“…[1][2][3][4][5][6][7][8][9] Recently, with the advent of proteomics and the use of mass spectrometry (MS) for peptide and protein identification, HPLC, a liquid separation technique that is easily coupled online to a MS, is playing a central role in the fractionation and separation of complex protein mixtures and digests. HPLC coupled to a MS allows, in addition to detection, accurate mass measurement that cannot be achieved by HPLC with UV detection.…”
This manuscript examines the effects of different experimental parameters on resolution, peak symmetry, peak width, and selectivity ( peak elution order) of peptides by micro high performance liquid chromatography. The experimental parameters are: mobile phase composition, flow rate, organic modifiers, ion-pairing agents, column temperature, the effect of packing material properties: particle size, particle porosity and reversedphase (RP) alkyl chain length, and column dimensions. When a mass spectrometer (MS) is used as the detector in micro-HPLC, certain experimental parameters such as mobile phase flow rate and buffer composition have to be adjusted in order to meet the requirements of the MS procedure employed. When electrospray ionization (ESI) is the selected MS mode of operation the mobile phase flow rate should be in * Correspondence: Haleem J. Issaq, nL min À1 and a volatile buffer should be used to achieve maximum sensitivity. Also, it was found that the elution order (selectivity) of peptides in RP-HPLC is affected by type and concentration of ion-pairing agent, organic modifier, column temperature, pH of the buffer, and the alkyl chain length of the derivatizing agent in RP. This manuscript includes work that has been done in our laboratory and is supplemented by data published by other researchers.
“…[1][2][3][4][5][6][7][8][9] Recently, with the advent of proteomics and the use of mass spectrometry (MS) for peptide and protein identification, HPLC, a liquid separation technique that is easily coupled online to a MS, is playing a central role in the fractionation and separation of complex protein mixtures and digests. HPLC coupled to a MS allows, in addition to detection, accurate mass measurement that cannot be achieved by HPLC with UV detection.…”
This manuscript examines the effects of different experimental parameters on resolution, peak symmetry, peak width, and selectivity ( peak elution order) of peptides by micro high performance liquid chromatography. The experimental parameters are: mobile phase composition, flow rate, organic modifiers, ion-pairing agents, column temperature, the effect of packing material properties: particle size, particle porosity and reversedphase (RP) alkyl chain length, and column dimensions. When a mass spectrometer (MS) is used as the detector in micro-HPLC, certain experimental parameters such as mobile phase flow rate and buffer composition have to be adjusted in order to meet the requirements of the MS procedure employed. When electrospray ionization (ESI) is the selected MS mode of operation the mobile phase flow rate should be in * Correspondence: Haleem J. Issaq, nL min À1 and a volatile buffer should be used to achieve maximum sensitivity. Also, it was found that the elution order (selectivity) of peptides in RP-HPLC is affected by type and concentration of ion-pairing agent, organic modifier, column temperature, pH of the buffer, and the alkyl chain length of the derivatizing agent in RP. This manuscript includes work that has been done in our laboratory and is supplemented by data published by other researchers.
“…The microcolumn HPLC technique makes it possible to prepare very high efficiency columns with, for example, 200,000 [7,8] or a million plates [9].The preparation of such a high efficiencycolumn with Hypersil ODs, etc. and its application to the separation of fulvic acid is now being studied in our laboratory and will be presented in a separate paper.…”
It must also be mentioned that when ammonium formate solution is used for bopindolol, no partial separation of any kind is observed. The separation factor for the pindolol derivative (a = 1.46) is approximately the same as that found by Hermansson for P-oxazolidone derivatives (a = 1.65) with an al-acid glycoprotein column.
SummaryIt has been shown that the Resolvosil column introduced by Allenmark is suitable forthe separation ofamino alcohols. Pindolol has been separated for the first time with this column. The separation of bopindolol has been reported forthe firsttime. Once more it has been pointed out that combined LClMS is a powerful tool for developing enantiomeric separation methods.
“…For example, fast separations are performed on columns typically 10-20 cm long (32), whereas high-resolution separations of complex mixtures will require column lengths of up to a few meters (26,27,(33)(34)(35). Some details of the preparation procedure have beenreported by many workers (14,26,33), but they vary considerably among laboratories.…”
Abstract. The packed capillary column is one type of microcolumn. It is used increasingly in liquid chromatography, supercritical fluid chromatography, and multidimensional chromatography. In order to attain high efficiency with packed capillary columns, reliable column preparation methods must be used. The development of the technique is summarized.
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