Nichols-Lab 402-Page 1 of 5 Liquid Extraction of Caffeine and Identification Using Mass Spectrometry and Gas Chromatography Abstract Caffeine is a widely used simulant to give people energy. The stimulant is found in many energy drinks, tea, and coffee. This experiment aims to successfully extract and identify a caffeine sample from coffee. This is done by liquid-liquid extraction to separate the caffeine from coffee then gas chromatography (GC) would separate the caffeine. After that it goes through mass spectrometry (MS) to identify the molecule as caffeine. These methods are very precise and give an exceptionally good idea of what a molecule is and how much of it was extracted. Using these methods, caffeine was successfully extracted and identified from the sample of coffee. The fragmentation from mass spectrometry was not as accurate as predicted, hinting at some error in the extraction process. Overall, the methods were highly successful at completing the goals. Introduction The goal of this lab was to take a caffeinated sample (coffee) and use various methods (MS and GC) to extract all of the caffeine from the liquid and correctly identify it as such. Caffeine can be extracted from coffee using a liquid-liquid extraction with ethyl acetate. This separates the caffeine from the water. Using the difference in densities, a separatory funnel can be used to create two distinct layers of ethyl acetate and caffeine on the bottom and everything else above it. After filtering the liquids, the ethyl acetate must be evaporated to leave only caffeine. After weighing the sample, ethyl acetate could be added to the beaker. At this point, the sample can be put into a gas chromatograph instrument to be fully separated. The GC works by injecting the sample into a column that is microns thin using a hypodermic needle. The interior of the column is coated in a non-polar, silicon based, material that allows the sample to travel through it without sticking. The column is placed into an oven where the sample is vaporized. The vaporized sample is pushed through the column using an inert gas. The sample vaporizes into two separate substances depending on its properties and as it is pushed through the exit, a signal will be output to show the abundance of the substance. The time it takes to get through the column will identify the substance. The substances will then be immediately put through the mass spectrometer where they are hit with high energy electrons, ionizing them so that it is able to measure the mass to charge ratio of the ion. Once the entering particles have been ionized, they flow into a magnetic field that directs them through an arc into a detector where a computer reads it m/z ratio1.This fragmentation is then read to identify the parent ion and which ions it broke in to. The mass spectrometer will then return a graph with peaks of the fragments which can be traced to their parent ion. Methods The experiment started by measuring 100 mL of coffee into a graduated cylinder which was then poured in an Erlenmeyer flask. Then, 2 g of sodium carbonate was added and dissolved into the flask. After the sodium carbonate had dissolved, the ethyl acetate was added and gently stirred for 10 minutes by swirling the flask. Once the time had passed and the contents were mixed, it was poured into a separatory funnel. After the ethyl acetate separated in 10 minutes and floated on top, it was collected into a pre-weighed 50 mL beaker. Once completely collected, the beaker was placed on a hot plate under the fume hood where it could be evaporated to leave only the caffeine in the beaker. Then the beaker was weighed to find the total mass of caffeine collected. Finally, ethyl acetate was added back to the beaker to dissolve the caffeine and a small amount of the sample was Nichols-Lab 402-Page 2 of 5 put into a vial to be injected into the GC. The full, in-depth lab instructions can be found in the Chemistry 110 lab manual on pages 11 and 122. Results Table 1: Quantitative analysis Identity of Sample Volume of Sample Used Mass of Empty Beaker Mass of Beaker and Caffeine Mass of Caffeine per serving Coffee (caffeine) 100 mL 27.370 g 28.840 g 14.7 mg / mL Table 2: Gas Chromatograph peaks with corresponding retention time and identity of molecules Peak 1 2 3 Retention Time (min) .803. .844 1.497 Identity of Molecule Diethyl Ether Diethyl Ether Caffeine Table 3: Mass spectrum of caffeine and assigned fragment ions Peak A B C D E F G m/z value 194.05 167.12 134.09 130.06 124.12 91.11 77.10 Identified Fragments C8H10N4O2 C7H9N3O2 C7H8N3 C7H4N3 C6H8N2O C6H5N C6H5 Table 4: Mass spectrum of ethyl acetate and assigned fragment ions Peak A B C D m/z value 88.04 86.00 83.96 51.00 Identified Fragments C4H8O2 C4H6O2 C4H4O2 C4H3 Discussion The first step in the extraction process involves liquid-liquid extraction with immiscible liquids. Liquid-liquid extraction is driven by the difference in chemical potential between the polar and non-polar liquids3. The solution wants to be at a lower energy state which is what prompts the transfer of solute. Diethyl ether was used as an organic solvent. Diethyl ether can form stronger bonds and a more stable state in the non-polar solvent. Ethyl acetate is immiscible in water, so it will form its own layer beneath the water after extracting the caffeine. Once the transfer of caffeine is completed, the liquids are transferred to a separatory funnel to complete the process. Gas chromatography is a powerful method that can separate mixtures and identify them based on Nichols-Lab 402-Page 3 of 5 retention times in the column. Solution is injected into the microscopic column and heated in an oven where the molecules separate based on boiling points. The column is coated in a non-polar silicon-based compound which ensures all the molecules injected will come out and not stick to the inside. Diethyl ether has a lower boiling point and will exit the column first which was seen in the first peak with a retention time of .844 minutes. The second and largest peak was caffeine based on its 1.497-minute retention time. Immediately after chromatography, the sample was bombarded with high energy electrons in the mass spectrometer. The multiple peaks show the possible fragments of caffeine. Each smaller peak can be identified as a fragment of caffeine (as shown above in table 3). The expected m/z value of caffeine is 194.05. The data collected had the highest m/z value of 167.12 which is a fragment of caffeine without CHN. Since this is still an expected peak resulting from caffeine, it can be concluded caffeine was identified, but in a small enough concentration where only fragments were identified. Conclusion In conclusion, liquid-liquid extraction, gas chromatography, and mass spectrometry are effective combinations for the separation and identification of a sample. The goals of the experiment were met in identifying and separating caffeine. There was some variability in the mass spectrometry of the experimental and expected values, this could be due to the lack of concentration of the molecule. There were 7 peaks identified in the mass spectrometer. While separating in the separatory funnel, some of the water remained with the diethyl ether due to human error. This could have caused variability in the mass spectrometry as well as diluting the sample making it less pure. Most of the fragments obtained are smaller than expected, which is due to an amount of diethyl ether being present lowering the mass charge ratio. The diethyl ether fragments are much smaller than caffeine which is what promotes the idea that it was present because of smaller caffeine fragments than expected. This lab successfully verified gas chromatography and mass spectrometry as methods to separate molecules from a substance. This can be useful in many fields and industries needing separation. For example, the coffee industry where caffeine needs to be extracted to produce decaffeinated coffee beans. Supporting Information Figure 1: Chromatogram experimental values Nichols-Lab 402-Page 4 of 5 Figure 2: Spectra experimental values E D F G B C A Figure 3: Spectra diethyl ether standard B D C A Nichols-Lab 402-Page 5 of 5 References 1. Mass Spectrometry https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/massspec/masspec1.h tm (accessed Mar 23, 2023). 2. Kautz, J.; Periago, J. Taking things apart…Putting things together In 110 Chemistry; Hayden McNeil Lab Solutions: Department of Chemistry, University of Nebraska-Lincoln. 17 November 2021. 3. Chaugule, A.; Patil, H.; Pagariya, S. International Journal of Advanced Research in Chemical Science 2019, 6 (9).
