Nicodem
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HPLC analysis of polar analytes with reverse phase columns
Does anybody have any idea on how to use a HPLC with a Nucleosil C-18 reverse phase column for detection of relatively polar ring substituted o- or
p-hydroxybenzoic acids and other hydroxy- and dihydroxybenzoic acid derivatives?
The problem is in that the retention times are too low, only a little more than the solvent front which means I can't get any useful separation. I
have tried changing the mobile phase compositions at anything between 70% to 40% acetonitrile and it barely affects the retention time (not enough, at
least). I have also tried to use 0.1% formic acid instead water to keep the pH of the mobile phase lower but it has no influence. Therefore the low
retention times are not due to the analyte being completely dissociated. Any advice would be appreciated.
I know this is not the appropriate column for these type of compounds, but it is the only one I currently have available. I don't have much experience
in HPLC based analytical chemistry, otherwise I just use it to follow the reactions, but this time I would need it for product quantification for
which I need complete resolution of the peaks.
…there is a human touch of the cultist “believer” in every theorist that he must struggle against as being
unworthy of the scientist. Some of the greatest men of science have publicly repudiated a theory which earlier they hotly defended. In this lies their
scientific temper, not in the scientific defense of the theory. - Weston La Barre (Ghost Dance, 1972)
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Klute
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You could try switching to a methanol/H20 system, or even somewhat acidic buffers (phosphate) to "salt out" those hydroxybenzoic acids.
I can ask at work to people working with calixarenes and other polyphenols if they have similar problems.
EDIT: do have acces to the following article? You migh find ideal eluants for 3-hydroxybenzoic acid..
3-Hydroxybenzoic Acid as an Internal Standard for the High-Pressure Liquid Chromatography Quantitation of Salicylic Acid in Plants*1
Analytical Biochemistry
Volume 289, Issue 1, 1 February 2001, Pages 99-102
[url=
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45BC750-DN&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view
=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=600cd3cd7e923166935d59643a123b98]Link[/url]
EDIT2: more revelant litterature
parabens (you could consider esterifying your acids for further analysis?)
Determination of combined p-hydroxy benzoic acid preservatives in a liquid pharmaceutical formulation by HPLC
Journal of pharmaceutical and biomedical analysis
2004, vol. 34, no1, pp. 207-213
Abstract
This paper describes a reversed-phase high performance liquid chromatographic (RP-HPLC) assay method for the determination of combined p-hydroxy
benzoic acid (ethylparaben (EP), methylparaben (MP) and propylparaben (PP)) preservatives in a liquid pharmaceutical formulation. The chromatographic
separation was achieved with potassium phosphate buffer (pH 7.05)-methanol (47.5:52.5, v/v) as mobile phase, a Spherisorb C18 column (250 mm x 4.6 mm)
and UV detection at 254 nm. The analysis time was <8 min. The method was validated with respect to linearity, precision, accuracy, selectivity,
specificity and ruggedness. The calibration curves showed good linearity over the concentration range of 2-140 μg/ml. The correlation coefficient
were >0.9999 in each case. The relative standard deviation (R.S.D.) values for intra- and inter-day precision studies were < 1%. The procedure
describe here is simple, selective and is suitable for routine quality control analysis and stability tests.
Use of acetate buffer:acetonitrile for reverse-phase analysis of p-hydroxybenzoic acid:
http://books.google.fr/books?id=1c9dq_10r4IC&pg=PA591&am...
Identification and Quantification of Phenolic Acids in Macrotyloma uniflorum by Reversed Phase-HPLC
Link
(phosphate buffer:MeOH)
[Edited on 16-3-2009 by Klute]
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chemoleo
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MeOH is a good suggestion. I rarely use it because my things stick better. You could also try H2O/THF.
Something to try:
Use triethylamine HCl, i.e. at 100 mM, pH 6 or so (keep acidic), in both the aqueous phase and the AcN phase. It works by caging unsaturated compounds
(dyes etc) such that it has different retention properties.
