Factors Affecting Resolution In HPLC.

In this slide contains Factors Affecting Resolution In HPLC and its criteria's.
Presented by: M.Sudheeshna. (Department of pharmaceutical analysis).
RIPER,anantpur.

1. 1   A Seminar as a part of curricular requirement for I year M.Pharm I Semester Presented by M.SUDHEESHNA (Reg.no:20L81S0713) Pharmaceutical analysis Under the guidance of Dr. P.Ramalingam M.Pharm , Ph.D. Director – R&D Division, Professor of pharmaceutical analysis and medical chemisry President – IPA local branch - anantapuramu FACTORS AFFECTING RESOLUTION IN HPLC

2. 2 Introduction Factors affecting resolution in HPLC Capacity factor , K’ Selectivity factor , α Column efficiency , N Tailing factor ,T Acceptance criteria How to improve resolution References Contents:

3. 3 HPLC is characterized by the use of high pressure to push mobile phase solution through a column of stationary phase allowing a separation of complex mixtures with high resolution. They travel according to their relative affinities towards stationary phase. More affinity – travels slower Less affinity - travels faster This technique is used for separation , purification, identification, extraction of compounds. Introduction:

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5. 5 Resolution is a function of difference in retention times of the two peaks (t RA and t RB ) and widths of the peaks at the base (W A and W B ). Baseline resolution is achieved when R = 1.5. There is a quantitative measure of resolution as shown in the equation below: R = t RA – t RB / 0.5( W A + W B ) Resolution:

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7. 7 Resolution can also be expressed in the Resolution Equation as a combination of the factors (separation, efficiency, and retention) that affect this value.

8. 8 Retention factor / capacity factor is the time the component interacts with the stationary phase ( t R ) relative to the time the component spends in the mobile phase ( t ). It is a measure of degree of interaction with stationary phase. K= (t R –t ) / t Where, t R = retention time of the peak t = column dead time Capacity Factor, K’:

9. 9 Influencing the capacity factor: Mobile phase strength : Increasing mobile phase strength – decreases k Temperature : Increase in temperature - decreases k

10. 10 As k grows longer its effect reaches a limit at a value of about 10.

11. 11 The separation factor is defined as  the ratio of retention factors (k). In addition to resolution, the separation factor (α) is also used as an indicator of the separation of two peaks. α = K 2 /K 1 A good selectivity for HPLC is 1.1, which allows a resolution of 1.5 to be achieved. Selectivity Factor, α

12. 12 Influencing the selectivity factor , α : Mobile phase type : type of interaction between m.p and analyte is critical to optimization of selectivity of system. Column type : bonded phase - better selectivity

13. 13 As α grows larger, its effect reaches a limit at a value of about 5.

14. 14 The efficiency of a column is reported as the number of theoretical plates (plate number),  N. It is the measure of sharpness of the peaks. A high value for efficiency indicates that more peaks can be separated. The number of plates will increase with the length of the column. Column Efficiency, N

15. 15 The calculation for efficiency is related to the peak width and is as follows: N = (t/w) 2 Where, t = retention time of the peak of interest W = peak width at the base. Similar to the measurement for resolution, the measurement for efficiency may also be performed using the peak width at half height: N = 5.54 (t / w h /2 ) 2

16. 16 where, W h/2  is the peak width as half height.

17. 17 Resolution will continue to increase as theoretical plates increase.

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19. 19 I t is defined as the distance between the front edge of the peak to the back edge of the peak divided by twice of the front edge of the peak with all measurements made at 5% of maximum peak height. Tailing factor is a measure of symmetry of a peak. Tailing Factor, T:

20. 20 It is measured by the following equation: T = W 0.05 / 2f where, W 0.05 = peak width at 5% height f = distance from peak front to apex point at baseline

21. 21 Acceptance Criteria:

22. 22 Increase N (efficiency) by: Increasing column length Decreasing particle size Reducing peak tailing Increasing temperature Reducing system extra-column volume How to improve resolution in HPLC

23. 23 Change α (selectivity) by: Changing column stationary phase Changing mobile phase pH Changing mobile phase solvent(s). Increase k (retention) by: Using a weaker solvent (changing polarity) Changing the ionization (polarity) of the analyte by changing pH Using a stronger stationary phase (changing polarity)

24. 24 References: K., Robards (1994).  Principles and practice of modern chromatographic methods . Haddad, P. R., Jackson, P. E. Amsterdam: Elsevier/Academic Y. V. Kazakevich and R. LoBrutto (ed.), HPLC for Pharmaceutical Scientists, 2007. Lindsay, S.; Kealey , D. (1987). High performance liquid chromatography. Patel A, Dwivedi N, Kaurav N, Bashani S, Patel S, Sharma HS,  et al.  Chemical analysis of pharmaceuticals: a review. J Med PharmInnov 2016;3:4-7.

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