Hi,

We have observed a small ghost peak that co-elutes with our active & the source of this peak has been traced to the buffer. Below are the HPLC conditions--

A = 80/20 25mM filtered potassium phos. monobasic/ACN

B = 100% ACN

Gradient--

Time A B

Init 91 9
5 min 91 9
35 min 46 54
35.1 91 9
45 91 9

Injection volume - 20ul
Wavelength - 210nm
Column - Zorbax Stablebond C8
Column Temp 40C

After much troubleshooting, the source of this peak was traced to the buffer. Using older lots of JT Baker Ultrapure Bioreagent, the chromatograms were clean. However, with recent lots of Jt Baker, Fisher HPLC grade & Fluka material, the peak is present. This peak is small (i.e. approx 0.08% peak area related to the active), but it's an issue, given its coelution with the active. We've run this method on systems that had never seen this active & the peak was seen, confirming that it's not stemming from carryover.

Does anyone have any ideas as to recent changes in production of this buffer material that may attribute to this peak?? Does anyone have any suggestions for possible resolutions?? The highest purity reagent is being used & we are filtering the buffer solution through 0.45u wetted Nylon filters.

Thanks much for your time.

Mike Penrose

Dear friend;

You may want to consider the following suggestion. In the old day in grad school we used to cleanup our water passing it through a bed of C18 material. This could be an old 10um 250x4.6mm column, or any other low pressure system such as those used in Flash Chromatography.

This way you could clean your buffer prior to use. Also, there are now "Empore" filters (3M) which contain c18 particles, I have not used them but they are likely to help. Obviously, whatever C18 packing you use it has to be cleaned with ACN first and then conditioned with Water.

It is always likely that the C18 bed or particles will get saturated with impurities retained on them (breakthough), however since you will be filtering only the buffer, this will happen only after a very large volume of it has been passed through.