Ultrafast sodium dodecyl sulfate micellar electrokinetic chromatography with very acidic running buffers.

A new micellar electrokinetic chromatography (MEKC) procedure has been developed that allows the use of the anionic surfactant sodium dodecyl sulfate (SDS) together with separation buffers at pHs as low as 1. The technique is based on the employment of a high molecular weight polyethylenimine (PEI)-coated capillary that provides strong cathodal electroosmotic flows at acidic pHs when SDS is added to the running buffer. The procedure is easy to implement since the capillary coating is done by just flushing a PEI aqueous solution through the capillary and the subsequent steps are the same as those for any MEKC protocol. Moreover, the coating renewal provides reproducible separations between injections (migration time RSD values lower than 1.82 and 3.44% were obtained for the same day and three different days, respectively). The good possibilities of this system are demonstrated by showing the separation of a group of eight polyphenolic compounds within a separation time shorter than 2 min. This procedure allows one to extend the optimization of SDS-MEKC separations to the very acidic pH range too.     

Separation of achiral and chiral analytes using polymeric surfactants with ionic liquids as modifiers in micellar electrokinetic chromatography

In this study, we report the use of ionic liquids as modifiers in the separation of achiral and chiral analytes in micellar electrokinetic chromatography. In this investigation, polymeric surfactants and ionic liquids were added to a low-conducting buffer solution. The polymeric surfactants used in this study were poly(sodium N-undecylelinic sulfate) and poly(sodium oleyl-l-leucylvalinate). The ionic liquids used in this study were chosen because of their high conductivity, hydrophobicity, and good solvating properties. Thus, it was expected that these ionic liquids would have the ability to assist in the separation of hydrophobic mixtures while maintaining adequate background current. Three analyte mixtures were separated using various buffer combinations of polymeric surfactant and ionic liquids. The ionic liquids were shown to improve the resolution and peak efficiency of the analytes while maintaining adequate background current.     

Prediction of retention in micellar electrokinetic chromatography from solute structure. 1. Sodium dodecyl sulfate micelles.

Like other chromatographic techniques, retention factor, k, in micellar electrokinetic chromatography (MEKC) is directly related to solute partition coefficient and the chromatographic phase ratio as k = Kphi. Unlike conventional chromatography, however, the phase ratio and partition coefficient can be accurately determined in MEKC for a given micellar pseudostationary phase. This means that retention factor in MEKC can be predicted for solutes with known micelle-water partition coefficients without any prior experimentation. In this paper, the use of this simple relationship for prediction of retention behavior in MEKC is examined. The principle of additivity of functional group contribution to partitioning is used to calculate the micelle-water partition coefficient, Kmw, for SDS micellar pseudophase. The micellar substituent constants for 20 functional groups (training set) were determined. Using these substituent constants, the Kmw and retention factors for a group of 80 neutral solutes (test set) were predicted. The linear plot of predicted versus observed log k had an R2 = 0.97 and a slope equal to 1.01. It is shown that the retention times (thus chromatograms) in MEKC can be predicted from the calculated retention factors after only one initial experiment to measure teo and t(mc) under the experimental conditions.     

Systematic Approach to Links between Separations in MEKC and Reversed-Phase HPLC

Retention factors and partition coefficients in micellar electrokinetic chromatography (MEKC) and reversed-phase high-performance liquid chromatography (RP-HPLC) are compared for a series of alkylbenzenes and substituted phenols. In both techniques, separations are based on partitioning between an aqueous phase and an alkyl phase. In MEKC, this was an SDS (C12) micellar pseudostationary phase, and in RP-HPLC an ODS 2 (C18) stationary phase. A nonporous silica (Micra 1.5-μm NPS), which has a low carbon loading, was used rather than a standard porous silica to avoid excessive retention in HPLC and to allow identical mobile phase conditions to be used in both separation modes. The average ratio of analyte retention factors, k(MEKC):k(HPLC), was found to be equal to the ratio β(MEKC):β(HPLC), where β is the phase ratio. This implies that partition coefficients, P, are similar in both MEKC and HPLC, since P = k/β, and that the dominant contribution to stability within each alkyl phase arises from hydrophobic interactions which are common to both separation media. Since partition coefficients are similar in MEKC and HPLC under aqueous buffer conditions, information on retention in one technique may be transferred to the other, provided that the phase ratios are known. In MEKC and HPLC, linear correlations of log octanol-water partition coefficients, K(ow), vs log k for the test compounds were transformed, knowing the phase ratio, to give log P values as a function of log K(ow). This allows quantitative links between MEKC and HPLC to be extended to include octanol-water partitioning. The addition of acetonitrile as an organic modifier over the concentration range 0-20% (v/v) was found to have a greater effect on k in HPLC than in MEKC. This could be a result of a decrease in the MEKC phase ratio due to an increase in the critical micelle concentration.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.     

