Chromatography Forum

By Alex on Tuesday, August 3, 2004 - 03:28 am:

Hello Chromatographers:

I have synthesized Leucine oligomers of different chain-lenghts (6, 8 and 10 Aminoacids) through solid phase peptide synthesis and made an ESI Mass spectrum to examine the purity.
The spectrum showed my main Product (Leu6) with an additional distribution of other chainlengths (Leu4-Leu5 and Leu7).
In order to purify the sample I tried following prodedures:

LC-MS (Agilent 1100)
Column: Reversed Phase with C18 chains (LiChrospher RP-18)
Samplesize: 10 µl
Flow: 0,3 ml/min
Room Temperature

Solvents:
Isocratic
1. 100 % MeCN
2. 90 % MeCN, 10 % H2O
3. 80 % MeCN, 20 % H2O
4. Same procedures as 1-3 with 0,1 % TFA

But the peakseparation is still to weak to use a preparative HPLC Column. Additionally leu oligomers are poorly solluble in different organic solvents. Best was TFA but then the sample flushes through the column without separation.

Every Hint/Help greatly appreciated!

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By Zelechonok on Tuesday, August 3, 2004 - 06:28 am:

This is typical application for mixed-mode separation.
You would have sufficient resolution among your oligomers if you take Primesep 100 column and RP condition with TFA as modifier. See for similar example in this link. http://allsep.com/makeChr.php?chr=Chr_009
The example does not have leucine, but it has isoleucine, which retain similarly. Your oligomers will be retained primarily by ion-exchange mechanism and they will be resolved from each other by difference in hydrophobicity.
Contact me directly if you have more questions

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By Zelechonok on Tuesday, August 3, 2004 - 07:43 am:

Another example of separation even more similar to yours.

http://allsep.com/makeChr.php?chr=Chr_070

This one is separation of lactic acids oligomers. They all have one carboxylic group, but different number of monomers in the chain. The column (Primesep B2 in this case) has positive charge, which provides retention. The selectivity mostly comes from complimentary hydrophobic interaction with mixed-mode SP.

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By Alex on Tuesday, August 3, 2004 - 07:59 am:

Thank you for your answer.
But I am not so sure if this will work. As you said, this is separation through ion exchange which would separate ionic/polar compounds. In the example it works because of the differend isoelectric points of the Aminoacids. In my case I have an unpolar peptide with Leucine varying only in chain lengt and less in terms of polarity.

Maybe I am wrong but I am still a newbie in this field.

Regards,
Alex

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By Zelechonok on Tuesday, August 3, 2004 - 09:23 am:

Ion exchange provides bulk of retention, but selectivity is result of small difference in hydrophobic properties of the oligomers (see second example with lactic acid).
Send us a sample, we will make a method for you if you are not convinced.

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By Uwe Neue on Tuesday, August 3, 2004 - 04:03 pm:

Alex,

Since your compounds are preferentially soluble in mobile phases with a high acetonitrile content, I recommend to try HILIC on a silica column. Use 90%, 80% and 70% acetonitrile to see where you got sufficient retention. Once you have retention, you can then manipulate the mobile phase with a buffer, if the selectivity is unsufficient.
The HILIC mechanism is due to the solubility of the polar functions in you molecule (amide and the end groups) in water, which is enriched on the surface of the packing. Another mechanism that may play a role is ion-exchange of the amine function with the surface silanols.

Anyway, if you can get it to work it is a cheap approach towards prep.

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By SIELC_Tech on Tuesday, August 3, 2004 - 04:14 pm:

Alex,

Unless you have a lot of time, please don't waste your time with HILIC. Check the following thread on chromforum:

"HILIC LC-MS serious peak broadening'problem"

LYR devoted a month trying to do HILIC for amino acids and I don't think that he had any luck.

Try our Primesep approach. Primesep 100 column with ACN-water-Sulfuric acid should give you mice separation at low organic (10-30%)

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By Uwe Neue on Tuesday, August 3, 2004 - 04:45 pm:

To Siel-Tech/Zelechonok:

From what I can tell by looking at the thread very quickly, the compound was only or preferentially soluble in water. Looks like a different beast than what we have here. Plus I think that a silica column would be a cheap solution that is readily available in many labs.

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By SIELC_Tech on Tuesday, August 3, 2004 - 05:15 pm:

Alex,

If you are looking for prep separation you can use ACN-water-TFA with Primesep columns (other choices are formic acid, ammonium acetate, ammonium formate ,etc.). Contact us if you need help (mail@sielc.com)

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By Alex on Wednesday, August 4, 2004 - 02:17 am:

Thank all of you for your fast answers.

Right now I will check the exact solubillity in different solvent mixtures MeCN/H2O first, to decide which suggestion fits best.

I will tell you how i will proceed...

Regards
Alex

Actually, I doubt HILIC is a proper alternative in this case. The relatively hydrophobic side chain of leucine and the length of the oligomers makes it questionable. Especially, a silica phase seems least suitable if HILIC is considered.

Please post any progress in this matter.

Just out of curiosity, since I don´t know anything about peptide synthesis. From advertising one gets the impression that it is a simple matter to control the reaction to get essentially 100% yield of what you want. In reality it is easier to do a prep. chromatography rather than to optimize the reaction??

Alex,

I think that you would be able to achieve much better resolution between your oligomers if you could use gradient elution (and your reversed phase column). I would start my gradient at let's say 50% MeCN:H2O (if your peptides are soluble) and then go up to 100% MeCN.

Also you are saying that your peptides are best soluble at TFA. Do you really mean 100% trifluoroacetic acid?

Finally at these high % of MeCN the contribution of TFA as ion-pairing reagent should be quite modest. Maybe it would be worthy to try some higher perfluorinated carboxylic acids (i.e. HFBA?)

Kostas