Column efficiency parameters

A presentation on column efficiency parameters in chromatography.. A part of gas chromatography in pharmacutical analysis..will be helpful for all mphrm students

1. COLUMN EFFICIENCY PARAMETERS PRESENTED BY SUBODH S SATHEESH MPHARM PHARMACEUTICS ECPS 1 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

2. It is expressed by the number of theoretical plates It is determined by the formula The number of theoretical plates is a measure of the “goodness” of the column If the retention time is high and peak width is narrow then it shows excellent chromatograms Column efficiency 2 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

3. where  tr  is the retention time measured from the instant of injection w  is the peak width W is determined by SD= σ ie w=4 σ 3 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

4. Resolution is the ability to seperate two signals In chromatography its the ability to seperate two peaks.ie seperation of constituents where   tr 1  and  tr 2  and  w 1  and  w 2  are the times and widths, respectively, of the two immediately adjacent peaks. If the peaks are sufficiently close w  is nearly the same for both peaks and resolution may be expressed as Resolution 4 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

5. Greater the distance more resolution and vice versa. 5 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

6. The rates of migration of substances in chromatographic procedures depend on the relative affinity of the substances for the stationary and the mobile phases Its the difference in time between the point of injection and the time of emergence of separation of component from the column. It is actually the time required for 50% of the component to get eluted. It is measure in minutes or seconds Retention time 6 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

7. 7 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

8. It is the volume of carrier gas required to elute components from the column to the time the peak maximum is obtained. Retention volume depends upon flowrate and retention time V R= t R - F C Retention volume 8 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

9. It is the ratio of partition coefficient of two components to be separated.   S= Kb/Ka = K’a / k’b = ( t b -t o )/( t a -t o) If peaks are far apart ie there is more difference in partition coefficient between compounds hence more seperation factor and viceversa Separation factor Less seperation factor More seperation factor 9 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

10. HETP is numerically equal to the column length divided by the number of theoretical plates in the column It varies from to one column to another as well as one solute to other The more efficient the column the better the resolution and the smaller the HETP. HETP=Length of column / no of theoretical plates HETP 10 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

11. Here we can see the effect of flowrate on HETP. An ideal flowrate happens in the minimal HETP. 11 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

12. A chromatographic peak should be symmetrical about its centre to follow gaussian distribution Asymetric factor is the measure of peak tailing or fronting. It is defined as the distance from the centre line of the peak to the back slope divided by the distance from the centre line of the peak to the front slope. Asymetric factor 12 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

13. The chromatographic peak in (a) is an example of tailing, which occurs when some sites on the stationary phase retain the solute more strongly than other sites. The peak in (b) is an example of fronting, which most often is the result of overloading the column with sample. For both (a) and (b) the green chromatogram is the asymmetric peak and the red dashed chromatogram shows the ideal, Gaussian peak shape . 13 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

14. Its function is to separate sample components to discrete peaks It should have reasonable chemical and thermal stability There are a lot liquid stationary phases available for gas chromatography. But there is no solvent that meet all the requirements of a perfect stationary phase LIQUID STATIONARY PHASES 14 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

15. Liquid phase should not permeate too deeply into the fine pores of the support structure as slow diffusion in and out of pores affects column efficiency It should be chemically inert It should be a good solvent for sample component  Liquid phase should have low volatility and high stability at elevated temperatures otherwise they can contribute to interference in analysis Characters of good Liquid StationeryPhase 15 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

16.   Stationary Phase Trade Name Max Temp Common Applications Dimethyl Polysiloxane OV – 1, SE – 30 350 o C Hydrocarbons, Polynuclear aromatics, PCB’s Poly(phenyl methyl) siloxane OV – 17 250 o C Steroids, Pesticides, Glycols Poly ( Trifluoro propyl dimethyl ) siloxane OV – 210 200 o C Chlorinated Aromatics, Nitro Aromatics, Alkyl substituted Benzenes Polyethylene Glycol Carbowax 20 M 250 o C Free acids, Alcohols, Essential Oils, Glycols 5% Diphenyl – 95% Dimethyl polysiloxane DB – 5 325 o C Flavors , environmental samples and aromatic hydrocarbons Typical liquid stationary phases 16 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

17. Nonpolar solutes like pentane butane etc can be effectively separated by nonpolar solvents like squalene . And polar solutes can be easily separated by polar solvents eg ; PEG . Boiling point is also a factor of consideration. among solutes with similar polarity if there is sufficient difference in BP effective separation can take place . Eg . Squalene min/max temp= 293/423 SE4 423/573 A solvent that could generate different partition ratios among solvents can only be useful in GLC Selection of solvents 17 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

18. H KAUR Instrumental methods of chemical analysis ninth edition 2013 ;1091-1092 Skoog holler crouch Instrumental analysis 2012 841-846 B K sharma Instrumental methods of chemical analysis twenty fourth edition 2005 c188-191 En.wikipedia.org gas chromatography Reference 18 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY

19. Many thanks MANY THANKS 19 PHARMACEUTICAL ANALYSIS-GAS CHROMATOGRAPHY