Ariel N. Grove HPLC & LC-MS Lab Report Introduction: Liquid chromatography is a method of introducing and analyte into a closed monitoring system in which the analyte is carried by the high pressure liquid medium through a system of detectors in order to measure levels or presence of a particular substance. HPLC stands for high pressure liquid chromatography, which is most commonly used on water based samples. Purpose: In this lab, we will detect caffeine in known samples of tea and will also measure the caffeine level in an unknown. We will create a calibration curve based off of the readings and compare both instruments to see which instrument better detects the analyte, which is caffeine. Procedure: prepared samples from a 1000 ppm stock solution of caffeine in distilled water (50, 40, 30, 20, and 10 ppm) HPLC 1. Before starting the HPLC, check to see that the degassed methanol and water levels are high enough (covering the silver intake tube) a. Degassing methanol or water (Only do if the levels are not high enough) i. Get a 2 L filtration Erlenmeyer Flask and place a stir bar in it ii. Connect the flask to the vacuum pump and place a rubber stopper on the top iii. Let run for 20 minutes (with the stir bar spinning) and then top off the methanol or water, and make sure all openings are sealed with parafilm 2. Make sure the computer is on, if not log on using the information on the post-it note on the computer 3. Examine both the water and methanol pump lines for any air bubbles a. Method for resolving air bubbles in the lines i. Locate the blue pump button on the instrument and turn the pump off ii. Connect the supplied syringe to the pumping line iii. Release the valve located on the bottom right by twisting it to the left iv. Pull the liquid into the syringe v. First close the valve, then remove the syringe 4. If necessary purge the lines with the following method a. Release the valve (same way as above) b. Hit the purge button on the instrument c. Let it run for a minute d. Shut off the valve, then promptly hit the purge button to stop 5. Before continuing turn the pump back on, and turn on the detector (box on top labeled SPD-10 AV) 6. To set the parameters follow this method: a. Click on “HPLC” icon on desktop b. A menu will pop up, choose “Shimadzu” c. Open the methods folder by clicking the methods icon, and set it to TSW.met d. On the tool bar near the top of the screen select the yellow icon button e. Next select parameters and set them as follows: i. Flow Rate A: .500 mL/min ii. Flow Rate B: .500 mL/min iii. Flow Rate C: 1.000 mL/min iv. Max Pressure: 6000 psi v. Min Pressure: 1 psi vi. Wavelength: 256 nm f. Select “Download” and click the [X] in the corner to close out the window 7. Running Instructions (run around 5 standard samples 2 or 3 times for best results) : a. Check that the black knob is switched to “Load” and not “Inject” b. Click the “run single” button c. In the file name box give you sample a descriptive name, and confirm the method as TSW.met d. Fill the syringe with your sample (you can fill it all the way) e. Insert the syringe into the hole, but do not inject it yet f. Select start g. The computer will prompt “Waiting for Trigger” in lower left corner h. Once the prompt appears, inject roughly 1/3 of the syringe (do not worry about putting too much in, extra flows out into the waste beaker), keep the syringe in the injection port i. Running the full 10 minutes in not required, run until your desired peak FULLY develops (usually 4-5 minutes) j. Click stop, and swiftly turn the knob back to “load” 8. Click the “Analyze” button, then “Report” and from the report menu, “Area %” 9. There will be several data points, but the most important are (note all 3 in lab book): a. Retention time b. Area under curve (might not be reliable for plotting a curve) c. Height (recommended for plotting curve) 10. Sometimes the peak is not recorded although you know it is the peak you want (based on retention time) a. Note: This is only required if your peak did not get recorded i. Select “Methods” tab ii. Select Graphic Events programming iii. Choose Manual Baseline iv. Select and hold to draw a baseline under the integration v. A box will pop up – select Integrate to Report vi. Click regenerate integration 11. To shut down the HPLC a. Go back a select “run single” b. Select “method” then select LOWFLOW.met i. Parameters should be: 1. Flow Rate A: .010 mL/min 2. Flow Rate B: .010 mL/min 3. Flow Rate C: 1.000 mL/min c. Click “download”, and error message may pop up, after that check the instrument screens (both A and B) to be sure that they say .010 mL/min d. Power down the SPD-10 AV (detector) e. Do NOT turn off the computer/log off or shut down the pumps f. Recheck both degassed containers to see if they need refilled, if they do follow the instructions at the beginning LC-MS 1. Before beginning, check that the degassed Methanol and Water levels are high enough (covering