Chromatography Forum

I see. I advise you to order some or all of those standards as individual chemicals so you can ascertain what their individual retention times are and determine whether or not you can detect them using your current instrumentation.

Do you intend to analyze cranberry juice? If so, then:
a) You'd better get a standard of quinic acid to analyze, and;
b) Get a nonendcapped C-18 column which which to analyze it.

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Check the retention times of the individual standards first.

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Hi Fruitjuice,

It is my opinion only; perhaps it is time to "Cut Bait" with the 4.6mm * 250 mm Altima 5um C8 column. From what you said above, you are observing peaks for malic (3.272min) and ascorbic (3.186min) acids that are far less than at retention factors of 2 (my calculation is that the values are 0.16 and 0.13 respectively), thus you are not separating either malic or ascorbic acid from the column void volume. Chilling the column may help, but it is not likely that it will help enough, so-to-speak.

Perhaps trying a Bio-Rad Aminex HPX-87H ion-exchange HPLC column at 60 degrees Celsius with 5 mM sulfuric acid as mobile phase would help--I've run this method myself for a subset of the analytes you're working with.

Another possible set of conditions to start with are here, from Agilent:

https://www.agilent.com/cs/library/appl ... 8720EN.pdf

I found this also, interesting pdf file:

http://www.cfs.gov.hk/english/programme ... s_2370.pdf

on page 12 is suggested using, in series, a C18 mm guard column, a 150 mm C18 analytical column, a 250 mm C18 analytical column and a Dionex OA (Organic Acids) column. Perhaps this is nearly the same as AOAC 986.13, I cannot ascertain this for myself. Seems a bit crazy to me to connect four columns to accomplish this separation, so I kept on looking for other alternatives...

...Restek's Allure Organic Acids phase seems also to be promising:

http://m.restek.com/pdfs/59530.pdf

http://www.restek.com/Technical-Resourc ... AR2062-UNV

These last two options seem to me to be best, though perhaps the Agilent column could be worked with a bit as well. Best Wishes in your continued method development!

Fruitjuice, you seem to be resisting mightily the implementation of the AOAC method that I listed earlier which works for most of these organic acids. If you want to reproduce someone else's results, then use their technique. At this point I suggest that you buy a nonendcapped C-18 column and try the separation with that.

@ Andy Alpert...your idea is reasonable to me, too. Just would like to have a look at the AOAC method for myself...the method I found above using the four columns seems a bit crazy.

Yes, why re-invent the wheel?

Glad you liked my suggestion. My post of Oct. 16th describes the AOAC method in detail.

@ Andy--my thanks!

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Fruitjuice, what the above two people are not stating outright, but which is crystal clear from your post and questions, is that you have absolutely no idea of what you are doing. Sorry to be so blunt, and I think you already know this, but you clearly have no formal training in HPLC analysis or method development and just seem to be going about this in a rather random way. At this time, you do not have the experience or training to understand what questions to ask or even how to interpret the advice being given to you. The fact that you do not yet understand the basics of how flow rate effects retention or what a "wavelength" is, or how temperature or column choice effect retention and/or separation simply means that you need both training and help from someone at your company with far more practical experience. No one on this group will be able to help you until you first understand the fundamentals of HPLC and learn how to follow a procedure (a pre-developed HPLC method in this case) provided to you.

We see this issue come up all the time on this and other web forums, but many members ignore the obvious clues and flood the untrained person with too much info that they are not ready to process yet. We could better serve these members, by instead suggesting they first read one of the classic books on HPLC analysis and most importantly.... work with someone local (at their company) to get some basic training before starting to run an analysis that they do not yet poses the skills needed to be successful yet. We all started out ignorant at one time. That is not a criticism, but a fact.