How to use these columns

Mobile phases Different types of mobile phases can be used. A mobile phase consisting of 5% 2-propanol in 20 mM potassium phosphate buffer pH 7.0 gives data in good agreement with literature data. Table 1 gives recommendations on pH and solvent content in the mobile phases. The mobile phase conditions should be choosen to suit the drugs to be tested, ie. for high protein binding drugs a mobile phase with higher eluting strength might be needed in order to reduce the retention times. Calculation of % protein binding Retention data (k´) is used to calculate the percentage of protein binding. A tm-marker is injected (an unretained compound). The retention factor (k´) for a drug is calculated by: k´= (tr - tm) / tm where tr = the retention time for the drug where tm = the retention time for a non-retained peak The % protein binding (P) is calculated by: P = 100 (k´/ k´+ 1)  Substance Bound in plasma * 100(k´/k´+1)  isoniazid 0 11.8  ethosuximid 0 9.2  primidon 19 31  folinic acid 40 65.9  carbamazepine 74 74.8  diltiazem 78 75.3  desipramine 82 86.8  propranolol 87 78.8  budesonid 88 85.4  indometacine 90 80.2  verapamil 90 74.4  imipramin 90.1 88.9  nortriptyline 92 89.2  sulindac 94 97.9  fluoxetine 94 81.7  amitriptyline 94.8 90.8  propafenone 95 81.4  carvedilol 95 92.6  paroxetine 95 87.3  omeprazol 95 74.2  nitrendipine 98 96.4  nicardipin 98.8 97.5  ketoconazol 99 96.3 * Data obtained from Goodman A. and Gilman A.G., The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill, New York, p.1712-1792 (1996).

Table 1  Conditions   Albumin columns   AGP columns   pH range :  5 - 7  4 - 7  Rec. conc.  2-propanol:  0 - 10%(v/v)  0 - 30%(v/v)*  normally 0 - 10%  Rec. conc.  acetonitrile:  0 - 10%(v/v)  0 - 30%(v/v)  normally 0 - 10% *Higher concentrations give high back pressure Correlation It is recommended to include a set of standard drugs to correlate the chromatographic data against published protein binding data. Literature data for plasma protein binding to use for correlation of binding to HSA can be obtained from Goodman A. and Gilman A.G., The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill, New York, p.1712-1792 (1996). Below is a plot with results obatined from chromatography of standard drugs on a CHIRAL-HSA column. The results are correlated to data from Goodman & Gilman. All the data are shown in the table below. Column: CHIRAL-HSA 50 x 3.0 mm Mobile phase: 6% 2-propanol in 20 mM potassium phosphate buffer pH 7.0 Flow rate: 0.5 ml/min Detection: UV 210 nm Return to main page