Method and apparatus for high pressure liquid chromatography
This modified HPLC instrument has the HPLC pump force only solvent through the serial flow line including a sampler, column, and detector; while buffer is discharged into the solvent from a syringe pump via a tee connection in the flow line downstream from the HPLC pump. A stepper motor drives the syringe pump at a discharge rate to yield an intended buffer/solvent dilution ratio compared to the steady HPLC pump flow rate. A pressure transducer detects variances of the flow line pressures, and servo controls the stepper motor for increasing the flow rate upon increased pressures and decreasing the flow rate upon decreased pressures, to hold the dilution ratio constant.
1. A method of operating a HPLC instrument having a constant flow rate pump, and a flow line from the pump serially through a sampler, column, and detector, the improved steps of discharging a pure liquid solvent from the HPLC pump under high pressures to provide a steady and constant flow rate through the flow line, sampler, column and detector;
separately discharging at a tee connection into the flow line between the HPLC pump and the column a concentrated buffer liquid that is at a flow rate that will provide an intended buffer/solvent dilution ratio, compared to the intended constant flow rate from the HPLC pump;
detecting any variance of the HPLC pump pressure in the flow line and responsive thereto of adjusting the rate of flow of the buffer into the line, by increasing it responsive to higher detected line pressures and decreasing it responsive to lower detected line pressures, operable toward maintaining the intended buffer/solvent dilution ratio constant.
2. A method of operating a HPLC instrument according to claim 1 , further including the steps of
compensating for any compressibility of the liquid buffer in having its pressure increased from atmospheric to the HPLC pump pressure, by
regularly and frequently determining the decreased buffer volume with the equation:
V=V 0 (1− K P ), where:
V is a volume of compressed buffer,
V 0 is a buffer volume at atmospheric pressure,
P is the end pressure,
K is compressibility factor of the buffer; and
increasing the buffer flow rate into the flow line responsive to its regularly determined decreased volume to maintain the intended buffer/solvent dilution ratio substantially constant.