Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.     

Separation of small peptides by electrochromatography on silica-based reversed phases and hydrophobic anion exchange phases

Peptide separations are regarded as a promising application of capillary electrochromatography (CEC) and, at the same time, a suitable model to elucidate its mixed separation mechanism when charged analytes are involved. In this paper, studies on the separation of small peptides (2-4 amino acids) on a Spherisorb octadecyl silane (ODS) phase at acidic pH and on a strong anion exchange (SAX)/C18 mixed mode phase at weakly basic pH are reported. For the ODS phase a comparison of CEC, capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) under identical buffer/eluent conditions is presented. The predicted retention factors for CEC under the assumption of simple superposition of HPLC retention and CZE migration matched the measured results for the peptides that had small retention factors in HPLC. For both types of stationary phases, a variation of the acetonitrile content in the mobile phase led to a wide range of retention factors, including negative values when co-electroosmotic migration was dominant. Though both the ODS and the SAX/C18 phase offer unique advantages, the SCX/C18 phase at pH 9 provides more flexibility to alter separation selectivity for the selected peptides.     

Capillary electroendoosmotic chromatography of peptides

This review focuses on the current state of peptide separation by capillary electroendoosmotic chromatography (CEC). When carried out under optimised conditions, peptide separation by CEC methods represents an orthogonal and complementary technique to micro-HPLC (micro-HPLC) and high-performance capillary zone electrophoresis (HPCZE). The origin of the selectivity differences that can be achieved with these three separation techniques (CEC, micro-HPLC and HPCZE), respectively are discussed, and the current limits of performance with CEC methods documented. Peptide separations by CEC methods with n-alkyl bonded silicas or mixed-mode phases are also illustrated. The development of different variants of CEC and pressurised CEC (also commonly referred to in the literature as electrically-assisted micro-HPLC) are examined. The potential of coupling CEC systems to mass spectrometers for real-time analyses of peptides or protein digests has been examined. Several future directions for the application of this technique in phenotype/proteomic and zeomic mapping of naturally occurring peptides and proteins are highlighted.     

Influence of temperature on the behaviour of small linear peptides in capillary electrochromatography

The influence of temperature, T, on the retention times, peak widths, peak symmetry coefficients and theoretical plate numbers of two small linear peptides, [Met5]enkephalin and [Leu5]enkephalin, has been studied with capillary electrochromatography (CEC) capillary columns of 100 microm I.D. and 250 mm packed length with a total length of 335 mm, containing 3 microm Hypersil n-octadecyl bonded silica. With increasing column temperature from 15 to 60 degrees C, the electroosmotic flow (EOF) and the column efficiencies increased, whereas the retention coefficients (Kcec) of both peptides decreased. A linear relationship was found between the EOF value and the square root of the temperature over this temperature range, with a linear regression correlation of 0.998. Non linear Van 't Hoff plots (In Kcec versus 1/T) were observed for these peptides between 15 and 60 degrees C, suggesting that a phase-transition occurred with the n-octadecyl chains bonded on the silica surface, affecting the CEC retention behaviour of these peptides. In CEC systems, the Kcec values of peptides incorporate contributions from both electrophoretic migration and chromatographic retention. Positive and negative Kcec values can, in principle, thus arise with these charged analytes. However, the Kcec values of the enkephalin peptides under all temperature conditions studied were positive with an eluent composed of water-50 mM NH4OAc/AcOH, pH 5.2-acetonitrile (5:2:3, v/v) and therefore the chromatographic component dominates the retention process with these small peptides under these conditions.     

Use of mixed-mode sorbents for the electrochromatographic separation of thrombin receptor antagonistic peptides

In this study, the thrombin receptor antagonistic peptide TRAP-1 and its alanine-scan analogues, TRAP 2-6, have been employed as probes to characterise the performance of C18/SCX mixed-mode capillary electrochromatographic (CEC) columns. It was found that the resolution of this group of peptides could only be achieved in a narrow pH range with phosphate-based running electrolytes. The influence of the running electrolyte composition, e.g. the buffer choice, the ionic strength, the pH and the organic solvent content, on the electroosmotic flow (EOF) of these mixed-mode CEC columns was investigated. In addition, the retention mechanism for this group of peptide probes in the electrochromatographic process was studied by examining the effect of varying the running electrolyte composition. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving both hydrophobic as well as ion exchange interactions. By modulating the running electrolyte composition, the hydrophobic or ion exchange components of the interaction process could be made to dominate the chromatographic retention of the peptides. Based on this strategy, a high-resolution separation of six closely related synthetic peptides was demonstrated with this mixed-mode CEC system.     

