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Column Chromatography

Column Chromatography. External Chromatogram. t RA. t RB. I. t R ' = t RA - t M. t R2 ' = t RB - t M. time. t M. A. B. System Suitability Parameters. Retention Time t R - Time from the injection to the emergence of the peak maximum,

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Column Chromatography

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  1. Column Chromatography

  2. External Chromatogram tRA tRB I tR'=tRA- tM tR2'= tRB- tM time tM A B System Suitability Parameters

  3. Retention Time tR - Time from the injection to the emergence of the peak maximum, - Used qualitatively for identification of compounds. (notice that as the solute is more retained the larger will be the tR value). Retention time of unretained substance (mobile phase) tM Time for unretained substance (mobile phase) to pass through the column Adjusted Retention Time tR’ tR’ = tR - tM Capacity FactorK’ Describes the migration rates of solutes through the column. K’ = tR - tM tM

  4. Selectivity factor ( should be more than one) - The larger the  the more selective the stationary phase and the more effective the separation. For a column separating two components A and B K´B (tR’)B (tR)B – tm  = ________ = ______________ = ______________ K´A (tR’)A (tR)A – tm Where compound B more retained than A. • Column resolutionRs(Rs should be at least 1 and 1.5 for complete separation) • 2 [tRB – tRA] • = ______________________ • WB + WA • Where compound B more retained than A. • The greater the separation of the zones from each other and/or the smaller the zone width the larger will be the resolution.

  5. Column Efficiency Column efficiency is quantitatively determined by calculating the number of theoretical plates N or H (HETP) • Number of theoretical plates N • N =16 tR2 / W2 • So as the retention time increases and / or the zone width decreases the more efficient will be the column . • Height Equivalent to Theoretical Plates (HETP) H • H = L / N • L is the column length

  6. Mobile Phase Composition Acetonitrile : Water 100 : 0 Acetonitrile : Water 80 : 20 Acetonitrile : Water 70 : 30 Acetonitrile : Water 50 : 50

  7. Mobile Phase Flow Rate 1 mL/min 2 mL/min 4 mL/min 8 mL/min

  8. Effect of concentration 10 ug/mL 20 ug/mL 30 ug/mL 40 ug/mL 50 ug/mL

  9. Elution Isocratic elution RS tR Gradient elution

  10. Column Length 250 mm Column Longer tR but Better Rs due to higher N 150 mm Column

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