Gas Chromatography-Mass Spectrometry Analysis (GC-MS) is a technique that incorporates chromatographic and spectroscopic techniques in separating and identifying constituents in a sample. According to Erika, Josefina and Maria (2011), the major goal of the technique is in quantifying an amount of a sample, which is done by comparing the relative concentrations among the atomic masses in the produced spectrum. The sample that needs to be quantified is organic and volatile, or semi volatile.
The GC part is used to separate is essential in carrying out separation techniques where mixtures are isolated from their constituents using a temperature-controlled capillary column. Smaller molecules in the instrumentation travel faster down the chromatographic column compared to larger ones which have higher boiling points (Erika et al., 2011). On the other hand, the MS part is important in determining the various components from their mass spectra. Each compound in a sample has a specific mass spectrum, which can be related with mass spectra records, and thus recognized (Erika et al., 2011). Furthermore, using of standards can result in quantifying the samples.
All states of matter can be analyzed by GC-MS. In case of liquids, the sample is injected directly into GC while for gases gas tight syringes are used. Solids are analyzed using solvent extraction, outgassing or pyrolysis (Erika et al., 2011). Outgassing is a technique where heat is applied to volatilize organic constituents and collected in a cold trap for analysis. Pyrolysis technique is where materials are introduced in the instrument by direct heat to break materials in a reproducible way.
The current application for GC-MS analysis is in: evaluating extracts from polymers, qualitative and quantitative analysis of volatile organic components, outgassing studies, thermal desorption, liquid or gas injections, and testing residual solvents. The future application is the use of MS instruments with quadruple ion trap analyzers in environmental analysis, which is easy in operation and can detect up to limits of parts per trillion; and portable GC-MS instruments will find more application when there is the need to rapidly identify a sample with certainty in case of an accident (Erika et al., 2011).
Sample to be analyzed using GC-MS
The sample to be analyzed is human sweat. Human sweat contains water which is of high percentage, mineral salts, and volatile organic components. The organic components in sweat are o-Cresol and p-Cresol, which are odorants; and Urea and Uric acid. However, according to CITE, sweat is not the main cause of body odor, but it is bacteria on the human body which feed on sweat. Fatty acids are also an important component of sweat and their amount in a particular individual determines his/her health status.
Human sweat has the main function of thermoregulation—body heat regulation. This is achieved through evaporation of sweat on the skin surface resulting into a cooling effect because of latent heat of evaporation of water (Sudhir & Ki-Hyun, 2011). This experiment will analyze the amount of fatty acids that can be extracted from an individual. The amount of fatty acids in a human is stipulated to be directly proportional to the body fat percentage (Sudhir & Ki-Hyun, 2011).
Fatty acids are to be determined in body odor because they are important constituents in body metabolism. Higher fatty acids are associated with obesity, a risk factor in cardiovascular diseases, hyperlipemia, and even diabetes (Barzantny, Brune, &Tauch, 2012). Therefore, there is a need for a non-invasive analysis of individuals at risk of obesity related diseases. The analysis is advantageous because many samples can be collected and that it is risk free in terms of infections. Sweat would be analyzed using GC-MS equipment and the quantity of fatty acids related to the body mass index of the individual.
Experiment
Equipment and experiment preparation
The analysis of sweat is to be carried out by a GC-MS equipment as shown in figure 1 below. The schematic of the equipment is as shown in figure 2 below. It involves a capillary column that represents a GC and a detector representing a mass spectrometer. Separation of sweat constituents will be done in a capillary column coated with an organic polysiloxane. Volume of sample to be injected in the machine should be a considerable amount ranging from 1mm3 to 3mm3. The flow rate of the gas in the gas chamber should be regulated at a rate that would allow for proper volatilization of the sample. Nunome, Tsuda, and Kitagawa (2012) recommends a range of 2-3 (cm3/min). The temperature of the MS detector and injector where sample is entered should be maintained at 2600C and 2500C respectively. Similarly, temperatures in the oven should be regulated at 300C for 60 seconds, and then increased by 1500C for 15 minutes. The next 4 minutes should have a temperature of 1800C and the last 2 minutes experience a temperature of 2200C. Therefore, the entire time of analysis should be between 20-24 minutes.
Detection after separation should be done in a selected ion monitoring mode. Furthermore, the peak on the MS should be looked at the 73 mark which detects all fatty acids. The MS operates efficiently in an electron impact ionization mode of 70eV (Nunome et al., 2012).
Figure 1: GC-MS machine
Figure 2: GC-MS schematic
Chemicals
The chemicals to be used should be all the fatty acids that are of analytical grade. They include: lauric acid, methyl decanoate, stearic acid, palmitic acid, Oleic acid, and myristic acid. All of which are to be used as Internal Standards.
