Lab Report on GCMS Microextraction

The lab report below was submitted as part of the coursework for CM3292 Advanced Experiements in Analytical and Physical Chemistry. Please do not plagiarise from it as plagiarism might land you into trouble with your university. Do note that my report is well-circulated online and many of my juniors have received soft copies of it. Hence, please exercise prudence while referring to it and, if necessary, cite this webpage.

CM3292 Analytical Experiment 3.1 GCMS Microextraction
To extract volatile compounds from an unknown using headspace solid-phase microextraction (SPME) and single-drop microextraction (SDME), and to separate and identify the extracted compound by gas chromatography/mass spectrometry (GC/MS).
Retention time using SPME-GCMS (min)
Retention time using SDME-GCMS (min)
The retention time for part A and part B is the same at 7.900 minutes. This is because the same operating conditions for the GC were used for both parts.
The 2 samples from parts A and B have similar mass spectra with peaks observed at m/z = 53, 68, 79, 93, 107, 121 and 136. This meant that both analytes have the same compound. After comparing the mass spectra with the NIST21 Library Search, it can be concluded that the unknown sample is limonene as the peaks of the sample limonene mass spectra match the obtained experiment results.

The limonene molecule undergoes electron-impact ionization, which involves high energy electrons and first removes one of the electrons in the π bond, leaving the carbon skeleton intact.  This gives the molecular ion (m/z = 136), which is also a radical cation.  It is easier to remove an electron from the π bond than σ bond, as the former is in a higher potential energy molecular orbital.  
                                                                     m/z = 136
From the molecular ion, we can determine the molecular weight of the compound. It had a low intensity in the mass spectra, which meant that it was unstable and had a tendency to break into stable, small fragments.
The cyclohexene ring of the molecular radical cation (m/z = 136) undergoes an alpha cleavage, losing a methyl radical to form a fragment vinylic cation (m/z = 121).
                                                                                                m/z = 121
The terminal alkene of the molecular radical cation (m/z =136) undergoes an alpha cleavage, losing a methyl radical to form another fragment vinylic cation (m/z = 121).
α cleavage

                                                                                                m/z = 121
Molecular fragment: C8H11+
The fragment cation (m/z = 121) undergoes a 1, 2-H shift before 2-bond cleavage, losing a neutral ethene molecule to form a smaller fragment vinylic cation (m/z = 93).
1,2-H shift

2 bond cleavage

                                                                                                                                   m/z = 93
Molecular fragment: C6H7+
The molecular radical ion (m/z = 136) undergoes a retro-Diels Alder cleavage to give a diene radical cation and a neutral diene molecule (both of m/z = 68).

Retro-Diels Alder cleavage

                                                                                m/z = 68
This was the peak with the highest intensity and was thus, assigned as the base peak, which also meant that it was the most stable fragment. This diene ion fragment was very stable due to it consisting a carbocation between the conjugate double bonds in the diene, making it resonance- stabilized and hence, it was much more stable than the other fragments and would not very likely fragment again.
The m/z = 68 diene fragment lost a methyl group to give a conjugated diene ion and a methyl radical. This peak had a medium intensity since it was rather stable due to conjugated double bond.
α cleavage

