Hello everyone,
I've been struggling the whole week to seperate a pic of Vitamine C, i will explain :
I am supposed to determine the quantities of ascorbic acid (vitamine C) , malic acid and citric acid , i obtain a pic of the sample that looks like two pics bonded.
I tried to contaminate an ascorbic acid standard de with once malic acid and once with citric acid, i've got the same pics as ascorbic acid ( nothing changed except the area cause i added a volume to it )
I've also injceted malic acid standard alone and citric acid standard alone but didn't obtained a proper pic ( probably not the suitable mobile phase)
My conditions were :
Device : UHPLC
Mobile phase : MeOH /KH2PO4 (80:20)
Column : C18
Flow : 0.24 ml/min
Injected volume : 0.7 ul
Wavelenght : 257 nm
I hope that i've been clear. Waiting for your suggestions
Hi Amel,
try a ProntoSIL C18 AQ column:
https://www.bischoff-chrom.de/prontosil-c18-aq.html
Regards
Markus
Your current HPLC method is not selective for the different samples. What is your Tzero? What is your K prime for ascorbic? Should be > 2.0. Looks like too much organic phase to start with. Everything is probably eluting at the Tzero.
Try a gradient, pH 2.5, starting with ~ 70% buffer / 30% organic then slowly increase organic over time. Be sure and use a DAD to monitor all wavelengths from ~ 210 to 300nm (so you do not miss any of the samples).
In the future, when asking questions about HPLC analysis, please include the detailed column information (length, ID, particle type and size, column full name and type), plus sample concentration and the brand and model of your HPLC system too. This basic info is always needed to discuss these matters. Thanks.
Dear Amel Benabidi , a regular RP column might not be the best of all choices to do the separation. I would put my money on using a column of a different - optimized for the purpose - selectivity. Such as a column of the type Acclaim OA (OA: organic acids). I even found the separation you are looking for under the URL given below.
I hope this helps.
All the very best for your research
Detlef.
See this application:
https://appslab.thermofisher.com/App/1653/fast-analysis-organic-acids-orange-juice-on-a-thermo-scientific-acclaim-organic-acid-oa-column
Check the websites of Waters, Agilent, Phenomenex, and Restek for methods for small organic acids. However, the chromatographic conditions for UPLC are not the same as regular HPLC. While the wavelengths are the same the mobile phases are stronger. You will have to experiment!
For reverse phase chromatography unless you are working with a refractive index detector or a size exclusion column starting your method development with isocratic conditions makes the process more difficult. Start with a gradient of water and either acetonitrile or methanol and a C-18 column and work your way towards a good separation. Use as little organic as the column will tolerate as your starting point. You may have to buffer your aqueous solution to get good separation.
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