HPLC - Designing a Method for a Sample or Series

Using TotalChrom software you can either design a method (one injection) or a sequence (several samples run with the same method). It is easiest to modify an existing method.

Things to Consider Before Starting

• Decide on the column and buffer system being used.
• If injecting >5mL of sample you need to designate one pump as a sample injection pump
• Decide of you are collecting fractions (for nonradioactive samples only).
• Decide on flow rate and operating pressure (see HPLC - Deciding on a Flow Rate and Pressure Settings)

New Method Setup

• Click on method and then Create New Method.
• Select HPLC stack
• Write something under method description (not required). Click Next.
• Select under template HPLC and fill in the blanks (not required). Click Next.
• Under Data Channels change sample rate if required (10pts/s is usually OK). Click Next.
• Under Pump Config set:
  • Ready Time: This is the amount of time of inactivity before the pump is turned off. The default is 500 min.
  • Standby Time: This is the amount of time of inactivity before the pump is set to the standby flow rate.
  • Standby Flow Rate: Typically 0.01 mL/min
  • Maximum Pressure: This should be the calculated max pressure for your column. See HPLC - Deciding on a Flow Rate and Pressure Settings.
  • Minimum Pressure: Leave at 0 psi.
  • Solvent Names: Fill these in with your buffers and/or samples
• Under Pump Program set:
  • Step 0 is for equillibration. Normally columns are equillibrated with > 10 column volumes of buffer. It is best to do this manually when connecting your column by going to Hands On on TotalChrom Navigator then setting the pump to a flow rate with 100% equillibration buffer and leaving it run for the desired volume. Set this to be 0.5 min of equillibration before each run.
  • For each subsequent step:
    • The time is the step time not the accumulated time.
    • Set the percentage of each buffer for that step. For a gradient set a target buffer percentage, then under slope set 1 for linear, -9.9 to -1.1 for a concave slope or 9.9-1.1 for a convex slope with a larger absolute number increasing the curvature.
    • Set a flow rate for that step.
    • Only select turn lamp off if you will only run one sample at a time (ie not a sequence).
  • Under the Detectors tab set the lamp to the desired wavelength (typically 260 nm) and set the time to be Program Time. There is no need to set autozero as this is hardwired to be done at the beginning of each run.
  • Under timed events, if collecting fractions set a TEV1 momentary pulse to start collecting fractions and a TEV2 momentary pulse to stop collecting fractions at the desired times of the run.
  • Click Next.
• Skip through the Process, Global Information, Component Defaults and Components unless required. For more information see HPLC - Processing and Reports
• Select Finish and save in the Methods folder for that column.