                                                                   m/z = 53

cannot be determined.  Also, the sample must be very volatile so that it can come into contact with the electron beam for EI.  Hence, this ionization method is not feasible for non-volatile compounds with high molecular weight.
One major disadvantage of GCMS is observed in this experiment. The column that was used cannot separate the optical isomers. As there is a chiral centre present in limonene, the molecule could be an R-isomer or S-isomer. Both of the will give the same fragmentation pattern.
(A)               Analysis of volatile flavour and fragrance compounds by headspace solid-phase microextraction (SPME) combined with gas chromatography / mass spectrometry
In this experiment, the SPME fibre is exposed to the headspace of the sample for 10 minutes.  It is assumed that this time is long enough for equilibrium to be established between the sample headspace and the extraction phase, such that the fibre does not adsorb more analytes. The polydimethylsiloxane fibre is suitable for the extraction of non-polar analyte. Stirring disrupts the concentration gradient in the sample and increases the analyte concentration in the fibre.  Hence, extraction is faster and more efficient when the sample is stirred.
(1) Untreated shampoo or fragrance sample cannot be simply injected into the GC/MS system for
(2) a) SPME fibre is expensive and reusing it helps to save cost. In addition, if quantitative analysis with standards were carried out, the fibre used should be kept constant.
     b) Problems associated with the reuse of SPME fibre include contamination with previous samples and decline in performance with increased usage.  Nonvolatile compounds may stay on the fibre and can be difficult to be removed.  Using headspace SPME will avoid the problem of nonvolatile compounds stuck on the fibre. To remove any possible contaminants on the fibre, the fibre should be cleaned after each extraction by leaving it in the GC injection port for a prolonged period at high temperature for complete desorption.
(3) Yes, it is possible to perform quantitative analysis with SPME using calibration plots obtained by repeated extraction of a series of standard solutions of different concentrations. Since the amount of analyte adsorbed can be quantified from the peak area in the GC/MS chromatogram, subsequent manipulations of the results can yield the quantity of analyte within the sample.  A lot of preliminary studies have to be done as exhaustive removal of analytes to the extracting phase does not occur in SPME, but an equilibrium is reached between the sample matrix and the extracting phase. Higher concentrations will take shorter time to reach equilibrium between fibre and headspace. Quantitative analysis result will only be valid before reaching equilibrium. As the calibration plots are not linear, the results may not be reproducible and accurate.

(4) Some of the disadvantages of liquid-liquid extraction are:
(5) Some of the advantages of SPME are:

(6) Highly volatile compounds that are sorptive are most suitable for headspace SPME analysis. Direct immersion SPME can be used to analyze non-volatile compounds.  In general, SPME is suitable for analysis of volatile and semi-volatile compounds in solid, aqueous and gaseous matrices. SPME has been used for wide range of applications, particularly in environmental, biological and pharmaceutical analyses.
(7) I would not use SPME for analyse of steroids in a human urine sample. This is because steroids are often involatile, which render analysis by headspace SPME impossible. As urine is a complex matrix, I would not want to dirty the expensive fibre by using direct immersion SPME. SPME may be used to analyse steroids after derivatization anof non-volatile steroids.

(8) Yes, SPME incorporates extraction, preconcentration and sample introduction into a single step.  Extraction and preconcentration occur during the sorption of analyte onto the stationary polymer. Sample introduction takes place when the analyte is thermally desorbed into the GC injector. The analytes are directly transferred from the fibre into the injection port of the GC and this minimizes loss that is prevalent in multi-step preparation. 

(B) Headspace single-drop microextraction (SDME) in combination with gas chromatography / mass spectrometry
(1) Direct immersion SDME cannot be used for a shampoo, fragrance or fruit sample because it can introduce suspended or soluble salts or solids that can contaminate the GC.  It can only be used for cleaned, filtered samples. 
(2) It is possible to perform quantitative analysis with SDME. However, problems involved in quantitative analysis with SPME are encountered in SDME too.

(3) Advantages of SPME over SDME are:
(4) Some of the advantages of SDME over SPME are:
  • SDME involves a solvent for extraction.  The range of pure or mixture solvent that can be used for SDME is more than the stationary phase (fibre) for SPME.  Hence SDME has higher selectivity and can extract more types of analytes than SPME.
  • SDME solvent (only a small quantity is required) is cheaper than SPME fibre.  SPME fibres are more expensive, have limited life-time and degrade with increased usage.
  • Sample carry-over in SDME is absent, as a new drop of solvent is used for each sample.
  • Solvent evaporation for SDME is faster than desorption in SPME in the GC injection port, tailing of peaks may result in SPME.

(5) Volatile, organic compounds are most suitable for headspace SDME analysis. For direct SDME, it can be used for semi-volatile compounds.

(6) Yes, I would use SDME, especially the headspace method, to analyse a human urine sample for volatile compounds. This method is simple, inexpensive, accurate and a low detection limit. The extracting solvent can be varied to selectively extract the analyte of interest.

(7) Yes, SDME incorporates extraction, preconcentration and sample introduction into a single step. 

(8) The sample may be heated to facilitate the extraction of analyte. Agitation of the sample by
The volatile analyte, which was found in the headspace of both samples, was successfully extracted by both SPME and SDME and analysed by GC/MS with a retention time of about 7.9 minutes. The identity of the analyte was determined to be limonene by comparing the acquired mass spectrum with the library mass spectrum.
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[5] Xu, L.; Basheer, C., Lee, K. H. Developments in single-drop microextraction. Journal of Chromatography A, 2007, 1152, 184-192.