I posted a query a few weeks ago regarding problems we were having with peaks in blank gradients.Thanks to all for the responses . We have done further work to eliminate kit, detergent contamination, column variability etc.

The system is 20cm C8 spherisorb 5um column, 238nm,30c, 35:65 CH3CN:0.05M NH4H2PO4 rising to 70:30 over 15mins and being held for 15 min.
Eventually we thought it must be the water quality. However, the problem doesn't seem to be consistent.
What I have noticed is the following. After having a few succesful blank runs, flushing with 50:50 CH3CN:H2O to store the column and then setting up the system again produces a 'contaminated' blank with large peaks ?! This took 3-4 runs to clear. I wouldn't have expected this. No samples were run. Is there something happening in the column on storage?

Our systems are on a regular service maintenance. The same thing happened on low and high pressure mixing systems.


Perplexed !!!!!

Selective breeding of a strain of bugs that like 50% ACN/water?

You might try adding 100ppm or so of sodium azide and see if that makes any difference. Of course, then you'd have to flush the azide out of the system, but that should be fairly quick.

Friend;

Sodim azide could help if the problem is related to bacteria, but it is likely to decrease the UV transparency at your wavelength (238nm).

I suggest you try to store your column in 100% Organic solvent (ACN, MEOH). Doing gradient runs it seems like no matter what the first chromatograms is never correct. However, if it takes 4 runs to clear or stabilize the system then you could have a problem.

Another way to speed up the system is to run 100% of your stronger mobile phase for a few minutes (15min) prior to doing your gradient runs.

You also might have some strongly bonded organic base or protein sticking to your column and dissolving slowly over the weekend period.
To wash off such a material you need high temperature, high ionic strength (0.2-0.5 Mol/L buffer but be careful at higher amount of organic not to have precipitation and pressure increase in the column. If the pressure goes up, stop pumping the organic eluent and wash with the aqueous buffer. If the temperatur is high enough (70-80°C) no precipitation will occure.
Make several quick gradients from 0-100% organic eluent. Repeat your 5 min gradients until there is a clear baseline.

Good luck!

Hi,

I have worked with gradients of this type and have had the same problems. We tried all sorts of water ranging from an in-house system to a Milli-Q system to bottled water. We never could pin point the problem. We tried washing columns, swithcing columns, reversing columns, you name it. We were a 24 hour facility so we would just keep running blank injections. This seemed to help but did not eliminate the problem. We spent years trying to resolve this issue. The method that we used was in place a good 6 years and we had no problems, then one day we were just plagued.

I know that this does not help your problem but I did want to let you know that it is not uncommon.

Is the peak large? If it is not to imposing you can just ignore it.

Mike

Have you tried replacing the pump seals ?