Hi,
I have a question about using HPLC to measure the ascorbic content in fruit/vegetable samples. The samples are fruit/veg puree and are treated with metaphosphoric acid (and metaphosphoric acid AND ethanol for Method 2) before they are centrifuged and filtered.
Please suggest what do you think is the best way get the ascorbic acid peak to appear cleanly/separately.
Method 1:
Mobile phase A: DI water + 0.1% TFA
Mobile phase B: Acetonitrile
Time A B
0 100 0
4 min 97 3
6 min 85 15
15 min 80 20
Flow rate 1 ml/min
sample method1 spike.pdf
sample method1 no spike.pdf
Note: the ascorbic acid peak is around 2.25 min.
Method 2:
Single mobile phase, phosphate buffer at pH 2.7
Flow rate 0.2 ml/min
0.2_0 ul.pdf
0.2_10 ul.pdf (spiked sample)
Note: in the files for method 2, the peak for ascorbic acid is the one at 11 min NOT the earlier peaks.
We use a rp column and an isocratic elution with 0.005 molar potassium dihydrogenphosohate adjustet to pH 2.3 with 85% ortho
Danke, Markus. Did your chromatograms come out cleanly? or was there other peaks merging with the ascorbic acid peaks? any papers I could refer to?
It came out clearly. We had no further interferences. We published results based on this method in Mitteilungen Klosterneuburg. But no further details on the method. Which column do you use ? Rp ?
Reverse phase, 4.6 mm x 15 cm Waters column and HPLC system. What was the column diameter you used coz it'll affect the flow rate and ascorbic acid comes out very fast. Your method sounds closer to the method 2.
What was the extraction procedure? because the method 2 was using metaphosphoric acid and then ethanol (with lots of centrifuging and filtering).
We could discuss in more detail via email at [email protected]
I have to really thank you for this! :)
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