HPLC Analysis of mAb 016 on Proteomix® SCX-NP5 Column at Different Flow Rates

HPLC Analysis of mAb 016 on Proteomix® SCX-NP5 Column at Different Flow Rates

Conditions

column Proteomix SCX-NP5, 25 cm x 4.6mm I.D., 5 µm particles, PEEK (Z777164)
mobile phase (A) 20 mM phosphate buffer, pH 7.5 and (B) A + 1 M NaCl
flow rate 1.5 ml/min
flow rate 0.8 ml/min
sample mAb 016 (11.8 mg/mL) 4 µl
detector UV, 280 nm

Description

Analysis Note This figure shows the how flow rate (F) affects analysis time and resolution during gradient analysis. When comparing the performance of different column formats or flow rates or gradient times, the rule of thumb is to keep the gradient volume, i.e. the product of gradient time and flow rate, constant. When gradient volume is kept constant, the compounds elute from the column in the same eluent composition.
Note that in this example the gradient time (tg) was reduced by the same factor as the increase in flow rate, thereby proportionaly increasing the gradient steepness, since gradient steepness is equal to the change in composition per unit time; in this case 6%B/16 vs. 6%B/30. It is now easy to see that the gradient volume was kept constant, since 30 min x 0.8 ml/min = 15 min x 1.6 ml/min = 24 mL. Clearly, the separation deteriorated substantially at the higher flow rate.
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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