the intake tubes) a. Degassing methanol or water (Only do if the levels are not high enough) i. Get a 2 L filtration Erlenmeyer Flask and place a stir bar in it ii. Connect the flask to the vacuum pump and place a rubber stopper on the top iii. Let run for 20 minutes (with the stir bar spinning) and then top off the methanol or water, and make sure all openings are sealed with parafilm 2. Make sure the instrument is on, it should be, but if not follow this method: a. Turn on the 2 roughing pumps b. Locate the mass spec power switch which is on the back right hand side (large black switch) c. Let it pump to a vacuum before use 3. Next the Nitrogen must be turned on a. Locate the Nitrogen tank and turn the knob on the top, it should read 40 psi b. Open the valve on the wall corresponding to Nitrogen c. Open the valve on the bench top and look at the pressure gauges to see that gas is flowing 4. The next step is to open the software a. Open Analyst Software 1.3.2 icon on the desktop b. Once the program starts up you will see a white box on the left c. In this box area click “Hardware Configuration” d. There will be a list of several things with checkmarks that have a yellow circle around them, this means they are deactivated e. Click “LCMS” f. Click “Activate Profile” then “Close” after the checkmark for LCMS turns green g. Look in the lower right hand corner, a status bar will be there with 3 icons h. If the first is red, check to see that the gas is properly flowing into the instrument, this should fix the issue i. Once all 3 are yellow, you are good to go 5. Next you must select “Build Acquisition Batch” which is in the same box as the Hardware Configuration a. Where it says “Set”, choose a name that you will recognize b. In the dropdown menu to the left of the “Method Editor” button select the method “Caffeine 2011” c. Then click “Add Samples” d. For “Sample Name” put something that will prefix all samples that you run, do the same for “Data File” e. In the box under “New Samples” type the number of samples you are running (the system can process 100 samples total) f. Select “Ok” g. A spreadsheet with your samples will appear. In “Vial Position”, type the position of each sample (Note: if you type 1 in the first sample, select the whole column, right click, and select “Auto Increment” the rest of the numbers will be filled in descending order h. Select the “Submit” tab, then click “Submit” 6. Now you are ready to run the sample using this method: a. Locate the top menu bar, select “View” then “Sample Queue” b. In the same bar click “Acquire” then “Ready” and then in the acquire menu again, select “Start Sample” c. If an error occurs saying the instrument is reserved for tuning, look for the “T” icon in the toolbar and unselect it d. You can view the progress of your samples by clicking “Open Data File” 7. After each sample runs you can analyze the data: a. On the chromatogram right click anywhere and select “List Data” (Note: we had an issue once were the list data menu did not come up, after right-clicking several move times in other spots, it appeared) b. A window will appear with 2 tabs, you want the second one, “Peak List” c. If needed the number of peaks can be minimized by righting clicking in the data window and selecting “TurboChrom Parameters…” set it so that you can still see your wanted peaks, but any noise is eliminated d. Record the retention time and height of peak e. When right-clicking on the chromatogram you can select “Show Spectrum” which will show the Mass Spec data. f. For Mass Spec to work you must hover over the peak in question with your cursor g. You may want to print your data, just switch the printer from the IR 8. Shutting down the instrument: a. Click “Hardware Configuration” again b. Select “LCMS” like before c. Click “Deactivate Profile” d. Close the window after the checkmark with the yellow circle appears next to the LCMS name e. Turn off the Nitrogen at all 3 places (bench, wall, tank) f. Close the program Calculations: Caffeinated Tea 279437=18534x+222204 X= 3.088 ppm Data/Results: All HPLC chromatograms are in lab notebook. Concentration 50 (ppm) Area 1173275 40 30 20 10 971732 750870 525784 469527 Caffeinated Tea 279437 HPLC Caffeine Concentration Curve 1.510 0 6 y=18,534x+222,204 Area 1000000 500000 0 0 20 40 Concentration (ppm) Conclusion: 60 The calibration curve based off of the HPLC results came out very nice, giving us the concentration of caffeine in one of the teas (rutters) was around 3.008ppm. This instrument was very easy to use and practically no problems during this section of the experiment. The tea could have been more dissolved but, human error occurred. When it was time to move onto the LC-MS, we were informed that this instrument had been having problems the past few weeks and that no experiments were to be run on the instrument. So we had nothing to compare our caffeine results from the HPLC too making this experiment incomplete.