scholarly journals Three Dimensional Liquid Chromatography Coupling Ion Exchange Chromatography/Hydrophobic Interaction Chromatography/Reverse Phase Chromatography for Effective Protein Separation in Top-Down Proteomics

2015 ◽  
Vol 87 (10) ◽  
pp. 5363-5371 ◽  
Author(s):  
Santosh G. Valeja ◽  
Lichen Xiu ◽  
Zachery R. Gregorich ◽  
Huseyin Guner ◽  
Song Jin ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Hydrophobic Interaction ◽  
Protein Separation ◽  
Three Dimensional ◽  
Hydrophobic Interaction Chromatography ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Top Down ◽  
Reverse Phase

Ion-exchange high-performance liquid-chromatography steps in peptide purifications

1982 ◽  
Vol 2 (10) ◽  
pp. 803-811 ◽  
Author(s):  
Hedvig Von Bahr-Lindstróm ◽  
Ulla Moberg ◽  
Jórgen Sjódahl ◽  
Hans Jórnvall
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Ion Exchange ◽  
High Speed ◽  
High Performance ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Complete Purification ◽  
Ion Exchange Hplc

Ion-exchange high-performance liquid chromatography (HPLC; on Ultropac TSK DEAE and CM) is compared with conventional soft-gel ion-exchange chromatography in identical peptide purifications. The results show that separating properties are similar, but as expected, ion-exchange HPLC has a much higher resolving capacity and a higher sensitivity, and allows a considerably shorter total separation time. The same buffer systems as for conventional ion-exchange chromatography can be used, including urea to solubilize large peptides, if care is taken not to exceed the pH limits set by the column matrix. A complete purification scheme by HPLC in the nanomolar range, utilizing exclusion, ion-exchange, and reverse-phase chromatographies, is given with a complex peptide mixture from a digest of a large protein. Similar steps as in conventional soft-gel schemes can be utilized. It is concluded that ion-exchange HPLC is a suitable complement to commonly used reverse-phase HPLC steps and that it permits high speed and sensitivity over wide ranges of peptide sizes and amounts.

Quantitative Amino Acid Analysis of Feedstuff Hydrolysates by Reverse Phase Liquid Chromatography and Conventional Ion-Exchange Chromatography

1984 ◽  
Vol 67 (5) ◽  
pp. 1024-1026
Author(s):  
Robert G Elkin
Keyword(s):  
Liquid Chromatography ◽  
Amino Acid ◽  
Ion Exchange ◽  
Soybean Protein ◽  
Amino Acid Content ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Reverse Phase Liquid Chromatography ◽  
Phase Liquid

Abstract Corn, soybean meal, and isolated soybean protein samples were acidhydrolyzed and analyzed for amino acid content by reverse phase liquid chromatography (LC) and by conventional ion-exchange chromatography (IEC) using an amino acid analyzer. The former method employed pre-column derivatization with orthophthalaldehyde (OPTA)/ethanethiol and fluorescence detection. In the LC procedure, glycine and threonine were not resolved, and proline and cyst(e)ine were not detected. In general, amino acid values obtained by LC and IEC compared closely within and across feedstuffs, and both agreed well with published amino acid composition data. The notable exceptions were aspartic acid, glutamic acid, and alanine. Results of this study suggest that reverse phase LC with pre-column OPTA derivatization can be applied to accurately measure primary amino acids in individual feedstuffs.

Review on the Application of Mixed-mode Chromatography for Separation of Structure Isoforms

2018 ◽  
Vol 20 (1) ◽  
pp. 56-60 ◽  
Author(s):  
Tsutomu Arakawa
Keyword(s):  
Ion Exchange ◽  
Mixed Mode ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Charged State ◽  
Partial Structure ◽  
Reverse Phase ◽  
Oligomer Formation ◽  
Structure Rearrangement ◽  
Disulfide Scrambling

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.

Rapid separation of bovine caseins by mass ion exchange chromatography

1989 ◽  
Vol 56 (3) ◽  
pp. 391-397 ◽  
Author(s):  
K. F. Ng-Kwai-Hang ◽  
J. P. Pélissier
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Rapid Separation ◽  
Rapid Isolation ◽  
Time Required

SummaryThe rapid isolation of major bovine caseins in gram quantities was investigated. Whole casein was precipitated from individual cow's milk by adjusting the pH to 4·6 and the precipitated casein was suspended in 4·5 M urea (pH 8·0) containing 0·02 M imidazole and 0·03 M β-mercaptoethanol, and bound on a QAE Zeta Prep 250 cartridge. Stepwise elution with the urea/imidazole β-mercaptoethanol buffer and varying amounts of NaCl gave five well resolved peaks, which were identified by polyacrylamide gel electrophoresis and fast protein liquid chromatography to be pure γ-casein, κ-casein. β-casein, β-casein and αs-casein, respectively. The ion exchange cartridge was regenerated by flushing with buffer containing 0·50 Μ-NaCl followed by equilibration with starting buffer before separation of next sample. The time required to run each sample including cartridge regeneration and equilibration was 4 hours.

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