Chromatography Forum

Hello

I'm doing an small asignment on HPLC.
The object of the asignment is to look at several methods of analysing amino acids in food by HPLC, and see if I can find any similarity between the methods. As in what kind of column is used, detection etc.

I have found some articles, but not many. If any of you know about some intersting web pages, and maybe tell me a bit about what is important to add in the discussion when it comes to comparing.
(the main object of the asignment is to show that I understand the basics of HPLC, not the aminoacids)

My problem is that this asignment must be handed in this Friday, and because of lot of other work I havent had the time to start this.

Hope there is someone here who could help me with this:)

Thanks

Regards
Giti

Giti,

There are lots of different ways to analyze for amino acids which can be grouped into four basic methods:

1. HPLC separation by with direct detection of the amino acids (two examples of this can be found at these links: http://www1.dionex.com/en-us/columns_ac ... s4732.html and http://www.alltechweb.com/productinfo/t ... /0040e.pdf )

2. HPLC separation with derivatization after the separation to render amino acids detectable by absorbance detectors (an example can be found at this link: http://www.pickeringlabs.com/catalog/CCG_SAAAC.php4 )

3. Derivatization prior to separation to render the amino acids compatible with HPLC reversed phase separations and detectable by absorbance detectors (examples can be found at this link: http://www.waters.com/WatersDivision/Si ... ids%20food )

4. Derivatization prior to GC separation (and example can be found at this link: http://www.alltechweb.com/literature/ca ... olumns.pdf )

Another detail is whether or not you are trying to analyze free amino acids (in which case you want to extract them from the food sample) or characterize all of the amino acids contained in the proteins present in the food (in which case you want to hydrolyze them to break the proteins down to their amino acid constituents)

Hi.
Thank you for the information.
Though now I feel more lost than before.
(by the way i wrote wrong, it has to be handed in on tuesday)

So i guess my way of limiting the asignment was not a good idea.
Any tips?

Giti

(thanks again)

Giti,

I'm not sure what you are looking for but here is a brief summary to go with the previous links:

The most common method of analysis of amino acids makes use of a reversed phase column. Because this retention mode works best when analytes are hydrophobic, amino acids are allowed to react with a reagent which makes them more hydrophobic and at the same time makes them absorb UV light so that we can use a UV detector since only a few of the amino acids absorbs UV light in its native form (in some cases we use a reagent that fluoresces for better detection sensitivity with a fluorescence detector). This is called precolumn derivatization because the amino acids are derivatized prior to injection into the instrument.

Two other common separation methods are cation exchange (at an acidic pH, all amino acids will be cations) and anion exchange (at an basic pH, all amino acids will be anions). After the amino acids are separated, they can be mixed the same reagents mentioned above to form either UV absorbing derivatives or fluorescent derivatives (this is called post-column derivatization because the derivative is formed after the analyte is separated) or if we choose a suitably basic eluent when performing anion exchange separations we can use electrochemical detection (since the most commonly employed method requires high pH for electrochemical detection on a gold electrode).

Chris

Hi.. now I am starting to working out an asignment here:) Thank you

One more question: So, if we talk about post column chromatografy we are talking about ion pair chromatograpghy?

And if I write something about that, I should also mention ionselcetivity, bufferselectivity etc?

All the differences makes me a bit confused.

Giti

Hi again Chris, hope you see the question.

Im thinking of mentioning something about differences in post column and pre column chromatography.
I think I have read that pre column are more prefered when it comes to analysing amino acids.
Can you give me some reasons?

Thank you so much for replying my questions.

Giti

Hi again..

Huh, I will never ever postpone asignments to the last days.
I have some final questions:

Pre column/post column? Reasons for that pre column is used

Flow rate: 1,0 ml/min - it seems like all methods I have seen have that flow rate. Any reason?

Column: 150 x 4,6 mm in diameter -- is that the best size?

Mobile phase: A mixture of methanol, acentonitrile triethylamine : These are solvents to make the column hydrofobic?

Derivatization reagents: OPA, FOMC or PITC - any differences in the quality?

UV or fluorescence? What is best

Thank you very much.

Giti

Pre column/post column? Reasons for that pre column is used

The good news: pre-column tagging does not require special equipment. The bad news: pre-column tagging makes the separation more complicated, because you have to deal with the exess tagging reagent.

Flow rate: 1,0 ml/min - it seems like all methods I have seen have that flow rate. Any reason?

No, it's just a nice, round, even number.

Column: 150 x 4,6 mm in diameter -- is that the best size?

There is no "best" size. There are tradeoffs among performance, pressure, cost, etc. This size is a typical "workhorse" column using present technology.

Mobile phase: A mixture of methanol, acentonitrile triethylamine : These are solvents to make the column hydrofobic?

No, but they affect how tightly sample molecules stick to the hydrophobic column. Visualize a piece of polyethylene plastic on which you had written something with a wax crayon. To take the wax off the plastic you might try water (not good, because the wax is hydrophobic). Methanol or acetonitrile would probably wash the wax off very quickly. The polyethylene is the stationary phase (column packing), the wax crayon is your sample, and the water/methanol/acetonitrile, etc are the mobile phase (solvent). The idea in HPLC is to get just the right balance of water + organic solvent to wash your sample off the column at the right speed.

Derivatization reagents: OPA, FOMC or PITC - any differences in the quality?

The choice is often dictated by prior sample workup chemistry.

UV or fluorescence? What is best

Depends. Fluorescence is more sensitive, UV is easier.

Giti,

You can also do underivatized amino acid separations on a C18
column, if you are using UV detector. Just use a column that won't
collapse under highly aqueous mobile phase and the following
mobile phase conditions:

http://www.silvertonesciences.com/modul ... TI083E.pdf

Hello everyone:)

Thank you all for your help:)

I really like this forum. Will be starting a 3 months project work on cancer soon, so I may come back but then my question will hopefully be a bit more advanced than my last question.

Giti:)