Predictions of micelle-water partition coefficients and retention in micellar electrokinetic chromatography from solute structure. 2. Fragmental constant approach

The fragmental constant approach (FCA) was used to calculate water-sodium dodecyl sulfate (SDS) micelle partition coefficients, K(mw), for uncharged solutes from their structure. Subsequently, the availability of K(mw) values allows prediction of retention factor, k, in micellar electrokinetic chromatography (MEKC) using the simple relationship k = K(mw)phi, where phi is the phase ratio. The FCA model describes a micelle-water partition coefficient as the sum of the partition coefficients of the constituent atomic/molecular fragments, measured by fragmental constant values, f (i), as well as correction factors to account for various "intramolecular effects" that cause deviations from the predicted partition coefficients as, log K(mw) = sum(n)(i=1)aif i+sum(m)(i=1)kiCm. The fragmental constants for a set of 41 fragments were determined using a training set of 229 aromatic solutes and 198 aliphatic compounds. The K(mw) of the aromatic compounds in the training set were determined by MEKC, while the K(mw) of the aliphatic solutes were estimated using the linear solvation energy relationship (LSER) for the SDS micelles. The fragments consisted of both aromatic fragments (i.e., directly attached to an aromatic ring) and aliphatic fragments. The FCA predictions agree nicely with the observed and LSER partition coefficient values, even for complex molecular structures such as beta-blocker drugs. The results show the great potential of the FCA for a priori prediction of retention behavior in MEKC from solute structure.     

Polymeric sulfated amino acid surfactants: a class of versatile chiral selectors for micellar electrokinetic chromatography (MEKC) and MEKC-MS

In this work, three amino acid-derived (l-leucinol, l-isoleucinol, l-valinol) sulfated chiral surfactants are synthesized and polymerized. These chiral sulfated surfactants are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation, and partial specific volume. For the first time the morphological behavior of polymeric sulfated surfactants is revealed using cryogenic high-resolution electron microscopy. The polysodium N-undecenoyl-l-leucine sulfate shows distinct tubular structure, while polysodium N-undecenoyl-l-valine sulfate also shows tubular morphology but without any distinct order of the tubes. On the other hand, polysodium N-undecenoyl-l-isoleucine sulfate (poly-l-SUCILS) displays random distribution of coiled/curved filaments with heavy association of tightly and loosely bound water. All three polymeric sulfated surfactants are compared for enantioseparation of a broad range of structurally diverse racemic compounds at very acidic, neutral, and basic pH conditions in micellar electrokinetic chromatography (MEKC). A small combinatorial library of 10 structurally related phenylethylamines (PEAs) is investigated for chiral separation under acidic and moderately acidic to neutral pH conditions using an experimental design. In contrast to neutral pH conditions, at acidic pH, significantly enhanced chiral resolution is obtained for class I and class II PEAs due to the compact structure of polymeric sulfated surfactants. It is observed that the presence of a hydroxy group on the benzene ring of PEAs resulted in deterioration of enantioseparation. A sensitive MEKC-mass spectrometry (MS) method is developed for one of the PEAs (e.g., (+/-)-pseudoephedrine) in human urine. Very low limit of detection (LOD) is obtained at pH 2.0 (LOD 325 ng/mL), which is approximately 16 times better compared to pH 8.0 (LOD 5.2 microg/mL). Another broad range of chiral analytes (beta-blockers, phenoxypropionic acid, benzoin derivatives, PTH-amino acids, benzodiazepinones) studied also provided improved chiral separation at low pH compared to high-pH conditions. Among the three polymeric sulfated surfactants, poly-l-SUCILS with two chiral centers on the polymer head group provided overall higher enantioresolution for the investigated acidic, basic, and neutral compounds. This work clearly demonstrates for the first time the superiority of chiral separation and sensitive MS detection at low pH over conventional high-pH chiral separation and detection employing anionic chiral polymeric surfactants in MEKC and MEKC-MS.     

