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HPLC Analysis of Proteins by Cation Exchange on Proteomix® WCX-NP5: Affect of pH

HPLC Analysis of Proteins by Cation Exchange on Proteomix® WCX-NP5: Affect of pH

Conditions

column Protemix WCX-NP5, 5 cm X 4.6mm I.D., 5 mu particles (Z777193)
column temp. 25 C
mobile phase [A] 20 mM EPPS, pH 8 with potassium hydroxide [B] 20 mM EPPS, 0.3 M potassium chloride, pH 8 with potassium hydroxide
mobile phase [A] 20 mM MES, pH 6 with potassium hydroxide [B] 20 mM MES, 0.3 M potassium chloride, pH 6 with potassium hydroxide; ([A] 10 mM potassium phosphate, dibasic, pH 7 with phosphoric acid [B] 10 mM potassium phosphate dibasic, 0.3 M potassium chloride, pH 7 with phosphoric acid;)
gradient 0 to 100% in 10 min, held at 100% for 5 minutes
flow rate 0.5 ml/min
pressure 1280 psi, 800 psi, and 1500 psi total pressure, respectively (88 bar, 55 bar, and 103 bar total pressure, respectively)
sample ribonuclease a, 0.38; cytochrome c, 0.15; lysozyme, 0.23; a-chymptrypsinogen, 0.23 g/L
injection 5.0 µl
detector UV, 280 nm

Description

Analysis Note Selectivity of proteins by ion exchange of proteins can be affeced by pH. The pH not only impacts the charge of the protein, but depending on column, it may also affect the charge of the stationary phase. As the pH is increased, the net charge of the protein becomes more negative (i.e. less positive). The retention data suggest that peaks 1 & 4 (ribonuclease A and lysozyme, respectively) are not fully deprotonated at pH 8; in contrast, peaks 2 & 3 (a-chymotrypsinogen and cytochrome c, respectively) appear to be fully deprotonated near pH 7 (going to pH 8 does not further reduce retention).
Categories Analytical Chromatography, Proteins
Featured Industry Life Science and Biopharma
Legal Information Proteomix is a registered trademark of Sepax Technologies
suitability application for HPLC

Materials

     
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