Chromatography parameters

Parameters used in HPLC and GC

1. CHROMATOGRAPHY PARAMETERS Mr K V NANDA KUMAR Associate Professor Department of Pharmaceutical Analysis Krishna Teja Pharmacy College, Tirupathi

2. Chromatogram A plot of the detector’s signal as function of elution time or volume . Chromatograms should be checked to ensure that chromatographic quality, including good peak shape, baseline drift, column bleed, sample carryover, and chromatographic resolution are within acceptable limits. 6/23/2021 2

3. Characterization of chromatogram A chromatographic peak may be characterized in many ways 6/23/2021 3

4. Retention time( tR ) Retention time ( tR ) is the time elapsed between sample introduction (beginning of the chromatogram) and the maximum signal of the given compound at the detector. Retention time is the time that a solute spends in a column or it can be defined as the time spent in the stationary and mobile phases. The longer retention time depends on the interaction of the analyte with the stationary phase. The stronger the interaction, the more will be the interaction time 6/23/2021 4

5. Retention Volume( Vr ) The volume of mobile phase needed to move a solute from its point of injection to the detector ( Vr ). Dividing the retention volume by the mobile phase’s flow rate, u, gives the retention time. 6/23/2021 5

6. Baseline width(w) Baseline width is determined by the intersection with the baseline of tangent lines drawn through the inflection points on either side of the chromatographic peak. Baseline width is measured in units of time or volume, depending on whether the retention time or retention volume is of interest. 6/23/2021 6

7. Void time(tm) & Void volume Besides the solute peak, figure also shows a small peak eluted soon after the sample is injected into the mobile phase. This peak results from solutes that move through the column at the same rate as the mobile phase. Since these solutes do not interact with the stationary phase, they are considered nonretained . The time or volume of mobile phase required to elute nonretained components is called the column’s void time, tm, or void volume. 6/23/2021 7

8. Essential Chromatographic Parameters 6/23/2021 8

9. Efficiency(N) Column efficiency is used to compare the performance of different columns. It is probably the most frequently cited parameter of column performance and is expressed as the theoretical plate number, N. Efficiency can be changed by altering the column length, particle size, or flow rate of the chromatographic system. The higher the number of theoretical plates in a column, the higher the efficiency of it, and after that, the more resolution can achieve. 6/23/2021 9

10. Retention factor, k Formerly referred to as capacity factor or k´ (k prime), the retention factor measures the period of time that the sample component resides in a stationary phase relative to the time it resides in the mobile phase. It is calculated from the retention time divided by the time for an unretained peak (t0). 6/23/2021 10

11. Selectivity or separation factor (α) The separation factor is a measure of the time or distance between the maxima of two peaks. If α = 1, the two peaks have the same retention time and co-elute. Selectivity can be changed by changing the mobile phase constituents or changing the stationary phase. Temperature may also be a factor in adjusting selectivity. 6/23/2021 11

12. Resolution (Rs) Resolution describes the ability of a column to separate the peaks of interest, and so the higher the resolution, the easier it is to achieve baseline separation between two peaks. Resolution takes into consideration efficiency, selectivity and retention To completely separate two molecules, R S must equal at least 1.5. 6/23/2021 12

13. Tailing factor If the peak to be quantified is asymmetric, a calculation of the asymmetry would also be useful in controlling or characterizing the chromatographic system. Peak asymmetry arises from a number of factors. The increase in the peak asymmetry is responsible for a decrease in chromatographic resolution, and precision TF is tailing factor (measured at 5 % peak height) B is distance from the point at the peak midpoint to the tailing edge, A is distance from the leading edge of peak midpoint to the midpoint 6/23/2021 13

14. Asymmetry factor AsF is peak asymmetry factor B is distance from the point at the peak midpoint to the tailing edge A is distance from the leading edge of peak midpoint to the midpoint 6/23/2021 14

15. Pressure The Pressure equation identifies five key factors that affect system pressure: solvent viscosity (h), flow rate (F), column length (L), column radius (r) and particle diameter ( dp ). It is a good idea to familiarize yourself with the pressure equation to understand these key contributors to system pressure. 6/23/2021 15

16. 6/23/2021 16