ABSTRACT
Synthetic oligonucleotides (ODNs) are routinely analyzed using capillary gel electrophoresis (CGE) for size-sieving based separations as well as electrospray mass spectrometry (ESI-MS) for identification. On-line coupling of these methods is therefore desired in order to combine the analytical capabilities provided by both methods. Performance of on-line CGE-ESI-MS systems is influenced by various parameters, and choice of optimal conditions is crucial for successful coupling experiments. In this study, we explore characteristics of the on-line coupled CGE-ESI-MS system for ODN analysis. Effects of CGE buffer concentration, capillary length, separation and orifice voltage on CGE separation and MS detection of a phosphodiester ODN mixture were examined. Attention was paid to the influence of the interface, such as geometry of capillary alignment, sheath liquid flow-rate and sheath liquid composition on performance of the system.
Subject(s)
Electrophoresis, Capillary/methods , Gels , Oligonucleotides/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Acetonitriles/chemistry , Analysis of Variance , Buffers , Electrolytes/chemistry , Electrophoresis, Capillary/instrumentation , Flow Injection Analysis , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/instrumentation , Time FactorsABSTRACT
A comparison of different separation methods (high-performance liquid chromatography (HPLC), capillary HPLC (CHPLC) and pressurized capillary electrochromatography (pCEC)) coupled on-line with mass spectrometry (MS) is undertaken using the separation of a crude extract of ergot fungus (secalis cornuti) as an example. New and simple setups for a two-dimensional CHPLC coupled on-line with electrospray ionization (ESI)-MS (2D-CHPLC-MS) as well as for capillary size-exclusion chromatography performed under pCEC conditions and coupled on-line with ESI-MS (CSEC-pCEC-MS) are shown. In addition, an improved method for column packing is presented.
Subject(s)
Chromatography, Micellar Electrokinetic Capillary/instrumentation , Electrophoresis, Capillary/instrumentation , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/standards , Chromatography, Micellar Electrokinetic Capillary/standards , Claviceps/chemistry , Electrophoresis, Capillary/standards , Electrophoresis, Gel, Two-Dimensional/instrumentation , Electrophoresis, Gel, Two-Dimensional/standards , Equipment Design , Ergotamines/analysis , Ergotamines/isolation & purification , Online Systems , Peptide Fragments/analysis , Peptide Fragments/isolation & purification , Proteins/analysis , Proteins/isolation & purification , Spectrometry, Mass, Electrospray Ionization/instrumentation , Spectrometry, Mass, Electrospray Ionization/standardsABSTRACT
Pressure-supported packed capillary electrochromatography (CEC) and packed capillary high-performance liquid chromatography (pHPLC) have been coupled on-line to electrospray ionization-mass spectrometry (ESI-MS) and coordination ion spray-mass spectrometry (CIS-MS). Separation of enantiomers of barbiturates and chlorinated alkyl phenoxypropanoates were performed on a permethylated beta-cyclodextrin stationary phase by pressure-supported CEC. For on-line detection with ESI- and CIS-MS, a modified sheath-liquid interface was used. CIS-MS is a universal, novel ionization technique which improves the selectivity as well as the sensitivity. Charged complexes were formed through the addition of central complexing ions such as silver(I), cobalt(II), copper(II), and lithium(I) to the sheath flow. Advantages of CIS-MS detection compared to the ESI-MS mode are discussed. In the CIS-MS mode, increased sensitivity and high selectivity was attained through different possibilities of complexation. The superiority of pressure-supported CEC compared to pHPLC in the hyphenation with CIS-MS is demonstrated.
