Dhruv
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HPLC peaks separation!!!
Dear Friends,
I am trying to seperate a compound having antifungal activity by Preperative HPLC on Agilent 1100 system using Thermo Hypersil Gold column. Solvent
A: 2% Acetic acid in DIW and Solvent B: Acetonitrile.
I got a peak with very good antifungal acivity. But when i try to analyze it further then i realized that its nothing but having 3 subpeaks. But the
3 subpeaks are so closely associated that i am not able to seperate them.
I tried different Solvent A and B combinations including
Solvent A Solvent B
1 Acetic acid 2% 0.5% AA in DIW n Acn (50:50)
2 Acetic acid 2% Acne 100%
3 Formic acid 0.5% Acne 100%
4 Orthophosphoric acid 0.2% Acne 100%
5 Trifluoroacetic acid 0.2% Acne 100%
6 Acetic acid 2% Meth 100%
7 Formic acid 0.5% Meth 100%
8 Orthophosphoric acid 0.2% Meth 100%
9 Trifluoroacetic acid 0.2% Meth 100%
But nothing is working.
Please suggest something
waiting for expert comments
with regards and thanks in advance
Dhruv
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unionised
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Do you know what any of the compounds are?
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Nicodem
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Isolate the peak containing the three compounds from your HPLC, obviously.
Check if the peaks separate at least slightly on silica TLC (or with an analytical normal phase HPLC, if you have one), trying various eluents and
ratios, obviously.
Then try using a normal phase preparative HPLC or just a normal column chromatography if you get a good enough separation on TLC.
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chemoleo
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try heptafluorobutyric acid, 0.1%, on a different column (one with different separation properties - i.e. if you used C18 so far, now go for C8/4 from
a different manufacturer).
HFBA often worked wonders for me.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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phlogiston
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The mobile phases you tried are all acidic. If your column can handle it, try an alkaline mobile phase. I routinely use 10 mM ammonium bicarbonate pH
9.5 (dissolve and adjust pH with ammonia).
Perhaps the three peaks are (stereo)isomers? If you are going to try different columns, why not include a chiral column?
-----
"If a rocket goes up, who cares where it comes down, that's not my concern said Wernher von Braun" - Tom Lehrer |
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ziqquratu
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Like Nicodem and chemoleo have said, trying a different stationary phase might be your best bet. A C5 or C8 might work, or perhaps something with
aromatic groups on the silica - and certainly, normal phase is definitely worth a go (although I'd probably skip the TLC and run it directly - you're
likely to get better separation by HPLC anyway). Ultimately, it will depend heavily on what your molecules are (which, I suspect, is what you're
trying to figure out by isolating them?)
Definitely look at the possibility of trying an alkaline mobile phase, as suggested by phlogiston. Also, try running with a slower gradient, over a
longer time.
@phlogiston, a chiral column would be unlikely to help in this case - chiral columns are awful to work with when you KNOW what your compounds are and
can look at the manufacturer's handbooks for similar molecules (it's rare that you could just grab a chiral column off the shelf and get decent
separation). Also, as he's getting some separation on a simple C18 column, the compounds are not enantiomeric (even if they are diastereo- or
otherwise isomeric, which is quite possible) so a chiral column would be an unnecessary complication.
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Ozone
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It really helps if I know what you are trying to separate from what.
O3
-Anyone who never made a mistake never tried anything new.
--Albert Einstein
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Dhruv
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For Unionized : Thanks for your comments and here are the further details you had asked for:
The compounds: In my opinion they are phenolic compounds as my initial extraction protocol includes ethanolic extractions.
For Nicodem : Thanks for your suggestions :
I will try to check on TLC with different eluents and ratios. Also will try to consider normal phase as an option as I have not tried it till now.
For Chemoleo: Thanks for your suggestions:
I will try heptafluorobutric acid, or use a different column. Hope it will work
For Phlogiston: Thanks for your comments:
I haven’t come across anyone reporting alkaline mobile phase for phenolic compounds but still I will try in case it helps. And I cant say if they
are isomers. Though I am planning to use a different column to see if it helps.
For ziqquratu: Thanks for your comments and suggestions:
I will definitely give a try to all the suggestions. Thanks a lot.
For Ozone:
I am fractionating the phenolic extract from a plant source. The antifungal activity is associated with one of the different peaks which I am getting
with Thermo Hypersil Gold column (having C18 like selectivity). That particular peak or fractions have further 3 subfractions which are not getting
separated and overlap each other. I have tried a lot by increasing the time and also using different solvent A and B combinations but all are of no
use. So needs suggestions of you all. Some of you have given some suggestions which I will try one by one. Still any other suggestion is welcome.
Thanks.
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methyl_ethyl
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You state the column in use has "C18 like selectivity", perhaps you can elaborate? Does the column incorporate some other functional group which
impacts the selectivity?
Is the stationary phase truly a silica bonded C18 alkyl chain or some type of hybrid which employs a mixed mode of separation.
Are you simply using a linear gradient?
As others have stated using stationary phases with shorter alkyl chains may have an effect on your selectivity.
Perhaps characterizing these peaks on the analytical scale will allow for proper resolution, and further elucidation of these multiple peaks.
Further characterization may provide useful information in support of scale up.
Are you able to adjust the temperature of your column? Increasing the temperature may have significant impact on resolution and selectivity of your
target analytes.
Describe your sample preparation procedure, what if these closely related compounds only differ in hydrophobicity due to the presence of sugar
moieties, however the primary structures are identical. Can you enzymatically or chemically cleave such structures in order to initially compare
"apples to apples"?
m_e
Unipolar Mania.... it\'s good for life
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Ozone
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Increasing the time will decrease resolution (Fickian diffusion leads to band-broadening, which is why components with longer Tr's always give broad
peaks. Ramping up the flow may improve the separation--up to a point, the peak HETP (high-equivalent to the theoretical plate) is at an optimum at
around 1.2 mL/min (van Deemter equation).
You may significantly increase the number of plates by using smaller particles, e.g. 3 um or less, if your pump can take the pressures. I can do 3 um
with an Agilent 1100 400bar set-up, but not at very high flows.
Alternatively, you can string together multiple columns for more plates, at the expense of peak-broadening and increased pressures.
I might suggest doing a pre-separation to get your fraction in bulk, and then working-up a separation for that. As nasty as it sounds, normal phase
(or an intermediate phase run "normal") might work if all-else fails. The upside is that method development is quick using TLC.
I will note, however, that many redox-active phenolics oxidize in air when removed from the bulk matrix. While stable in the complex mixtures, you
product may oxidize when in dilute solutions without the ROS-scavenging protection of its relatives.
Best of luck,
O3
-Anyone who never made a mistake never tried anything new.
--Albert Einstein
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