By Agata Wroblewska on Thursday, May 20, 2004 - 06:00 am:

Help! How to detect shikimic acid by HPLC in the solution with glycerol, etanol and acetate?

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By Constantine Sychov on Thursday, May 20, 2004 - 06:22 am:

Hi, Agata! Do you have any problem? The standard system is - 250 mm C18 phase with high carbon loading, eluet - 10-50 mM phosphate buffer pH by H3PO4 to 2.6-3, DETECTION 204 (210) nm. Shikimic acid elutes after tartaric, as far as I remember

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By X.L. on Friday, May 21, 2004 - 01:44 pm:

I tried Acclaim OA (4x250 mm)column, shikimic acid elutes out after oxalic, tartaric, malic, formic,partically resolved from lactic, and elutes before acetic acid (baseline resolved). Conditions are 50 mM phosphate buffer at pH2.6 to 2.8, adjusted with phosphoric acid. Detection: UV, 210 nm.

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By Anonymous on Friday, May 21, 2004 - 02:51 pm:

Shikimic, glycerol, ethanol, and acetic are readily separated by dilute sulfuric acid eluent using any number of the available ion-exclusion columns on the market. Suppliers include Bio-Rad, Shodex, Transgenomic, Varian (Metacarb), Hamilton, Supelco, Alltech, etc.

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By X.L. on Friday, May 21, 2004 - 02:59 pm:

I should mention that Acclaim OA column is a reversed-phase silica based column. From the product information at Pittcon2004, 12 hydrophilic organic acids (oxalic, tartaric, malic, formic, iso-citric, lactic, acetic, citric, succinic, fumaric, and cis & trans aconitic acids can be baseline resolved isocratically within 10 min.

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By Constantine Sychov on Sunday, May 23, 2004 - 05:54 am:

Mmm, I carried this separation on 250mm LUNA C18. I think C18 phase must be 1) with rather high carbon loading 2)unendcapped

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By A.Mouse on Sunday, May 23, 2004 - 12:18 pm:

These are very polar compounds. As Constantine has mentioned, you need to work with a mobile phase that is fully aqeuous. Standard modern highly-coated C18 phases do not work well in such a mobile phase. There are several other possibilities of phases that a compatible with 100% water. One is an unendcapped phase. Another is a fully endcapped phase with a low coating, such as the Atlantis phase. The third possibility is a phase with an embedded polar group. I have seen claims (with examples) that the second type of phase gives the highest retention in water.

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By Constantine Sychov on Monday, May 24, 2004 - 06:30 am:

Hi, A.Mouse!

You mean that "low coating" is C8 coating or low carbon loading? If second - "the highest retention in water" looks like nonsence.
It seems to me, the use of phases with polar groups for separation of polar substances is overestimated by manufacturers. To separated acids rather cheap 300$ unendcapped C18 phases suit best. Average lifetime under mentioned above conditions is about a year.

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By X.L. on Monday, May 24, 2004 - 09:51 am:

As A. Mouse pointed out, a standard modern highly-coated C18 colums is usually not compatible with 100% aqueous, due to "de-wetting". If it didn't dewet, a C18 column should exhibit higher retention than the polar-embedded RP column, or the C18 column with hydrophilic end-capping or without end-capping, (provided that the same raw silica is used for all three bondings).

To test the aqueous compatibility of a column,the "stop-flow" test can be used. Here is one way to do it.

Condition: 25mM phosphate buffer, pH3, at RT to 30 C, flow rate - 1 mL/min, Detected at UV 254 nm.
Mix: cytisine, uracil, and thymine.

Protocol:
1.First wash the column of interest with 70%ACN/30%H2O for 20 to 30 column volumes
2.Then equilibrate the column with above aqueous mobile phase for 20 column volumes before injecting the sample mix.
3. Stop the flow for 10 to 20 minutes
4. Resume the flow with aqueous mobile phase.
5. Repeat 2 to 4 for more cycles and monitor the tetention time change.

In general, a C18 column with endcapping suffers 50% retention drop after the first cycle and a C18 column without endcapping gives more small retention time drop. A good column for this application should give highly reliable retantion, say retention time change is less than 3% after 50 cycles of stop-flow cycles. However, if you don't stop flow during use, non-endcapped or under-bonded C18 can last very long time before you can observe dewetting.

Last point is that "dewetting" is reversible if column bed is not mechanically disturbed. Purge the dewetted column with 70%ACN/30%H2O for some time will re-wet the pore, making the column useful again.

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By Uwe Neue on Saturday, May 29, 2004 - 10:52 am:

Sychov, I just made a similar statement in another post. Many standard C18 columns show less retention in water than in water with a bit of organic solvent. These columns also show the behavior just described by X.L. Unendcapped C18s, like some of these modern AQ columns or old-fashioned ones like Resolve C18, do not show this problem. Also, some modern C18s that have been specifically designed for the retention of polar compounds, such as Atlantis dC18, do not have this problem and give maximum retention in water.