p*******y
发帖数: 133 | 1 HI I am doing an LC method for a drug containing a tertiary amine to couple
with MS.
Amines are known to be pretty difficult with LC.
I use TFA as ion pairing agent (A: 0.1% TFA inwater, B ACN), and the
gradient is 5% to 95% organic in 2min on a small 10cm C18 colume. I got two
peaks with similar peak area after injecting a pure compound solution in
water/acn. First eluting at solvent front and the other eluting at 2 min.
When I dissolve the compound in 0.1% TFA, I got only 1 peak at 2min. I | Y****r
发帖数: 3473 | 2 you need to change to a different buffer (slightly basic) system | k******n
发帖数: 133 | 3 I am not an expert. several suggestions:
1. Use End capped column
2. add amine modifier
3. increase pH, for example, use BEH column and run RP at pH 8.0
4. Swith to non_RP chromatography
couple
two
【在 p*******y 的大作中提到】
: HI I am doing an LC method for a drug containing a tertiary amine to couple
: with MS.
: Amines are known to be pretty difficult with LC.
: I use TFA as ion pairing agent (A: 0.1% TFA inwater, B ACN), and the
: gradient is 5% to 95% organic in 2min on a small 10cm C18 colume. I got two
: peaks with similar peak area after injecting a pure compound solution in
: water/acn. First eluting at solvent front and the other eluting at 2 min.
: When I dissolve the compound in 0.1% TFA, I got only 1 peak at 2min. I
| m********r
发帖数: 127 | 4 I am not a LC expert, just my two cents.
In my experence, some compounds with amine show a bit tailing or broading,
not double peaks, due to the non-specific interaction of the amine group to
the silicon beads. Transitions between amine forms are too fast to be
captured by UV or MS. I never used LC-MS before, but I had some experience
with compounds with amine show decent peak shape in HPLC with UV detection
even without any modifier.
Are you sure when the sample in water/acn solvent, the double | m********r
发帖数: 127 | 5 Oh, btw, is ur sample ionizable? What's the pka? Maybe try 20mM phosphate or
acetate buffer as mobile phase?
I had some bad memory of water make my ionizable compounds silly in HPLC. | p*******y
发帖数: 133 | 6 Thanks a lot for your suggestion, Manchester
The reason I believe that the first peak is my compound is that when I
dissolve everything in 0.1% TFA, the first peak disappears and the peak area
for the second peak increased accordingly. When I inject different
concentrations of real samples, the relative ratio of peak area from the
first and second peaks stays roughly the same. I had previously worked with
a smaller amine compound and had similar results.
This is kind of weird for me as well be
【在 m********r 的大作中提到】
: I am not a LC expert, just my two cents.
: In my experence, some compounds with amine show a bit tailing or broading,
: not double peaks, due to the non-specific interaction of the amine group to
: the silicon beads. Transitions between amine forms are too fast to be
: captured by UV or MS. I never used LC-MS before, but I had some experience
: with compounds with amine show decent peak shape in HPLC with UV detection
: even without any modifier.
: Are you sure when the sample in water/acn solvent, the double
| e**********y
发帖数: 8 | 7 You were seeing a "break-through" of your compound on the column, which is
not unusual for hydrophillic compounds. That is, your analyte was not
completely focused on the column head, therefore some of it came off at the
solvent front (not retained), whereas the rest eluted at the proper
retention time. The solution to is problem is to (1) make your injection
solvent weaker by diluting your extracted sample in more water; and/or (2)
hold your gradient at low organic for some time (say 15-30 seco
【在 p*******y 的大作中提到】
: Thanks a lot for your suggestion, Manchester
: The reason I believe that the first peak is my compound is that when I
: dissolve everything in 0.1% TFA, the first peak disappears and the peak area
: for the second peak increased accordingly. When I inject different
: concentrations of real samples, the relative ratio of peak area from the
: first and second peaks stays roughly the same. I had previously worked with
: a smaller amine compound and had similar results.
: This is kind of weird for me as well be
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