5.6: GC-MS Procedure and instructions for data analysis

In this experiment, you will extract residual compounds present on paper currency and quantitate the amount of cocaine on each bill using GC-MS.

Materials

• Paper currency: bring your own if possible - the money is not destroyed by the extraction process. We will provide bills if you do not have your own.
• Methanol, analytical grade
• 4 ppm solution of doxepin (Internal Standard)
• Serial dilution of cocaine including internal standard (this will be prepared for you)
• 15 mL centrifuge tube for each dollar bill extraction
• 3 mL syringe and 0.2 µm filter to filter dollar bill extracts
• Agilent CG-MS with Mass Hunter Software
• Two 10 mL beakers; one for syringe wash methanol, one for waste
• 10 µL syringe for injecting samples

Create a standard curve

The standard solutions contain 0.6 ppm doxepin internal standard and cocaine concentrations between 0.1-5 ppm. These solutions are prepared for you. Each analysis takes about 12 minutes.

How to use your time: You should start running the standards using the operating instructions, then prepare your extraction samples while you are waiting between standard injections. After you have created your extraction sample, use the time between injections to compile and analyze your data using the instructions for data processing in section 5.9.

With the software open and the correct method running (see operating instructions in section 5.8), inject at least four of the standards using quantitative injection technique; you can choose which standards to use based on your pre-lab reading. 

1. Methanol blank
2. 0.1 ppm cocaine + 0.6 ppm doxepin in methanol
3. 0.5 ppm cocaine + 0.6 ppm doxepin in methanol
4. 1.0 ppm cocaine + 0.6 ppm doxepin in methanol
5. 2.5 ppm cocaine + 0.6 ppm doxepin in methanol
6. 5.0 ppm cocaine + 0.6 ppm doxepin in methanol

During any waiting time while samples are running, begin data analysis using the instructions in Section 5.8. 

Ideal milestones for lab progress

Plan to use the entire scheduled time for this experiment. The following is an ideal timeline for progress in this 3-week module. However, real lab work does not always adhear to ideality. Instrument and sample issues are expected, and flexibility is delibrately built into the schedule. You should process the data as it is being collected so that issues are recognised immediately. If you expect a problem with the instrument or samples, stop immediately and notify your instructors.

Suggested timeline (ideal):

• Week 1: By the end of the first lab meeting, you have...
  • A complete calibration curve (methanol blank + four standards = 5 injections) 
  • One of the standards is run in triplicate  to gauge the amount of variation between sample runs (2 additional injections)
  • your extraction sample is run in triplicate (3 injections)

This is 10 injections at 12 minutes each, or about two hours of time if you are efficient. Use time between injections to work on data analysis. Before you leave lab on Day 1, be sure to collect and anayze your data. In other words, create your calibration curve as your data is being collected. If the calibration is going well, you may use any additional time to continue making replicate injections. 

• Week 2: By the end of the second lab period, you have...
  • At least two replicates for each of the calibration points. If time allows, you should inject all of the standards three times total (ie two times on Day 2).
  • At least one additional injection of the sample. If time allows, inject twice.
  • Comparison of the data from each day to confirm it is consistent.
• Week 3: If you run into issues on the first two days, this period can be used for writing. However, if you do run into issues or have not met the milestones above, then it is expected that the time in Week 3 is used for data collection.

Ideally, by the end of Day 2 in lab, you would have triplicate data points for each standard solution. However, you can decide when you are satisfied that your standard curve is appropriate to estimate the amount of cocaine present. If not, the third lab meeting should be used for additional data collection. Diagnose any issues with your standard curve using the data analysis methods outlined in Section 5.8.

Instructions for quantitative injection

It is important that the same volume be injected each time if the quantitative precision is to be as good as possible in this experiment. Please inject between 1-2 \(\mu L\) of sample; these instructions are for exactly two  \(\mu L\) injections.

