1. Purification of immunoglubin by ion exchange chromatography Bahiya Osrah Osrahb@gmail.com

3. Principle Technique or process used in seperation of ions and polar molecules based on their affinity to ion exchanger Separation based on charge (competitive) Aim: purification and separation of protein (charged molecules or ionizable)

4. Prepare anion exchanger column

5. Your colors will be different!

6. Components of ion exchange chromatography 1.Sample 2.Mobile phase (carries the smple onto column ex: Tris HCL buffer or buffer salin solution 3.Stationary phase (agarose or cellulose beads with covalently bonded functional groups (+ve or –ve) depends on the sample to be separated ex: DEAE (cross linked dextran gel) 4.Column 5.Analyte ( individually seperated components)

7. Principle Ion exchange chromatography retains analyte molecules on the column based on ionic interaction The stationary phase surface displays ionic functional groups that interacts with analyte ions with opposite charge The elution occurs by increasing the concentration of similary charged molecules that will displace the analyte ions from the stationary phase (competitive)

8. Types of Ion Exchange chromatography Cation exchange to separate positive charged cations Stationary phase: -ve Elution: increase PH  to make the analyte more negative  elute Increase Salt conc (+ve) to compete with the analyte Anion exchange To separate negative charged anions Stationary phase: +ve Elution: decrease PH  to make the analyte more positive  elute Increase salt conc (-ve) to compete with the analyte

9. Types of Ion Exchange chromatography

10. +ve net charge-ve net charge PH< 7 Acidic PH> 7 Basic Some Proteins net charge with different PH PH effect

11. Salt effect

12. Anion exchange

13. Procedure Step1: preparation of stationary phase 2gm of DEAE sephadex + 70ml water Adjust PH=7 Add 1ml of NAOH + 1ml water  to remove any negative molecules attached to the matrix Add tris HCL buffer Adjust PH=8  to make the stationary phase +ve

14. Procedure Step2: separation of immunoglobulin Sample: serum 1.Add the DEAE to 10ml syringe 2.Add 1 ml of sample to the DEAE 3.Add 12.5 ml of Tris-HCl buffer 4.Collect 5 fractions (2.5 ml for each tubes) 5.Add different concentrations of salt (sodium chloride) from low  high 6.Collect 10 fractions for each concentration

15. Increasing salt concentration Aim: to compete with the –ve charged analyte and spead up the elution 5 tubes 10 tubes  20mmol/l 10 tubes  50mmol/l 10tubes  100 mmol/l 10tubes  150 mmol/l 10 tubes  200mmol/l  take 0.5ml sample  + 0.75 biuret  Incubate for 15 min at 37C  Read Abs at 540nm

16. Anion exchange

18. Results Tube number Abs

19. Readings of ELISA StandardAbsorbance 2000 mg/ml1.99 2501.25 1500.75 350.25 150.14 50.08 sample0.0360.040 +ve high0.430.47 +ve low0.110.17 -ve0.010.05