Also you should definitely have a low pH, particularly with acidic compounds- I found that salts of acids stick very badly if the pH of the solvents
is not low, it ends up in the flowthrough. 0.1% TFA, 0.1 % heptafluorobutyric acid (the best for resolving something that can't be ressolved
with TFA) or 0.1% H3PO4 are all good acids that worked well for me for very acidic compounds.
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Ozone
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Yep. The TFA (or other acid) works by protonating any free siloxane groups you have. This prevents interaction of them with your analyte which
prevents tailing and sharpens the peaks (thus increasing resolution). The TEA can also be interesting (I have used a dicyclohexylamine:formic acid ion
pairing reagent to separate small carboxylic acids on C18). Take care, some C18 is not bound very well and pH ~2 will strip the phase off. The
stabilbond (Agilent) has a range from 2-10, which is great for phenols. I have had bad luck with other columns.
The solvent system will only effect the selectivity (a). What you have is a capacity (k) problem, and that is intrinsic to the stationary phase.
That's why modifying it (the stationary phase) might help. Your organic concentration is OK (<~1% organic and the phase collapses dropping your to
capacity to nil and everything will whiz right on through).
Still, the hydroxyphenyl acids are difficult (and a crummy old column will not help). My results with caffeic acid have been mixed
(3,4-dihydroxycinnamic acid) and difficult to reproduce.
There is also a temperature dependence (effecting the rate of mass-transfer in and out of the phase)! If your capacity is too low, try using a column
heater. Bring it up slow, there is usually a sweet spot ~30-40°C with C18.
Cheers,
O3
-Anyone who never made a mistake never tried anything new.
--Albert Einstein
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Nicodem
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Thanks a lot for all the good advices. I will try a few suggestions, MeOH/H2O, TFA or the suggested buffers, and so on, and see how it works. I will
consider anything that will give at least 30 more seconds of reproducible retention time as success.
I'm glad to see there are members with more experience in using HPLC here. Up to now I only used it as a bit of a more sophisticated TLC. 
…there is a human touch of the cultist “believer” in every theorist that he must struggle against as being
unworthy of the scientist. Some of the greatest men of science have publicly repudiated a theory which earlier they hotly defended. In this lies their
scientific temper, not in the scientific defense of the theory. - Weston La Barre (Ghost Dance, 1972)
Read the The ScienceMadness Guidelines!
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Klute
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HPLC is a wonderfull science in itself! I'm lacking decent experience with the more sophisticated equipement, as we routinely use GC rather than
HPLC, but I really like this technic, it really has a special charm I think. A bit tedious compared to GC though.
\"You can battle with a demon, you can embrace a demon; what the hell can you do with a fucking spiritual computer?\"
-Alice Parr
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unionised
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I may be missing something, but why stop at 40% ACN? Anyway, the move to MeOH will not only give better retention, but it gets round the shortage of
ACN.
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Nicodem
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I didn't try less than 40% because there was less effect on prolonging the retention time than I hoped for, while the pressure rose too high (due to
the viscosity of water). But since it's an old column I might just give it a try even if it collapses. It will at least give me an excuse to buy a new
one, since this one is pretty used anyway. By the way, what type of column would be most appropriate for such polar benzoic acids (possibly containing
also other polar functionalities like phenolic and aliphatic OH groups and amide links, and only one C6 to C10 lypophilic hydrocarbon group)? I'm
nearly clueless about various column types and their appropriateness for various analytes.
PS: Acetonitrile shortage does not affect my lab, yet.
Klute: I wish I could use GC instead, but unfortunately my products are not volatile at all and they commonly decompose on heating. Otherwise, GC is
so much better. It has practically no problem with lack of resolution and the FID detector gives the product ratio almost identical to the molar one
as long as the compounds have similar MW (not to even mention the possibilities whn having a GC-MS). Certainly the method of choice... if your
products are volatile enough and thermally stable. 
[Edited on 18/3/2009 by Nicodem]
…there is a human touch of the cultist “believer” in every theorist that he must struggle against as being
unworthy of the scientist. Some of the greatest men of science have publicly repudiated a theory which earlier they hotly defended. In this lies their
scientific temper, not in the scientific defense of the theory. - Weston La Barre (Ghost Dance, 1972)
Read the The ScienceMadness Guidelines!
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