Hi everyone,

I'm trying to analyze Glycine by a HPLC method at 200nm. No matter what I do, It seems that it will elute at the solvent front. Please advise me if you have any suggestion.

Look here:
http://www.wako-chem.co.jp/siyaku/info/chromato/pdf_app/peptide/01.htm

I found this by searching Yahoo under glycine analysis by HPLC

I had the opposite problem! I developed an assay for a drug and the pharmaceutical preparation which I initially used as a standard contained glycine. The glycine eluted at 100 minutes (very annoying as the RT of the drug was 13 mins!).

I was using a Phenomenex Luna C18(2)with approx. 30% MeOH in phosphate buffer, pH 3 so try a C18 with considerably more organic modifier unless you want to spend weeks on your analysis!! :o)

Good Luck!

Ann

Glycine is the smallest amino acid. It does not stay on C18 column well, or does not retain at all. I wonder if you want try normal phase, like silica column, ACN/Water mobile phase, isocratic.

Or use ion-paring reagent, for example NFPA?

Or maybe try a C30 column?

What decetor do you use? UV? MS?

You might have to derivatize glycine, like what the Japanese did.

For ann: what you saw was not glycine.

Yes, the best way is to derivatize Gly. and to measure it easy in RP. You MAY try NP (amino for example) and AcN/water if you really have nothing else to do :)) pure science, theoretically? it MAY work but it must not :)
Ann -- evidently, you`re wrong, you saw an admixture.

C Sychov and Anon#3

I can only report my experience. I did not merely assume that the 100 minute peak was glycine based on the drug formulation. I suspected that it may be glycine and therefore, injected a glycine standard. The injection of the glycine standard solution produced a single peak also at 100 mins (there were no peaks in blank injections of mobile phase or sample diluent). When (on the same day) I assayed a pure drug standard (i.e. without glycine) there was no 100 min peak. I was using UV detection not MS so, of course, other than the RT, I cannot confirm that the 100 minute peak was glycine but I think you will agree that analysis of blanks and pure standards as reported above provided good evidence that it was.

I have used the Luna C18 for many applications and have found it to be much more retentive than many other C18 phases. Different bonded phases make big differences.

Best regards

Ann

Ann,

What you saw (the 100min peak) is very ikely (99.99% sure) just some impurity in the sample, or column bleeding. Try a new Luna column, see if you can get the same peak or not.

If 30% MeOH retains glycine at 100min on the Luna column, no one will use it for reversed phase separation.

Hi Anon

If it is an impurity, why did it appear exclusively in samples containing glycine? Why did it not appear in samples which did not contain glycine? If it was something bleeding from the column, why did it not appear in blank samples? I would happily agree with you if it were not for the above questions.

BTW I have no requirement for assaying glycine, I came across it as a component of a pharmaceutical formulation (where I was assaying the drug compound rather than the components of the formulation). As for trying it on a new Luna column, I evaluated this assay on several (dedicated i.e. used for this assay only) Luna C18 columns (same phase, same length, different bore) and had the same results.

As I said, I have used the Luna C18 columns for several different applications. In combination with the stationary phase, mobile phase composition, pH and analyte chemistry obviously dictate the results of the chromatography so I don't see how you can make a general statement that if 30% MeOH retains glycine, the column is no good for RP HPLC?

I can understand that you are suspicious of my results because they obviously contradict with your experience [and that's your prerogative, I take no offence!:o)]. However, I remain confident that the 100 minute peak was glycine.

Thanks for taking the time to reply.

Best wishes

Ann

ZIC™-HILIC is a suitable alternative. The zwitterionic character of glycine makes it difficult get any retention on a reversed phase column unless you use ion-pairing or derivatization.

By using HILIC mode you may easily solve the problem. Since you need to use detection in the low UV range a phosphate buffer is recommended.

A ZIC™-HILIC column (5 µm, 150x4.6 mm) will give you a k'-value in the range of 2 to 6, while using 65% to 85% acetonitrile and 10 mM phosphate buffer as mobile phase. An isocratic run works well, however that is depending on your matrix.

Small and tricky peptides like gly-his-lys may also be retained on the column by this technique.

Hi Anon #1,

You can easily retain glycine by using ion-pairing chromatography.

You may want to have look at Petritis et al. J. Chromatogr. A, 961, (2002), 9-21. "A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids.

In this article I use 0.5 mM pentadecafluorooctanoic acid (PDFOA) in water and compare 7 different detectors (ELSD, UV (210 nm), RI, NMR, CD, MS, MS-MS, CLND) as this mobile phase was compatible with all these detectors. The method separates all polar proteogenic amino acids (Asp, Asn, Ser, Gly, Gln, Cys, Glu, Thr, Ala, Pro) and even more like Tau, Hyp etc. under isocratic conditions.

I also quantify a peptide hydrolysate (Gly-Gly-Asp-Ala) so you have some results of Gly quantification as well.

Depending on the chemistry of your hydrophobic column and its length equilibration times varying but are pretty long (about 3 hours for a 12.5-15 cm) but the method is isocratic so it is not a problem.

For the UV I found 210 nm to give the best noise to signal ratios with this mobile phase. You can monitor your column equilibration by monitor your breakthrough curve of your UV when the PDFOA reach the outlet of the column.

For more information about the use of perfluorocarboxylic acid as ion-pairing reagents for the separation of underivatized amino acids you may look at the original article : Petritis et al. J. Chromatogr. A 833 (1999), 147-155. You may also want to use the column that gave me the best results (see articles).

