Improved reversed-phase high performance liquid chromatography columns for biopharmaceutical analysis

Ricker, Robert D.; Sandoval, L.A.; Permar, Bernard J.; Boyes, Barry E.

doi:10.1016/0731-7085(95)01627-9

Cited by 11 publications

(4 citation statements)

References 6 publications

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“…and instrument (LTQ) has probably been reached. To overcome such limitations, one could employ any or all of the following: longer microcapillary column, longer run time and/or altered gradient, , different solid-phase material, or high mass accuracy instrumentation …”

Section: Resultsmentioning

confidence: 99%

Shotgun Analysis of Integral Membrane Proteins Facilitated by Elevated Temperature

Speers

Blackler

2007

Anal. Chem.

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The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30-80 degrees C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized. This report demonstrates that microLC analysis of membrane proteomic samples significantly benefits from the application of heat. Enriched membrane and membrane-embedded peptides (the latter obtained by membrane shaving) were analyzed by microLC-tandem mass spectrometry (MS/MS) from 20 to 60 degrees C using a standard reversed-phase material. Maximal protein and hydrophobic peptide recovery was obtained at 60 degrees C. The membrane-shaving method employed, a recently optimized version of the high pH/proteinase K protocol, provided significant integral membrane protein enrichment: 98% of identified proteins were predicted to have at least one transmembrane domain (87% to have at least three), and 68% of peptides were predicted to contain transmembrane segments. Analysis of this highly enriched sample at elevated temperature increased protein identifications by 400%, and peptide identifications by 500%, as compared to room-temperature separation. Given that most microLC-MS/MS analyses are currently conducted at room temperature, the findings described herein should be of considerable value for improving the comprehensive study of integral membrane proteins.

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Section: Resultsmentioning

confidence: 99%

Shotgun Analysis of Integral Membrane Proteins Facilitated by Elevated Temperature

Speers

Blackler

2007

Anal. Chem.

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“…To assay all analytes, the mass spectrometer was operated at unit mass resolution and set to selectively monitor the MRM transitions for the [ All of the chromatography experiments described herein were performed on a 2.16100 mm Agilent Zorbax 1.8 l SB-C18 rapid resolution LC-MS column. The Zorbax column was selected for this application because of its excellent chemical and thermal stability [34]. With the exception of the temperature experiment, the flow rates for all other method development experiments were 0.75 mL/min at a column set temperature of 1008C.…”

Section: Lc-ms/msmentioning

confidence: 99%

Stereoselective quantitation of a serine protease inhibitor using LC‐MS/MS at elevated column temperature

Shen¹,

Merka²,

Dreyer³

et al. 2008

J of Separation Science

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A LC-MS/MS method using a LC column packed with sub-2 micron particles and elevated column temperatures was validated for the quantitation of SCH 503034 diastereomers (SCH 534128 and SCH 534129) in human plasma. The method was validated over the concentration range of 2.5 to 1250 ng/mL. Inter-assay precision, based on percent relative deviation for n = 18 replicate quality controls, was 4.5% for SCH 534128 and 4.9% for SCH 534129. Inter-assay accuracy based on n = 18 replicate quality controls was +/- 7.8% for both SCH 534128 and SCH 534129. The method involved the novel application of ion pairing reagents to increase the stereoselectivity of the separation. Temperature, types of ion pairing reagent, and concentration of ion pairing reagent were all found to play significant roles in the resolution of the SCH 534128 and SCH 534129 diastereomers on a LC column packed with sub-2 micron particles. Specifically, a sensitivity increase of five-fold was demonstrated by increasing the column temperature. Without sacrificing resolution, the run time was significantly shortened when the column temperature was elevated to 100 degrees C.

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“…Development in stationary phases has provided effective means to improve protein separation . Silica supports with different chemically bonded phases, ethyl (C 2 ), butyl (C 4 ), octyl (C 8 ), octadecyl (C 18 ), or cyanopropyl are all applicable . Short alkyl chain bonded phases (C 2 , C 4 , C 8 ) are more often used than long chain phases (C 18 ), on account of undesirable peak tailing or lower recovery caused by the latter .…”

Section: Introductionmentioning

confidence: 99%

Evaluation and comparison of n‐alkyl chain and polar ligand bonded stationary phases for protein separation in reversed‐phase liquid chromatography

Ding

Guo

Xiao

et al. 2014

J of Separation Science

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Protein retention is very sensitive to the change of solvent composition in reversed-phase liquid chromatography for so called "on-off" mechanism, leading to difficulty in mobile phase optimization. In this study, a novel 3-chloropropyl trichlorosilane ligand bonded column was prepared for protein separation. The differences in retention characteristics between the 3-chloropropyl trichlorosilane ligand bonded column and n-alkyl chain modified (C2, C4, C8) stationary phases were elucidated by the retention equation l nk=a+cC(B). Retention parameters (a and c) of nine standard proteins with different molecular weights were calculated by using homemade software. Results showed that retention times of nine proteins were similar on four columns, but the 3-chloropropyl trichlorosilane ligand bonded column obtained the lowest retention parameter values of larger proteins. It meant that their retention behavior affected by acetonitrile concentration would be different due to lower |c| values. More specifically, protein elution windows were broader, and retentions were less sensitive to the change of acetonitrile concentration on the 3-chloropropyl trichlorosilane ligand bonded column than that on other columns. Meanwhile, the 3-chloropropyl trichlorosilane ligand bonded column displayed distinctive selectivity for some proteins. Our results indicated that stationary phase with polar ligand provided potential solutions to the "on-off" problem and optimization in protein separation.

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Improved reversed-phase high performance liquid chromatography columns for biopharmaceutical analysis

Cited by 11 publications

References 6 publications

Shotgun Analysis of Integral Membrane Proteins Facilitated by Elevated Temperature

Shotgun Analysis of Integral Membrane Proteins Facilitated by Elevated Temperature

Stereoselective quantitation of a serine protease inhibitor using LC‐MS/MS at elevated column temperature

Evaluation and comparison of n‐alkyl chain and polar ligand bonded stationary phases for protein separation in reversed‐phase liquid chromatography

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