH range and the magnitude of the change(as expressed in flow-rate)is large on either side of this small range.These observations may be explained in the following way.It is known that the ionization of primary,sccondary and tertiary amines in PEI is almost linearly related to pH between pH 4 and 10.i.e PEI is not totally ionized until pH is dro oupsI of nd contrast,ioniz et d to greater road titrat 深ayna含 ace of the capillary causing the than amine groups and the negative electrical potential of the silica surface projects through the polyamine coating into the solution and establishes a positively charged double layer.Between pH 7.9 and 8.0,the surface is isoelectric and electro-osmotic flow is zero.Although surface charge density is changing substantially on either side o this isoelectric point,electro-osmotic flow is almost constant.This shows that electro-osmotic flow is only weakly coupled to charge density. As the molecular mass of the polymer is increased to 1800,electro-osmotic flow at low pH is faster than with PEI 6.This is due either to an increase in the positive
CE OF PROTEINS 13 6.0 8.0 2.0 4.0 6.0 6.0 10.0 12.0 PI-I Fig. 2. Plot of electro-osmotic flow as a function of pH for three sizes of polyethyleneimine: PEI 6 (Mr 600); PEI 18 (M, 1800); PEI 200 (MC 20000). Influence of polymer size on electro-osmotic flow The influence of polyamine molecular mass on electro-osmotic flow over the pH range from 3 to 11 is shown in Fig. 2. There is a strong correlation between the electro-osmotic flow-rate, direction of flow and polymer size. For low-molecular-mass polymer (PEI 6), the electro-osmotic flow moves from the negative to the positive electrode at low pH, indicating a positively charged wall. However, as the pH is increased, there is a sharp reversal in direction of the flow between pH 7.9 and 8.0. Flow reversal is the result of a switch in the net charge on the wall from positive to negative within that very small pH range. This is unusual in that the change occurs abruptly over a small pH range and the magnitude of the change (as expressed in flow-rate) is large on either side of this small range. These observations may be explained in the following way. It is known that the ionization of primary, secondary and tertiary amines in PEI is almost linearly related to pH between pH 4 and 10, i.e. PEI is not totally ionized until pH is dropped to 4 or lesr?. This is the result of the very high charge density in the polymer and electronic interaction between adjacent amine groups. In contrast, ionization of surface silanols begins at pH 3 and is not complete until the solution pH is raised to greater than 8. Again the broad titration curve is due to high charge density and electronic effects occurring at the surface. Below pH 7.9, positively charged amine groups dominate at the surface of the capillary causing the zeta potential to be positive. Above pH 8.0, surface silanol groups are more abundant than amine groups and the negative electrical potential of the silica surface projects through the polyamine coating into the solution and establishes a positively charged double layer. Between pH 7.9 and 8.0, the surface is isoelectric and electro-osmotic flow is zero. Although surface charge density is changing substantially on either side of this isoelectric point, electro-osmotic flow is almost constant. This shows that electro-osmotic flow is only weakly coupled to charge density. As the molecular mass of the polymer is increased to 1800, electro-osmotic flow at low pH is faster than with PET 6. This is due either to an increase in the positive