Separation of Glycerol and Sorbitol HPLC

Hello, I work at Dynalene Inc., and we have an Aligent HPLC. We have a raw material that contains glycerol and sorbitol and would like to use HPLC to get the concentrations of each for quality control. However, we have run in to a few problems and would like your technical assistance.

Experiment: First, glycerol was held constant at 1% and sorbitol varied in the range of 0-1% with the rest of the solution made the same as the eluent to make a calibration curve for sorbitol (x=weight % sorbitol and y=peak area). Then, sorbitol was held constant at 1% and glycerol varied in the range of 0-1% with the rest of the solution made the same as the eluent to make a glycerol calibration curve. Then, the raw material was diluted 40x to get sorbitol and glycerol in the 0-1% range and the peak areas from that HPLC were used with the calibration curves to get the concentrations of glycerol and sorbitol. This worked out well; however, we want to be able to test the final product which contains 2% of the raw material.

Unfortunately, when I tested the final product (no dilution) the peak areas were much higher than the 40x dilution of the raw material. 40x dilution of the final product still had higher peak areas than 40x dilution of raw material. 100x dilution of the final product still had higher peak area for the sorbitol peak than the 40x dilution of raw material, but lower peak area for glycerol. Still the concentration of glycerol would come out to around 50% (after multiplying by 100 to account for the dilution), when it should be less than 1%. Do you have any ideas as to what is causing this? What should I do to fix it?

Thank you,

Victoria Madison

  • Hi Victoria


    The higher peak areas for the product can really only come from a few places...


    Assuming that the product can't contain 50% glycerol, then could the additional peak areas come from another component of the product that co-elutes with your target analytes?


    You don't say which detector you are using, but could some method parameter have changed that causes the analytes to give a bigger response.


    Doesn't sound likely, but are you injecting a larger volume of sample of the product?


    Do the product and the raw material have the same viscosity?  Could you somehow be not injecting the volume of the standards/raw materials that you think you are because they are so viscous?


    Can you tell us more about the HPLC system and method?  What detector are you using?  What injection volume does the method use?


    As usual, more questions than answers!



  • Andy,

    Thank you for your response.

    The final product can't contain 50% glycerol, as it only contains 2% of the raw material that is part glycerol, sorbitol, and water. The final product does contain ethylene glycol, so it could be that elutes at the same time.

    The UV-VIS detector was used. No parameters were changed.

    The sample volume injected is always the same, 20 microliters.

    I don't think either the product or raw material are viscous enough for the full volume not to be injected.

    Hope the further information helps.


  • Hi Victoria,


    I would inject an ethylene glycol standard to see if that's what you're seeing... I suspect that is the problem you're experiencing!


    You haven't said what column and mobile phases you're using, so I don't know what changes you might be able to make to alter the selectivity of the separation.


    It may be that you need to choose a different separation mechanism.  I wonder if a separation on something like Agilent's HiPlex H columns would work for you, but I would recommend that you check with your local Agilent representatives.


    Kind regards


  • The mobile phase is 80:20 acetonitrile and water. The column is a Luna amino (NH2) column 250x4.6mm from Phenomenex. I can try ejecting an ethylene glycol standard either Friday or Monday. Thanks for your help!

  • Some other projects took priority, but I did finally get the chance to try that. It turns out ethylene glycol eluted at the same time as glycerol and benzotriazole eluted at the same time as sorbitol. So I will have to work on the method to hopefully separate all of them.

  • This question has been marked as assumed answered.

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