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. I. Effect of the mobile phase

The high-performance liquid chromatographic (HPLC) behaviour of two different styrene-divinyl-benzene-based reversed-phase (RP) columns was evaluated using crude acetic acid extracts from normal and diabetic human pancreata as samples. Acetic acid gradients in water and acetonitrile gradients in triethylammonium phosphate (TEAP) and trifluoroacetic acid (TFA) were used as mobile phases, and comparisons were made with a silica-based C4 column. When two different polymeric RP columns were eluted with acetic acid gradients in water, surprisingly similar HPLC profiles of the pancreatic extracts were obtained. Elution of the polymer-based columns with acetonitrile gradients in TFA or TEAP resulted in changes in the polypeptide selectivity of these columns, in parallel with that of a silica-based C4 column eluted under similar conditions, indicating the general usability of polymeric columns for RP-HPLC of peptides and proteins. The pronounced difference in composition between normal and diabetic samples, which also was demonstrated after size-exclusion chromatography (SEC) on a silica-based and an agarose-based high-performance SEC column, was found to be related to the different ischaemia times for the two types of pancreata.     

Rapid separation of peptides and proteins by isocratic capillary electrochromatography at elevated temperature

The use of capillary electrochromatography (CEC) for the separation by isocratic elution of synthetic peptides, proteins as well as the tryptic digest of cytochrome c has been demonstrated. The monolithic porous stationary phase was prepared from silanized fused-silica capillaries of 75 microm I.D. by in situ copolymerization of vinylbenzyl chloride and ethylene glycol dimethacrylate in the presence of propanol and formamide as the porogens. The chloromethyl groups at the surface of the porous monolith were reacted with N,N-dimethylbutylamine to form a positively charged chromatographic surface with fixed n-butyl chains. Results of studies on the influence of temperature and mobile phase composition on the retention and selectivity of separation by CEC demonstrated the feasibility of rapid polypeptide analysis and tryptic mapping at elevated temperature with high resolution and efficiency. Typically the chromatography of a tryptic digest of cytochrome c took about 5 min at 55 degrees C and 75 kV/m with hydro-organic mobile phases containing acetonitrile in 50 mM phosphate buffer, pH 2.5. For peptides and proteins plots of logarithmic k'cec against acetonitrile concentration were nonlinear, whereas Arrhenius plots for the mobilities were nearly linear. Comparison of the separation of such samples under conditions of CEC and capillary zone electrophoresis (CZE) indicates that the mechanism of separation in CEC is unique and leads to a chromatographic profile different from that obtained by CZE.     

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.     

Separation of peptides on mixed mode of reversed-phase and ion-exchange capillary electrochromatography with a monolithic column

The mixed mode of reversed phase (RP) and strong cation-exchange (SCX) capillary electrochromatography (CEC) based on a monolithic capillary column has been developed. The capillary monolithic column was prepared by in situ copolymerization of 2-(sulfooxy)ethyl methacrylate (SEMA) and ethylene dimethacrylate (EDMA) in the presence of porogens. The sulfate group provided by the monomer SEMA on the monolithic bed is used for the generation of the electroosmotic flow (EOF) from the anode to the cathode, but at the same time serves as a SCX stationary phase. A mixed-mode (RP/SCX) mechanism for separation of peptides was observed in the monolithic column, comprising hydrophobic and electrostatic interaction as well as electrophoretic migration at a low pH value of mobile phase. A column efficiency of more than 280,000 plates/m for the unretained compound has been obtained on the prepared monoliths. The relative standard deviations observed for t(0) and retention factors of peptides were about 0.32% and less than 0.71% for ten consecutive runs, respectively. Effects of mobile phase compositions on the EOF of the monolithic column and on the separation of peptides were investigated. The selectivity on separation of peptides in the monolithic capillary column could be easily manipulated by varying the mobile phase composition.     

