Anal. Chem. 1994,66, 2038-2046
Capillary Zone Electrophoresis Separations of Basic and Acidic Proteins Using Poly(vinyl alcohol) Coatings in Fused Silica Capillaries Martin Gllges,? Marla Helen Kleemlss, and Gerhard Schomburg’ Department of Chromatography and Electrophoresis, Max-Planck- Instttut fur Kohlenforschung, 45470 Mulheim, Germany
Poly(viny1 alcohols) (PVA) of a molecular weight of about 50 000 were applied to the modification of fused silica surfaces for the separation of large charged molecules such as proteins. Basic and acidic proteins could be separated in PVA-modified capillaries at highly optimized and analytically suitable performance with regard to efficiency, zone symmetry, and resolution. PVA can be used in the “dynamic” mode as an additive to the buffer medium or as a water-insoluble ”permanent” coating on the fused silica surfaces which can be achieved by a simple procedure of thermal immobllization. Permanent PVA coatings proved to be stable for series of separations over a wide range of pH of the buffer medium and also to suppress the electroosmotic flow, even at higher pH values. They were applied to the resolution of protein glycoforms. Proteins are complex molecules which are characterized by their different pl values. They can be separated in CZE according to their different electromobilitiesusing buffer media which have appropriate ionic strengths and a suitable pH for high migration velocities of basic or acidic proteins. Separation performancein analytical CZE is considerably impaired by intermediate or irreversible adsorption of the proteins at the surfaces of the fused silica capillaries. Peak widths are thereby increased and symmetry of analyte zones is distorted. An extensive theoretical treatment of the influence of analyte wall interaction on the performance of electromigrative separations has recently been published by Schure and Lenhoff.’ More or less “irreversible” adsorption of proteins may contaminate the capillary surface. By this adsorption, the electroosmotic flow (EOF) of separations within such capillaries may also be changed considerably with the consequence being poor reproducibility of migration times.2 A nonuniform {-potential over the length of the capillary due to analyte adsorption may cause additional band broadening by local variation of the EOF.2,3 To avoid these negative effects, the surfaces in the most commonly used fused silica capillaries must be deactivated for suppression of this highly undesirable wall interaction of t
Present address: SmithKline Beecham Pharmaceuticals, Old Powder Mills,
Nr.Leigh, Tonbridge, Kent TN11 9AN, England. (1) Schure, M. R.;Lenhoff, A. M. Anal. Chem. 1993,65, 3024-3037. (2) HjertCn, S.; Kubo, K. Electrophoresis 1993, 14, 390-395. (3) Towns, J. K.; Regnier, F. E. AnaLChem. 1992, 64, 2473-2478.
2030
Anal!icalChemistty, Vd. 66,No. 13, July 1, 1994
charged and also uncharged molecules. Two different approaches are suitable to prevent analyte/wall interactions: (1) dynamic coating by smaller ionic, zwitterionic, or nonionic molecules and especially by low concentrations of certain water-soluble nonionic and ionic polymers. These “modifiers” are contained in the running buffer and are strongly adsorbed on the walls. ( 2 ) permanent coating with small and large molecules chemically bonded to the surface or otherwise immobilized as thin or thicker films on the capillary walls. Water-insoluble or even soluble polymers which are strongly adsorbed on the surface may be suited for permanent surface modification. The addition of polymers to the buffer was first applied by HjertCn mainly to suppress the electroosmotic flow and to stabilize the separated zones by increasing the viscosity of the buffer medium? Dynamic modification with respect to surface adsorptivity can be achieved by additonof hydroxylic polymers such as poly(viny1 a l c o h ~ l ) ~or. ~cellulose deri~atives.~.’ Cationic polymer^^^^ have also been found effective as buffer additives. To obtain permanent or stationary coatings, polar polymers can only be applied if they are strongly adsorbed on the surface,1° cross-linked, or covalently bonded” (e.g., by anchoring via silanization of the surface silanols) in order to avoid dissolution in the buffer medium. Combinations of different coating procedures such as silanizationand adsorption of detergents for wall deactivation in fused silica capillaries have been successfully applied as well.l2 Another aim or also a not necessarily desirable consequence of surface modification by such methods is the variation of the electroosmotic flow, which might be completely suppressed but can also be decreased, increased, or even reversed. The speedup of analytical separations and the reversal of migration direction of certain analytes for reasons of detection at the appropriate side of the capillary may be important. The EOF can be completely suppressed by nonionic, hydroxylic, and preferably polymeric modifiers.13 This work will deal with poly(viny1 alcohol) as a dynamic and permanent modifier (4) HjertCn, S . Ark. Kemi 1958. 13, 151-152. ( 5 ) Gilga, M.;Husmann, H.; Klcemiss. M.H.; Motsch, S. R.;Schomburg, G. J . High Resolut. Chromatogr. 1991, IS, 452457. (6) Belder, D.; Schomburg, G . J. High Resolut. Chromatogr. 1992,15,686-693. (7) Lindner, H.; Helliger, W.;Dirschlmayer, A.; Jaquemar, M.; hschendorf, B. Biochem. 1. 1992,283,467-471. (8) Wfktorowicz, J. E.; Colbum. J. C . Electrophoresis 1990, 11, 769-713. (9) Wiktorowicz, J. E. US Patent 5 015 350, 1991. (10) Bentrop, D.;Kohr, J.; Engelhardt, H. Chromatographfa 1991,32, 171-178.
