HPLC Procedure 8 - 2005 Use Gloves and safety glasses for handling buffer/mobile phase – (Mobile phase is toxic and flammable) Column Type: Luna 5 micrometer, NH2 100A Turn on both power strips to apply power to the pump, column heater and detector. Turn on detector power switch located on front of detector 15 – 20 minutes before running samples. Turn on pump power switch; make sure the flow rate on the pump is set to 00. Make sure the attenuator on the detector is set to 8X. Check that the solvent select knob is set to position 2 and the waste knob is set to waste. These knobs are located on the pump module. 1. Check system set-up a. Check buffer/mobile phase reservoir (min of 540 ml) b. Verify LOAD/INJECT switch is set to LOAD, Verify Injection pin is in injection port and that port is locked. c. Verify reference outlet and column outlet drains are draining into disposal container (buffer/mobile phase is toxic and flammable) d. Remove reference inlet and reference outlet caps e. Flush reference cell by filling syringe with mobile phase – use large gas type syringe with metal fitting on the end - with 5 ml buffer/mobile phase (min) f. Thread syringe into reference inlet located on the left side of the differential refractometer (R401). g. Inject buffer/mobile phase into reference inlet. Verify complete priming of the reference cell by observing buffer/mobile phase exiting the reference outlet. h. Replace reference inlet and outlet caps i. Remove column outlet cap WARNING – Failure to remove cap will cause an overpressure of column and column failure! j. If many samples need to be analyzed, the reference cell may need to be primed again after several hours of running. 2. Priming the Pump a. To prime pump inlet – open solvent drawoff valve located on lower front panel of pump and drain buffer/mobile phase into a small waste flask. Check mobile phase inlet line to insure no air bubbles are in line. (do not change “waste and solvent” knob) b. Tighten solvent drawoff valve. c. Verify pressure switch is set to 2000 PSI. (check range setting knob) d. Thread syringe into left side bypass valve. e. Flip bypass valve to the right. This will send mobile phase through the reference side. f. Set flow rate to 0.4 mL/min g. Turn on pump at pump power switch. ( Verify power strip is on ) h. Draw buffer/mobile into syringe for one full cycle of pump (one full cycle is indicated by the pump piston indicator travel) you may not pull liquid into the syringe – the purpose of this is to pull solution into the pump. i. Set bypass valve to left position, this runs mobile phase through the detectors with the sample. j. Remove syringe. k. Empty syringe into disposal container. l. Set flow rate to 1.8 ml/min m. Verify pump prime by observing steady pump pressure on gauge. If oscillating pressure is observed – re-prime 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 3. Column Preparation a. Verify buffer/mobile phase is exiting column outlet into disposal container b. Set flow rate to 3 mL/min. If pump stops and reset button lights depress reset button, if pump stops again contact lab manager. c. Turn on column heater at plug strip, it has no power switch of its own. d. Verify temperature is set to 40 C (see bottom of column holder) e. Wait for column to achieve steady state temperature (about 30 minutes for temperature stabilization and flushing of system). 4. Software Set-up (Done during system flushing) a. Turn on computer b. Enter username (analab) and password (Letmein!) c. After computer boot double click Diamir icon. d. Enter username (analab) and password (Letmein!) e. Click on “Systems” tab (lower left hand corner of window) f. Check the Waters HPLC box g. Click on Acquisition pull-down menu – select monitoring baseline h. In the pop-up window select method for analysis (pull down menu). If you are running glucose choose the sugar test.METH method. i. Verify appearance of baseline on chromatograph chart. If error occurs, close chromatograph window by clicking on red circle. Repeat steps g-i. j. Verify baseline is stable (channel 1). k. Verify scale is set to –200K to 200K. (right click on scale to change) l. Click (red X circle) to stop software and prepare for sample run. 5. Software Set-up for Sample Run a. From Acquisition pull-down menu select quick start b. Choose sample method (same method as step 4.h) – click O.K. c. Change file name on Injection pop-up window. d. Edit description e. Note sample parameters, change if required. f. Click start button. g. Verify System Status as “Waiting for Injection”.6. Sample Injection a. Referring to load / inject valve in step (2e) verify valve handle is in the load position. And flow rate is 3ml/min. If valve handle is not in load position the plug will be forced out upon release by 2000 PSI. b. Select the Hamilton 10 micro liter syringe. c. Release the injection port plug by turning the bottom lever down. d. Remove the round silver port plug and place it in the hole in the valve handle. e. Draw 10 micro liters of sample into the Hamilton syringe. f. Insert syringe fully into sample port. g. Inject sample. h. Replace round silver port plug into port. i. Turn bottom lever up until lever is tight. j. Turn upper switch to “Inject” position. This will automatically start data acquisition. k. Wait 30 seconds. l. Turn upper switch to “Load” position. m. This should start the data acquisition. The display should change from “Waiting For Injection” to “ Running” n. If you cannot see your data, right click on the display and zoom in/out as needed or hold the right mouse button down and the move the cursor vertically over the graph. 7. Clean Syringe a. Rinse syringe thoroughly with DI water until clean. 8. Chromatograph Data Acquisition a. Wait until sample clears the column (returns to baseline) as software collects data. b. Once sample has returned to baseline select stop button, the red one with a white X. c. From the file tab go to ->open->chromatograph d. Select your file, click open. e. Choose results option located at the top left menu box. f. To print chromatograph click on printer icon on top toolbar. A/B box should be set to A. g. Save