Peptide separation in hydrophilic interaction capillary electrochromatography

Separation of small peptides by hydrophilic interaction capillary electrochromatography (HI-CEC) has been investigated. The negative surface charge of a hydrophilic, strong-cation-exchange stationary phase (PolySULFOETHYL A) provided a substantial cathodic electroosmotic flow (EOF). The influence of acetonitrile content, ionic strength, mobile phase pH as well as applied voltage on the migration of the peptides was studied. Possible retention mechanisms of the peptides in HI-CEC were discussed. It was found that hydrophilic interaction between the solutes and the stationary phase played a major role in this system, especially when mobile phases with high acetonitrile content were used. However, an ion-exchange mechanism and electrophoretic mobility also affect the migration of the peptides in HI-CEC. Elution order and selectivity was proved to be different in HI-CEC and capillary zone electrophoresis (CZE), thus revealing the potential of HI-CEC as a complementary technique to CZE for the separation of peptides. Efficiency and selectivity of HI-CEC for the separation of peptides were demonstrated by baseline separating nine peptides in 6 min.     

Effects of G-quartet DNA stationary phase destabilization on fibrinogen peptide resolution in capillary electrochromatography

The migration of fibrinogen peptides in capillaries coated with G-quartet-forming DNA oligonucleotides in open-tubular CEC (OTCEC) was studied, in order to investigate factors affecting the retention of peptides on G-quartet DNA stationary phases. At 25 degrees C, the peptides eluted in the same order in OTCEC using a two-plane G-quartet DNA stationary phase as in CZE, including two peptides that were completely overlapped. It was found that baseline resolution of the coeluting peptides could be achieved in the OTCEC experiment, but not in CZE, at run temperatures of 35-40 degrees C. A stationary phase formed by a scrambled-sequence oligonucleotide that does not form a G-quartet did not provide any resolution of the two coeluting peptides, even at the higher temperatures, indicating that some destabilization of the G-quartet enhances resolution but that some degree of G-quartet structure is necessary. The effects of destabilization were further explored through variation of the cations (sodium or potassium) used in attachment of the G-quartet oligonucleotide to the capillary surface and in the mobile-phase buffer. Resolution was lower when a more stable, four-plane G-quartet stationary phase was used, supporting the conclusion that some flexibility in the G-quartet structure facilitates differential interactions that resolve otherwise coeluting peptides. The increase in peptide resolution upon destabilization of the G-quartet structure could prove to be an important factor in the application of G-quartet DNA stationary phases for nonaffinity-based separation of native proteins and peptides.     

Life-time studies with capillary electrochromatography columns operated under different conditions

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k(cec), peak symmetry coefficients, lambda(sym), and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 microm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 microm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 - 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors (k(cec) values) of these n-alkylbenzenes, acquired with an eluent of (25 mM Tris-HCl, pH 8.0,)-acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k(cec) values of the n-alkylbenzenes for one set of replicates (n=6) with one CEC capillary column was < 0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60,000, whilst the observed N values for all new CEC capillary columns were > or = 40,000 for n-octyl bonded silica capillary columns and > or = 50,000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, lambda(sym), of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1,000 injections were always in the range 0.95-1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.     

Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. II. Separation of amino acids and peptides on sulfonated naphthalimido-modified silyl silica gel

The potential of 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP) as a mixed-mode stationary phase for capillary electrochromatography (CEC) was investigated for the separation of charged analytes, taking four amino acids (tyrosine, phenylalanine, tryptophan, histidine) as model analytes. The elution process of these charged analytes in CEC with SNAIP was dominated by a combination of both electrophoretic process and chromatographic process involving hydrophobic as well as electrostatic interactions. In order to study the retention mechanism, the CEC retention factor k* and the velocity factor ke* were measured for the amino acids, which allowed the assessment of the respective contribution from the differential processes underlying the separation. Migration and retention could be mediated by changing various mobile phase compositions, including buffer pH, buffer concentration, and concentration of organic solvent. Based on the results obtained by separation of the amino acids, the separation of eight peptides (Gly-Val, Gly-Phe, Gly-Ile, Gly-His, Gly-Lys, Lys-Lys, Gly-Gly-Gly, Gly-Gly-His) was attempted. A good separation was achieved under an isocratic elution with a mobile phase consisting of 35 mM phosphate buffer (pH 3.8) and 40% methanol.     

