Chromatography Forum

Dear All
I did the analysis of drug in plasma by using liquid-liquid extraction for extraction of drug. I used the mobile phase Phosphate buffer (25mM;pH 7.0) with acetonitril in the ratio of 35: 65% v/v. I used this column first time after purchasing . As i washed the column with overneight with water followed by Acetonitrile. After two days i again started with the same column and same mobile phase but the system pressure is going high. I checked the system pressure without column and it is found to be acceptable.
Also i following procedure for extraction of drug first by protein precipitation then followed by liquid-liquid extraction.
What may be reason for increasing back pressure?
What care i should take for avoiding this problem ?
Suggestions will appreciated.

Filter samples and or use a guard column prior to injection.

Apparently you are not getting rid of all proteins, some of these could be precipitating on your frits, etc. If this is the case you should get rid of more proteins via ultrafiltration or SPE.

Sometimes is useful to flush column in reversed order.
(But take care if supplier allow it. I heard that sometimes is used different frit porosity on inlet and outlet and sorbent may be partially flushed away).

It is usually best to wash the column with the same water/organic ratio as your mobile phase. (Dolan has several articles discussing this, so you can do a search.) In my experience with peptides, I washed with 35/65 ACN/water, and never had problems.

Never left your column (if it is reversed-phase) in 100% acetonitrile, even after overnight water washing. I used such buffers very often and always left column in acetonitrile:water as 80:20%.
Some reversed-phase columns do not like pH 7.0. For such columns we used saturated pre-columns inserted between pump and injector.