Preparation of standards
The listed chemicals should be used as six stock standards. They are to be dissolved in a 70% ethanol-water solution to yield a known concentration preferably 100µM. Mixed solutions are then to be prepared by mixing in a small centrifuge and then evaporation to dryness. The residue should then be dissolved by adding 15mm3 internal standards. The sample (1-3mm3) is then injected into GC-MS for analysis.
Sweat Preparation
The middle finger should be washed with tap water for 10 seconds and then wiped using a clean tissue containing 70% ethanol-water solution. Thereafter, the finger should be rinsed with distilled water for approximately 15 seconds. A waiting period of 20 minutes should then proceed after which sweat on the surface of the middle finger should be collected in a small vial. An inhaler is sprayed inside the vial and then contents in the vial to be dried in a centrifuge evaporator at 900C. A small sample of internal standard is added in the sample and then 1-3 mm3 of the sample injected into the GC-MS machine. The experiment should be repeated for individuals of different ages, and sex. It is important that the participants should not have eaten in the last 12 hours.
Analysis and expected Results
Analysis will be carried out by looking at the relationship between the GC peak and the fatty acid concentration through analyzing standard samples. The more linear the calibration curve, the higher the correlation between fatty acids and body weight (Barzantny, Brune, &Tauch, 2012). Calibration curves can be obtained by diluting mixed standard solutions to different concentrations and then using software like SPSS or Excel in coming up with a trend for each standard that has a regression index also called a correlation coefficient. For accurate standards, the correlation coefficient should be nearer to 1 for each standard.
GC-MS analysis
Internal standards for each fatty acid that will be analyzed, will record a peak based on their respective retention times. The presence of peaks should signify presence of fatty acids in human sweat, and the respective retention time directly relate to the length of fatty acids. According to Nunome et al., (2010), the amount of fatty acids in the sweat should be directly proportional to the amount under the skin.
For proper analysis, sweat samples are to be collected from individuals fasting. The results after analysis should indicate the presence of all the six fatty acids with those being obese recording higher concentrations of fatty acid and more secretion of sweat (Nunome et al., 2010). Furthermore, in analyzing the fasting factor, sweating rate should increase with fasting time mainly because fatty acids in sweat are released from adipose tissues into sweat through blood during lengthy fasting.
Conclusion
The combination of gas chromatography and mass spectrometer has resulted in a crucial analytical technique in quantifying minute concentrations of samples. The gas chromatography part (GC) is an equipment that is used in isolating individual substances from a mixture, while the mass spectrometer part (MS) entails identification and quantifying the isolated samples. The GC part works by using a chromatographic column where smaller molecules which are more volatile travel faster down a column while larger ones move slower hence resulting in a separation. MS on the other hand works by relating the mass spectra of a substance with a standard one hence identifying the sample. Therefore, components that can be analyzed using this technique are volatile organic compounds or semi-volatile all of which are from the three states of matter.
In this experiment, the functioning of GC-MS instrumentation can be determined through analysis of human sweat. Human sweat consists of a large proportion of water, organic constituents, and nitrogenous compounds like urea. Organic components contain the volatile part of sweat and it is when bacteria on human skin eat the organic parts that an odor occurs.
The specific component that can be analyzed is fatty acids in the volatile organic component category. Fatty acids especially in sweat of humans starving (preferably 12 hour starvation) can be proved to be directly related to the body fat and BMI of an individual. The GC-MS isolates fatty acids from other organic components, and then the MS quantitatively determines the concentrations of different fatty acids in human samples.
The experiment is essential because it enables many experiments be carried out at the same time as it is not time consuming, and that it is less risky health-wise as it does not involve operations. Therefore, if the experiment becomes a success, then the application of GC-MS of being able to qualitatively and quantitatively analyze volatile organic components, and carrying out liquid injections will be achieved.
References:
Barzantny, H. H., Brune, I. I., & Tauch, A. A. (2012). Molecular basis of human body odour formation: insights deduced from corynebacterial genome sequences. International Journal Of Cosmetic Science, 34(1), 2-11. doi:10.1111/j.1468-2494.2011.00669.x
Erika R., Josefina P., & Maria, C. (2011). Gas chromatography/mass spectrometry and pyrolysis-gas chromatography/mass spectrometry for the chemical characterisation of modern and archaeological figs (Ficus carica). Journal Of Chromatography A, 12183915-3922. doi:10.1016/j.chroma.2011.04.052
NUNOME, Y., TSUDA, T., & KITAGAWA, K. (2010). Determination of Fatty Acids in Human Sweat during Fasting Using GC/MS.Analytical Sciences, 26(8), 917-919. doi:10.2116/analsci.26.917
Sudhir Kumar, P., & Ki-Hyun Kim, (. (2011). Human body-odor components and their determination. Trends In Analytical Chemistry,30784-796. doi:10.1016/j.trac.2010.12.005
