Shifting Retention times/Peak areas

[pgdow2]

Hi,

I have an Agilent 1100 system, I am running a gradient mobile phase through a c18 column. I am failing system suitability repeatedly. When I examined my start/End pressures I am experiencing significant drops in pressure - after column conditioning the pressure seems stable at 160 bar, but after a few injections the pressure drops to as low as 115 bar. I have also witnessed the pressure climbing back up at the end of the run for the remaining Supelco injections - any ideas?

[sassman]

It is normal for the pressure to drop during a gradient run since the organic part of your mobile phase has lower viscosity than water. Do you have a sufficient equilibration period at the end/beginning of your run? If you can give more details about your method, it might help us to diagnose the problem.

[pgdow2]

My gradient is as follows:

Solvent A Water
Solvent B Scetonitrile
Sovent C 1% TFA

Time (Minutes)----Flow (mL/Min.)---------%A----%B----%C
N/A------------------------1.25----------------65----25-----10
1.0------------------------1.25----------------65-----25-----10
25.0-----------------------1.25----------------45-----45-----10
30.0-----------------------1.25----------------45-----45-----10
35.0-----------------------1.25----------------65-----25-----10
40.0-----------------------1.25-----------------65----25-----10
120.0----------------------0.00-----------------65----25-----10

Conditions are:

1.25mL/min
30min run time
10min next inject delay

Pressure changes we have witnessed are as follows:

---------------------------------------Pressure (Bar)
---------------------------Start Pressure End Pressure
Equilibration - 40mins---------161--------------162
Column Condition 160mins---161--------------163
Standard Inj 1------------------161-------------144
Standard Inj 2------------------160-------------141
Standard Inj 3------------------157-------------139
sample matrix inj 1------------155-------------137
sample matrix inj 2------------152--------------135
sample matrix inj 3------------150-------------135
sample elution inj 1-------------150------------134
sample elution inj 2------------149------------134
sample elution inj 3------------149-------------134
sample @ 5micrograms--------147-------------133
sample @ 5micrograms--------146-------------113
sample @ 5micrograms---------122-----------113
sample @ 5micrograms----------119-----------112
sample @ 5micrograms----------118-----------109
sample @ 5micrograms----------115-----------131
Sample @ 3micrograms-----------137----------121
Sample @ 3micrograms-----------130----------118
Sample @ 3micrograms----------146-----------128
Sample @ 7micrograms-----------133----------116
Sample @ 7micrograms---------121------------131
Sample @ 7micrograms------------137----------117
Standard Inj 1------------------123--------------136
Standard Inj 2-------------------151--------------139
Standard Inj 3------------------155-------------140