Atmospheric pressure photoionization for enhanced compatibility in on-line micellar electrokinetic chromatography-mass spectrometry

Atmospheric pressure photoionization (APPI) is presented as a novel means for the combination of micellar electrokinetic chromatography (MEKC) and mass spectrometry (MS). The on-line coupling is achieved using an adapted sheath flow interface installed on an orthogonal APPI source. Acetone or toluene is added as dopant to the sheath liquid to enhance analyte photoionization. It is demonstrated that with APPI signal suppression and interferences by the surfactant sodium dodecyl sulfate (SDS) and nonvolatile buffers can be circumvented. This implies that MEKC conditions can be selected independently from MS detection. Moreover, it is shown that both polar and apolar compounds can be photoionized, thereby also facilitating the analysis of compounds that are not amenable to electrospray ionization. Consequently, the MEKC-APPI-MS system can provide effective separation and detection of compounds of diverse character in one run using background electrolytes containing up to 50 mM SDS. Concentration limits of detection derived from extracted-ion traces (full scan mode) of test compounds were approximately 1 microg/mL, and the detection sensitivity remained unaffected during 1 day of continuous use. Overall, the system features are very favorable for applications such as drug impurity profiling as is illustrated by the analysis of mebeverine and related compounds (both charged and neutral) at the 0.25% (w/w) level.     

Electrokinetic chromatography using thermodynamically stable vesicles and mixed micelles formed from oppositely charged surfactants

The electrokinetic chromatography (EKC) of a novel mixed surfactant system consisting of oppositely charged surfactants, sodium dodecyl sulfate (SDS) and n-dodecyltrimethylammonium bromide (DTAB), was investigated. The chromatographic characteristics of large liposome-like spontaneous vesicles and rodlike mixed micelles formed from the mixture were explored and compared with those of SDS micelles. Separations of a series of n-alkylphenones showed that the spontaneous vesicles provided about a 2 times wider elution window than SDS micelles. Both vesicle and mixed micelle systems were found to provide larger methylene selectivity than SDS. The different elution order of a group of nitrotoluene geometric isomers with DTAB/SDS spontaneous vesicles and SDS micelles pseudostationary phases suggested the possibility of different separation mechanisms with these two systems. Comparisons of polar group selectivity, retention, and efficiency were made between vesicles, mixed micelles, and SDS micelles. The correlation between the logarithms of the retention factors (log k') and octanol-water partition coefficients (log P(ow)) for a group of 20 neutral compounds was also studied with DTAB/SDS vesicles. Spontaneous vesicles have great potential as a pseudostationary phase in electrokinetic chromatography.     

Polysodium N-undecanoyl-L-leucylvalinate: a versatile chiral selector for micellar electrokinetic chromatography

Dipeptide micelle polymers are a new class of polymeric surfactants of which the polysodium undecanoyl-L-leucylvalinate (poly-L-SULV) was found to be a broadly applicable chiral selector for micellar electrokinetic chromatography. This negatively charged dipeptide micelle polymer is a high molecular weight compound with large countercurrent mobility, zero critical micelle concentration, low aggregation number, and high solubility in water or water-organic solvents. In an extensive chiral screening program, enantioseparation of 75 racemic compounds was tested with poly-L-SULV as chiral pseudostationary phase in neutral pH and basic pH background electrolytes. A total of 58 out of 75 racemic compounds could be resolved after choosing an appropriate concentration of poly-L-SULV. Although anionic chiral analytes are difficult to resolve using poly-L-SULV, the percent success rate for chiral resolution of cationic (77%) and neutral (85%) racemates was very high. Aspects regarding electrostatic, steric, hydrophobic, and hydrogen-bonding interactions of this dipeptide micelle polymer with various classes of chiral analytes are discussed.     

