When a chromatographer execute the analysis on HPLC or GC, he expects for proper peak shape. The proper peak shape is measured by its symmetry.
The symmetry should not be grater than 2.
Figure:-1 Symmetrical peak shape.
Some time chromatographer face improper peak shape i.e. peak tailing, peak fronting, shoulder peak, split peak, peak broadness which are the not symbol of good chromatography.
Figure:-3 Tailing and Fronting peak
The cause of these peak shape distortions are as follows:-
1- Sample solvent not compatible:- If sample solvent is not compatible with mobile phase then peak shape distortion observed. The cause of incompetent solvent is as analyst use different solvent for diluent preparation or if diluent got contaminated and respective contaminant interact with analyt components. Hence precautions for solvent selection is most important.
2- Blockage of column frit:- The mobile phase flows in column in linear way and sample also flow along with mobile phase but when any foreign particles (i.e. buffer deposition or clotting of impurities on column frit) block the flow path then sample got spread. Hence some amount of sample reach latter and analyt compounds also travel delay. Due to this, peak getting shoulder or splitting.
Figure:-4 Showing introduction of sample in column Inlet (a) under normal conditions (b) with partial block frit.
To resolve the frit blockage, back wash the column with 30-50 ml of strong organic solvent. If still blockage not resolved then replace the frit with new one. The use of gard column is suggested to avoiding blockage of column frit.
3- Dead volume in flow line:- The peak broaden case occurred due to dead volume in flow line. If flow tubing are not properly connected between sample injection unit, column and detector flow cell from where samples pass, then peak broaden occurred.
To remove dead volume, at time of new peak tubing installation press the tubing in joint for such way as it reach to the connection properly and there should no gaps between tubing and joint.
4- High volume of sample:- The detector is too sensitive and if high volume of sample injected, then detector gets saturated and gives broad flat top peaks.
Figure:- 5 Broad flat top peak.
To resolve broad flat top peak, we should reduce the sample concentration or injection volume.
Abhishek Singh is the author of Pharma technical support. He is pharmaceutical professionals, having experience of more than 12 years in Quality.