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I've been working on what should be a simple enough HPLC assay for
caffeine using a C18 column, acetonitrile, NaH2PO4, water mobile
phase. I got useful sensitivity and was waiting for more samples when
I used the system for another assay... maybe the problem. However,
after going back to the caffeine assay I thought the resolution had
dropped some. I had some old (old, reports dated 1995...) unused,
sealed in the box columns so I thought I'd try a 'new' one. Worked
great for a day or so, go ahead collect samples to assay, adjust
acetonitrile between 8 and 15% to move the peaks into the 'right'
place. Now, the resolution has gone, a few days later. I've just
tried a second 'new' column. This one had probably dried out... no
better
The question is, what is the shelf-life of C18 HPLC columns? If it is
somewhat less than 12 years ;-) then I need to buy a new one...
I think I'll try running 100% acetonitrile while waiting for some
answers...
Any thoughts, thanks
David
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The following message was posted to: PharmPK
David, if not the column check:
- detection wavelength
- detector lamp (how many hours of use?)
- detector cell for leaks
- integrator settings
- injection volume/loop size/reproducibility
--
William R. Wolowich, Pharm.D.
Interim Chair and Assistant Professor
Department of Pharmacy Practice
College of Pharmacy
Nova Southeastern University
[Had tried different wavelengths with the other assay but back where
I started, 275 nm. Lamp is old. The solvent front spike seems about
the same so I don't think a cell leak is the problem but I may get to
look into that... just using a recorder for now, same injection
volume using an autoinjector. May not have great reproducability but
solvent front peak and low resolution caffeine/theophylline (IS)
peaks are consistent. Better before, bad now. Thanks - db]
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The following message was posted to: PharmPK
You are advised to use an old column for method development and if
you have accomplish method
Development of course with Validation then you can comfortably
purchase a new column.
Shelf life of column not specific like tablets depends how you have
used and maintained them.
Cleaning columns sometimes is a challange although HPLC literature
recommends 50:50 methanol: water. There are situations where it does
not work so one has to design the appropriate organic solvent.
For instance if you are working with methanol : phosphate buffer as
the mobile phase the best organic solvent to clean the column is
simply water 100%.
s.o.o
[Started with a used column, moved to a 'new' column which was better
for a day or so but not now. The 'new' columns have been setting in a
cupboard unopened for some time, 12 years. I'll try column cleaning
but the 'new' columns haven't seen many samples. Thanks -db]
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David,
Am I reading your notes right: you are using a C18 column in normal
phase? Phase collapse can occur in 100% aqueous solution for even
"fresh" ones. Only some "fresh" C18 columns can be used with 5% (10%
is better) organic.
Regards,
Frank Bales, Ph.D.
[Reverse phase, i.e. 5-15% acetontrile with some NaH2PO4 in water. I
did 'wash' it with 100% acetonitrile and I seem to have good
resolution (for now) with the second 'new' column using 10%
acetonitrile. - db]
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Dear Prof. Bourne,
Regeneration would be suitable for the columns of your case.
This can be done by `washing' the columns in the following sequence.
Hexane (non polar), Acetonitrile, Water and Acetonitrile.
Regards.
Suresh
[Thanks for the suggestion, I washed with acetonitrile (100%) and on
Friday afternoon the column was much better. I washed with 10%
methanol before shutting down for the day. Hopefully the column will
be useful on Monday - db]
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If the column was simply in the Cupboard for twelve years and never
been used then it is almost as good as new. Just check the column
pressure usually should be around or less than 1000 PSI.
Of course if the column pressure is above 3000 PSI however much you
clean it then you have to
discard it.
Where you are cleaning with 50:50 methanol water and the column
pressure appears not to come down. Try this two possibilities:-
100% methanol for 24 hours.
100% water for 24 hours
See what happens.
s.o.o
[10-15 min of acetontrile (100%) seemed to make a big difference and
I was able to get good peaks and even a reasonable standard curve.
I'm hoping Monday will see a positive progression. Column pressure is
fine. I did have cavitation at the pump head but was able to clear
that OK. Outlet flow seemed OK most of the time so I don't think that
was The problem. It will interesting to see if the second 'new'
column holds up and if the first 'new' column can be used. Thanks - db]
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