Chromatography Forum

I am facing the high pressure problem with follwoing chromatographic conditions even with barnd new column.

Please help me.


Information:
phosphate 2 with pka value of 7.9
Mobile phase-A pH: 7.0 Potassium phosphate buffer (6.8g in 2000lit)
Mobile phase-B:methanol, and tetrahydrofuran (39:11)
column: waters Symmatry C8, 15cm x 4.6mm x 3.5um
injection volume: 20ul
flow rate: 1.0ml/min
temperature of column: 40 degC
detector: UV 250m
Prepare a mixture of methanol and water (1:1).
HPLC:Agiment 1200 series

Time MP A MP B
0 80 20
50 50 50
65 50 50
75 40 60
80 80 20

How high is your pressure? (A rough estimate tells me that you should expect between 2500 and 3000 psi.)

For a similar system which uses phosphate buffer and methanol in gradient and 1ml/min flow rate, pressure fluctuates from 800 psi (97% methanol) and 2000 psi (97% buffer) for me.

How high is the pressure for your system? cause a brand new column should not ideally be the culprit for high back pressure... check your tubing as well...

Mukul

Your mobile phase is aggressive - high back pressure generating
Plus the smaller particle size 3.5 uM.

The method conditions are generating this high pressure.

Run the conditions (mentioned on manufacturer testing) with similar conditions and check the back pressure. Compare with the specs.