Hplc parameters, factors affecting resolution

HPLC Parameters

1. HPLC Parameters, Factors affecting Resolution By dhineshkumar v Im.Pharm (pharmacology) PSG COLLEGE OF PHARMACY 1

2. Contents Chromatogram Chromatographic parameters for HPLC Factors affecting resolution PSG COLLEGE OF PHARMACY 2

3. Objectives At the end of this presentation, listeners will be able to Identify the components of chromatogram List and explain the chromatographic parameters with respect to HPLC Explain the factors affecting resolution PSG COLLEGE OF PHARMACY 3

4. Chromatogram PSG COLLEGE OF PHARMACY 4 A chromatogram is a pictorial record of the detector response 

5. Chromatographic parameters Retention time Adjusted Retention time Retention volume Retention or factor Selectivity factor Theoretical plate Column efficiency Asymmetry peaks Resolution PSG COLLEGE OF PHARMACY 5

6. Retention time Difference in time between the sample injection and appearance of peak maxima. The retention time is given by the symbol t R Different compounds have different retention times. Retention time may vary depending on: Pressure used Nature of the stationary phase composition of the mobile phase Temperature of the column  PSG COLLEGE OF PHARMACY 6

7. Adjusted retention time It is a measurement between retention time( and unretained time (   PSG COLLEGE OF PHARMACY 7

8. Adjusted retention time Example: Retention time of a compound ( = 5 min Unretained time ( )= 1 min Then, the adjusted retention time( ) will be = 4 min jump   PSG COLLEGE OF PHARMACY 8

9. Retention volume The volume of mobile phase that passed through the column from the point of injection to the detector is called as retention volume.   PSG COLLEGE OF PHARMACY 9

10. Retention factor It is defined as the ratio of adjusted retention time ( and unretained time ( Example: Adjusted retention time ( = 8 min unretained time ( = 2 min Then, Retention factor (K) Higher the K value, greater the resolution .   PSG COLLEGE OF PHARMACY 10

11. Retention factor PSG COLLEGE OF PHARMACY 11 K value 1-10. Good separation <1.0 Poorly retained >10 Too long to separate and broaden the peaks

12. Selectivity factor (Separation factor) Separation factor is defined as the ratio of the partition coefficient of two components to be separated OR ratio of adjusted retention time of two components K B , K A = partition coefficient or distribution constant of substance B and A , = retention time of the substance B and A = unretained time Higher the selectivity factor, greater the resolution   PSG COLLEGE OF PHARMACY 12

13. Theoretical plate Theoretical plate is an imaginary or hypothetical units of column where distribution of analytes between stationary phase and mobile phase has attained equilibrium. It is otherwise called as functional unit of the column Ideal column has theoretical value more than 2000 Higher the theoretical plates, higher the efficiency of column   PSG COLLEGE OF PHARMACY 13

14. Column efficiency It is also known as plate count, or number of theoretical plates. Narrow peaks take up less space in the chromatogram and thus allow more peaks to be separated PSG COLLEGE OF PHARMACY 14 Retention time Peak width efficiency

15. Column efficiency High equivalent theoretical plate (HETP) HETP = = Higher the column efficiency, higher the resolution   PSG COLLEGE OF PHARMACY 15 HETP Column efficiency

16. Peak asymmetry Ideal chromatographic peaks are symmetrical, if asymmetrical peaks are obtained then it is said to be inappropriate There are two types of asymmetrical peaks, fronting and tailing PSG COLLEGE OF PHARMACY 16 Tailing is due to saturation of stationary phase Fronting is due to more active adsorptive site in the stationary phase

17. Asymmetric factor( A f ) Thi s is measured to find the symmetry of the peak. If the peak is symmetrical then the value will be 1. But practically it is very difficult to get. Ideal A f value according to IP is 0.95-1.05   PSG COLLEGE OF PHARMACY 17

18. Resolution Resolution is defined as the difference in retention times between the two components divided by the combined widths of the elution peaks. It is otherwise defined as the measure of the extent of separation of two components and the baseline separation achieved Ideal resolution value should be greater than 2   PSG COLLEGE OF PHARMACY 18

19. Factors affecting resolution Resolution is affected by three important parameters, they are 1. Selectivity (separation factor) 2. Efficiency 3. Retention (capacity factor) PSG COLLEGE OF PHARMACY 19

20. Current FDA values for the validation of chromatographic methods PSG COLLEGE OF PHARMACY 20 PARAMETER LIMIT Retention factor K ≥ 2 Resolution value > 2 Tailing factor T ≤ 2 Theoretical plate (efficiency) N > 2000 PARAMETER LIMIT Retention factor K ≥ 2 Resolution value Tailing factor T ≤ 2 Theoretical plate (efficiency) N > 2000

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