Separation and identification of etodolac and its urinary phase I metabolites using capillary electrochromatography and on-line capillary electrochromatography-electrospray ionisation mass spectrometry coupling

Capillary high-performance liquid chromatography (capillary HPLC), pressure-assisted capillary electrochromatography (pCEC) and capillary electrochromatography (CEC) were performed in the same capillary packed with 5 microm octadecylsilica (C18) as stationary phase. These three separation modes were compared from the viewpoint of peak efficiency and separation selectivity in order to critically evaluate the advantages which CEC may offer compared to capillary HPLC for the solution of practical biomedical problems. The separation of the non-steroidal anti-inflammatory drug etodolac (ET, 1) and its phase I metabolites, 6-hydroxy etodolac (6-OH-ET, 2), 7-hydroxy etodolac (7-OH-ET, 3) and 8-(1'-hydroxyethyl) etodolac (8-OH-ET, 4) was selected as an example. Baseline separation of all compounds was achieved in different modes and conditions. The effect of pure electrophoretic separation mechanism on the overall separation selectivity observed in CEC has been shown. A high electroosmotic flow (EOF) was observed in C18 packed capillary even at pH 2.5 in various buffers. Furthermore, these separations were coupled on-line with electrospray ionisation mass spectrometry (ESI-MS) and the parent drug and its metabolites were identified in urine. For the coupling of CEC with ESI-MS a laboratory-made electrophoretic device was used in order to overcome some technical disadvantages of commercial instrumentation.     

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.     

Separation of tryptic peptides of native and glycated BSA using open‐tubular CEC with salophene–lanthanide–Zn2+ complex as stationary phase

Open‐tubular CEC (OT‐CEC) with a new stationary phase, salophene–lanthanide–Zn²⁺ complex, has been applied to the separation of tryptic peptides of native BSA and BSA glycated by glucose and ribose. Glycation of proteins (non‐enzymatic modification by sugars) significantly affects their properties and it is of great importance from a physiological point of view. Separation of tryptic peptides of glycated BSA by CZE was poor because of their strong adsorption to the bare fused silica capillary. An improved separation of tryptic peptides of both native and glycated BSA was achieved by OT‐CEC in the fused silica capillary non‐covalently coated with salophene–lanthanide–Zn²⁺ complex, which suppressed the adsorption of peptides to the capillary and via specific interactions with some (glyco)peptides enhanced selectivity of the separation. Significant differences have been found in OT‐CEC analyses of tryptic hydrolysates of native and glycated BSA. In OT‐CEC‐UV profile of tryptic peptides of native BSA, 44 peaks could be resolved, whereas a reduced number of 38 peaks were observed in the profile of tryptic peptides of glucose‐glycated BSA and only 30 peaks were found in the case of ribose‐glycated BSA. The developed OT‐CEC can be potentially used for monitoring of protein glycation.     

Capillary electrochromatography of basic compounds using octadecyl-silica stationary phases with an amine-containing mobile phase