(ll)Bntin,G.M.;Chang,J.P.;Kuhlmann,R.H.;Zcgcrs,K.;Kraak,J.C.;Poppc, H.J. Chromatogr. 1989,471, 429436. (12)Towns. J. K.; Regnier, F. E. Anal. Chem. 1991, 63. 1126-1132. 00082700/94/03662038$04.50/0
0 1994 Amerlcan Chemical Society
which is characterized by its applicability over a wide range of pH between 3 and 10. A procedure for the application of PVA has been developed which leads to a permanent hydroxylic coating without involvement of covalent bonding to the silica surface. The procedure of the generation of permanent PVA coatings is quite simple because no chemical reactions for covalent bonding of the modifying molecule, e.g., by silanization are required. The generationof permanent PVA coatings is based on the unique propertyof poly(viny1alcohols) to become water insoluble by thermal treatment at temperatures of up to 160 OC. At higher temperatures, decomposition of the polymer begins.l4
EXPERIMENTAL SECTION Dynamic Coating Using Poly(viny1 alcohol) as Buffer Additive. The water-soluble,non-UV-absorbing polymer PVA is added to the running buffer at low concentration (0.05% (w/w)). By flushing the system with a few capillary volumes of the PVA-containing buffer, a thin polymeric layer is formed on the capillary surface by adsorptionfrom the buffer solution. Such a polymeric layer reduces protein adsorption to a great extent and improves the separation performance at pH values not higher than 4. A PVA stock solution is freshly prepared by dissolving 5% (w/w) PVA (MW 50 000,99+% hydrolyzed, purchased from Aldrich) in boiling water. PVA-containing buffer solutions of 0.05% (w/w) are obtained by adding the required volume of stock solution to a measured volume of a conventional phosphate buffer. The actual separations have been performed after flushing the capillary for a short time with running buffer (typically 90 s) before and with pure water (typically 2 min) after each separation. Purge times depend on its inner diameter and on the vacuum applied at the end of the capillary. Stable and reproducible separation conditions are realizable by purging the capillary with a few volumes of water, followed by renewal of the coating by rinsing with running buffer containing PVA. Similar experiments have also been performed with dextran, hydroxyethyl cellulose, and hydroxypropyl methyl cellulose for comparison to PVA. For investigation of the modifying properties of PVA, separations with dynamic wall modification have been performed in fused silica capillary tubing of different origins. Capillary materials supplied from different producers as well as different lots from the same producer show varying surface adsorptivity. For this work, capillaries purchased from MicroQuartz (Munich, Germany) and Polymicro Technologies (Phoenix, AZ) were compared. Generation of a Permanent PVA Coating. About 1.2 m of a 50-pm4.d. capillary that is to be coated is connected with a cut of shrinking tube to a 3-m cut of capillary of the same internal diameter. The 3-m cut acts as a flow resistance (throttle). For the setup used, see Figure 1. A 10% (w/w) solution of PVA (MW 50 000,99+% hydrolyzed, Aldrich) in water is forced through both capillaries under a nitrogen pressure of 2.0 MPa using a specially designed device which has been described in a previous p~blication.~ After both (13) HjertCn, S.Chromatogr. Rev. 1967, 9, 122-219. (14)Encyclopedia of Polymer Science and Engineering; J. Wiley & Sons: New
York, 1989;Vol 17, pp 167-198.
PNSSUm
rhrlnklng lube
1.2 m I50 pm to be coated
3mI50pm thmttb Capiky
magnotic diner
Figure 1. Production of poly(vlnyl alcohol) coated capllhrles.
