I have gotten the Vitamin C to stick to a C18 using methanol at 0.5 mL/min, but it always comes out in a blob when I run a food sample. I have set the UV to 243, 250 and 260 to try and phase out the blob and leave the Vit C, but no dice...
Hi! We analyze vitamin C in juice using buffer pH3.2: acetonitrile (95:5), and detection at 265nm and get nice peaks. You could try!
Hi! We analyze vitamin C in juice using buffer pH3.2: acetonitrile (95:5), and detection at 265nm and get nice peaks. You could try!
Hi, I used 0.1% formic acid as mobile phase to determine vitamin C in foods (fruits and vegetables). If you need more information you can send me a message.
I send you my article so you can read it.
Article An improved and fast UHPLC-PDA methodology for determination...
What is your food matrix? you may need some clean-up prior to injection.
You may use a Hydrophilic Column and refer to "Yuegang Zuo, et al. (2014) Hydrophilic interaction liquid chromatography: Fundamentals and applications. In: Yuegang Zuo (Eds.), High-Performance Liquid Chromatography (HPLC): Principles, Procedures and Practices. Nova Science Publishers, Inc."
Vitamin C (ascorbic acid) is water soluble. It is essential to use aqueous solvent (buffer) to get it retained on the column. There are few columns that offer adequate retention w/o an ion-paring reagent. We use the Synergy Hydro RP with monochloroacetic acid pH 2.7 and ~3% methanol or acetonitrile. 243nm is the optimum wavelength. Samples should be extracted into aqueous solution. Dithiothrietol or TCEP should be added post-extraction as reducing agent to convert the dehydroascorbic acid to ascorbic acid to measure the total.
By using a sodium dihydrogen phosphate buffer (100 mM) adjusted at pH 2.5 as mobile phase, UV detection at 226 nm and a specific column for organic acids we have obtained good results for vitamin C analysis in foodstuff.
You should look at the following application note:
http://kb.mtc-usa.com/article/AA-00832/0/
Can use zorbax column with 0.1 % orthophosphoric acid in water as mobile phase A and acetonitrile, as mobile phase B. Draw the calibration curve of standard ascorbic acid and run the diluted sample.
On the C18 column (254nm), you could try a mobile phase at pH ~2.6 with 0.2% metaphosphoric acid/methanol/acetonitrile (90:5:5, V/V/V) at 0.7 mL/min and 25 degree Celcius. The stability of vitamine C from the C18 column should decrease with increasing temperature. This may take care of the problem. See the attached papers.
Which HPLC column are you using?
Which food matrix do you have?
You definitely need to do some sample clean-up ahead of time. Solid Phase Extraction can be helpful. I have attached a method summary for solid phase extarction of vitamin C in some food matrices. If you need more details and more assistance regarding your specific food matix, email Agilent sample preparation technical support team at "[email protected]".
Another link!
http://www.nutrientdataconf.org/PastConf/NDBC17/6-1_Vanderslice.pdf
Try with Aminex HPX-87H BioRad. It's very good. Movil phase H2SO4 0.005M @220 nm
This Paper may be useful for you.
http://www.ccsenet.org/journal/index.php/jfr/article/download/16740/11224
Roche method for determination od Vit C in Food is Photometric Titration with 0,1 N Jodine Solution. If you are interested I can send you the protocol. BR
Vit C determination by HPLC as presented in "ASEAN Manual of Food Analysis" may help in solving your problem. Please see it in http://www.inmu.mahidol.ac.th/aseanfoods/doc/ASEAN%20Manual%20of%20Food%20Analysis.pdf page 141. Best wishes. Prapasri
Dear Jonathan,
quantification of ascorbic acid by HPLC is affected by on-column degradation of it into dehydroascorbic acids.
See http://www.sciencedirect.com/science/article/pii/S0889157504000262 (Chinnici et al., 2005) for further information. There are some tips that could help the correct determination of ascorbic but they are not fully decisive. Regards
Dear Mr. Decenzi, vitamin C is very unstable. It suffers degradation on line in liquid chromatography. If you measure at 260 nm or major wavelenght, the ideal pH is above 5.0. On the order hand, at 243 nm, the best pH is 2.0. We published an article in Analytica Chimica Acta that may help you. The article is: "Use of column with modified silica for interfering retention in a FIA spectrophotometric method for direct determination of vitamin C in medicine". Although, the method was applied in flow injection analysis, it can be adapted for liquid chromatography. You can download in my profile. I wait to have helped you.
Dear Decenze,
please see the attached file" Vitamin C quantification using reversed- phase ion pairing HPLC", may be useful for you. Regards
Dear Decenze,
For C18 reversed phase column, you can try with the mobile phase: Acetonitrile/ 10mmol/l Phosphate buffer(pH7.0) = 50/50, with a flow rate of 1.0ml/min and detect UV210nm.
Hello. I believe that you may need to do some sample prep. Attached is a paper that may help.
Working in the food manufacturing industry I have used the above sample prep using the metaphosphoric acid for pH and taka diastase which breaks down the starches in food. I also used a Carbohydrate LC column to analyze. Vitamin C is unstable and will degrade of a short period of time, so analysis in a few hours of preparation is important. Calibration standards will also degrade quickly so using a new curve every time is important.
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