Separation and detection of explosives on a microchip using micellar electrokinetic chromatography and indirect laser-induced fluorescence

A new approach for sensitive detection on a microfabricated chip is presented. Indirect laser-induced-fluorescence (IDLIF) was used to detect explosive compounds after separation by micellar electrokinetic chromatography (MEKC). The detection setup was used in an epifluorescence configuration with excitation provided by a near-IR diode laser operating at 750 nm. To achieve indirect detection, a low concentration of a dye (5 microM Cy7) was added to the running buffer as a visualizing agent. Using this methodology, a sample containing 14 explosives (EPA 8330 mixture) was examined. Concentrations of 1 ppm of trinitrobenzene (TNB), trinitrotoluene (TNT), dinitrobenzene (DNB), tetryl, and 2,4-dinitrotoluene (2,4-DNT) could be detected with S/N ratios between 3 and 10. Analyses showing 10 peaks, with plate numbers on the order of 60000, were completed within 60 s using a 65 mm long separation channel. The three isomers of nitrotoluene (2-, 3-, and 4-nitrotoluene) were not resolved. Additionally, the two nitramines (HMX and RDX) could only be detected at much higher concentrations, likely due to the low fluorescence quenching efficiencies of these compounds. The analysis method was also used to separate and detect nitroaromatic compounds in extracts from spiked soil samples. The presence of 1 ppm (1 microg of analyte/1 g of soil) of TNB, DNB, TNT, tetryl, 2,4-DNT, 2,6-DNT, 2-NH2-4,6-DNT, and 4-NH2-2,6-DNT could readily be detected. In the interest of increasing the sensitivity of the analysis, various on-chip injection schemes were evaluated. It was found that a 250 microm double-T injector gave a 35% increase in peak signal compared to a straight-cross injector, which is less than expected based on injected volume.     

Liquid-phase microextraction of phenolic compounds combined with on-line preconcentration by field-amplified sample injection at low pH in micellar electrokinetic chromatography.

This paper describes a novel method that applies field-amplified sample injection (FASI) in micellar electrokinetic chromatography (MEKC) with a low pH background electrolyte (BGE). Six phenolic compounds prepared in water or NaOH solution were used as the test analytes. Sample was injected electrokinetically after the introduction of a plug of water. During the injection, the water plug was pumped out of the capillary inlet by the electroosmotic flow, and the phenolic anions migrated very quickly in the direction of the outlet. When the anions reached the boundary between the water plug and BGE, they were neutralized and ceased moving. Thereafter, MEKC was initiated for the separation. This on-line preconcentration method could be conveniently coupled with a liquid-liquid-liquid microextraction procedure, in which a hollow fiber was used as an extraction solvent support to extract the analytes from the water sample. The acceptor phase consisted of 8 mM NaOH. After extraction, the extract was analyzed directly by MEKC, as described.     

Identification of PCR products using PNA amphiphiles in micellar electrokinetic chromatography

We present a method to identify single-stranded PCR products of varying lengths by hybridization of n-alkylated peptide nucleic acids (PNA amphiphiles) to the products, followed by separation with micellar electrokinetic chromatography (MEKC). These end-attached PNA amphiphiles (PNAA) partition to nonionic micelles in the running buffer (Triton X-100), linking the tagged DNA to the micellar drag-tag. This linkage shifts the electrophoretic mobility of a tagged component away from both untagged DNA and tagged DNA of different lengths. The mobility of the tagged DNA is established by its extent of partitioning to the micelle phase as well as its size relative to the attached micelle. A model is presented that can be used to determine the length of an unknown oligomer given an experimentally obtained mobility. We find that the collective action of micelles that transiently attach to the tagged DNA impart about the same hydrodynamic drag as covalently bound "drag-tags" of a similar size. With the use of the PNAA-MEKC method, PCR products of 88, 134, 216, and 447 bases are clearly resolved in less than 5 min. To our knowledge, this work represents the first use of surfactant micelles as drag-tags to separate DNA in capillary electrophoresis. Furthermore, the PNAA tag only attaches to DNA containing a target sequence, helping ensure that only the desired PCR products are analyzed.     