capillaries have been completely filled, the coating solution is slowly discharged from the first capillary at a decreased nitrogen pressure of 0.15 MPa. During this procedure, the throttle capillary coupled to the outlet of the capillary to be coated effects a nearly constant flow of the coating solution through the first column, so that the polymer film which is deposited on the walls will be more homogeneous over the entire length of the capillary. The final immobilization of the PVA coating is achieved by heating the capillary to 140 OC under a gentle flow of nitrogen for several hours. Experiments have been performed with capillary tubing from different suppliers. Selection of Test Compounds for Dynamically and Permanently PVA Coated Capillaries. Experiments on separation performance were done with separate test mixtures of either basic or acidic proteins, with variation of ionic strength and pH of the bdfer system, to investigate the influence of dynamic and permanent surface modification by PVA. The test sampleswere composed accordingto the following criteria and mainly with regard to the unambiguous assessment of the efficiencies and symmetries evaluated from the peaks obtained: (1) The mixturesshould contain a variety of proteins of either low or high pZ. (2) The homogeneity or heterogeneity due to the absence or presence of various glycosylated derivatives of the single test proteins should be known. (3) The test proteins and the test samples composed should be stable during storage when longer series of test experiments are to be performed. (4) Impurities of the proteins should only be present at low concentrations;they should be resolvable from the main component at least under optimized performance of separation. (5) The main components of the applied test mixtures of proteins should be resolvable even at minor separation performance as achieved in poorly optimized systems. Chemicals. Phosphoric acid (for biochemical applications), cacodylic acid, and 2-amino-2-methyl-1-propanol (AMP) were obtained from Fluka (Neu-Ulm, Germany). The buffer solutions with AMP were adjusted with cacodylic acid to the desired pH. Sodium dihydrogen phosphate and sodium chloride were from Merck (Darmstadt, Germany). Benzyl alcohol, dextran (average MW 2 000 000), and poly(viny1alcohol) (hydrolysis grade 99+%, average MW 50000) were purchased from Aldrich (Steinheim, Germany). Hydroxyethyl cellulose (Cellosize WP 40, middle viscosity) was from Fluka; hydroxypropyl methyl cellulose was from Sigma (Deisenhofen, Germany). Protein samples were from Sigma or were purchased as denoted in the legends of the figures. AMlytlcal m k b y , Vol. 66, No. 13, July 1, 1994
2089
Instrumentation. CZE separations were performed with homemade equipment which had been described in earlier publication~.~~J~ On-column UV detetection was achieved using a Spectra Physics Model Spectra-100 (Darmstadt, Germany) operating at 214 nm. All electropherogramswere registered at 5 mAU full scale. High voltage was generated by a FUG HCN 35-35000 unit (Rosenheim, Germany). Positive potential at the injection side will be referred to as positive polarity. Sample introduction was done by electromigration. The separation capillary was cooled by a thermostated water cycle.
RESULTS AND DISCUSSION Different test mixtures of acidic and basic standard proteins and natural protein samples were separated. Series of repetitive separations were performed for the assessment of the reproducibility of migration times and the ruggedness and stability of the separation system in general. "Dynamic" Surface Modification by PVA and Other Hydroxylic Polymers. The highly hydroxylic poly(viny1 alcohol) is very effective with regard to the suppression of analyte/wall interactions of basic proteins even when applied in low concentration (0.05%) as additive in buffers at lower pH. Poly(viny1alcohol) is a nonionic hydroxylic polymer and is synthesized by hydrolysis of poly(viny1 acetate). The efficacy of such "dynamicn adsorptive coatings regarding protein separations may generally be interpreted as related to the shielding of the acidic silanol groups by an adsorbed polymeric layer. Hydrogen bridge bonding between the surface silanols and the hydroxyl groups of the PVA or other hydroxylic polymers may be strongly involved in the fixation of layers which are suited to suppress Coulombic interaction of the silanols with the charged molecules of analytes. The analytical application of "dynamic coating" with hydroxylic polymers suffers from a strong dependence of its efficacy on the surface properties of the fused silica material." At pH >5, dynamic coating is not effective at all due to insufficient, i.e., too weak, adsorption of the nonionic hydroxylic polymer on the negatively charged surface. In Figure 2, two separations of the test mixture containing the selected basic proteins are shown. For these separations the dynamic surface modification with PVA has been applied at the two different pH values of 3.0 and 3.5. At the higher pH of 3.5 in separation B, the migration times of the basic proteins are longer due to the decreaseof the molecular charge densities of the proteins. EOF Suppression by Adsorption of Hydroxylic Polymers such as PVA. Surface deactivation by adsorption of a hydroxylic polymer such as PVA is accompanied by nearly complete suppression of the electroosmotic flow up to pH values of 8. At a pH of 10, the EOF begins to increase considerably. See curve B in Figure 3. Curve A in Figure 3 illustrates the characteristic dependency of the EOF in fused silica capillaries on the buffer pH without modification by PVA adsorption. At pH >5, the already high EOF increases (15) Lux, J. A.; Yin,H. F.; Schomburg, G.Chmmorographia 1990, 30, 7-16. (16) Yin, H. F.; Motsch, S. R.; Lux, J. A.; Schomburg, 0. 1.High Resoluf. Chromafogr. 1991,14, 282-284. (17) Klccmiss,M. H.; Gilgcs, M.; Schomburg, G. Elecfrophoresls 1993,14,515-
522.
2040
4
-.I
lo
"
"
I
'
1
.
1
u
I
'
L ,
'
15 lo 15 mln -0 2. Separatkm of basic proteins in a capillary dynamically coated wtth poly(vinyl alcohol). Sample: (1) cytochrome c, (2) lysozyme, (3) trypsin, (4) trypsinogem, and (5) ar-chymotrypslnogen;0.2 mg/mL each. CapMary: 57cm effective length, 70cm total length; 50-pm i.d. Buffer: 70 mM sodium phosphate: (A) pH 3.0; (B) pH 3.5; 0.05 % (w/w) PVA (MW 50 000, 99+ % hydrolyzed,Akkich). Conditions: 30 kV (429 V/cm), (A) 33 PA; (B) 34 pA 20 O C . Injection: 10 kV. 5 s.Detection: UV, 214 nm. [I 09 mWs]
1
1
.............................
