Column 4.6*250 mm , 5 micro.

are there guard column or no?

Sounds like you may be washing the column using the reverse of what you are suppose to. "the washing starts with 0.2 ml/min with increase in the ratio of water".

• What is the normal pressure seen on YOUR HPLC with this mixture and that same column? Pressure is relative. You need to compare it to something under identical conditions to know how much higher than normal it actually is.

For a RP column, you would wash it with a less polar solvent (i.e. Methanol or ACN), not water. Start the wash gradient at 50/50 (ACN/Water), then ramp it over 10 minutes (at 1 ml/min) to 95% ACN/ 5% Water, hold for 15 minutes, then stop. The idea here is to INCREASE the organic solvent, not water over time to reduce any retention. Next run the same gradient, but this time use Methanol/Water, not ACN/Water (same %'s as before). This may help to remove any fouling. Methanol has very different solubility properties than ACN so even though it is slightly weaker than ACN in strength, it superior solvation properties may help dissolve and flush out more material. If this fails to reduce the back-pressure, even stronger solvents such as IPA or THF could also be tried.

BTW: You did not provide any information about your HPLC system. The problem may be caused by the HPLC system and NOT the column. For example, does your HPLC system have a clog or restricted frit or filter inline? What is the backpressure of the same gradient method you use if the column is replaced by a zero dead volume fitting? At 1.00 ml/min with 50/50 ACN/Water, you might expect a backpressure of less than 20 bars with NO COLUMN inline.

There is a guard column dr mehaya. It's pressure 200psi

Dr william, the normal pressure at this ratio is ~1700 psi.

Actually, the whole system and filters were checked.

OK, so if I understand you correctly, the HPLC back-pressure under initial conditions with the column used to be ~ 1,700 psi and now it is ~ 2,400 psi under the exact same test conditions. Is that correct? If so, please try the gradient wash mixtures mentioned before to elute off as much of the fouling from the column as possible. *Use high percentages of organic, but not 100% organic or you may precipitate them out onto the column. If you are unable to remove the fouling then dispose of the column and purchase a new one. HPLC columns are disposable, consumable materials. They have a finite lifetime and should be disposed of when they no longer perform well.

A few ideas to reduce the chances of this happening in the future: (1) Please always follow up each analysis with a proper column wash cycle. You should incorporate a solvent mixture which is stronger than your mobile phase in the wash solution. Make sure you use solvents which fully dissolve EVERYTHING. The goal is to flush off any debris or strongly retained material. Failure to include a proper wash method may result in column fouling, poor results, retention time variations, distorted peak shapes and other problems. (2) Be sure and fully dissolve all samples in the mobile phase before injecting them. (3) Filter samples through an appropriate filter membrane before injecting.

One really important question that has not been asked... What types of samples are you injecting into this column?

What is the brand and model of column? Was the column ever run under basic conditions?

Some columns run above pH 7.5-8 can have high back pressure as the silica dissolves and particles clog the frit. since some columns are designed for these conditions, this may not apply hence the question about what C18 column is used. If the column was damaged in this fashion, it will never work well again.

Kromasil c18 is used.

"What types of samples are you injecting into this column?"

2ry amine.

pressure with guard column 200 psi indicated there is no problems in tubes and other HPLC parts before guard column

so, i recommended to exchange your column with equivalent one (c8 or c18 ) and check , may be your column is damage or blocked

I concur with Dr William suggestions. But i want to add a little to what he advised. I experienced similar problem in the past and what i did was prolong washing for over 24hrs by changing the solvent ratio from 5 to 95%, methanol:distilled water and vice versa, in between. I also replaced methanol with acetonitrile for some hrs in between. Thesame procedure was repeated by installing the column in the reverse position. Eventually the pressure dropped from 18 to less than 10 mpa and all the system suitability parameters returned to normal.

You may want to try this technique also. Hope it will work for u too. If it doesn't, check the system parameters that indicate column efficiency viz. Plate counts, resolution value, capacity factor, tailing factor....if they are not within acceptable range, then u have no option but to replace the column.

Thanks for your help.

I did your advices but it seems that the column must be replaced.

That is OK. Columns are consumable items. After you obtain the new column, find a suitable standard and develop a QC method to characterize the column for future reference. Please do not use the column vendor's "QC" std to do so. Those compounds are poor choices. We usually recommend you choose a pure sample/std which is related to your own applications. When column performance is questioned, simply setup and run your own column "QC" method to obtain new data and compare it to the original data.

You can also improve the overall lifetime and performance of the column (and HPLC system) by following the suggestions made earlier.

Thanks, dr. William