Chromatography Forum

hello sir,
we have developed a method for the drug,the method seems to be good while analysing the samples continously ,the peak shapes are good and everything is ok,but we face the problem when we repeat the same analysis after washing the column with water.
The problem is the peaks are going wrong shapes that is they are getting splited and tailing peaks, we face the same problem with two columns.the columns are waters symmetry and x-terra
we are using potassium di hydrogen phosphate buffer with ph 7.8,the ph has been adjusted with 2% KOH solution.
can any one plz explain me what the problem is


regards malleswar

Dear Malleswar
The problem is not with your method or column.It is with your washing procedure.You are using 100% water for washing the column that is very wrong.Use some % of solvent(ACN is best one) to water (50:50,65:35) for washing the column.

Water is very polar.In your column you have the c18 or c8 linkages which are non polar.Just think what happens when a very polar solvent move on non polar bed,Repulsion takes place your stationay bed collapsed.Analytically it is known as PHASE COLLAPSE.If you use c18 its effect is more when compared to c8

Regards
KIRANREDDY MUNNANGI

Oh, Dugra, Dugra, dark you are.

Class of substance (chemical or pharmacological group), the type
(RP -grupp?) of the column ...

One can strongly doubt that phase collapse has anything to do with dewetting. The latter causes a retention time lowering. The problem here seems to be a simple matter of patience, the column is not equilibrated.