3
4
5
6
7
8
9
10pH
Flguro S. pH dependence of electroosmotic flow. (A) Unmodified capillary: (B) PVA-modMed capiwary (dynamically coated). Marker: benzylalcohd.CapiYary: (A)41-~meff~length,54-~mtotallength; (8)13-cm effective length, 70cm total length, 50-pm 1.d. (Pr). Buffer: (A) 20 mM sodium phosphate: (B) 20 mM sodium phosphate, 0.05% PVA. Conditions: 35 kV; (A) 388 Vlcm, 19-45 PA; (B) 500 V/cm, 15-35 p A 20 O C . Detectbn: UV, 214 nm.
strongly and becomes 6 times higher than with dynamic PVA suppression. Influence of Iodc Strength of Buffers on Performance of Protein S e p ~ t i o n ~It. is well-known that the ionic strength of buffers has a strong influence on the suppression of protein adsorption.18 Too high ionic strengths are not favorable because of the high currents arising at the commonly applied electric field strengths. In analytical practice and when using liquid cooling of the capillary, electrical currents up to 100 pA are generally acceptable. (18) Grccn, J. S.:Jorgenson, J.
AnelytcalChemktry. Vd. 66, No. 13, July 1, 1994
I 2
W.1.Chromorogr. 1989, 478, 63.
l
~
,
T
11 4
34
I
~
,
,
?
4
lo
lib
5
i
,
,
lo b lo Flgure 4. Influence of fused silica material on dynamic coating with PVA. Sample: (1) cytochrome c, (2) lysozyme, (3) trypsin, (4) trypsinogen, and (5) achymotrypslnogen;0.2 mg/mL each. Capillary: 57cm effective length, 70cm totallength; 50-pm i.d. (A) MlcroQuartz, Mirnchen, Qermany; (B) Polymlcro Technologies, Phoenix, AZ. Buffer: 50 mM sodium phosphate; pH 3.0 0.05% (w/w) PVA (Mw 50 000, 99-k % hydrolyzed, Aldrlch). Conditions: 30 kV (429 V/om); (A) 32 PA; (B) 26 PA; 20 "C. Injectbn: 10 kV, 5 8. Detection: UV, 214 nm.
Influence of Surface hoperties of Fused Silica Tubing on Protein Separationswitb Dynamic Surface Modification Using Different Types of Hydroxylic Modifiers. A significant influence of the surface properties of fused silica capillary tubing on the efficacy of deactivation by PVAor other nonionic hydroxylic polymericbuffer additives was observed. Compare also ref 17. In Figure 4 are shown CE separations of the basic protein mixture which were obtained with capillary tubing originating from MicroQuartz and from Polymicro Technologies. With a 5 0 mM phosphate buffer, the peak widths and symmetries of the strongly basic cytochrome c and lysozyme are not optimal with the material originating from Polymicro Technologies and could be considerably improved with a buffer of the slightly increased ionic strength of 75 mM. The efficiencies calculated for all peaks except trypsinogen were higher than 1 0 0 0 000 theoretical plates/m at the latter separation, The four electropherogramsA-D of Figure 5 are separations of the basic protein test sample performed with different commercially available hydroxylic polymers. With fused silica tubing from one lot of the Polymicro Technologies product, PVA was less effective as modifier in comparison to hydroxyethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC), and dextran (DEX). In separation C of Figure 5, it can be recognized that HEC is not sufficiently effective as a modifier to effect good separation efficiency for the lysozyme peak. With HPMC (separation D) this peak has the optimal shape, however, whereas with dextran (separation B) a peak broadening similar to that with HEC is observed. Thedextran modifier has, in spite of its high molecular weight (2 million), too high a solubility in water and may therefore be less strongly adsorbed to thesurface than HPMC. With HPMC, separation efficiencies of 1.5 million plates/m could be achieved.
1
"PMC
I
rll;l
-~\ ,
@
"gi
@
0 lo
HEC
5
~
l
15
lo
15
Flgure 5. Hydroxylic polymers for dynamic coating. Sample: (1) cytochrome c, (2) lysozyme, (3)trypsin, (4) trypsinogen, and (5) cuchymotrypslnogen A; 0.2 mg/mL each. Capillary: 57cm effective length, 70cm total length; 50-pm 1.d. (PT). Buffer: 50 mM sodium phosphate; pH 3.0; 0.05% (w/w) potymerlc additive; PVA, poly(viny1 alcohol); DEX, dextran; HEC, hydroxyethyl cellulose; HPMC, hydroxypropyl methyl celblose. Condltions: 30 kV (429 Vlcm), 28 PA; 20 "C. Injection: 10 kV, 5 s. Detection: UV, 214 nm.
