Please can someone tell me whether I should be using ion pairing agents or pH to increase retention on a C18 column. At a pH of 3.5 and 140mg OSA, 5-HTP runs at 3min but L-DOPA runs at 1.4min (not very practical when I come to samples)

You may find some aspects on this problem in a recent thesis from Uppsala University written by Anna Törnkvist.

http://publications.uu.se/uu/fulltext/nbn_se_uu_diva-3306.pdf

Beside that, I imagine that HILIC is a suitable mode to get retention for L-DOPA.

thanks Einar. I checked Dr Tornkvist's thesis but it did not actually discuss how to manipulate the position of L-Dopa once you know where it is. I'm afraid I do not know what HILIC means.

The chemical name for DOPA is alpha amino beta 3,4 dihydroxybenzene. Would this suggest that it would ionise and therefore be susceptible to pH change or might it react with an ion pairing agent?

I have not made a lit search, but expect that there are numereous refs available.

Anyway, ion-pairing should be possible at an acidic pH value. Nowadays, I consider ion-pairing to be less interesting and often HILIC is an alternative.

In HILIC you use a hydrophilic stationary phase and a mobile phase containing a high content of polar organic modifier (commonly acetonitrile).

Typical isocratic conditions;
70 % ACN
30 % 2-5 mM formic acid

More info
http://www.sequant.com/products/2700.shtml

I would think that 70% ACN would not be the best choice for ECD.

Actually 70% ACN is fine for an ECD as long as your buffer is soluble.

Regarding the original question, you can certainly add more OSA or use SDS to increase retention times. To improve selectivity, increase the ion-pair concentration while also increasing your ACN concentration. This will cause the bases to retain longer while causing the (non-ion pairing) acids and neutrals to elute earlier.

You can retain your compunds without using ion pairing reagent on our Primesep columns. We don't haave this particular application but based on the structure of your compounds I believe you can do it. I am going to order DOPA and 5-HTP and try to develop a method on one of our columns. You can check the following link for separation of amino acids and other polar compounds without ion pairing reagent (retention time from 3 min to 20 min or more, depending on mobile phase.
http://allsep.com/makeChr.php?chr=Chr_020
http://allsep.com/makeChr.php?chr=Chr_009
You can visit our website at www.primesep.com for more details or call us. Or just send email inquire for you problem mail@allsep.com

Isn't TFA an ion pair reagent?

e.g.

Gustavsson et. al., J. Chromatogr. A, 937 (2001) 41-47

Ion-pairing properties of TFA became a typical misconception. TFA to some extend is ion-pairing but very poor one. Retention of polar compounds increases slightly with TFA vs. H2SO4 at the same pH on reverse phase column.
In article that you mentioned the authors compare MS ESI intensity with different fluorinated additives including TFA. In the table of retention factor they even didn’t show data for TFA. Even heptafluorobutanoic acid shows only 0.6 K for tyrosine on C18. I assume that TFA shows even less.
On Primesep 100 column for example tyrosine’s K=2.4 at H2O/MeCN/TFA-70/30/0.05 %. Clearly it is not TFA that is doing ion-pairing effect but column itself.
In addition, the TFA with negative charge will interact very poorly with stationary phase which is also possesses negative charge. Instead, the repulsion effect of TFA ions will be observed on Primesep acidic columns. Very quick equilibration of the column is observed when switching from one acid to another or when acid gradient is used d (1-2 column volumes only).

We have had so much trouble with TFA that I wonder why anybody would use it unless it was ABSOLUTELY necessary?? (The only thing that worked was the anal. of oxalic acid on graphitic carbon columns with TFA/H2O, and that only with a very tightly controled TFA conc.).

I used to use TFA with much success. Usually I get 100% more retention compared with acetate at medium pH to low H.

The TFA in our case (Primesep columns) is one choice among many. You can also use acetic, formic, sulfuric, phosphoric etc. acids and ammonium acetate or formate. You just need certain ion strength of mobile phase (usually 0.05-0.02% of acid or 5-20 mmol of ammonium acetate or formate). Visit www.primesep.com for more details.

AllsepTech, thanks for the thoughtful reply.

Jimmy: Double retention at the same pH? What´s your analyte? What about your additive concentrations? I have seen claims that TFA pairs (ion) with proteins, making them more hydrophobic...

I work on small molecules. Ocassionally we use TFA at medium pH to achieve required resolution. On one project, we used TFA and acetate buffer, pH 4.6. It worked well. Probably both acetate and TFA acted as ion pair. Without TFA, we would have had to use much more buffer, which would have higher background.

Jimmy

According to my data (unfortunately a bit scarce) TFA absorbs more strongly (at~250nm) than AcOH in aqu. solution. NaOAC absorbs less than HOAC, so matters are complicated. But: Do you, Jimmy, have some data on the background intensity versus concentrations of the two species?

Responding to the request on HPLC Method for L-DOPA and 5-HTP.
As I promised before to "Anonymous" person on this board, we have developed a method for separation of L-DOPA (3,4-hydroxyphenyl)-alanine and 5-hydroxytryptophan. It took us less than an hour to develop LC/MS compatible method. We kept in mind that some people did not like the idea of TFA in the mobile phase. So we employed ACN-water-formic acid. Here are the details

Column: Primesep 200 (4.6x150 mm)
Mobile phase ACN-water-HCOOH=20/80/0.1, 1 ml/min
Retention for L-Dopa 6.8 minutes
Retention for 5-HTP 13.1 minutes.
If you want to shorten you run time just go to shorter column or increase amount of formic acid or ACN in the mobile phase.
If you need actual chromatogram, please send your request to mail@allsep.com

For other applications using our mixed mode columns please visit www.primesep.com

How other amino acids retain at this conditions?

Cliu,
Please check our website for the retention of several amino acids using our Primesep 100 column. You can make conclusions by comparing your structures with the one in this application.

http://allsep.com/makeChr.php?chr=Chr_009

Anybody have a reliable method for the quantitation of serotonin in reaction mixture during synthesis (tryptophan-5-hydroxytryptophan-serotonin)? I have tried RP on C18 and need something better.

I guess that you don't get retention?

Check this link:
http://allsep.com/makeCmp.php?cmp=Cmp_100