RP-HPLC bubbling advice needed

[prishly]

Hi all.

I have a RP-HPLC snag. While a sample containing 30% glycerol is being run through a Synergi MaxRP-C12 column with a linear acetonitrile gradient (water phase being Tris-0.05%TFA, pH 8.5) and the gradient reaches 45-50%, pressure goes down and a bubble forms blurring a substantial part of the picture. Glycerol-free samples run with no trouble. Glycerol is essential for solubility of proteolysis fragments in the sample, sample pH is 8.0 and is needed for proteinase K proteolysis. It would be great if someone could help sort this out. Thanks in advance.

[prishly]

Sorry, glycerol concentration is actually 7.5 % since I dilute sample 4 fold before chromatography

[Bruce Hamilton]

What do you dilute the sample with?. If glycerol is essential for solubility of the proteolysis fragements, what happens to those when the glycerol is separated by the column?. I suspect they may drop out of solution and help cause the bubble.

Try to improve your sample preparation process, but if you can't, you could put a little backpressure on the detector ( short length of narrow bore tubing at the outlet ) to prevent the bubble forming. Take care not to exceed the detector cell maximum pressure.

Bruce Hamilton

[prishly]

Thanks for replying. The problem is, bubble forms even if we run samples without proteolytic peptides, just the buffer with glycerol. I don't think it's the proteolytic fragments going out of solution because subsequent blank runs do not reveal any remaining peptides.

[HAZMAT]

you could put a little backpressure on the detector ( short length of narrow bore tubing at the outlet ) to prevent the bubble forming. Take care not to exceed the detector cell maximum pressure.

Bruce Hamilton

Even better: http://www.optimizetech.com/opti-shop/i ... qm5rdmvv45

/plug

[Jade.Barker]

Glycerol-free samples run with no trouble. Glycerol is essential for solubility of proteolysis fragments in the sample...

I am interested in your problem since we get glycerol up to 8-10% (naturally occurring) in some of the samples we process. I’m a novice, so forgive me it these ideas are elementary or non-applicable:

Glycerol is used in manufacturing foam products… It seems it is quite good at forming and sustaining bubbles. Just for fun; mix a little mobile with some glycerol and shake it. How foamy does it get? If you check it at different proportions you can identify the “foamyâ€

[Kostas Petritis]

A couple of things: I guess that you are buying proteinase K that is already dissolved in glycerol? You could buy the lyophylized form. I am not sure that glycerol helps that much in keeping your peptides in solution so you can do without it. Proteinase K works really well with SDS and urea which can help with protein/peptide solubilization.

If you do not want to go there, glycerol is also very hydrophilic and it will be washed out reversed phase type columns. I suggest that you keep isocratically in the initial conditions longer so that glycerol has been completely flushed out from your columns and tubing (i.e. 10 column volumes or more if throughput is not a problem). You will then operate as if virtually you had no glycerol in our system.

Another way is to increase the injection volume so you can further dilute your sample while injecting the same amount of sample. In that case the viscosity of your sample will go down and you won't experience the sudden pressure drop and hopefully the bubble...

[Uwe Neue]

I am not sure if I understand the problem. You say that suddenly the pressure goes down due to a bubble. I would think that this would be a bubble in the pump, but the pump does not see any glycerol. What am I missing?