With capillary tubing originating from the MicroQuartz, HEC proved to be absolutely unsuitable as a modifier even at pH 3, using a buffer with 20 mM sodium phosphate 3 0 mM NaCl; all peaks of the basic protein mixture showed a strong tailing and peak broadeningin general, especially those of the most basic cytochrome c and lysozyme. HEC has to be considered to be less polar than PVA because of the lower molar content of hydroxyl groups and the steric properties of the molecule, which consists of glucose units. HEC usually reduces the EOF as effectively as PVA and can also be applied successfully to chiral separations of small m0lecules.~J9With more complex proteins, multimodalanalyte/wall interactions give rise to poor separations using HEC as modifier. It will be discussed below that different surface properties of tubing material have no significant influence on the performance of protein separations if the permanent PVA surface modification is used. Reproducibility of Migration Times and Separation EfficienciesObtained witb Dynamic Surface Modification. The relative standard deviations (RSD) of migration times and average theoretical plate numbers N (m-l) given in Table 1 were determined from 35 runs during several days with dynamic PVA modification of the capillary walls. The capillary was filled with water and stored overnight. After each separation the capillary was flushed with about 1 mL of water and than filled with the PVA-containing buffer. The duration of rinsing had no significant influence on migration times and efficiencies.
+
(19) Snopck, J.;sOini, B.: Novotny, M.;Smolkova-Keulemansova,E. 1.Chromorogr. 1992,609, 1-17.
Table 1. Relatlve Standard Doviation d Mlgratkn Tlnm a d Average EtllCIench~sN wlth Dynamk PVA ModlficcrHon protein RSD (%) no N (m-1)
cytochrome lysozyme trypsin trypsinogen a-chymotrypsinogen
1.5 1.2 1.3 1.3 1.3
35 35 34 34 35
873 OOO
1
947 OOO 1 073 OOO 836 OOO 1026 OOO
2
5
* n = number of injections (for conditions, see Figure 8).
Permanent PVA Coatings for Protein Separations. Dynamic surface modification with PVA is effective only at lower pH. At medium and higher buffer pH, the capillary surfaces have more negative charges from stronger dissociation of the SiOH groups, probably because of this stronger negative charging, the adsorption of PVA molecules and the suppression of analyte/wall interaction is weakened. Fixation of the modifying layer by covalent bonding of PVA as, for example, achieved after prior epoxysilane derivatization of the surface cannot be done. PVA is only solvable in water, and the fixing reaction cannot be effectively executed in aqueous solution because the water would react with the epoxy group as well. Moreover, the formed covalent Si-oSi bond will not be stable at pH >8. It is known that thin layers of PVA turn pseudocrystalline with thermal treatment. The solubility in water and the swelling properties of such films are strongly decreased.14J0 Prior to heat treatment the polymer chains in films of PVA are largely in disorder. The mobility of the polymer chains increases with temperature, so that they can assume orientations of higher order.14 The PVA molecules become more strongly associated by hydrogen bridge bonding, and water molecules cannot penetrate into the microcrystalline domains. The formation of microcrystalline regions depends on the temperature and the grade of hydrolysis type of PVA applied. Inadequately hydrolyzed PVA contains residual acetate groups, which would disturb the desired strong association of the polymer chains. Therefore PVA products of the highest grade of hydrolyzation (99+%) should be applied. Temperatures beyond 150 O C should not be used for the thermal immobilization because PVA starts to decompose at 160 OC. In this way, PVA coatings of the capillary surfaces can be immobilized without thecommon methods of covalent bonding. In our experiments, such coatings proved to be stable in separations even at higher pH (up to 10). The PVA coatings were generated as described in the Experimental Section. It is important that the displacement of the excessive viscous aqueous solution of PVA from the capillary is executed slowly in order to obtain PVA films which are thick enough. Immobilization is achieved by heating to 145 O C during the vaporization of the residual water and possible volatile decomposition products of the polymer under a gentle gas flow. The permanently coated surfaces exhibited a much lower adsorptivity against proteins than untreated surfaces. Addition of hydroxylic or other modifiers to the buffers is not necessary with the permanent coatings. (20) Kcnncy, J. F.;Willcockson, G.W.J. Polym. Sei., Purr A-I 1966, I , 679-698.
2042
Analyticai Chemistry, Vol. 66. No. 13, Ju& 1, 1994
I
lo
.
'
'
'
I
'
16
l
lo
~
~
15 min
Fi@ure6. Dynamic and permanent coating with poly(viny1alcohol). Sample: (1) cytochrome c, (2) lysozyme, (3)trypsin, (4) trypsinogen, and (5) cu-chymotrvpsmrtogem A. CapHlary: 57cm effective length, 70(A) 0.05 % (w/w) cm total length; 50-pm 1.d.; porymicro~ed~ldogies; PVA as buffer additive, (B) thermally hnmobllired PVA. Buffer: 50 mM sodium phosphate; pH 3.0. C " s : 30 kV (357 V/cm), 28 p k 20 O C . Injection: 10 kV, 5 s. Detection: UV, 214 nm.
