GC-MS Method

CHEMICALS PROVIDED (FOUND ON SHELF UNLESS OTHERWISE NOTED): 

• • Unknown water sample (get this from me)

  • 1 M HCl solution

  • 100 mg/L bisphenol-A stock in methanol (kept in the refrigerator, MSDS)

  • 100 mg/L deuterated bisphenol-A in methanol (internal standard, kept in the refrigerator, MSDS)

  • 100% methanol (MSDS)

  • 5% methanol in water

  • Sodium sulfate anhydrous (MSDS)

  • BSTFA + 1% TMCS (kept in the freezer, MSDS) 

  • Pyridine (MSDS)

    • • Safety information: Flammable liquid and vapor. Harmful by inhalation, in contact with skin and if swallowed. May cause skin, eye, and respiratory tract irritation. Use personal protective equipment. Keep away from open flames, hot surfaces and sources of ignition. Use only under a chemical fume hood. Wash off immediately with plenty of water for at least 15 minutes. Immediate medical attention is required. Rinse immediately with plenty of water, also under the eyelids, for at least 15 minutes. Immediate medical attention is required. Move to fresh air. If breathing is difficult, give oxygen. If breathing is difficult, give oxygen. Do not use mouth-to-mouth resuscitation if victim ingested or inhaled the substance; induce artifici.

  • Hexane (MSDS)

REQUIRED VOLUMETRIC GLASSWARE:

• • 25-mL volumetric flasks (4)

  • 10-mL glass pipet

SPECIAL EQUIPMENT (FOUND ON SHELF UNLESS OTHERWISE NOTED):

• • pH meter with buffer solutions (found above the balances)

  • 100-1000 μL adjustable micropipette (next to the sink in 306)

  • 10-100 μL adjustable micropipette (next to the sink in 306)

  • SPE C-18 tubes (4)

  • Solid-phase extraction manifold and vacuum pump (next to sink in 306)

  • 25-mL Erlenmeyer flasks (4)

  • 4-inch test tubes (4)

  • MICROVAP nitrogen evaporator with water bath (in the hood in 306)

  • Pasteur pipettes (4)

  • Cotton

  • Wire rod

  • Screw-cap test tubes with caps (4)

  • Test tube vortex mixer

  • Microwave oven (between the fume hoods in 306)

  • 2-mL autosampler vials with caps (4)

PROCEDURE:

Note: It would be a good idea to begin heating the water bath shown in step C-1 below. Fill the Pyrex baking dish ~80% full with tap water and put it on the hotplate.  Put the test tube rack and thermometer in the water and begin heating. You'll want to hold the water temperature around 50oC so you'll need to adjust the hotplate temperature from time to time while you're completing parts A and B.

A.  Solution preparation

• 1. The unknown solution needs to be in a pH range of 5.5-6.5. Calibrate the pH meter with pH 7 and pH 4 buffers, measure the pH of the unknown sample solution, and adjust the pH accordingly with drops of 1 M HCl if needed. (Stir well. It should only take 1-2 drops.)

  2. Obtain four clean, dry 25-mL volumetric flasks and label them 1-4.

  3. SAMPLE: Using a clean 10-mL glass pipet, deliver 10 mL of your unknown water sample into each of the four flasks.

  4. Obtain the small vials of BPA standard solution and deuterated BPA internal standard solution from the refrigerator. (You should find the vials in a well-labeled beaker.) Don't mix them up or your experiment will be ruined!

  5. STANDARD ADDITION: Using the 10-100 μL micropipette (with a new tip), deliver 0, 50, 100, and 150 μL of BPA standard solution into flasks 1-4, respectively.

  6. INTERNAL STANDARD: Using the 100-1000 μL micropipette (with a new tip), deliver 400 μL of deuterated BPA internal standard into each of the four flasks.

  7. Fill each flask to the mark with ultrapure water, cap, and mix well.

  8. Return the BPA standard solution and the deuterated BPA internal standard solution to the refrigerator. MAKE SURE THE CAPS ARE SCREWED ON TIGHT!

B. Solid-phase extraction

1. Obtain four 500-mg SPE tubes, label them 1-4, and fit them on slots on top of the SPE manifold. Make sure all the drain valves are closed (horizontal) and that the clear plastic bucket is in place below the drains. Connect the manifold to the vacuum pump if it is not already connected. Make sure the white base containing the tubes sits squarely on the glass box to ensure a tight seal. 

2.  CONDITIONING: Condition the tubes by adding 3 mL of 100% methanol to each. (Note: 3 mL is when the liquid is at the top of the tube.) Open the valves to drain the methanol and turn on the pump.

3.  Similarly, add 3 mL of ultrapure water to each tube. When draining the water, make sure to close the valve before all the water reaches the top of the solid bed since we do not want the SPE tubes to dry out.

4.  Obtain four 25-mL Erlenmeyer flasks, label them 1-4, and pour all of your sample solutions into the the corresponding flask. (It's ok to use an extra 1-2 mL of ultrapure water to rinse the volumetric flasks into the Erlenmeyer flasks.) 

