Peak split on only 1st compound

[Mike H.]

What are the less common reasons the first peak of a gradient could split? We're running a basic TFA/MeoH MP gradient and are seeing that the first peak splits. MS suggests it's one compound. We are using MP as the diluent. Also we have noticed that if we drop temp. to 40° from 55°, the peak split goes away. Also we have eliminated degradation since we aged a sample at 55°for 30 min and tested it. It splits at 55°, but doesn't at 40°.

How likely is it that the MP temp being ambient and the column temp being 55° could cause a problem? Has anyone experienced this? If so what did you do about it? thx

[Gerhard Kratz]

Temperature difference MP vs. column temperature can cause that. MP bottles in an water bath set at the same temperature will eliminate that problem.

[Vlad Orlovsky]

Your peak might be too close to the void and you have a mismatch between your diluent and mobile phase initial point of gradient. I would suggest to move peak or/and may be try to inject less.

[Gaetan Glauser]

Do you have an active pre-heater ahead of your column?

[Mike H.]

No, but it actually was one of the common problems. Solvent diluent was too strong for MP. After I switched to MeOH/Water no peak split. I'm an idiot.

[Consumer Products Guy]

No, but it actually was one of the common problems. Solvent diluent was too strong for MP. After I switched to MeOH/Water no peak split. I'm an idiot.

Good.

Smaller injection volume also can remedy this. We frequently extracted products with solvents "stronger" than the mobile phase.

[Vlad Orlovsky]

We sometimes prepare a much higher concentration of sample is some totally different than MP diluent (waxes in hexane, lipids in THF/EtOH) and inject 0.5-2 uL. Mixed-mode columns can tolerate a bigger discrepancy between MP and diluent.