HILIC is also an alternative in order to obtain Gly retention. I have achieved retention personally with columns like Astec NH2, Polyhydroxyl ethyl aspartamide (from PolyLC) etc... Other "polar" columns should work as well.

About Ann statement(s): I would have to agree with the others that is unlike to achieve Glycine retention with the mobile phase and columns described. I have tried different reversed phase columns with as much as 100% water and haven't achieve any retention for Gly (maybe a polar embelled column or Atlantis type could maybe offer some retention but I do not have any personal experience).

Hope the above helps,

Kostas

Hi Kostas,

Thank you for all the useful information.It gives me lots of hope. I'm just wondering what mobile phase you were using with that NH2 column. I'v tried a polar embedded column but it didnt help.

Anon#1

I wonder if a C30 column can retain Glycine or now....

Anon #3

Hi again,

Actually as I wanted to test how many amino acids I can separate in a single run I have studied gradient elutions. I started with 80% acetonitrile and went down to 30% in 20 min(balance water). By using a 150 x 4.6 Astec Amino column and 0.8 mL/min I had retention time of about 12 min for Gly. I guess that you can have decent retention for Gly by using 70% ACN (but you can define the optimum easily - it will depend of your matrix as well).

If you choose to use the Polyhydroxyelthyl aspartamide you might want to use about 50 mM of salt to improve peak shape.

You might want to check our publication: Petritis et al. J. Chromatogr. A 913 (2001) 331-340. Simultaneous analysis of underivatized chiral amino acids by LC-ESI-MSMS using a teicoplanin chiral stationary phase.

This publication deals with the chiral analysis of amino acids (so I haven't included glycine for evident reasons) but the retention of amino acids is obtain through HILIC (I say retention and not selectivity between D and L).

Even this column can retain glycine (I have tested it) but it would have been a very very expensive solution ;-).

Concerning the polar embedded columns. Just courious if someone could test the column Atladis with glycine. I am really curious to see if it provides any retention (I have heard that retains even more hydrophilic compounds than glycine...).

About the C30 suggestion of Anon #3. I find unlike to have retention but not impossible. I have tested some of the most hydrophobic C18 columns that exist out there (according the interesting article of Uwe Neue J. Sep. Sci. 2003, 26, 174-186 "Characterization of HPLC packings") like the Purospher RPe 18 with no retention under even 100% water and with or without TFA.

Hope the above helps,

Kostas

For Ann,
I aggre with the previous mentioned statements that what you saw in 100 min Rt was not glycine. The latter has the ability to make hydrogen bonds. Maybe what you saw down there was a very complex hydrogen bonding molecule that involved glycine and other than your drug molecules. That complex molecule has an increased hydrophobic character combined with a relative polar mobile phase makes it to elute at 100 min Rt.

For Kostas Petritis,
You are using 100% water with a hydrophobic C18 column, it is resonable that get no retention for glycine, the water elutes glycine rapidly.
Try 90% AcCN and above to see what's happening.
Decreasing the polarity differece between the column and the mobile phase increases the k's of the polar component, such as glycine

Hi Chempara,

Actually what is propably going to happen with higher than 90% ACN, is that glycine is going to precipitate due to misibility problems.

I believe that what really happens with polar compounds when you increase your organic solvent concentration so much with a C18 column, is that you use your residual silanols as a polar binding spot, in a hydrophilic interaction chromatography way.

But there are much more adequate columns to do so (peak shapes are terrible if you try it with C18 columns).

Chempara,
Most C18 columns are not wetted by water. This is correct. However, the Atlantis dC18 has been specifically designed to get around this problem. The issue is fundamentally the wetting angle on the surface towards water. It can be manipulated, if you understand what the mechanism is. The Atlantis dC18 is also not a weakly retaining packing material. On average, it exhibits more retention than a standard C18.
The bottom line is that Atlantis dC18 can be used without difficulty in 100% water and has a shot on retaining glycin.
My first bet though would be to use the Atlantis Silica HILIC column. It is a polar packing used for hydrophilic interaction chromatography, similar to the NH2 column and the other polar columns mentioned above. The advantage of a silica column is that it does not bleed, which could be a problem at 200 nm.

You may want to take a look at the Alltech Prevail C18, which is water-wettable and seems to be highly stable at low pH. Alltech has an application number 9449 in their brochure 460 for underivitized amino acids and it looks like good retention (almost 1.5 min beyond the void) at pH 1 with a 100% aqueous mobile phase and 250mm which should help. This approach may be a lot easier than some of the more complicated suggestions. Try www.alltechweb.com

Hi last anonymous,

I just browsed the applications that you described and if you take a closer look you'll see that they use not one but two ion-pairing reagents in their mobile phase; one is heptafluorobutyric and and the other is TFA. This approach is the same as the one I described earlier and actually is not easier in any way.

I just wanted to clarify the C18 column by itself it can not retain glycine.

Kostas

HI KOSTAS,
I'M THIKING THAT IF THE FIRST DILUTION IS MADE IN WATER AND A SECOND ONE IN A MOBILE PHASE REACHING CONCENTRATION AT ABOUT 100mcg per ml MIGHT SOLVE THE PROBLEM OF GLYCINE'S PRECIPITATION.
THIS IS A WAY THAT EUROPEAN PHARMACOPOEIA USES TO BYPASS MICSIBILITY PROBLEMS.
ALSO SPECIAL ATTENTION MUST BE TAKEN CARE IN ADJUSTING THE pH OF THE MOBILE. THE AMINO GROUP HAS TO BE UNCHARGED IN CASE OF USING ORDINARY C18 COLUMNS.