Electrophoretic Systems Separation Strategies

To optimize the separation of peptides, the experimental conditions have to be adjusted to emphasize differences in the charge-to-mass ratios of the analytes.

Apart from external parameters like electrical field, capillary dimensions (length, inner diameter) and temperature, separation is mostly influenced by the electrolyte. Intrinsic variables like type of buffer, mobility, ionic strength, pH and buffer additives determine electrophoretic and electroosmotic mobility.

In the first place selectivity in the analysis of pep-tides is controlled by pH. Altering the acidity of the separation medium affects both the charge of the peptide and the ionization of the capillary wall, resulting in the change in EOF.

The hysteresis-like course of the EOF shows the greatest variation in the pH range of approximately 5-7, i.e. near the dissociation constant of the silanol groups. For pH values below 3 or greater than 9, the influence of the superimposed EOF can be neglected and the migration of the peptide is almost independent from the EOF.

In acidic media (pH & 2) both basic and acidic residues of the peptide are protonated. Selectivity is attributed to the number of positive-charged ammonium groups in the chain resulting in different charge densities. Analytes migrate with the EOF. In high pH buffers (pH & 10), deprotonation of terminal and side chain ammonium groups (His) induces negatively charged species (presence of carboxylate groups) which migrate in the opposite direction to the EOF. At higher pH values the side chain amino groups of arg and lys are the only ones affected.

Optimization of pH values below 2 and above 12 is difficult to achieve since the limiting values of mobility are reached. Furthermore, due to the high conductivities of protons and hydroxyl ions, high currents accompanied by Joule heating are generated. For practical purposes selectivity control for peptides with a majority of acidic moieties is mainly achieved in the range of pH 3-6 while basic residues are mostly affected at pHs around 10.

Additionally, isoelectric points of the peptides have to be included in the optimization strategy.

If peptides are obtained by chemical or enzymatic digests of proteins the cleaving agent has to be considered, e.g. trypsin cuts at the C-terminal side of lys and arg respectively. Thus fragments contain an excess of acidic residues. Selectivity can be easily affected in acidic media. Cleavage at aromatic or aliphatic side chains is performed with chymotrypsin or pepsin, yielding fragments with both acidic and basic residues and optimization can be extended to the full pH range (Figure 6).

Frequently used electrolytes for peptide mapping are phosphate, citrate and acetate as acidic buffers while borate or TRIS/Tricine are mainly applied under basic conditions. Phosphate and citrate are buffers that can be used over a broad pH range due to their multiple association constants. Borate exhibits very low conductivity compared to phosphate and other buffers. Buffer concentrations in the range of 10 mmol L"1 to approximately 100 mmol L"1 can be used. The electrolytes used should not possess any UV absorbance at low wavelengths.

An increase in ionic strength generates sharper peaks (zone focusing) due to the drop of the electrical field at the sample-electrolyte boundary and sample loading capacity can be increased. High ionic strengths induce high electrical currents and the in crease of Joule heating can give rise to band broadening.

Dispersive effects caused by the interaction with the capillary wall are usually not a problem with peptides but larger species can exhibit characteristics similar to proteins in that they tend to adsorb at the capillary wall.

High ionic strength, extreme pH values and buffer additives competing in adsorption with the peptides are strategies of optimization which can be adapted from protein analysis. At extreme pH values, peptides and the capillary wall are equally charged so electrostatic repulsion diminishes adsorption. Coated capillaries have been used to suppress this phenomenon.

High salt content in the sample may destroy the separation efficiency of the electrophoretic system so sample preparation steps must remove the high ionic strength in the sample.

Enhancement in selectivity can be attained if an additional equilibrium is superimposed on to the elec-trophoretic process. Mostly the additives used for this are complexing agents which interact with specific groups of the peptide.

As for amino acids, metal ions can be employed for the separation of peptides and histidine-containing peptides especially interact with zinc salts. Separation of two histidine dipeptides (l-l, d-l ) can be attributed to favourable steric arrangement of the histidine residues in one isomer.

Cyclodextrins form dynamic inclusion complexes with hydrophobic parts of the peptide, e.g. with amino acid residues containing aromatic rings like phenylalanine. The mass of a complexed analyte is

15 20

Time (min)

Figure 6 Tryptic digest of a haemoglobin variant separated by CZE. Capillary: poly(vinyl alcohol) coated fused silica capillary 50 ^m i.d., 50/57 cm, buffer: phosphate 50 mmol L_1; pH = 2.5; E = 526 V cmT1, 214 nm; injection 0.5 psi, 5 s.

increased in this way and lower charge-to-mass ratio results in decreased mobility.

Ion-pairing reagents like short chain alkylsulfonic acids are particularly applied to adjust selectivity for hydrophobic peptides. Concentrations below the critical micellar concentration are used. The mechanism is based on the interaction between the hydro-phobic surface of the peptides and the hydrophobic alkyl chain. Depending on the hydrophobicity of a peptide, different amounts of alkylsulfonic acid are attracted. Charge-to-mass ratios of the individual peptides are influenced to a different extent leading to the separation of the species.

A second approach to impart selectivity to large peptides with identical mobilities but different hydro-phobicities is the use of ion-pairing reagents above their critical micellar concentration (CMC). This technique may also be used for peptides differing in neutral amino acids such as ala, val, leu or ile. MEKC takes advantage of the partitioning of the peptides between the electrolyte and the pseudo-stationary phase of the micelles. Hydrophilic moieties of the peptide interact with the outer polar sections of the micelle whereas hydrophobic parts are situated in the inner hydrophobic sphere. These peptide-micelle aggregates possess a different mobility compared to the electrophoretic mobility of the peptide in free solution.

Types of surfactants employed are divided into anionic, cationic and nonionic micelle-forming reagents. Because of the different charges, different migration directions are obtained. Negatively charged SDS, one of the most frequently used additives, migrates counter to the EOF and is used in concentrations up to approximately 150 mmol L_1.

Common positively charged reagents are cetyl, dodecyl and hexadecyltrimethylammonium salts. These reagents invert the EOF at concentrations below the CMC so that as a consequence the polarity of the applied electrical field has to be reversed.

The addition of organic solvents such as methanol, ethanol, acetonitrile or tetrahydrofuran can provide selectivity for closely migrating peptides. These changes can be mainly attributed to solvation of side chains and variations in dissociation of the functional groups of the peptide. Additionally the EOF is modified due to altering the £- potential and the increase in buffer viscosity which generates a lower EOF and lower currents. In this way separations have been established for peptides differing in only a single neutral amino acid.

Peptides, especially large peptides with protein-like characteristics, sometimes tend to adsorb at the capillary wall. Beside the possibilities for avoiding dispersive effects mentioned above, the addition of amino-

or diamino compounds like diamino-pentane, butane or morpholine can diminish the peptide-wall interaction. Competing equilibria in the electrostatic attraction between analyte-silanol and amine-silanol groups suppress the adsorption of the peptide. Another approach to reduce adsorption is derivatization of the silanol groups with an uncharged polymer (coated capillaries).

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