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Dear all,
I have an analytical issue for you. I am trying to find eluting
conditions in LC/MS of a NCE having a lipophilic centre and an
imidazole moiety (pKa around 6-7) and a log P around 3.6.
Now making the eluent acid (CH3COOH, HCOOH), with cosolvent it comes
out too quickly and it tails in a C18, 2.1 x 150 mm column, 5 um.
CH3CN is better than CH3OH because it tails a lot in isocratic mode.
Buffer acetate pH 6.0 is better either in isocratic mode with CH3CN
or in gradient mode enriching in CH3CN in a short (3 min) time.
However, the peak tails less, but tails still. Has anyone any
suggestion on how to improve my elution? Thanks. You are always my
final call.
Federica
Federica Vacondio
federica.vacondio.-a-.unipr.it
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Dear all,
The following message was posted to: PharmPK
I always want to know in what the molecule of interest is soluble.
However,
it has been noted on some occasions that by increasing the
temperature of
elution (test for stability issues) the peak shape may improve. As the
molecule has at least one ionizable group within the aqueous range,
buffering the system to various pH's will affect the elution profile
(and
the solubility of the entity).
James Ladd
James Ladd
Scientist Erimos Pharmaceuticals
840 main campus Drive suite 3700
Raleigh, NC 27606
jladd.-at-.erimos.com tel: +1 (9198215204) EXT2816
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The following message was posted to: PharmPK
Dear Federica,
I assume the logP value you mention refers to that of the neutral
compound. Do you use ESI or APCI? What is your mobile phase flow rate?
Do you happen to have another detector (e.g. DAD) in-line? What is the
composition of the mobile phase at the start (initial conditions)?
I hope you are not overloading your column because this can cause really
bad peak tailing. This can be compound specific. Is your column very
old? Do you use a guard-column?
I would expect a compound with a logP of around 3.6 to show some
retention on a C18 column. We routinely use ammonium formate (45 mM pH
4.5) and MeCN/water and this works well with most of our compounds (also
with positive ESI LC/MS). Gradient usually works better than isocratic.
With 'difficult' compounds we often simply use a different type/brand of
column and you'd be surprised what a difference that can make.
Not easy to solve these problems!
Frederik Pruijn
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.aaa.auckland.ac.nz
--
Dear Federica,
Further to my earlier post, I forgot to ask you what your compound is
dissolved in, at what concentration, and how much you do inject?
Kind regards,
Frederik Pruijn
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The following message was posted to: PharmPK
Hi Federica,
If you follow some thumb rules of HPLC method development, you should be
able to fix your problem. I am listing some, if not all, below:
1. Whether the analyte is basic/acidic, always use a mobile phase with
pH 2 units above or below analyte's pKa.
In your case, as imidazole moieties will have a pKa around 7.0 (weak
base) LogP is no longer an indicator of retention of your compound as it
ionises and LogD at the pH of your mobile phase is all that matters.
Since you cannot use a mobile phase of pH 9.0 (2 pH units above pKa),
you can use acidic mobile phase with pH below 5.0 (2 units below pKa). I
suggest you to use a pH of 4.0. Ammonium acetate (pH range 3.8-5.8) or
ammonium formate (pH range 2.8-4.8) buffers should do well at this pH
(also ideal for the MS). You can try different ionic strengths ranging
from 10mM - 50mM. Once you prepare buffer of required strength, adjust
the pH to 4.0 using acetic acid/formic acid for ammonium acetate and
ammonium formate respectively.
Refer the following link for more details of mobile phase buffers:
http://www.sigmaaldrich.com/supelco/the_reporter/reporter_19_02.pdf
2. Injection volume less than 5% of the flow rate is ideal. For eg. If
you are using a flow rate of 1.0ml/min, 50uL would be desirable.
3. Always see that the sample solution that you inject onto column have
similar or less percentage of organic phase than in the mobile phase.
For eg. if your mobile phase (isocratic) contains 50% organic phase, see
that your final sample that you inject onto column will have 50% or less
organic. In case of gradient methods, keep the organic similar or less
than the initial mobile phase (start of run) organic composition.
4. Always maintain a minimum of 10-20% aqueous composition in the mobile
phase. When using gradient method, see that your initial mobile phase
has at least 20% aqueous phase. This helps maintain peak shape.
5. Always see that the analyte retention time is not less than 4-5 min
especially when you are using medium (10-15cm)/long (25-30cm) columns.
Too fast an elution leads to tailing.
6. Most importantly, allow your column to equilibrate well in the mobile
phase (it can be 30min or couple of hours depending on
analyte/column/mobile phase type) and use RP columns that are end capped
(especially when using for basic compounds)
7. In RP chromatography, increase aqueous phase composition to increase
retention time of the analyte.
8. Use of ternary mobile phases (buffer, acetonitrile and methanol) some
times helps improve peak shape.
9. Always make one change to your method each time as it gives better
understanding and avoids confusion. For eg. in your case if you decrease
pH (as your compound is basic) you'll reduce retention time and if you
increase aqueous composition you'll increase retention time. Suppose if
you do both at one time, you might not see any change in retention time
which obviously gives your wrong impression. So, avoid that.
10. You can adopt different approaches for same effect. For eg. to
increase retention time you can either increase aqueous composition, use
a longer column or use a same length column with higher carbon loading
etc. But, you got to chose an approach that suits your situation best.
11. Sequence of events that you can adopt in HPLC method development for
NCEs:
a. select a column (based on your compounds' logP/logD and other
structural features)
b. select a mobile phase buffer (based on your compounds' pKa, for
neutral compounds pH doesn't really matter)
c. Work on mobile phase composition (buffer vs organic)
d. make finite changes to your method (such as use of acetonitrile
instead of methanol or use of both, increase or decrease in aqueous
composition etc)
Hope this helps.
Kasiram.
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)