Chromatography Forum

I know that 95% of labs working on derivatization and RP separation of amino acids use fluorescence or other types of detection, but what I've got at my disposal is just a good old UV.
Does anyone else look at Amino Acids by UV?
I'd be willing to trade advice on my method development for some graphics work (I've done some graphics for analytical chem articles, mostly instrumentation diagrams and concept images, some journal covers, etc.)

More details on my situation:
Looking to quantify taurine, arginine, lysine, and theanine in beverages.
I have an old PE series 200 HPLC which is capable of precolumn derivatization (but requires sample volumes of at least 400uL)
I've tried using OPA/3ME for derivatization, with borate buffer, using a gradient of phosphate buffer and organics mobile (45:45:10, ACN:MeOH:H2O).
I'm not getting any peaks, and I don't even know where to start in terms of improving things. It could be that the derivatization is failing, it could be the detection is not sensitive enough, it could be a separation problem, a whole host of things.

Any help you can offer would be greatly appreciated.
Many thanks!!

Courtney

What concentration are your amino acids? I've used the Waters AccQ tag system with UV absorbance instead of fluorescence, and it worked fine, but obviously not with as high a sensitivity.

Here are methods for analysis of underivatized amino acids without IP reagent and with UV detection:
http://www.sielc.com/Application-Amino- ... sules.html
http://www.sielc.com/Compound-A-Methylserine.html
http://www.sielc.com/Compound-Proline.html

Amino acids are retained by combination of cation-exchange and reversed-phase mechanisms. You can use any UV transparent acid and corresponding buffers and UV 200-210 nm.

We use the Waters picotag process (pre-column derivatization) and analyse by HPLC-UV.

We came out quite well in a recent collaborative study we participated in (participating labs analysisng the same samples).

I would avoid any post-column derivatization methods due to the variability.

If the direct UV detection is not sensitive enough the FMOC-Cl method was by far the best in my hands, also, one can buy most of the derivatized amino acids to use as standards for both UV and fluorescence.

If the direct UV detection is not sensitive enough the FMOC-Cl method was by far the best in my hands, also, one can buy most of the derivatized amino acids to use as standards for both UV and fluorescence.

From amino acids, only serine, tryptophan and phenylalanine are aromatic fragments with conjugated electrons. Other amino acids are visible only in the carboxyl group at 200-220 nm where the UV absorbing a huge number of substances.

So that direct UV is suitable only for mixtures of pure amino acids. But you ,ColaChemist , (seem to nick) work in area food products ..

We are talking about chromatography and not UV spectra. Chromatography is all about selectivity and the task is to find suitable column and conditions to separate compounds even if the all are "visible" at 200-220 nm. We developed a lot of methods for underivatized amino acids in various formulations and never considered "UV activity" of other components as an issue. UV method for underivatized acids is less sensitive then derivatization methods, but beauty of it is that you don't need to do an extra step and it is compatible with LC/MS and prep separations.

Interesting discourse, thanks for your replies.
I agree that in my case, with varying levels of matrix interference, it's probably not ideal to go without derivatizing.
I will also say that I'm loathe to adopt a system with proprietary reagents, as then I'm left at the mercy of the company distributing it, and have less ability to adapt it.

Question:
As a proof of principle before I try to optimize separation, I'd like to set up a standard UV experiment (no separation) to confirm I'm actually derivatizing, and that I'm using an optimal ratio. Anyone have suggestions on this? I'm concerned about the lack of separation, and even though I intend to scale this up quite a bit, is there any chance I'll be able to detect derivatization by a benchtop UV-Vis?

lmh: the concentrations will vary on the beverage, but I'm trying for the moment to look at concentrations between 100 and 500 ppm. I would hope I would be able to detect at these levels.

In running your experiments you will depend on ComEd to exclusively power your equipment, in water company to exclusively supply water and gas company to heat your labs and don't forget government to print money for your salary

A little Friday's humor.

If you get the FMOC standards as I said you run them, get peak(s), then run the aminoacids which you derivatized yourself . . . . It is that easy. You can also do this without a column, initially, but you have to be aware of "method peaks".