Chromatography Forum

Has anybody achieved separation of Norleucine and Leucine Using RP-HPLC without derivatization. Is it posible to separate them with High-resolution MS detector!??


thanx

There would be numerous ways to separate and detect these two isomers, derivitised or not. Probably the most important criterion is what levels of detection and quantitation are you aiming for?

Have you worked on AAs before, and what have you tried so far with this particular separation?

Hi resolution MS detector won't do it as they are isomers and not isobars (such as Lys and Gln). You can separate them by using heptafluorbutyric acid or nonaflouropentanoic acid or tridecafluroheptanoic acid (higher homologues for sure I have to see my notes if HFBA will work). For the first two you need to start by high aqueous conditions (almost 100%) and do a gradient...

So, I checked my data and as you can see in the chromatogram below with 0.1% HFBA and a Merck Purospher RPe 12.5 x 0.46 cm, 5 um you are able to separate Leucine from Norleucine (along with several other amino acids). Resolution is not as great but probably by going to higher homologue perfluorinated carboxylic acids (or a smoother gradient) you can get better resolutions. You may want also to try porous graphitic carbon which provided pretty good resolution for Leu and Ile (but I didn't try Nleu). The selectivity of the isomers changes in the porous graphitic carbon column. The conditions in the chromatogram are 100% water to about 30% ACN all including 0.1% HFBA...

So this kind of ion pairing agents would not do anything to the background noise of the ms? Isnt there a possibility of using compounds with masses lower than 100 or even 50?

Leucine, isoleucine, and valine have all been separated on Unison UK-Amino:

http://www.silvertonesciences.com/files/TI311E.pdf

There is a possibility that UK-Amino could work for this separation.

These ion pairing reagents are negatively charged and won't give any signal in positive ion mode (except maybe from any contained impurities) but they will decrease your analyte signal modestly due to ion-suppression. Actually these amino acid in MS/MS give very strong signals so I wouldn't worry about sensitivity (even in their underivatized form). You can indeed go lower than m/z 100, my the lowest m/z I have detected is Glycine among amino acids (m/z 76) and ethanolamine being the lower MW compound (m/z 62) I ever did with quadrupole type instrumetns. I have extracted ion currents for all these amino acids (actually the method that I describe was used in order to analyze simultaneously 76 underivatized amino acids in LC-MS/MS).

I can include some supporting info beginning of September but right now I am about to leave for Las Vegas and won't be posting for a while...

Very Usefull information, Kostas!
The thing is that I am using single MS with optimized AGC stettings. And Yes the amino acids under positive mode do give prety strong signals, but for m/z of the norleucine I get three peaks and two of them are overlaping. the Ile is easyly separated only with formic acid as an additive. could I use 3ClAcetic acid with the same success as your preposition... thanx

<cold sweat> Don't do it! No, really, don't! It will probably work fine, but only if you never, ever, intend to work in negative mode MS ever in your life, even in 20 years' time, might you be OK to use perfluorinated acids in your MS system. Otherwise don't even think about it. No. </cold sweat>

I used nonofluoropentanoic acid in two LC-MS systems for separation of amino acids (but not norleucine), and it worked fine (beautifully, in fact). But in negative mode, I could see nothing but nonofluoropentanoic acid for a month. I had to take the entire spray chambers of both instruments to pieces and clean every bit individually, and all through the waste drain from the spray chamber before I got the contamination down to a level where negative MS was possible at all. I could still see the contamination 3 months later. I still have glassware in the lab labelled "263-free", in memory of the trouble it caused.

At the time, I looked at various message boards on how to get rid of it. Recommendations varied from "try alternating 100% organic and 100% water at a high flow rate for a few days" to "keep an LC-MS system just for ion pair reagents".

Oh, TCA might be fine. I got the feeling the problem was that nonofluoropentanoic has such a good hydrophobic tail it sticks to any plastic items like glue.

I got the feeling the problem was that nonofluoropentanoic has such a good hydrophobic tail it sticks to any plastic items like glue.

Anecdotally, I thought that was why many people claim once you use TFA in your mass spec, it's there for ages/life.

okay, what about TCA? will it work? will it do any damadge?

TCA won't work because it is not volatile.

About the use of HFBA my experience was different. Previous posts imply that on line degasseurs will bound HFBA (or I guess higher homologues) and then leach them for really long time. When I used different perfluorinated carboxylic acids without an on-line degasseur and a triple-quadrupole (API-300) in positive mode, cleaned the source and then tried negative mode I didn't observed any relevant ions.