Separation of Standard Proteins with Permanently PVA Coated Capillaries. Separations of basic and acidic test proteins at buffer pH of up to 10were executed in permanently PVA coated capillaries under the same conditions as with the dynamic method except that PVA as modifier was not contained in the buffer. The two CZE separations of basic proteins in Figure 6 were obtained with capillary tubing which did not prove to be suited for application with "dynamic" PVA surface modification. With the permanent PVA coating, even for cytochrome c and lysozyme, efficiencies of 900 000 plates/m could be achieved with the same tubing material. These results show that the strong adsorptivity of the tubing material and the related analyte/wall interactions are very effectively suppressed by immobilized PVA layers. The separation of Figure 7 was obtained with a permanently coated capillary of Polymicro tubing with half the sample load of the system compared to the previous separations. The plate numbers are specified on the peaks. Capillary tubing from different manufacturerscan be given permanent PVA coatings and the varying surface properties will not influence the resulting suppression of protein/wall interaction. Separation of Acidic Proteins. Separations of three acidic proteins are shown in Figure 8. Electropherogram A was obtained with dynamic PVA modification at pH 3. Electropherogram B was obtained with a permanent PVA coating and showed the highest separation efficiency, with theoretical plate numbers of 670000 m-1 for @-lactoglobulin B and 940 000 m-l for a-lactalbumin. Separations of this quality could not be obtained in bare fused silica or with any of the other dynamic and permanent coatings described. Protein Separations in the Medium-and High-pH Range. In permanently PVA coated capillaries, reproducible separations could be achieved even at a pH of 5.5 (see Figure 9) in which the strongly basic lysozyme and cytochrome c appear as symmetricaland narrow peaks, although the plate numbers (300 000-850 OOO m-') were somewhat lower as with separations at pH 3. Neverthelessthe a-chymotrypsinogenappears in separation B as a symmetrical peak even at long migration
~
N
-
10 nF'
1851
1
1 239 000
I
L I
1
I
I
I
I
10
min Figure 7. Etficlenclesobwith a poly(vlnyialcohd)coatedcapillary. Sample: (1) cytochrome c,(2) lysozyme, (3) trypsin, (4) trypsinogen, and (5) achymotrypsinogen. Capillary: 5 7 t m effective length, 70cm total length; 75-pm i.d.; thermally immobilized PVA (MW 50 000, Aldrich). Buffer: 50 mM sodium phosphate; pH 3.0. Codtlons: 30 kV (357 V/cm), 89 pA; 20 "C. Injection: 10 kV, 2.5 s. Detection: UV, 214 nm. Number of theoretical plates per meter calculated from width at haifheight.
I A
;o
,
I
.
.
'mk;
'lb
T
I
,
,
is A
Figure 8. Separation of acidic proteins in a capillary dynamically (A) and permanently (8) coated with poly(vlnyi alcohol). Sample: (1) &Iactogiobuiin, (2) &lactoglobulin A, and (3) a-iactalbumin; 0.3 mg/ mL each.Capillary: 57cm effectivelength, 7 k m total length; 50-pm i.d.; (A) bare fused sHIca, dynamic modificatbn by PVA; (B) thermally immobilizedPVA (MW 50 000,99+ % hydroiyzed, Aldrlch). Buffer: (A) 70 mM sodium phosphate, 0.05% (w/w) PVA, (B) 70 mM sodium phosphate; pH 3.0. Conditbns: 30 kV (429 V/cm), 32 MA; 20 "C. Injection: 10 kV, 4 8. Detection: UV, 214 nm.
times of 40 min. The longer migration times at the still suppressed EOF at this pH are the consequence of the lower mobility of the proteins in buffers of higher pH and ionic strength because of the decrease of effective molecule charging. Acceptable separations of acidic proteins can be achieved even at pH 10in PVA-coated capillaries; see Figure 10.Acidic proteins are negatively charged at this pH and the EOF is still strongly suppressed; therefore, these separations had to be executed with reversed polarity of the electrical field. Phosphate buffers could not be applied for such separations, because at these pH values, the systems are beyond their buffering range. Protein elution was impossible with borate buffers; therefore, the amino alcohol (2-amino-2-methyl- 1-
h
L,
1-
01
...........................................
.
