Periodic baseline spikes, Pharmacia FPLC

[Johte]

Hello all,

System is run by a LCC-501 and contains two P-500 pumps. The detector is a UV-M II. FRAC-100 fraction collector. REC-102 chart recorder. The instrument is located in a 4 C cold room.



On my chromatograms, I am getting a periodic spike up and down and I cannot seem to locate the issue. I have removed the column and re-tighten all the positions and flushed liters of water.

I checked the flow by running a program for 10 mL at 0.25 mL/min into a graduated falcon 15 mL tube and the final volume was right on the mark.

The chart scale also seems to be at rate of 0.8 of what it should be. 10 mL at 0.1 cm/mL comes out to about 0.8 cm. The manual mentions calibrating the pulses from the pumps but does not mention how to do so.

If anyone is familiar with this instrument and has any ideas for either of these problems I would appreciate the insight.

Thank you.

[tom jupille]

As a first guess, those look like air bubbles passing through the flow cell. If this were my problem, the first think I would do would be to check all the connections on the *intake* side of the pumps. A slightly loose fitting can allow the pump to aspirate a small amount of air. You might also have to degas your buffers (not usually required for FPLC, but . . . ).

Additional thoughts: did the problem appear suddenly (and if so, did it coincide with a specific event like a new column or replacing tubing or something like that) or has it come on gradually (in which case, try a new batch of buffers and see if that makes a difference).

[Andy Alpert]

Is your equipment plugged into isolated outlets? If not, then maybe there's a piece of heavy equipment somewhere on the same circuit that is cycling on and off at the intervals coinciding with the current spikes.

[Johte]

Tom, I can trace the issue back to before my time in 2011. Buffers are always degassed. And connections post pump were checked recently. I will look into the pump seals.

Andy, the FPLC has been located in a 4 C cold room which has been known to have some power issues. However, all parts of the FPLC are on a single power strip. I can try moving each piece to a different outlet but if it's the cold room itself I don't know if much can be done about that.

Thanks for the assistance.

[bunnahabhain]

Are these tick markers from the fraction collector? Some instruments mark the switch to next collection vessel by giving back a pulse to the detector signal. Check if these two events correlate.

[Johte]

Are these tick markers from the fraction collector? Some instruments mark the switch to next collection vessel by giving back a pulse to the detector signal. Check if these two events correlate.

bunnahabhain, The light tick marks you see starting approximately 3 cm from (0,0) are fraction collector marks. The fraction collector does send a signal to the chart recorder but the spikes occur whether the fraction collector is running or not.

Thanks for the input.

[tom jupille]

It looks like the image has three injections and the beginning of a fourth (right?). And are the runs isocratic or gradient (looks like isocratic to me).

The negative dips begin 2 or 3 chart divisions from the left, and end 4 or 5 divisions from the right. If this were due to aspirated air (my original guess), I would expect the spikes to appear consistently. If it were due to injected air, I would expect the pattern to be somewhat repeatable from injection to injection.

At this point, I'm leaning toward Andy's suggestion of an electronic problem. One way to diagnose that would be to shut off the flow but leave the detector and recorder on (maybe 24 hours -- obviously at a slow chart speed). If the problem is electronic noise, the spikes should show up. If they *don't* show up, you have at least eliminated that possible cause and can go back to looking for air bubbles.

Of course, if you are doing prep work on this system rather than quantitation, then the spikes are, at worst, an annoyance rather than a problem (especially if they have been there for 6+ years!). As such, the best approach may be to just live with it.