Characterization of chemical selectivity in micellar electrokinetic chromatography. 4. Effect of surfactant headgroup

The influence of surfactant headgroups on migration behavior in micellar electrokinetic chromatography is examined. Using linear solvation energy relationships (LSER) and functional group selectivity studies, the effect of six anionic headgroups on chemical selectivity is characterized. The sodium dodecyl surfactants of the sulfate [SO4-], sulfonate [SO3-], carboxylate [CO2-], carbonyl valine [OC(O)NHCH(CH(CH3)2)CO2-], and sulfoacetate [OC(O)CH2SO3-] anions are investigated. Solute size and the hydrogen-bond-donating ability of the micellar phase play the most significant roles in solute retention in all of the surfactants studied. While solute-micelle hydrogen bonding plays a dominant role in the observed selectivity, the dipolarity and polarizability of the micellar phase also have a small influence. The results also suggest that the hydrogen-bond-accepting ability for surfactants is inversely proportional to the proton acidity (pKa) of its headgroup. The observed hydrogen-bond-donating ability and dipolarity of surfactant systems are believed to be a result of the water that resides near the micelle surface.     

A universal concept for stacking neutral analytes in micellar capillary electrophoresis

Unlike recent studies that have depended on manipulation of separation buffer parameters to facilitate stacking of neutral analytes in micellar capillary electrophoresis (MCE) mode, we have developed a method of stacking based simply on manipulation of the sample matrix. Many solutions for sample stacking in MCE are based on strict control of pH, micelle type, electroosmotic flow (EOF) rate, and separation-mode polarity. However, a universal solution to sample stacking in MCE should allow for free manipulation of separation buffer parameters without substantially affecting separation of analytes. Analogous to sample stacking in capillary zone electrophoresis by invoking field amplification of charged analytes in a low-conductivity sample matrix, the proposed method utilizes a high-conductivity sample matrix to transfer field amplification from the sample zone to the separation buffer. This causes the micellar carrier in the separation buffer to stack before it enters the sample zone. Neutral analytes moving out of the sample zone with EOF are efficiently concentrated at the micelle front. Micelle stacking is induced by simply adding salt to the sample matrix to increase the conductivity 2-3-fold higher than the separation buffer. This solution allows free optimization of separation buffer parameters such as micelle concentration, organic modifiers, and pH, providing a method that may complement virtually any existing MCE protocol without restricting the separation method.     

A fluorescence detection scheme for capillary electrophoresis of N-methylcarbamates with on-column thermal decomposition and derivatization

This paper describes a fluorescence detection method for N-methylcarbamate (NMC) pesticides in micellar electrokinetic chromatography (MEKC) separation. Fulfillment of the fluorescence detection hinged on the discovery that quaternary ammonium surfactants (particularly cetyltrimethylammonium bromide, CTAB), besides serving as hydrophobic pseudophases in MEKC, are also capable of catalyzing the thermal decomposition of NMCs to liberate methylamine. Thus, a multifunctional MEKC medium consisting of borate buffer, CTAB, and derivatizing components (o-phthaldialdehyde/2-mercaptoethanol) was formulated, which allowed first normal MEKC separation, subsequent thermal decomposition, and finally in situ derivatization of NMCs. With careful optimization of the operation conditions, fluorescence detection of 10 NMC compounds was achieved, with column efficiencies typically higher than 50,000 and detection limits better than 0.5 ppm. The present work represents an unprecedented effort in capillary electrophoresis (CE), in which an intact capillary was consecutively utilized as chambers for separation, decomposition, derivatization, and detection, without involving any interfacing features. The success in the implementation of such a detection system resulted in strikingly simple instrumentation as compared with the traditional postcolumn fluorescence determination of NMCs by reversed-phase HPLC. Similar protocols should be workable in the determination of a wide range of pesticides and pharmaceuticals in CE formats.     

