Hi,

I'm a graduate student trying to separate C4 to C10 homologous series of n-alkylamines on RPLC. As the UV detection does not work for alkylamines, what type of detector should be used?

One of the HPLC column catalogs suggests "fluorimetric detection: 7-60/7-54, 3-72/4-76 filters". I would be grateful, is someone can answer what this means.

Raj

Raj,

The easiest way to separate and detect amines if you must use LC for their separation is by derivatization. An old and easy to use standby is OPA (ortho phthalaldehyde). The procedure can be found in many texts and probably on several vendors websites. These give both a UV absorption and a fluorescent method of detection. There are other reagents to form other derivatives, again try some of the vendors websites such as piercenet.com, alltechweb.com, and sigma-aldrich.com. Hope these are some good leads for you,

Regards,
Mark

Mark,

Thanks for the leads - will follow them up. However, there is one hitch... what happens if I intend to inject the RPLC eluant into the MS? The noise due to the derivatizing agent would be too high! Guess - I need to split the flow to the MS before derivatizing the eluant.

Anyway, thanks for the time and consideration.

OPA derivatization of amines can be performed prior to injection. Some of the derivatives are not terribly stable but autosamplers with the capability of mixing reagents just prior to injection reportedly provide excellent results in such applications (this is hearsay, haven't tried it myself). I understand that Agilent supports determination of amino acids with their instrumentation using this technique so you might want to contact them. Of course, the other option would be to use conductivity detection. In this case no derivatization would be required. Generally, in this case the analytes are better separated by ion pair chromatography or cation exchange chromatography.

Hi,

One more question on these alkylamines separation... I tried to separate them in RPLC with water/acetonitrile/acetic acid and used post column derivatization with OPA and was able to detect them with a UV detector at 340nm - the problem was severe tailing!! Will substituting acetic acid with TFA solve the problem.

If there is no acid in the water/ACN mobile phase - will it aggravate the tailing problem?

Raj,
Since you don't want to add anything to your mobile phase, you could try electrochemical detection. You might get by with no changes to your eluant. Ion exchange methods usually require addition buffers and ionic strength adjustors. Electrochemical detection sometimes has a step curve in developing methods. Don't give up easy, set your instrument so you can change parameters and shoot injections quickly, e.g., take out the column during development.
Jim

You should not get tailing with a good modern HPLC column and simple formic acid as the buffer. BTW, why do you think that the tailing is from the column and not from your post-column derivatization device?

Raj,
Uwe has a point. Tee a pump between the column and the PCR and double the mobile phase flow. If your problem gets better you have void-volume mixing in the PCR.
Jim

Jim and Uwe,

Thanks for the leads... will check it out...

Jim and Uwe,

The expt did not work!! With a RI detector, I found that amines are not retained much on a RP column. So separating them in RPLC is not easy.

However, I have one more question:- How does one go about detecting aldehydes? I checked with UV detector and they dont show up. Doesn't the carbonyl group act as a chromophore?

I would try DNS-Cl, DBS-Cl or FMOC-Cl derivatization prior to injection. None of these reagents require severe conditions and they work well in water, give a strong response on UV and fluorescence. Also they are easy to separate on almost any C18. The hydrolysate of the reagent elute near the void volume. Tosyl chloride works the same way. For more details see Handbook of derivatives for chromatography (pages 159-205).
For aldehydes I would try phenyl hydrazine derivates, chapter 6,8,9 in the same book.

If thye are not retained enough, use less acetonitrile.
Carbonyls will show up in the low UV, but derivatization gives you more sensitivity.