Effect of temperature, wavelength, pH, Ion pair reagents and organic modifiers' concentration on the elution of cystatin C. Stability of mobile phase

Abstract

Robustness of an analytical chromatographic method for separation of cystatin c hasbeen verified. Changes in many parameters were carried out, such as, wavelength,column oven, mobile phase composition, chromatographic column.Imperative changes have altered the efficiency of the chromatographic separation;such changes include pH alteration of the mobile phase as well as alkyl sulfonatemolarity changing.All robustness conditions showed no major effect on the chromatographic separationof the analyte except with the changes related to TFA and alkyl sulfonate ion pairreagents. Peak area RSD, asymmetry and No. of theoretical plates were < 0.7%, < 1.2 and > 10,000, respectively. Results obtained using mobile phase after 6 months of storagehave proven its stability and possibility of use. Gradient elution mode was utilized toelute cystatin c with a UV detection of 224 nm. Ace and Waters C8 (150 x 4.6 mm i.d., 5μm) as chromatographic columns were used. © 2011 Nova Science Publishers, Inc. All rights reserved.