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Hi
I want to know what are the possible places where the drug can be
stuck or remain in HPLC system and how to over come the problem.
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Drug can most definitely be stuck in or on the column. I've overcome
this problem by running fresh moblie phase or 100% acetonitrile
through the system for a few hours. Just make sure you check what type
of column you have and what can safely be run through it before using
100% acetonitrile.
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The following message was posted to: PharmPK
Frits are notorious.
Head of the column
Stator/rotor low and high pressure sides
Injector loops
Transfer lines
Injector needles-in and outside
Needle port(s)
Rinsing/Washing plumbing as recommended by the vendor
Replacement of stators/rotors and needle ports as recommended or more
frequently if you identify a problem
Use of a pre or guard column (sacrificial)
Replacement of plumbing
Transfer/ valve lines to detector
You can also reverse the column and flush
[Not sure if I like the last one if you have any dead space in the
column, but then if you do the column is probably ready to be replaced
- db]
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Hello,
Do you mean late elution or no elution?
Retaining of drug molecules depends on the nature if your drug,column,
mobile phase, pH...
If you are using an RP column, you can flush with 10-15 column volumes
of 100% ACN or ACN:Methanol combination. If it is a NP column, you can
increase your water content.
If it is a late elution, you can optimise your mobile phase
composition and also adjust the pH basing on its pKa such that drug
will be predominatly in the ionized form
Late rentention can also occur if you use some ionpairing agents.
Thanks
Ravi Talluri
DMPK
Sai Advantium Pharma, Pune
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Contamination (carryover) is common in case of high concentration
samples and long bioanalytical batches. I had experienced
contamination in terms of carryover from HPLC injector, where the
injector rotar seal had to be replaced to overcome the carryover of
analyte. To overcome the carryover from such analytes you can optimize
the rinsing solution and the rinsing cycle time. Although due to inert
nature of HPLC components this problem is very rare.
I hope you are not concerned about late eluters; as discussed by
others they can be removed by flushing your column with high organic
proportion mobile phase.
Regards,
Kuldeep Sharma
DMPK Lab.
Jubilant Biosys Ltd.
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The following message was posted to: PharmPK
Hi, John,
Please tell us a little bit more in order to help you. Type of column,
mobile phase, do you run a gradient?, which HPLC system, analyte acidic
or basic (provide approx. pKa and approx. log P), do you use injector
rinsing step...
The most probable place of sticking is the column itself (the largest
surface area in the whole system) or perhaps also the injector
components and tubing, but this is less likely, especially if you use
high % organic in each gradient.
You can determine the source very easily - just take out part after part
(for example column, pre-column, conection tubing etc) and replace it
with a zero dead volume coupler and a fresh or new parts (unused tubing,
column...etc) and then run zero volume injections.
Please let us know your results.
This is becoming an ever more important aspect of method development
especially with the new ultra-sensitive LC QQQ MS systems with femto and
ato gram detection levels.
Regards,
Jurij Trontelj
Faculty of Pharmacy
University of Ljubljana
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