HPLC Procedure

PPE – Gloves for handling buffer/mobile phase

  1. Check system set-up
  2. Check buffer/mobile phase reservoir (min of 540 ml)
  3. Verify reference outlet and column outlet drains into disposal container (buffer/mobile phase is toxic and flammable)
  4. Remove reference inlet and reference outlet caps
  5. Fill syringe with 5 ml buffer/mobile phase (min)
  6. Thread syringe into reference inlet
  7. Inject buffer/mobile phase into reference inlet. Verify complete priming of the reference cell by observing buffer/mobile phase exiting the reference outlet.
  8. Replace reference inlet and outlet caps
  9. Remove column outlet cap WARNING – Failure to remove cap will cause an overpressure of column and column failure!
  1. Priming of Pumps
  2. Turn on pump at plug strip
  3. Prime pump inlet – open solvent drawoff valve and drain buffer/mobile phase. Verify all air is removed from mobile phase inlet line.
  4. Tighten solvent drawoff valve.
  5. Thread syringe into bypass valve
  6. Flip bypass valve switch to the right
  7. Set flow rate to 0.4 mL/min
  8. Turn on pump at pump power switch
  9. Draw buffer/mobile into syringe for one full cycle of pump (one full cycle is indicated by the pump piston indicator travel)
  10. Set bypass valve switch to closed position.
  11. Remove syringe.
  12. Empty syringe into disposal container
  13. Set flow rate to 1.8 ml/min
  14. Verify pump prime by steady pump pressure. If oscillating pressure is observed – reprime pump – repeat steps c-m)

Column Description:

Column is a Luna 5 micron NH2 100 angstrom

Mobile Phase: 80% Acetonitrile/20% Water

Flow rate: 3 mL/min

Temperature: 40C

Injection Volume: 10 microLiters

  1. Column Preparation
  2. Verify buffer/mobile phase is exiting column outlet into disposal container
  3. Set flow rate to 3 mL/min
  4. Turn on column heater at plug strip
  5. Verify temperature is set to 40 C (see bottom of column holder)
  6. Wait for column to achieve steady state temperature (about 30 minutes for temperature stabilization and flushing of system).
  1. Software Set-up (Done during system flushing)
  2. Turn on computer
  3. Enter username (HPLC) and password (5503)
  4. After computer boot double click Diamir icon.
  5. Enter username (HPLC) and password (5503)
  6. Click on “Systems” tab (lower right hand corner of window)
  7. Check the Waters HPLC box
  8. Click on Acquisition pull-down menu – select monitoring baseline
  9. In the pop-up window select method for analysis (pull down menu)
  10. Verify appearance of baseline on chromatograph chart. If error occurs, close chromatograph window by clicking on red circle. Repeat steps g-i.
  11. Verify baseline is stable (channel 2).
  12. Verify scale is set to –200K to 200K. (right click on scale to change)
  13. Click red circle to stop software and prepare for sample run.
  1. Software Set-up for Sample Run
  2. From Acquisition pull-down menu select quick start
  3. Choose sample method (same as step 4.h)
  4. Change file name on Injection pop-up window
  5. Edit description
  6. Note sample parameters
  7. Click start button
  8. Verify System Status as “Waiting for Injection”
  1. Sample Injection
  2. Verify upper switch is in the load position.
  3. Select the Hamilton 10 microLiter syringe.
  4. Release the injection port plug by turning the bottom lever down.
  5. Remove the round silver port plug and place on a clean dry Kimwipe.
  6. Draw 10 microLiters of sample into the Hamilton syringe.
  7. Insert syringe fully into sample port.
  8. Inject sample.
  9. Replace round silver port plug into port.
  10. Turn bottom lever up until lever is tight.
  11. Turn upper switch to “Inject” position.
  12. Wait 30 seconds.
  13. Turn upper switch to “Load” position.
  1. Clean Syringe
  2. Rinse syringe with DI water until clean.
  1. Chromatograph Data Acquisition
  2. Wait(up to 12 minutes) as software collects data.
  3. From the Diamer program window select file->open->chromatograph
  4. Select saved file
  5. Choose results option just below method pulldown window located at the top left menu box.
  6. Print chromatograph
  1. System Shutdown
  2. Put away syringe
  3. Turn column heater at the power strip switch
  4. Turn the pump power off (at pump and at power strip)
  5. Dispose of mobile phase in organic waste receptacle
  6. Shutdown computer.