The separation and detection of the major protein-protein and protein-metal complexes of erythrocytes directly from cell lysate under native conditions has been accomplished for the first time using capillary electrophoresis electrospray ionization-mass spectrometry (CE/ESI-MS). All three major protein-protein and protein-metal complexes in human red blood cells (RBCs) with a concentration dynamic range of approximately 3 orders of magnitude were successfully detected. Intact complexes detected in lysed RBCs included carbonic anhydrase II (CAII-Zn at approximately 0.8 amol/cell) complexed with its zinc cofactor, carbonic anhydrase I (CAI-Zn at approximately 7 amol/cell) complexed with its zinc cofactor, and hemoglobin A (Hb-tetramer at approximately 450 amol/cell)a tetramer formed by two alpha-beta-subunits and four heme groups. The average molecular weights measured for these complexes were consistent with their theoretical values within 0.01% mass accuracy. The use of Polybrene as a self-coating reagent in conjunction with ammonium acetate at pH approximately 7.4, narrow capillary for high separation efficiency, and forward polarity CE to avoid acid production at the tip of the capillary were overriding experimental factors for successful analysis of protein complexes. Diluting the lysed blood sample in ammonium acetate for a minimum of 6 h before injecting the sample into the CE was essential for obtaining the mass accuracy consistent with their theoretical average molecular weights. At physiological pH, the mass spectrum of the electrophoretic peak of Hb-tetramer included a small amount of the monomers and Hb-dimer. The migration time and peak profile of these species were almost identical to that of the tetramer, indicating that they are formed from decomposition of the Hb-tetramer during the ESI process. A separate electrophoretic peak for the Hb-dimer was only detected when the pH of the BGE was lowered from 7.4 to approximately 6.6. Running CE in forward polarity mode was essential for detection of the intact Hb-tetramer as well as CAI-Zn and CAII-Zn complexes. Under forward polarity mode, CE outlet/ESI shared electrode acts as the cathode of the CE circuit and the anode (positive voltage for positive ions) of the ESI circuit, thereby maintaining approximately neutral pH at the CE outlet/ESI electrode. In addition, under forward polarity mode, CAII-Zn and CAI-Zn migrated ahead of Hb-tetramer, avoiding being masked by 562x and 64x, respectively, molar excess of Hb-tetramer.

Capillary electrophoresis has been applied to quantitate nucleotide degradation in fish tissues, to provide a basis for determining the K value, an indicator of fish freshness. The three major compounds, inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx) were distinctively separated at 416 V/cm applied potential, 100 mM CAPS buffer, pH 11. There was a good correlation between the peak area and the nucleotide concentration. By using a short distance (22 cm) from the sample entrance to the detector, the identification and determination of these compounds in each sample were completed within 15 min. The results obtained correlated very well with those obtained by enzymatic assays. The capillary was completely regenerated with 1 N NaOH, to dissociate all bound materials from the capillary wall, mainly cations in the fish extract. This provided the same silica surface for repeated runs, resulting in reproducible electropherograms.

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Cyclodextrin-modified capillary electrophoresis has been developed for separation and analysis of benzene and its derivatives. The procedure used a mixture of negatively charged sulfobutyl ether-beta-cyclodextrin (SB beta CD) and neutral hydroxypropyl-beta-cyclodextrin (HP beta CD) to effect differential distribution (partitioning) of the aromatic hydrocarbons between the buffer and CD phases. In 80 mM phosphate buffer, containing 15 mM SB beta CD and 5 mM HP beta CD, benzene, toluene, ethyl benzene and three xylene isomers (BTEXs) were well resolved with a number of theoretical plates well above 100,000, for 50 cm of effective length. Some halogenated benzenes were also observed to separate well from the BTEX components to indicate their suitability as an internal standard for BTEX analyses. Equilibrium complexation models were used for investigating the effect of the cyclodextrin(s) at different concentrations and temperature on the electrophoretic mobility.