Chromatography Forum

Hello All
I am using a 100 x 4.6 mm 3 micrometer Perfectsil Target ODS-3 C18 column to measure drugs in plasma samples, my mobile phase is 65/18/17 0.01M K2HPO4/methanol/acetonitrile pH=7.5. For two weeks its back pressure is more variable and higher than normal. It seems to be partially clogged then I decide to clean the column. I applied the following protocols to clean up but it wasn't not useful and the pressure didn't decresse.
• Flush the column with 20 column volumes Water
• Flush the column with 20 column volumes Acetonitrile
• Flush the column with 5 column volumes Isopropanol
• Flush the column with 20 column volumes Heptane
• Flush the column with 5 column volumes Isopropanol
• Flush the column with 20 column volumes Acetonitrile

Please let me to know how I should go about it ?
Thank you

1. Did You flush in the direction of the flow or switched flow to opposite direction? Sometimes wash is more effective when using back flush, it is recommended to have lower flow than during measurements.

2. Quite often there is just no way to restore column - You have no choice other than change to the new one..... Maybe changes in the sample prep would prolong the life span of the column...

Thank you very much for your consideration
I have flushed it in the direct and opposite flow direction but the problem wasn't solved,
about sample prep., the drug was extracted by diethyl ether then organic phase was dried under nitrogen. The residue was dissolved in the mobile phase. I think this is a safe prepration protocol.
considering that I bought the column later, do you have any other recommendation?
Thanks in advance

with what do you filter your samples and mobile phase?

I only filter my mobile phase with 0.22micrometer sartrious membrane filter

How many injections have you made on this column? It may be that the column has reached its life expectancy, like R13 said.

Well, I have no solution to save the column.

I do not work with plasma samples anymore for some time now, but I would not consider extraction with diethyl ether as really "safe" as it is extracting quite many other plasma components (I think You can see some sediment in the test tube when dry) and there is no filtration of the sample as during SPE.

In such cases we always used pre-columns and changed them after several measurement sequences.

For some methods with liquid/liquid extraction we did centrifugate the HPLC vials before puting them into HPLC (and did set up injector to take liquid well above the bottom of the vial with "bottom sensing" off for Agilent HPLC).

Honestly if you tryed that and it didnt work i would say game over....are you sure its the column ? it could be the connections around it...i had the same problem and it turned out it wasnt the column at all but an outlet cable from my detector...anways good luck

Very thanks of my dear friends
yes I check all of the connections around, surely my problem is thanks to column clogged or contamination
I have other questions:
1- How can I find that high backpressure of the column (c18) is related to the packing deterioration or contamintion? in the other hand, how I can check the integrity of column packing?
2- Do you think flushing with different concentration of NaOH or phosphoric acid is useful? Are they safe for c18 column?

Very thanks of my dear friends
yes I check all of the connections around, surely my problem is thanks to column clogged or contamination
I have other questions:
1- How can I find that high backpressure of the column (c18) is related to the packing deterioration or contamintion? in the other hand, how I can check the integrity of column packing?
2- Do you think flushing with different concentration of NaOH or phosphoric acid is useful? Are they safe for c18 column?

To me problem is the frits on the column, if you replace it you may get better results (possible peak symmetry will be worse than the new column). Packing deterioation generally causes system suitability problems not high pressure

During acid and base flush you have to be careful not to have a pH below 1.5 and over 7.5

yes it is right, but based on this study "Chemical stability of Reversed Phase HPLC silica under NaOH regeneration conditions" The columns were subjected to alkaline elution condition, and it is recommended none of the C18 modified materials shows any Si-leakage at 1 mMNaOH (pH 11). Experimental conditions:
•The column was equilibrated with 10 column volumes of 100 % ethanol.
•The column was purged with 11 column volumes of Ethanol (99.5 %) / 1 mMNaOH (aq) 50:50 (v/v)
•The column was purged with 10 column volumes ethanol / H2O / HAc 10:90:0.2 (v/v/v).
•The column was purged with 11 column volumes 100 % acetonitrile.
Do you any one experience this like conditions ?
thanks alot

Never hurts to use an inline frit between the injection port and the column. After so many hrs of operation/injections, take a look at that frit, and you may be surprised at the garbage this simple device has collected that would have otherwise clogged your columns inlet

Two suggestions:
- Start thinking of HPLC columns as disposables. When the performance gets worse and/or backpressure increases try backflushing. If this doesn't help and the column isn't usable anymore, thank the column for its services, throw it away and use a new one. When I think back how many hours I've spend trying to revive columns and consider the success rate, the price of a new column doesn't seem to be that high anymore .
- Use precolumns with plasma samples!