The capillary electrochromatographic (CEC) analysis of basic compounds on octadecyl-silica stationary phases (Hypersil ODS and Spherisorb ODS I) was studied. A basic drug (fluvoxamine) and one of its possible impurities were used as test compounds. With an eluent of acetonitrile-phosphate buffer (pH 7.0), the compounds could be baseline-separated; however, broad and tailing peaks were obtained. To minimise detrimental interactions with residual silanol groups, the pH of the mobile phase was lowered to 2.5, but the plate numbers were still quite low (<2.6x10(4) plates/m). Addition of a masking agent (hexylamine or triethylamine) to the mobile phase resulted in much better peak efficiencies (ca. 1x10(5) plates/m). Therefore, the influence of the amine concentration and pH of the mobile phase on the CEC performance (peak width, peak tailing, electroosmotic flow, selectivity) was investigated in detail. Highest efficiencies (2.8x10(5) plates/m) could be obtained with the Spherisorb column, while the Hypersil column offered a better selectivity. Furthermore, the results show that the residual silanol groups are (at least partly) responsible for the separation of the basic compounds and that the amount of injected sample has an unusually large effect on the peak efficiency. The usefulness of the system for impurity profiling was demonstrated with a mixture containing fluvoxamine and its stereoisomer (a possible impurity) at the 0.1% level. The general effectiveness of amine additives in CEC was illustrated by the separation of a mixture of five structurally different basic drugs yielding plate numbers in the 1x10(5)-3x10(5) plates/m range. Comparison with capillary electrophoretic analysis revealed a unique selectivity of the CEC system which is based on both electrophoretic mobility and chromatographic partitioning.     

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF). A neutral bulk monolithic stationary phase was then prepared by in situ copolymerization of vinylbenzyl chloride (VBC) and ethylene glycol dimethacrylate in the presence of 1-propanol and formamide as porogens. Benzyl chloride functionalities on the monolith were subsequently hydrolyzed to benzyl alcohol groups. Effects of pH on the EOF mobility of the column were measured to monitor the completion of reactions. Using a column with this design, we expected general problems in CEC such as irreversible adsorption and electrostatic interaction between stationary phase and analytes to be reduced. A peptide mixture was successfully separated in counter-directional mode CEC. Comparison of peptide separations in isocratic monolithic CEC, gradient HPLC and capillary zone electrophoresis (CZE) indicated that the separation in CEC is governed by a dual mechanism that involves a complex interplay between selective chromatographic retention and differential electrophoretic migration.     

Theoretical models of separation selectivity for charged compounds in micellar electrokinetic chromatography

Equations and theoretical models for MEKC separation selectivity (α(MEKC) ) were established to explain a change in separation and electrophoretic mobility order of fully charged analytes, in which α(MEKC) is related to the dimensionless values of mobility selectivity in CZE (α(CZE)) and retention selectivity (α(k)) in MEKC, and where α(CZE) and α(k) are defined as the ratio of electrophoretic mobility in CZE and the ratio of retention factor (k) in MEKC for two charged analytes, respectively. Using four alkylparabens as test analytes, excellent agreement was found between the observed α(MEKC) and the proposed α(MEKC) models of test analytes in MEKC over a wide range of SDS concentrations and values of k. For example, in comparison with CZE separation of charged analytes, MEKC separation can enhance separation selectivity up to the maximum value when the selectivity ratio (ρ) is greater than 1.0 (ρ=α(k)/α(CZE)), while lower separation selectivity is obtained with ρ<1.0 (α(CZE) >α(k) >1).     

Use of electrokinetic measurements for characterization of columns used in capillary electrochromatography

The separation mechanism in capillary electrochromatography (CEC) is a hybrid differential migration process, which entails the features of both high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE), i.e., chromatographic retention and electrophoretic migration. The focus of this paper is on the use of electrokinetic data, such as current, electroosmotic flow (EOF) and column efficiency measurements, that are readily available, for an improved understanding of CEC separations. A framework is presented here for the use of this data for evaluation of a variety of performance parameters including, conductivity ratio, interstitial EOF mobility, porosity, and zeta potential. This framework is applied for characterization of two monolithic columns with different chemistry that were manufactured in-house. The above-mentioned performance parameters were calculated for the two columns and it is found that the poly(VBC-EGDMA-SWNT) monolithic column with the GPTMS-PEI coating offers a significantly improved flow distribution in comparison to the poly(VBC-EGDMA) monolithic column. This observation is confirmed by performing separation of peptides on the two columns and height equivalent of a theoretical plate (HETP) measurements on the resulting peaks. It is shown that following our approach leads to an improved understanding of the separations achieved with the columns and to better column design.