Abstract
Robustness of an analytical chromatographic method for separation of cystatin c has been veri fi ed. Changes in many parameters were carried out, such as, wavelength, column oven, mobile phase composition, chromatographic column. Imperative changes have altered the ef fi ciency of the chromatographic separation; such changes include pH alteration of the mobile phase as well as alkyl sulfonate molarity changing. All robustness conditions showed no major effect on the chromatographic separation of the analyte except with the changes related to TFA and alkyl sulfonate ion pair reagents. 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 storage have proven its stability and possibility of use. Gradient elution mode was utilized to elute cystatin c with a UV detection of 224 nm. Ace and Waters C8 (150 x 4.6 mm i.d., 5 μ m) as chromatographic columns were used.
Highlights
Cystatin C (CC) is composed of 120 amino acid residues with a molecular weight of about 14 KDa and two intra-chain disulfide bonds
CC has a leader hydrophobic sequence which enhances the binding with the hydrophobic surface of the stationary phase. 1-hexane sulfonic acid sodium salt reagent was used to bind with the stationary phase instead of the analyte [5]
Close retention time of CC peak, RSD < 0.7%, No of theoretical plates was > 10,000 and the asymmetry factor was < 1.2, all prove the stability of mobile phase after six months of room temperature storage. (Table 2)
Summary
Cystatin C (CC) is composed of 120 amino acid residues with a molecular weight of about 14 KDa and two intra-chain disulfide bonds. Ion pair liquid chromatography technique (Cation analysis) has been used for separation and quantification of CC, incorporating trifluoroacetic acid (TFA) and 1-hexane sulfonic acid sodium salt in the mobile phase. Two ion pair reagents were included in the mobile phase.
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