Chromatography Forum

Hi, I am trying to separate a mixture of two substrates with HPLC and need help with it. I have a reverse phase C18 column (3.9x300, 10u) and the products come out very quick (<5min) with little separation.

Is there a set protocol or something I can follow to improve the separation? It my little experience, changing gradient usually solve the problem, and I am not sure what eles I can do. I am running at 1mL/min, so with column vol of ~3.6 mL, the materials seem to be going right through. Increasing water content too much shuts off my HPLC for the back pressure.

As you might imagine, I have never officially study analytical chemistry, I just want to have reasonable separation.

Thanks -

At 1 mL/min, the backpressure on this column should be very small. Please tell me about the backpressure that you are measuring. It is possible that your column is clogged, or it is possible that there is an issue with the tubing in the instrument, or it is possible that the backpressure limit is set too low. What is the backpressure if you run the same flow rate without the column?

Thank you for replying. The back pressure of the column depends on the content of water. If runs from 60:40 ACN:H2O to 90:10 ACN:H2O, the pressure is between 500-700 psi. At 50:50, it reaches ~860 psi. At 20:80, it goes over 1000 psi, and (because I am setting limit to 1000 psi), it shuts off.

If no column is attached, the pressure is very very low. (<20psi).

The column is new, and we have not injected many samples yet other than the substrates. So I think the column is Ok. I am actually not sure what the typical backpressure should be for this column.

1000 psi as an upper limit for your pressure is low, personally I would set an upper limit of 3000. If the column is new it should easily cope with a higher pressure than 1000 psi.

The information sheet supplied with the column should give you some information on the pressure range the column is capable of withstanding.

As JGK mentioned, the column can be used without difficulty to 6000 PSI. So you can set the pressure limit much higher. Most people set it to 3000 bar. but for reasons other than the column.

I agree, expecially for a 300 mm column. I typically use an upper pressure limit of 5000. With 90% water in your mobile phase, there is no way you will be able to keep your pressure under 1000. It will be fine if you up the pressure limit to 4000 or even 5000. Also run a couple of controls each containing just one of the substrates, and extend your run time to make sure those are the peaks your are looking for. Your real peaks could be appearing downfield and you might not be seeing them because your runs are so short. If this is the first time you are doing this test, you need to make absolutely sure that those are your peaks of interest.

Thank you everyone! I've changed the limit to 5000 and tried gradient from 10 to 90% ACN:water. Pressure dis go over 1000 but not as high as initially thought would be (700-1200 psi). I could not find any information on what the "normal range" of the pressure should be with the sheet came with the column.

Retention time, however did not change as much (3.5 min went back to ~5 min) - but I have not tried too many methods yet. In addition, we have more serious reproducibility problem now. Ex., run1: Rt = 7.2 min; run2: Rt = 4.5 min (same method (10:90(ACN:H2O) to 2.5 min, then gradient to 90:10 over 10 min), the same sample). Not sure how to resolve this. Any suggestion please?

Could it be the (1) bad column (again, the column is out of the box, but...) (2) wrong column.

The substrate in use is an authentic sample we bought from Aldrich. We are using a mix of the two substrates. We have been running 30 min initially, so the peak we see should be the right peak.

At the end of the gradient run, you need to program some time to bring the column back to the initial conditions of the gradient. Don't forget, the gradient is generated in the pump, and there is volume between the pump and the column, and there is volume inside the column as well.

A typical rule of thumb for reequilibration in a gradient is to use the gradient delay volume plus about 5 column volumes to get back to initial conditions.

Also, if you want to further increase retention of your peak, you can go to a more polar starting condition. However, investigate the proper reequilibration of the column first.

Now it is consistent after runnign ~30 min before injection.
But does people really let it equilibrates 5 vol of the column vol? WIth 3.9x30, the column vol is ~3.6 mL and x5 = 18 mL. With 1 mL/min, you equilibrate for 18 min after each run?

I am already at 5:95 (ACN:H2O) start. Well, but I did not fully equilibrate the column till today so all the previous runs are not accurate representation... Thanks again for the input!

The column is good to over 6000 psi. You can run without difficulty the separation at 2 mL/min and reduce all your retention times 2-fold. You need to reduce all gradient segments including the reequilibration times by the same factor 2 to get the identical chromatogram, just 2 times faster.