Analysis of organic acids using HPLC-PDA

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

26 posts Page 1 of 2
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Not likely. A C8 column has a totally different retention mechanism (reverse phase) than columns which are used commonly used to resolve organic acids. These columns are typically a polymer of Divinyl Benzene Styrene (DVBS) in the Hydrogen form. See the Waters/Agilent/Phenomenex websites.
AOAC method 986.13 for organic acids analysis involves the following:

Columns: A 250x4.6-mm column and a 150x4.6-mm column connected in series.
The columns are ODS-1.
Mobile phase (isocratic): 100 mM KH2PO4, pH 2.50
Flow rate: 0.4 ml/min (with 5-µm, 100-Å material, the backpressure was 1700 psi).

ODS-1 is nonendcapped. Endcapped versions of C-18 are fine except for the separation of tartaric and quinic acids; separation of these requires a nonendcapped material. The above combination affords a good separation of tartaric, malic, and citric acids (in order of elution). I don't know about ascorbic, benzoic and sorbic acids. This method was in regular use by a food analysis lab of my acquaintance.
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
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No; C-18 only, I'm afraid. Whether or not it's endcapped depends on whether or not you ever expect to have to analyze quinic acid in the presence of tartaric acid, as in cranberry juice.

The AOAC method's mobile phase is 100% aqueous; no organic solvent is used.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Deleted
Since this is an isocratic method, then using just the column that's 25 cm. long will result in separations that are 62% as good as what you'd get with 40 cm of column length (= a 25- plus a 15-cm column). Since you seem to be resisting the purchase of any new columns, then my advice is to try the 25-cm column alone. If you're getting some separation but want to get better separation, then you can always go ahead and buy that additional 15-cm column later.

I expect that the AOAC method was an ad hoc one that some guy in a lab developed with whatever columns he happened to have on hand. It worked, and he proceeded to enshrine the method.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Deleted
Andy Alpert wrote:
I expect that the AOAC method was an ad hoc one that some guy in a lab developed with whatever columns he happened to have on hand. It worked, and he proceeded to enshrine the method.


I always figured similar for most USP, ICH other "official methods".
The molar extinction coefficient at 254 nm would be quite low for most of these organic acids, and I'm surprised that anyone would recommend it. Detection at any wavelength between 210 and 230 nm would be OK.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Deleted
Detection: Do you mean 210-400 nm all inclusive using a diode array detector? There's no point in doing that. Any single wavelength in the range 210-225 nm would work.

It's possible that all of your standards would be resolved using a particular column of the type that you describe, but you should get at least some separation between some of them. Were they all retained and just coeluted? If they weren't even retained, then I'd try some other company's C-18 column or else check to make sure that the column you're using really is a C-18 column. That could involve a call to the manufacturer.

Regarding the baseline noise: Is your amplitude of detection set to a very sensitive setting? That shouldn't be necessary. If not, then there may be some problem with your detector or some other instrumentation. The easiest way to troubleshoot that would be to put your column on a different HPLC system entirely and see if the detection problem disappears. If it does, then you can start to troubleshoot the original system systematically.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Deleted
All of these compounds have carboxylic acid groups and will absorb light adequately in the range 210-230 nm. There is no point in screening over the wavelength range 192-400 nm. This is a standard AOAC analysis, not a research project!

Your concern about sensitivity and concentration of the analytes is misplaced until you get them separated from each other. It sounds as though you're able to see peaks for them. The fact that you "guessed" that the components of the mixed standard all coeluted means that you haven't run them individually. I would do that next, in order to ascertain exactly what's eluting when and to confirm that I can detect it at all with your current instrumentation.

Where did you get this mixed standard from?
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Deleted
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