Peaks combining?

Hello,We're trying to set up a method that can simultaneously analyze the 12 water soluble vitamins.  So far our best one produces an interesting phenomenon.  When Vitamin C is run at 1 microL inj of 0.05 mg/mL, a single peak is observed at 0.9min.  This peak has an area of ~300,000 and a height of 0.14 AU.  When Thiamine is run at 1 microL inj of 0.05 mg/mL, a single peak is observed at 0.5min.  This peak has an area of ~550,000 and a height of 0.50 AU.  The interesting part is when 1 microL inj of a mixed sample of 0.05 mg/mL VitC + 0.05 mg/mL Thiamine is run, there is a single peak observed at 0.5min.  It's area is ~900,000 and height is 0.85 AU.  Any thoughts as to what might cause this?Attached are chromatograms of the 3 runs, as well as gradient details.  A is Water + 10mM Ammonium formate + 0.1% Formic acid.  B is MeOH + 10mM ammonium formate + 0.1% formic acid.Thank you


  • Have you considered the possibility that thiamine is binding ascorbic acid?  Perhaps some change in pH would separate them.  Also you might want to look at different wavelengths for analyzing the two.

  • Thank you for the help.  We attempted another method which replaced the two mobile phases with equivalents lacking 10mM Ammonium formate (so meOH + 0.1% formic, water + 0.1% formic) and we get seperation with Thiamine at ~0.5min and vitC at ~1min despite them combined in matrix.  We've also since identified additional vitamins using this method; Pyridoxine, Thiamine, Ascorbic acid, Folic acid, PABA, Riboflavin, and nicotinamide.  Biotin and pantothenic acid were also attempted but did not seem to absorb at this wavelength (270).  Hope this helps future vitamin-seekers.

    Column: HSS 100mm

    Temp: 40C

    Inj: 1microL of a 0.005 mg vitamin/mL cocktail


  • Biotin and pantothenic acid have virtually no absorbance at 270nm. Attached are UV spectra for the two of them. You can see them at 205nm.

  • Hmm, we actually did try that using dual channel 270/205.  The 205 had horrible background noise, probably because methanol absorbs readily at 205.  The original concentration for the cocktail (0.005) was chosen based off a Waters method.  Standard additions (where concentration of a particular vitamin was roughly doubled) to cocktail did not seem to "bump up" any peaks in 205.  Attached are chromatograms of the cocktail, cocktail+Biotin, and cocktail+pantothenic acid at 205 nm.

    Thank you all for the help so far,


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