Gas Chromatograph-Mass Spectrometer
Determination of Caffeine in Beverages using SPME-GC-MS
- Caffeine-trimethyl-13C3 solution
- Caffeine-containing beverage (or bring your own)
- Caffeine/benzoic acid unknown solution (get this from me)
Required Volumetric Glassware:
- 250-mL volumetric flask
- 50-mL volumetric flask (5)
- 25-mL glass pipet
- 15-mL glass pipet
- 10-mL glass pipet (8)
- 5-mL glass pipet
- 2-mL glass pipet
- SPME apparatus (with 7-μm polydimethylsiloxane coating bonded to a fused silica core)
- 100-1000 μL adjustable micropipetter
- Very small magnetic stir bar
- Large glass vials with caps (7)
- SPME apparatus preparation.
There is nothing you need to do to prepare the SPME fiber, but find me so that I can show you how it works. It's easy, but I want to make sure everyone is familiar with how to operate the device. (You must be very careful not to break the fiber.)
- Caffeine stock solution preparation.
Accurately weigh ~0.25 g of caffeine (normal – not isotopically labeled) and quantitatively transfer to a clean 250-mL volumetric flask. Add ~200 mL of DI water and swirl the flask to dissolve the caffeine. You may need to sonicate the solution for complete dissolution of the solid. After all the caffeine has dissolved, dilute the flask to the mark with DI water.
- Standard Preparation.
- Obtain five 50-mL volumetric flasks and label them 1-5. Using the appropriate pipet, transfer 2, 5, 10, 15, and 25 mL of your caffeine stock solution to flasks 1-5, respectively. These are your standard solutions.
- Obtain five clean, dry glass vials and caps, and label them 1-5. Use a 10-mL pipet to transfer 10 mL of each standard solution into its respective vial.
- Use an automatic micropipet to spike each standard solution with 200 μL of the isotopically-labeled caffeine stock solution, then screw the caps on each vial.
- Sample Preparation.
- Pour approximately 50 mL of beverage into a 125-mL Erlenmeyer flask. If it is carbonated, remove the carbon dioxide by sonicating the flask for approximately 5 minutes (or until there is no more effervescence).
- Obtain another glass vial and cap. Use a pipet to transfer 10 mL of the beverage into the vial.
- Use an automatic micropipet to spike the sample with 200 μL of the isotopically-labeled caffeine stock solution, then screw the cap on the vial.
- Obtain another glass vial and cap, and repeat steps 2 and 3 with the unknown caffeine/benzoic acid solution.
- Instrument Preparation.
- If necessary, remove the autosampler tower from the GC injector, set it on the table beside the instrument, and replace it with the black SPME holder. Make sure the holder is placed securely over the injector nut so that it doesn’t tip over.
- Open the GCMS 6890 software by double-clicking the icon on the desktop.
- Click the Method menu and select Load.
- In the dialog box that appears select the Spme2.m folder and then click Ok.
- The Instrument Control window allows you to change all the various conditions for your GC-MS analysis. We’ll start by setting up our GC temperature program. Click on the Oven icon and choose Edit GC oven.
- Set the temperature program to begin at 50°C for 2.0 min, then ramp at 40°C/min to 280°C and hold for 2.0 minutes. After your screen looks like the image below, click the OK button.
- Next we’ll set up the MS parameters. Click on the MS icon and select Edit MS SIM/Scan…
- In the MS SIM/Scan Parameters dialog box, set the Solvent Delay at 3 min, and select SIM as the acquisition mode (Acq. Mode) as shown below. Then click the Edit SIM Params button.
- When the Edit SIM Parameters dialog box appears enter the m/z value for caffeine (194) in the m/z box and then select add ion. Repeat this for the 13C-labeled caffeine (197). Any other ions in the Edit Ion box should be deleted by selecting them and clicking Delete Ion(s). When complete, the box should look like the image below. Click the Close box.
- Data Acquisition.
- Set the SPME needle depth at 3.6 and clamp the entire device on a ring stand over a magnetic stirrer.
- Remove the cap from vial #1, add a very small magnetic stir bar, and place the vial in the center of the magnetic stirrer.
- Lower the SMPE device into the vial until the needle is just below the liquid level. Being sure to keep the vial in the center of the stir plate, clamp the SPME device in place on the ring stand.
- Slowly push down the SPME plunger to expose the fiber to the solution. Make sure the fiber is not in danger of contacting the stir bar or the walls of the vial. Lock the fiber in place using the Z-slot, and turn on the stirrer to begin stirring the solution. Stir for 10 min (set the timer so you don't forget). During this time you can complete steps 5-7 below.
- In the Instrument Control window click the green arrow to begin the set-up for data acquisition.
- In the Acquisition – Sample Information window enter your operator name, a name for the data file (only 8 characters are allowed), and a name for your sample. Click the Start Run button.
- After clicking Start Run, the message box shown below should appear:
We are performing a manual injection, so press the Prep Run key on the GC and wait for the Pre Run light to come on.
- Stop stirring after 10 min, and slowly retract the fiber back into the needle.
- The following steps should now happen quickly:
- With the needle height still at 3.6, carefully insert the SPME needle into the GC injector. (Make sure that the needle is touching the rubber septum of the injector before you insert the needle. You should sense the “sponginess” of the septum.)
- Press the Start button on the GC (see the picture above).
- Carefully push down the plunger on the SPME device to expose the fiber to the injector oven.
- You should now be acquiring data. A dialogue box will appear asking if you want to override the solvent delay. You can ignore it or select NO. (The solvent delay tells the instrument to leave the MS detector off until the specified time has elapsed. This is especially important if we are performing a normal injection of solutes dissolved in a large volume of solvent like dichloromethane. A large concentration of solvent could cause such a large signal at the detector that it could actually do damage to the detector. So in those cases we generally keep the detector off for a few minutes until the solvent passes through the system.)
- The entire run should take a little less than 10 min. After 3 minutes have elapsed, you can retract the fiber and pull the SPME device out of the injector.
(BE SURE TO RETRACT THE FIBER BEFORE REMOVING THE DEVICE!) Allow the SPME device to cool for 2-3 minutes before proceeding to the next sample.
- Repeat steps 1-11 for the rest of your standards, your beverage sample, and the unknown solution. While the next sample is being prepared, follow the steps in the Analysis section to measure the peak areas for the data you just created. Use this data to make a rough calibration plot in Excel by plotting the m/z ratio (194/197) versus the number of mL used to create the standard (2, 5, 10, 15, or 25 mL). You should see a linear relationship. Also make sure the peak ratios from your samples fall between those from your standards.
- Make sure to show me your calibration plot before you shut down the system. After I have seen your data, close the GC-MS software. Don’t save your modified method.
- All waste solutions can be poured down the drain.