5. LOADING: Gradually begin loading your sample solutions 1-4 into the corresponding SPE tube, using disposable pipets to deliver the sample to the tubes. Turn on the vacuum and open the valves. Keep adding the solutions into the tubes until all of the 25 mL of sample has passed. Once finished, rinse each flask with 1-2 mL of ultrapure water and pass this through the column as well.

6.  WASHING: Wash each SPE tube with 3 mL of 5% methanol (make sure it's the 5% solution and not pure methanol), then open the valves completely and let the tubes dry for 10 min under vacuum.  

7.  Turn off the vacuum and wait for a moment to release the vacuum seal. Replace the plastic bucket from the manifold with the test tube holder. Obtain four 4-inch test tubes and label them 1-4. Position each test tube underneath its corresponding SPE tube, making sure the outlet tubes fit into the test tubes. 

8.  ELUTING: With the valves on the manifold closed and with the vacuum pump off, add 3 mL of 100% methanol to each SPE tube. Turn on the vacuum and open the valves slightly to keep the flow rate to a minimum retention. Be sure to collect all of the eluent in the test tubes. 

C. Drying

1. Use a Sharpie to mark the halfway point on each test tube. Place the test tubes in the MICROVAP evaporation setup as shown in the picture. With the water at ~50oC, turn on the nitrogen to maintain a gentle flow rate in the test tubes until the solutions have evaporated to the halfway mark (VIDEO).  Keep the temperature bath going when you're finished because you'll do more evaporation in steps C-4 and D-3 below.

2.  Prepare four drying tubes to get rid of any residual water in your solutions. First, obtain four Pasteur pipettes and label them 1-4. Tear off a small wad of cotton and use the wire rod (on the shelf) to push it to the bottom of the first pipette (left side of figure below). Then pour in ~0.5 g of anhydrous sodium sulfate. (It works really well to cut off both the top and bottom of a disposable pipet and use this as a funnel as shown on the right side of the figure below - credit goes to Kiley Schroeder '23 and Kailee Vigen '23 for this remarkable feat of engineering.) Repeat this procedure for the remaining three pipets.

3.  Obtain four screw-top test tubes with screw caps and label the tubes 1-4. Clamp one of the Pasteur pipettes onto a ring stand with the empty test tube labeled #1 below it. Use a disposable pipette to transfer the remaining eluent from sample solution #1 into the Pasteur pipette. Add ~1 mL of 100% MeOH to rinse the original test tube and another 1 mL to wash the drying pipette to elute all of the BPA. Use a rubber pipette bulb to push out as much solution as possible. Repeat for the other three samples.

4.  Place all four test tubes back into the MICROVAP and evaporate the solutions to complete dryness under nitrogen at ~50oC. Watch this VIDEO to see how to set up the nitrogen flow. (While waiting for the solvent to evaporate, begin setting up the software as described in Section E below. You can also do part of the cleanup in Section G.) When you are finished evaporating, just switch off the nitrogen flow with the toggle switch on top of the evaporator. You'll need to use it again below.

D. Derivatization.

1. Take the BSTFA + 1% TMCS out of the freezer. (The vial should be in a well-labeled beaker.) Make sure to return it to the freezer as soon as you are finished using it.

2. Wearing gloves and working in the hood, use the 100-μL micropipette to add 50 μL of BSTFA + 1% TMCS  and 10 μL of pyridine into the empty test tubes. (Be sure to use a different tip for the BSTFA/TMCS and the pyridine.) Screw the caps on the test tubes and vortex each for 1 min with the test tube vortex mixer. (Pyridine smells awful, so make sure the bottle is capped tightly before returning it to the shelf.)

2.  Place the test tubes in a 100-mL beaker, take the beaker to the microwave oven, and microwave the samples for 5 min on medium-high power.

3.  Put the test tubes back into the MICROVAP and evaporate to complete dryness under nitrogen at ~50oC. (When you are done, make sure to close all the valves you opened in step E4 above.)

4. Once the tubes are dry, unplug the hotplate and switch off the thermometer.

5. Use the 100-1000 μL micropipette to add 1000 μL of hexane to each test tube.  (Be quick; the hexane tends to drip from the tip.) Cap and vortex each test tube for 15 s to mix well.

6. Obtain four GC autosampler vials and caps. Label the vials 1-4 and use disposable pipets to transfer each sample solution from the test tube into its corresponding GC vial. Quickly screw on the vial caps to prevent any evaporation of the hexane.

E.  Software Setup

1. Open the software by double-clicking the GCMS1 icon on the desktop. (If you need to log onto the computer, the username is Admin and the password is 3000hanover. )

2.  Click the Method menu and select Load Method... 

3.   Find the method called BPA SPE Template.m and click OK. (Notice the file path in the image below.)

4.  Go back to the Method menu and click Save Method As... to save the template method with a new name specific to you and your partner.  For instance I might call mine Mark's BPA method.m.