@
Glycoproteins consist of proteins with defined amino acid sequences but variable glycan structures. The relative portions of the different glycoforms of a certain protein depend on the environment in which the protein has been glycosylated, so that based on the pattern of the separated glycoforms of the protein, conclusions on its origin can be made. In conventional electrophoresison slab gels (SDS-PAGE) and also with HPLC methods, the appropriate resolution of glycoforms of proteins cannot be achieved. With the high separation efficiencies of CE, it is possible to resolve the glycoforms of not too complex populations in spite of their minor mobility differences.22-26 With such proteins the suppression of wall adsorption is absolutely necessary when the maximum efficiency has to be achieved. This will be demonstrated with separations of ribonuclease B, pepsin, and ovalbumin. The separation of a test mixture of ribonuclease A and B and three separations of commercial ribonuclease B preparations are shown in Figure 12. Grossman et al. separated two forms-B1 and B2-of ribonuclease at high pH in an unmodified capillary. These authors considered the two glycoforms to contain oligosaccharides consisting of two units of N-acetylglucosamine and either one or six units of mannose.25 With a PVA-coated capillary, ribonuclease B could be well resolved into five peaks. The peak pattern is nearly identical to that obtained by Rudd et al. under completely different condition^.^^ The migration sequences of ribonuclease A and B and probably also that of the glycoforms were reversed in this separation. Rudd applied a system with a phosphate/ borate buffer (pH 7.2) and 50 mM sodium dodecyl sulfate (SDS) as additive. The five peaks were correlated with (22)Novotny. M.; Sudor, J. Electrophoresis 1993,14, 373-389. (23)Kakehi, K.; Honda, S. Methods Mol. Biol. 1993, 14, 81-97. (24)Rudd, P.M.; Scragg. I. G.; Coghill, E.; Dwek, R. A. Glycoconjugate J . 1992, 9, 86-9 1. (25) Grossman, P.D.;Colburn,J. C.;Lauer, H.H.Anal. Chem. 1989.61, 1186 1194. (26)Landers, J. P.;Oda, R. P.; Madden, B. J.; Spelsberg,T.C.Anal. Eiochem. 1992, 205. 115-124.
2044
AnalyticaiChemistry, Voi. 66,No. 13, July 1, 1994
15 io i5 min Flgure 12. Purlty of commercial ribonuclease A preparations. Sample: (A) mixture of (1) ribonuclease A and (2) ribonuclease B); ( 5 D ) ribonuclease A preparationspurchased from (6) Pharmacla, (C) Sigma (Type I-AS), and (D) Fiuka. Capillary: 57cm effective length, 70cm total length; 50-pm 1.d.; thermally immobilizedpoly(vinylalcohol). Buffer: 100 mM sodium phosphate, pH 3.0. Conditions: 35 kV (500 V/cm), 62 pA; 20 OC. Injection: (A) 15 kV, 5 8; (6-D) 25 kV, 5 s. Detection: UV, 214 nm.
glycoforms with five to nine mannose units. The correlation was derived from results obtained with other analytical methods. The reverse migration sequence is yet not fully understood by us, but may be due to complexation of the saccharides with the borate. Separations E D of Figure 12 were performed to characterize the purity of commercial ribonuclease A products and prove the suitability of CE for analytical separations of such glycosylated proteins. The highly acidic single-chain phosphoprotein pepsin has a molecular mass of 34 500 and consists of 324 amino acids. It is a protease occurring in the stomachs of vertebrates. The active pepsin A is released autocatalytically from pepsinogen and exists in several glycoforms.26 These glycoforms could be separated at pH 8.5, as shown in Figure 13. The necessary selectivity could only be achieved with high ionic strength buffer. A separation with a similar pattern but lower resolution has been published by Landers et al., who used putrescine as additive at pH 9.26 The reverseelution order is the consequence of the positive polarity of the electrical field and relatively slow separation by migration against a high EOF. The separation of Figure 13 was obtained with negative polarity against a very low EOF. Separation of the Glycoforms of Ovalbumin. Ovalbumin is a glycophosphoprotein consisting of 385 amino acids with a molecular mass of 43 000. The heterogeneity of this protein is caused by oligosaccharides which are linked to asparagine 293 and two phosphorylation sites at serine 68 and serine 344. As shown in Figure 14, the albumins from chicken and turkey eggs were separated at pH 3 and 9.5. Different peak patterns
I
I
I2
1s
20 min
Flgurr 13. Separation of pepsin glycoforms. Sample: pepsin from porch8 stomach mucosa, 2 mglmL. Capillary: 5 7 4 m effective length, 7 k m total length; 50-pm i.d.; thermally ImmobUlzed PVA. Buffer: 100 mM sodium phosphate, pH 8.5. Conditions: -25 kV (357 Vlcm), 97 p A 20 O C . Injection: 10 kV, 4 s. Detectlon: UV, 214 nm.
pH3
@
pH9.5
L
4
I
"
'
,
t
,
,
,
,
#
l
15 10 min Figure 14. Heterogeneity of egg albumins at low and high pH. Sample: (A, 6) albumin from chicken egg; (C, D) albumin from turkey egg. Capillary: 5 7 4 m effective length, 70cm total length; 50-pm i.d. (w); thermally immobiiitedpoly(vinyiaicohoi). Buffer: (A, C) 150 mM sodium phosphate, pH 3.0; (B, D) 60 mM AMPD/HsP04, pH 9.5.Conditions: (A, C) +30 kV (429Vlcm). 73 FA; (B, D) -35 kV (500 Vlcm), 9 p A 20 O C . Injectlon: (A) 5 kV, 5 s; ( E D ) 15 kV, 5 8. Detection: UV, 214 nm.
are obtained by reversal of the surface charging of the proteins at these pH values. The pZ of the protein is about 4.7; at pH 3 the glycoforms are separated as positively charged species whereas at pH 9.5 they are negatively charged. At this pH the EOF is strongly reduced as well; therefore, the polarity of the electrical field had to be reversed.
