In-line coupling of low-pressure short capillary electrochromatography columns to electrospray ionisation mass spectrometry.

A new in-house designed and constructed injection valve for capillary electrochromatography (CEC) based on a rotating injection part with compartments for the eluent as well as for the sample has been coupled to a mass spectrometer via a sheath flow electrospray ionisation (ESI) interface, using short capillary columns of 15 cm length. The CEC columns were packed with 3 microm C(18) bonded silica particles, and a mixture of peptides was analysed using an ammonium acetate/acetonitrile eluent. A significant increase in the signal-to-noise ratio was obtained when the peptides were dissolved in water with the same content of organic modifier as in the eluent with an addition of 0.5% (v/v) acetic acid. When the CEC analysis was performed without any additional pressure, the separation current sometimes dropped tremendously due to bubble formation, caused by different permeability in the first and packed part of the column causing an extremely low electroosmotic flow. The separation current was restored to its original value by applying only 7 bar at the inlet of the CEC column, and the separation performance for the test peptides was recovered. A comparison of the CEC performance of peptides in pure CEC mode and in low-pressure CEC mode is reported.

Separation of basic solutes by reversed-phase capillary electrochromatography.

The separation of basic solutes at low pH by capillary electrochromatography (CEC) has been investigated. The feasibility of separation of basic solutes by CEC was demonstrated. Influence of operational parameters, solvent composition, pH, temperature on retention and selectivity of the separation of a mixture of basic, neutral and acidic drug standards has been investigated. The observed elution behavior has been modeled to account for both chromatographic retention and differential electrophoretic mobility of the solutes. This model was verified experimentally. It is demonstrated in this work that the elution window of solutes in reversed-phase CEC is expanded to range from -1 to infinity.

Towards the column bed stabilization of columns in capillary electroendosmotic chromatography. Immobilization of microparticulate silica columns to a continuous bed.

This article discusses a novel method generating a continuous bed inside the CEC column. The column bed composed of microparticulate reversed-phase silica is completely immobilized by a hydrothermal treatment using water for the immobilization process. This process eliminates the manufacture of frits of both ends of the column and all problems associated with their preparation. Fundamental studies on operational parameters will be presented such as the dependence of the immobilization on the column temperature, the type of stationary phase and the column back pressure. The immobilized CEC columns show the same high column efficiency as packed columns with frits.

Advances in capillary electrochromatography.

This paper presents practical aspects of capillary electrochromatography (CEC). Preparation of capillary columns, including terminating frits and immobilization of the packed bed, is described in detail. Longevity and reproducibility of CEC columns is demonstrated. Instrumental aspects, sample introduction, sensitivity of detection, and gradient elution are discussed in detail. A number of examples that illustrate the versatility of CEC, in particular, for providing an isocratic separation alternative to conventional gradient separation in HPLC, are given.

Recovery of biologically active enzymes after HPLC separation.

The mass and activity recovery of eight different enzymes (two monomeric, six oligomeric) with molecular masses between 25,000 and 240,000 daltons were tested after HPLC separation on three different HPLC instruments (two with stainless steel and one with titanium flow paths). Most of the tested proteins are known to be sensitive to heavy metal ions. Eight wide pore, ion-exchange columns, two size-exclusion columns and two hydrophobic-interaction columns were used. Both stainless steel and glass column hardware were used in all three separation modes. The elution times were between 8 and 12 minutes. In almost all cases, the activity recovery was between 90% and 100% compared with a control sample incubated in the chromatographic elution buffer for the same time at the same temperature. A severe activity loss (about 30%) was observed with only one ion-exchange column and one enzyme. Neither the column hardware nor the material of the HPLC equipment had any negative effect on the activity recovery of the enzymes tested.

Fast separation of biological macromolecules on non-porous, microparticulate columns.

The use of high-performance liquid chromatographic columns for the separation of proteins and nucleic acids is gradually increasing in biochemical laboratories. The efficiency of these columns for such separations has been much lower than that achievable for the separation of smaller molecules. Non-porous microparticulate packings are the logical answer one arrives at after consideration of the chromatographic behaviour of proteins. Non-porous stationary phases are described for the separation of proteins, peptides and nucleic acids. The stationary phases used are TSK-Gel NPR-C18, TSK-Gel NPR-DEAE, TSK-Gel NPR-SP and HYTACH MicroPell C18. A number of fundamental properties of columns based on these sorbents were evaluated, such as permeability, retention behaviour towards small and large molecules, load capacity and stability. Instrumental requirements for these columns are discussed and some applications described.