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HPLC

Now this may sound like an incredibly stupid question, but I've run a load of HPLC samples, and got the chromatograms and values for each sample. My problem now is, where on earth do I start with analysing them?

Any helping hints or starting points are massively appreciated :smile:

Reply 1

cpchem

Original post by ManicMetalhead

Well, it depends on what you've been HPLCing and how, doesn't it?

Normal-phase, reverse phase? What compounds have you been running? Visualising by fluorescence at 254nm?

Reply 2

ManicMetalhead

Original post by cpchem

Well, it depends on what you've been HPLCing and how, doesn't it?

Normal-phase, reverse phase? What compounds have you been running? Visualising by fluorescence at 254nm?

Capsaicin (hot compound in chilli's)

Reverse phase,
UV detector set to 280nm
1ml/min
7 minute run time

Ran a few standards of pure capsaicin, and then made up and ran solutions of each chilli I had.

What else is there?

Reply 3

cpchem

Ah, so you're doing quantitative work rather than trying to work out what compounds you've got. Nice one.

So, if you've done several runs of pure compound at varying concentrations, you should be able to plot a calibration curve (concentration of capsaicin against the integral of the peak).

Reply 4

ManicMetalhead

Yeah, the calibration curve I can do, I just wasn't sure what to do with my results afterwards. I kinda get to the calibration curve, then hit a block and don't know what to do from there.

Reply 5

cpchem

Original post by ManicMetalhead

Yeah, the calibration curve I can do, I just wasn't sure what to do with my results afterwards. I kinda get to the calibration curve, then hit a block and don't know what to do from there.

Then you start working in reverse. You've got samples of unknown capsaicin concentration, but you know (from the HPLC traces) the peak integrals. Use your calibration curve to go backwards - from the integral of the capsaicin peak, you should be able to read a concentration from the graph.