I
15
-
~
20
~
mln
~
I
~
Flgurr 15. Separatkm of chicken egg whtte protekrs. Sample: & M e n egg white, l0-fdddiluted with buffer, filtered through 5-run membrane fitter; (1) lysozyme, (2) conalbumin, and (3)ovalbumin. Capillary: 57cmeffecthrelength,70cmtotailength; 50-pmi.d.;thennallylmnoMlhed PVA. Buffer: 150 mM sodium phosphate, pH 3.0. Conditbns: 30 kV (429Vlcm), 78 pA; 20 O C . Injectkm: 15 kV, 5 8. Detection: UV, 214 nm.
For similar separations, Landers et a1.26applied a borate buffer at pH 8 with putrescine as additive. They achieved an even better resolution as in Figure 14 probably because of complexation of the borate with the saccharides. The pattern of a separation performed with a phosphate buffer at pH 9 was very similar to our separation B in Figure 14, but with reversed migration order as already mentioned above. In the work of Landers et al., it was proved by enzymatic dephosphorylation that the heterogeneity of the ovalbumin originates from glycan components and the protein naturally occurs as a diphosphate ester only. The enzymatically cleaved oligosaccharidescould be identified by H0nda.~39~'Nine major components were found which contained between 7 and 11 monosaccharide units such as mannose, galactose, and N-acetylglucosamine. Chicken egg white was separated at pH 3 under the same conditions as for the separation of the commercialovalbumins. The sample was diluted 10-foldwith buffer and filtered through a membrane with 5-pm pore diameter; for the obtained separation, see Figure 15. The major peaks could be correlated by comparison with other standard separations. Lysozyme appears as sharp peak because it is not glycosylated. The ovalbumin appears with a pattern by resolution of the glycoforms as in Figure 14A. The different components of the inhomogeneous conalbumin could not be resolved under these conditions except for a shoulder at the front of the peak. Similar separations were also tried by McCulloch in commercial polysiloxane-coated capillaries (CElect).28 These separations with phosphate/borate buffers at pH 8 were not successful as regards the resolution of the glycoforms. (27) Honda, S.; Makino, A.; Suzuki, S.; Kakchi, K. Anal. Biochrm. 1990, 191, 228-234. (28) McCulloch, J. D.J . Liq. Chromatogr. 1993, 16, 2025-2038.
Am!~tlcaIChemisby, Vol. 66,No. 13, Ju& 1, 1994
2045
~
Conclusions on the Application of Dynamic and Permanent PVA Coatings. In comparison to most of the methods of permanent surface modification for protein separations which involve covalent bonding to the surface, the dynamic method allows for separations of the same or better performance but unfortunately only at low pH. Dynamic modification is simple to employ and does not require elaborate chemical reactions in such narrow-bore capillaries. The dynamic modification can be refreshed by automated rinsing procedures. PVA layers could be fixed to capillariessurfaces by thermal immobilization without covalent bonding to the surface as achievable by silanization procedures. The fixation is effected by strong adsorption at the surface and also by strong intermolecular interaction between the polymer chains under formationof pseudocrystalline regions. In the low-pH-range, separations of the same quality as with the dynamic PVA coating were possible without addition of PVA to the buffer. Negative influences of surface adsorptivity related to the different qualities of commercial capillary tubing that occur at dynamic modification are effectively eliminated by thermally immobilized PVA. With the permanent PVA coating, the application range of the capillaries could be extended to nearly the whole pH range in which proteins are stable. Separations at low and medium pH could be performed at high efficiencies, whereas in the alkaline range, lower efficiencies were observed. Because of the completely suppressed EOF in the pH range between 3 and 10,very long migration times arise in separations
2046
Analytical Chemistry, Vol. 66, No. 13, July 1, 1994
of basic proteins with buffers in the neutral pH range; the separation of acidic proteins is not possible under these conditions, because they are not charged and therefore have a low electromobility. The suppression of the EOF was favorable at separations above pH 8. High selectivities were achieved on the basis of differences between the net mobilities of the proteins. In application to practical samples, the components of inhomogeneous proteins that are characterized by different degrees of glycosylation could be resolved because of the high separation efficienciesachievable by good shielding of the capillary surface and the corresponding avoidance of analyte/wall interaction. Such separationscannot be achieved in conventional electrophoresis or liquid chromatography. With PVA-coated capillaries, the relative portions of the singleforms of a population could be directly determined in the case of proteins with a single glycosylation site. With acidic proteins the distribution of the different glycosylated forms could also be evaluated by separation in the same capillary either at low pH as positively charged or at high pH as negatively charged ions. It seems to be desirable to further improve the stability of the PVA coatings, e.g., by cross-linking for separations at pH of 8 and higher. Received for review February 4, 1994. Accepted April 11, 1994.' *Abstract published in Advance ACS Abstracts, June 1, 1994.
