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questions to a fast analysis - flow cell and data rate
Posted: Mon Oct 26, 2009 9:25 am
by dagizoli
Hi Guys!
I would like to analyze a mixture of two ketone's. A need a very short analysis time (under 1 minute). I developed a method, but the resolution is not sufficient. How can I arrive better resolution, with a 10 micro liter or a 2 micro liter flow cell? Do I need a better detector (40-80 Hz) for a fast HPLC or can I use a conventional (10Hz) detector?
Any other advise, how can I make it better?
thank you!
Best regards
dagizoli
Re: questions to a fast analysis - flow cell and data rate
Posted: Mon Oct 26, 2009 10:06 am
by virtu
Dimensions of column? Volume of cell? Width of peaks?
If volume of column<200mkl - 2mkl (cell)
20-40 data points / width of peak (data acquisition!!!)
Posted: Mon Oct 26, 2009 10:18 am
by dagizoli
dimension of column: 50x4,6 mm Phenomenex Luna C18 3 micron
Volume of cell: 2 microliter
Width of peaks: it is a good question... (overlaping) the width of two peaks is 32 sec, the selectivity factor is 1,3...
Posted: Mon Oct 26, 2009 11:24 am
by danko
What is the applied flow rate? Also the retention times for the 2 peaks. The selectivity looks fine!
Best Regards
Posted: Mon Oct 26, 2009 12:50 pm
by dagizoli
Hi Danko,
so the flow rate was 2 ml/min, and the 2 retention times are: 30 and 39 sec, but it wasn't a baseline separation....
thank you.
best regards
dagizoli
Posted: Mon Oct 26, 2009 12:59 pm
by danko
Are you sure you have any significant retention? Did you calculate K (the retention factor)?
Best Regards
Posted: Mon Oct 26, 2009 1:14 pm
by dagizoli
Sorry, I forgot to write, that I developed this method on an other HPLC. This configuration was really good there. The main difference is the UV-detector.
So, please take these informations into consideration when you answer my question.
Posted: Mon Oct 26, 2009 1:47 pm
by Chromatographer2010
If you were using an HPLC before you probably had significant gradient delay time. You could confirm this by putting an isocratic hold in your new gradient or starting with a lower initial concentration of organic and possibly a lower final concentration.
If your running isocratic then you probably have excessive bandspreading caused by void volume external to your column. You might want to re-swage your fittings since different column manufacturers using different connection variables such as tubing distance past the ferrule and ferrule angle.
The band spreading could also be causing a problem in gradient if the peaks are eluting in the isocratic portion or if the void is after the column.
Posted: Mon Oct 26, 2009 2:30 pm
by dagizoli
Thank you, dear Chromatographer2010!
I use an isocratic method, so I will check my external dead volume.
Posted: Mon Oct 26, 2009 3:08 pm
by danko
Dagizoli,
Remember the retention factor.
Best Regards
Posted: Mon Oct 26, 2009 4:54 pm
by virtu
to Dagizoli:
if 10Hz - approx. 160 data point/width of peak
Volume of cell - OK
Are you change column?
take attention:
1. vol. of injection
2. volume (and "death vol.") of chrom. communications.
3. time constant of detector
Posted: Tue Oct 27, 2009 7:51 am
by dagizoli
Hi virtu,
the column is the same.
vol. injection is the same (2microliter)
time constant is 0,1 sec (it is the minimum)
death vol. --> I must check this again....