5.  Again from the Method menu, select Edit Entire Method...  Check the boxes for Method Information and Instrument/Acquisition, then click OK.

6.  Enter your name(s), the date, and any other information about the experiment in the Method Comments.  Make sure the Data Acquisition and Data Analysis boxes are checked.

7.  Make sure the Inlet and Injection Parameters dialogue box looks like the image below.  This is where we tell the software that we're using the autosampler for injection and the MS for detection.  

8.  Scroll through the Edit Method Parameters window and make sure each parameter setting is the same as in the images below. The click OK.

9.  The GC Edit Parameters allows us to configure a number of different settings, most of which we don't need to deal with. But we will pay attention to the Inlets and Oven settings. Begin by selecting Inlets from the menu on the left and set the Inlet Mode to Splitless.  This means that the He carrier gas stream is not split and all of the sample is carried onto the column.  (It's actually more common to run in Split mode where only about 1-10% of the sample is carried to the column.)

10.  The Oven settings window is where we set the temperature program for the GC oven.  We want to start at a temperature of 95 oC for 1 min to condense the sample at the beginning of the column. Then we'll release the sample by raising the temperature at 45 oC/min until we hit 230 oC which we'll hold for 1 min.  Finallly, we'll raise the temperature to 280 oC at 25 oC/min. Create this program by entering the parameters shown in the following window and then click Apply.

11.  You should now see the new temperature ramp reflected in the graph at the top of the screen:

12.  Click OK to continue, then skip the Display Signals window leaving all of the boxes unchecked.

13.  The next window is where we set up single ion monitoring (SIM).  Use the image below to see where SIM is selected, and where the m/z values are entered.  Remember that 357 is used for BPA and 368 is the deuterated BPA used as the internal standard. 

14.  Click Ok when the Select Monitors window appears, then click OK to save your method.

F.  Data Acquisition.

• 1. 1. Place the four vials into the trays on  the GC-MS autosampler, and make a note of the locations.  For example, in the figure below the vials are in slots 1-4 of tray 1.

2.  Under the Sequence menu, select Load Sequence, then open SPE BPA Template.sequence.xml.

3.  Again from the Sequence menu, select Save Sequence As..., then save it with a filename unique to you.  

4.  Once more from the Sequence menu, select Edit Sequence.  A template should appear.   

5.  Notice that the template is set up to run each sample in order. If you've loaded your samples into different sample holes and/or a different tray than what's indicated, make sure to either move the vials or change the settings in the table.

6.  Load the method file you created above into the Method File slot for each run. You can do this simply by choosing it in the first line, then right-clicking on it and choosing Fill Down. Also enter a unique filename for each run. [NOTE: Although I didn't do this in the image below, it would be helpful if you created your own folder in the Data Path so it will be easy to find your data when the sequence is complete. Just click the ... box in line 1 of the Data Path column and choose Make New Folder. Create your folder and fill down as before to fill in the rest of the column.]

7. Click OK to exit the sequence table, then select Run Sequence from the Sequence Menu. The dialogue box below will appear. Enter information about your data in the Sequence Comment box and your name in the Operator Name box, then click Run Sequence to begin data acquisition. 

8. Your first sample should begin to run. If the "Override solvent delay" dialog box appears, click "No."

G.  Clean up

1. 1. Throw all used SPE tubes and micropipette tips in the trash.

   2. Pour the aqueous BPA waste from the SPE plastic bucket and any leftover sample solution into the "aqueous BPA waste" container in the hood.

   3. Pour the organic BPA waste from your GC vials into the "organic BPA waste" container in the hood.

   4. Dispose of the Pasteur pipettes and empty autosampler vials in one of the glass waste containers.

   5. Clean all test tubes and 25-mL Erlenmeyer flasks with soap and water, giving 2-3 final rinses with RO water, and leave them inverted on a test tube rack next to the sink to dry.

   6. Place all used glass pipets in the "Pipets to Be Washed" bin. 

   7. Rinse all volumetric flasks 5-6 times with tap water and then 2-3 times with RO water, and return to the shelf. (Use acetone to wash off any markings.) Similarly rinse all glassware from your drawer and return it to your drawer.

   8. Clean all bench top and hood areas. Use the sponge in your drawer to clean your bench area.

H.  Data retrieval

1. Open the Qualitative Navigator software by double-clicking the program icon on the desktop.

2.  When the Open Data File window appears, find your data, select all the data files together, and click Open.

3.  From the Chromatograms menu, choose Extract Chromatograms.

4.  When the Extract Chromatograms window appears, make sure to set the Type as SIM and also make sure the Integrate when extracted box is clicked, then click OK.

5.  From the File menu, select Print → Analysis Report...

6.  In the Print Analysis Report window, make sure to choose Microsoft Print to PDF as the printer, then click OK.

7.  Choose a location to save the report as a PDF file, then open it to verify that all your data is there. Close the program (you don't need to save anything once you have your report) and make sure to upload the report to your LabArchives notebook.