questions to a fast analysis - flow cell and data rate

[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

[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!!!)

[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...

[danko]

What is the applied flow rate? Also the retention times for the 2 peaks. The selectivity looks fine!

Best Regards

[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

[danko]

Are you sure you have any significant retention? Did you calculate K (the retention factor)?

Best Regards

[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.

[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.

[dagizoli]

Thank you, dear Chromatographer2010!

I use an isocratic method, so I will check my external dead volume.

[danko]

Dagizoli,

Remember the retention factor.

Best Regards

[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

[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....