1. Wear gloves when handling the syringe.
2. Clean the syringe by drawing up and expelling into waste several full syringe volumes of solvent (the same solvent used in the sample solutions).
3. Empty and dry the syringe by pumping the empty syringe up and down several times.
4. Move the syringe plunger the the 2 \(\mu L\) mark to fill the syringe with a plug of air.
5. Draw up about 2 \(\mu L\) of sample solution by moving the plunger to the 4 \(\mu L\). This is most easily done by drawing up excess solution and then pushing the plunger back to the goal mark.
6. Dab the tip of the syringe to remove excess sample. Then, pull the syringe plunger back so that you can see the air on both sides of the solution.
7. Measure the volume of solution. This is most easily done by pushing the plug of solution exactly to the zero calibration mark and measuring against the graduated markings. 
8. Record the volume of solution to be injected.
9. Inject by pushing the plunger all the way to the zero mark (thus injecting the solution and the air plug). Then determine how much was actually injected.
   1. Position the needle in the guide of the injection port.  Pierce the rubber septum.  Smoothly inject {t<1 second}; withdraw the syringe smoothly.
   2. Draw back the syringe plunger and measure any remaining sample volume.  RECORD this value and subtract it from the initial total volume.  This result is the volume you actually injected.  If necessary, use the volume injected to normalize the areas of all peaks for each run.

Preparing the analyte samples

You should prepare your sample for analysis while you are also running the series of standards on the GCMS.

1. Fold each bill accordion style, with about 12 folds. Insert the folded bill far into a 15 mL centrifuge tube.
2. Use a digital pipette to add exactly 5.00 mL of methanol into the tube with the folded bill.
3. Invert the tube for at least 10 minutes.
4. Using tweezers, carefully remove the bill from the tube. Try to leave as much of the methanol in the tube as possible.
5. In a 1-4 mL volumetric flask, fill part way with the extraction solution, then add enough internal standard solution (4 ppm doxepin in methanol) to reach a final concentration of 0.6 ppm doxepin. Fill to the calibration mark with the extraction solution.
6. Use a 3 mL syringe with a 0.2 µm filter to filter the contents of the volumetric flask into a clean glass tube that can be sealed to prevent evaporation.

Treatment of Data

The general criteria for writing and submitting lab reports are found in the orientation module. Specific additional expectations for this module are given below.

1. Include the following results in the lab report: (assume general expectations apply, e.g. label plots and figures with titles, axes titles, sample calculations, etc)
   1. Standards
      1. A good chromatogram of each standard sample (peaks labeled with r.t. and identity of each compound of interest). Ideally, this would be one figure with chromatograms stacked on the same X axis. Don't give us several pages of individual chromatograms, please!
      2. A mass spectrum of cocaine and doxepin (with labeled peaks).
      3. Results of the library search of one standard solution.
   2. Calibration
      1. Calibration plot of \(C_A\) (cocaine concentration) vs \(\frac{S_A}{S_{IS}}\) including a linear fit with y-intercept, slope, and errors.
      2. Derived relative response factor (\(K\)) and its associated error.
   3. Quantitation
      1. How much cocaine is detected on each bill analyzed in units of \(\frac{\mu g}{mL}\) (ppm) and  \(\frac{\mu g}{\text{bill}}\)
      2. Include concise error analysis.
   4. Identification of other compounds on the bill
      1. What compounds, if any, were detected in analyte solutions that were not present in the standard solutions? Provide the results of the library search.
      2. Provide the Mass Spectra of notable compounds detected.
2. In your discussion, be sure to address points from the pre-lab assignment, and.. 
   1. Discuss the relationship between boiling points, molecular structure, and retention time of the compounds.
   2. Explain why doxepin appears as two peaks, and suggest how we might increase separation cocaine from doxepin.
   3. Discuss the fragmentation pattern you observed and how it supports assignment of the compound. Address how molecular structure yield the observed mass ions.
   4. Discuss and interpret the results of the library search on your dollar bill

References

1. McLafferty, F. Interpretation of Mass Spectra; 3rd ed.; University Science Books: Mill Valley, CA, 1980.
2. Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Interpretation of Organic Compounds; 4th ed.; Wiley: New York, NY, 1981.
3. Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, Fifth Edition; Harcourt Brace: Philadelphia, 1998; 591-621.
4. Heimbuck, C.A.; Bower, N. Teaching Eperimental Design Using a GC-MS Analysis of Cocaine on Money: A Cross-Disciplinary Laboratory. 79, 10, 2002.