Chromatography Forum

I'm having serious baseline issues with SEC. The column is new and the mobile phase is 30% methanol 70% phosphate buffer. There is no noise without the column. The phosphate buffer is filtered through a 20 micron membrane. The column guard doesn't make the noise better or worse.



Lately the noise has been worse than this.

Thanks.

can you say the type of the SEC?
MeOH at these amounts is not recommended for use in a lot of SEC columns

Standard shipping solvent is 0,05% NaN3 + 0,1M Na2SO4 in 0,1M phosphate buffer pH 6,7! What is your buffer concentration? Is it a silica based SEC column? Salt concentration should be <0,5 M! When changing solvent I would recommend a flow rate at 0,3ml/min. What is your flow rate and what back pressure do you monitor.

It is important to know what type of SEC column this is and whether the chromaogram you are showing is with or without an injection. Also, how much is this noise relative to the response you get for your analytes?

Hi everybody. The SEC columns used are biosep-sec-s4000 followed by biosep-sec-s2000 with pH 7 and 50 mM phosphate. Its 70% buffer 30% methanol.

the UV looks fine but the RI has very high noise. peaks from noise are like 10% the area of the sample peak (approximately). there is no noise with just water.

this is at 248 nm also. solvents degassed by He sparge and there is a degasser on the instrument.

we've tried:

filtering buffer through finer membrane (0.2 micron)
getting new columns (columns now are brand new)
long equilibration times
using a precolumn/column guard (made no difference in baseline)
yelling at problem (good stress relief; no closer to solving problem)


Could decreasing the buffer concentration help?

SEC retains small molecules (like buffer salts). could this be a result of buffer salt retention and elution not being completely uniform?

are you premixing or using 2 bottles?

premix.

You said the UV was fine but the RI had high noise. If you run the method without injection, will the RI trace be still noisy? It will be good if you can post the chromatograms with and without injection.

Yes, the smaller the analyte, the long it is retained on a SEC column. Therefore, if your sample contains a host of stuffs such as salts, surfactants, and etc in addition to the analyte, the RI trace will be much more complex than the UV trace.

So when I use waters 2695 with RI detector there is not nearly as much noise as with agilent 1100 series with RI detector.

is your instrument next to ventilation?

Yes. The ventilation makes no difference however. The waters 2695 with RI detector is right next to it as well and the baseline is fine.

There is some difference between the agilent with RI SEC setup and waters with RI SEC setup. The baseline is fine on the waters system but on agilent system it is horrible. I know waters is like a cadillac and agilent is like a dodge but agilent ought to still produce a stable baseline.

The agilent RI detector is also fine when only water is passing through, but once the 70% pH 7 phosphate buffer 30% meoh solution is passing through there is noise. This doesn't happen on the waters instrument.

Purging the RI is a must. Possibly something to look into. If both systems have been purge adequatley then it's just the sensitivity of the instruments. My experience Agilent instruments provide more sensitivity.

i would check some of the settings on both systems in their methods.
maybe there is a difference that makes the noise on the agilent.
is it only a noise problem, or is the drift also higher?

The dift doesn't seem to be a problem. Both instruments have had the reference cell purged adequately as far as I know.

what about the settings in the instrument method?
are they equivalent?
is the sampling rate the same?