Shear effects in a micellar solution near the critical point

This paper is concerned with theological properties of a phase-separating mixture in the presence of macroscopic shear flow. Specifically, we have investigated the shear viscosity of a nonionic micellar solution in shear-induced quasi-stationary states, where a dynamical balance between the growing concentration domains and the suppression by shear is established. During the phase separation, when concentration domains of the two phases with viscosity difference are formed, the measured viscosity of all mixtures as a function of temperature is found to satisfy to a good approximation a novel theological equation proposed by Onuki. In addition, we have determined that the observed characteristic temperature related to the crossover from the shear-affected regime to the shear-unaffected regime depends on the shear rate asS ^p , withp=1:vz≃0.51, wherev andz are universal critical exponents associated with the range and decay rate of the critical fluctuations. This power law depdendence onS. expected for a mixture at the critical composition, appears to be valid also for all off-critical mixtures. Paper presented at the Fourth Asian Thermophysical Properties Conference, September 5–8, 1995, Tokyo, Japan.     

Study of mixed micelles and interaction parameters for polymeric nonionic and normal surfactants

Surface tension (ST) measurements were carried out on various binary mixtures of the "normal" surfactants, such as nonionic surfactant, hexaethylene glycol mono-n-dodecyl ether(C12EO6), and cationic surfactant, tetradecyltrimethylammonium bromide (TTAB), and polymeric copolymer, Pluronic F127, F127(PPO)-g-PVP, and F127(PEO)-g-PVP. In all cases mixed micellar aggregates were formed and critical micellar concentrations of binary mixtures containing different mole fractions of the surfactants were measured using surface tension measurement. In the region where mixed micelles are formed, the interaction of two "normal" surfactants and three "polymeric" nonionic surfactants showed synergistic behavior and the results were analyzed using a interaction parameter, beta, which characterized the interaction in the mixed micelle and introduced by a regular solution theory. The regular solution theory can be applied to describe the interaction between TTAB and C12EO6, and graft polymeric surfactants systems. The results discussed in this paper indicated that regular solution theory has broader extent of application.     

Simultaneous enhancement of separation selectivity and solvent strength in reversed-phase liquid chromatography using micelles in hydro-organic solvents

The role of micelles and organic solvents as the modifiers of the aqueous mobile phase in reversed-phase liquid chromatography (RPLC) in controlling retention and selectivity is discussed. Elution strength increases in RPLC with an increase in organic solvent or micelle concentration. Simultaneous enhancement of separation selectivity with elution strength in the hybrid eluents of water-organic solvent-micelles was observed. This selectivity enhancement occurs systematically, i.e. peak separation increases monotonically with volume fraction of organic solvent added to micellar eluent, and is observed for a large number of ionic and nonionic compounds with different functional groups and for two surfactants (anionic and cationic). For two test mixtures, 13 amino acids/peptides and 15 phenols, it is shown that a better separation and shorter analysis time are observed at stronger hybrid eluents. This selectivity enhancement can be attributed to the competing partitioning equilibria in micellar LC systems and/or to the unique characteristics of micelles to compartmentalize solutes and organic solvents.     

Separation of closely related large peptides by micellar electrokinetic chromatography with organic modifiers.

Large peptides with similar electrophoretic mobilities were separated by micellar electrokinetic chromatography (MEKC) with organic modifiers. [Leu13]motilin and [Met13]motilin differ by only one neutral amino acid residue. Because the electrophoretic mobilities of these peptides are almost identical, these peptides were not separated by capillary zone electrophoresis (CZE). Such large peptides have not been separated by conventional MEKC either, because they interacted strongly with the micelle. However, they were completely separated by MEKC when an organic solvent was added to the micellar solution. Some insulins, larger peptides than motilin, from different origins, which have very similar electrophoretic mobilities, were also successfully separated by the same technique. The size of peptides which were separated without organic modifiers was examined.