By L. on Friday, July 30, 2004 - 11:53 pm:

Hi. I am currently trying to separate a racemic mixture of a compound on a chiral column. However there are three peaks, instead of the expected 2 peaks.

My sample had been sent for numerous analysis and proved to be of high purity. Is there any other reasons for the existence of a third peak, besides sample impurity?

Thanks in advance.

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By Zelechonok on Saturday, July 31, 2004 - 05:22 am:

It is possible that you molecule has more than one chiral center.
In this case you may see more than two peaks on chiral column.

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By Constantin Sychov on Saturday, July 31, 2004 - 10:08 am:

If you use UV-detector even small admixtures can cause intensive detector response. For example, when analyzing reaction mixture of phenylallylmethanol, the product is just two little peaks near high peak of benzaldehyde (however benzaldehyde is admixture).

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By B.Buglio on Saturday, July 31, 2004 - 05:25 pm:

Could you be looking at a meso compound
(asymmetric cabon w a plane of symmetry)? In such
a case you would see three peaks (all having the
same elemental formula).

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By L. on Sunday, August 1, 2004 - 11:00 pm:

Hi. My compound is an acid, has only one chiral center and is not a meso compound.

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By L on Sunday, August 1, 2004 - 11:08 pm:

Hi. My compound is an acid, has only one chiral center and is not a meso compound

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By L. on Sunday, August 1, 2004 - 11:12 pm:

Sorry for the double post.

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By anon on Monday, August 2, 2004 - 02:28 am:

What about the UV spectra of the peaks? Do you have acceptable retention and the peak shape for every peak?

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By B.Buglio on Monday, August 2, 2004 - 06:16 pm:

Your latest info eliminates my idea. For
clarification tho - was suggesting
that the compound might have a d,l AND meso form

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By anonymous on Tuesday, August 3, 2004 - 12:12 am:

There is a possibility for rotational isomers in your compound?

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By b on Tuesday, August 3, 2004 - 12:15 am:

Ther is a possibility for rotational isomers in your compound?

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By Zelechonok on Tuesday, August 3, 2004 - 06:09 am:

Try to reduce or increase concentration/injection volume of your sample. See if peaks ratio would change. If it does then you may have injection problem.

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By L. on Tuesday, August 3, 2004 - 08:06 pm:

Anon: Retention and peak shape are acceptable.

B.Buglio: I don't think my compound has a meso form.

b: I don't think there's possibility for rotational isomers in my samples.

Zelechonok: Thank you for the suggestion. I will try that.

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By Anon on Wednesday, August 4, 2004 - 02:32 am:

UV spectra of peaks?

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By abc on Wednesday, August 4, 2004 - 06:22 am:

Hi,
Please answer:
Why the peak elution order change in HPLC separation of chiral compound by increasing the temperature

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By Zelechonok on Wednesday, August 4, 2004 - 07:37 pm:

Chiral separation usually involves complex interaction of the analyte with embedded chiral selector. There are several different molecular forces involved. Each force can have different temperature dependency. As result resolution of optical isomers is temperature dependant and seldom switch of elution order is observed.

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By Benjamin on Tuesday, August 10, 2004 - 05:59 am:

Dear L.

There is another possibility that has not been mentioned in this discussion. Sometimes molecules present a special type of Cis-Trans isomerism due to restricted rotation around a particular bond, such as the case of Proline-containing aminoacids. In these cases the molecule seems to yield multiple signals even though chemically can be of high purity.

Something you may want to try is to collect each peak obtained in your separation and reinject each one separately. If you see multiple signals of similar Tr's (compared top original separation) in one or more of the fractions, then it is likely your molecule has type of isomerism.

Good Luck,

Benjamin