Peak analysis questions

[Cheeba333]

Hi,

This is my first time in the forum so I am not sure if this is the right place to ask this question. If not, it would be nice if you could direct me to the proper subforum.

So I am trying to analyze the ascorbic acid content of a sample which has been extracted using a 2.5% Metaphosphoric acid buffer in methanol. For my analysis, I ran my extraction buffer as a blank besides the samples themselves and the standards. The blank yields a peak that has a retention time close to the retention time of my standard. What is puzzling is that the peak from the blank run disappears when the standard (which has been diluted with the buffer) is analyzed, but reappears when I am analyzing the samples themselves. What could be causing the issue? Thank you

[dap]

Try to eject minimal volumes of this solutions and compare results. Imho the solvent is too acid and/or too organic. Please also post chromatography conditions.

[Cheeba333]

I've attached a photo of the comibned chromatograms from my blank (blue), standard (red) and a sample (green) for reference.

http://imgur.com/a/QPExV

Here is an image of the blank chromatogram http://imgur.com/a/pHAmM

As for the conditions, I am using C18 reverse phase column (fitted with a precolumn), with 0.1% formic acid as weak solvent and 100% methanol as the strong solvent. The flow rate is 1.2ml/min, with a gradient elution. Sample injection is set to 20ul and detector is set to 245nm.

[dap]

Please try 2 uL of same solutions.

[HPLCaddict]

I'd suppose the peak at ~1 Min. is the solvent front. The ascorbic acid in your standard elutes at ~1.35 Min which is way too early. This correspond to a capacity factor k of less than 1. For "decent" chromatography, the capacity factor should be at least 2 - meaning with a dead time of ~1 Min the retention time of your analyte should be at least 3 Mins. You CAN get away with less than that, but this might cause all sorts of trouble - like peaks being very sensitive about the sample solvent, which probably is the case here.
Apart from that, the peak shape of your ascorbic acid peak in the standard is far from being acceptable.
That said, reversed-phase with a C18 and formic acid/methanol is not really optimal for such a polar molecule like ascorbic acid. You might want to try ion-pairing or HILIC. I'd suggest to browse the application databases of column manufacturers, there are literally hundreds of protocols out there.