CHM3120L GC LAB REPORT

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Feb 20, 2024

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Onukogu 1 Gas Chromatography: Analysis of Caffeine in Coffee CHM3120 L 11/27/2023

Onukogu 2 Introduction Gas chromatography is an analytical separation method used to analyze volatile substances in their gas phase. For this technique, the components of the sample are first dissolved in a solvent and then vaporized to separate the analytes by dividing the sample between two phases (a mobile and a stationary phase). 1 The mobile phase which is usually a chemically inert gas such as He, N 2 etc., so that it does not react with the sample, acts to carry the molecules of the analyte through the heated column. The stationary phase is either a liquid or a solid and does not move through the column like the mobile phase instead it is contained in the column. The components of the sample are then separated based on the attraction to the two phases. The components with relatively weak attraction to the stationary phase elute from the column rapidly while the components with a good attraction to the stationary phase elute later. 2 The separation in gas chromatography is done based on the attraction to the stationary phase and the temperature of the mobile phase. As a result, the temperature can be manipulated so that molecules with higher boiling points can also elute from the column while molecules with lower boiling points elute first. 3 A detector such as the flame ionization detector (FID) is used to detect the molecules as they separate. An Ideal detector should have adequate sensitivity, good stability and reproducibility, high temperature range, short response time, linear response, and non-destructive to sample. The FID is the most common gas chromatography detector; however, it is destructive to the sample, and can only be used for quantification of hydrocarbons. 4 Hydrocarbons release ions and electrons in flame (when burned), these released electrons are attracted to a positively charged filament and when they strike, there is a change in current that is flowing through the filament which is what gets detected. 5 For this experiment, the caffeine content from 3 different types of coffee (light roast, dark roast, and espresso) will be separated and quantitated from the other coffee ingredients using gas chromatography. The external standardization method is used to quantitate the caffeine. In this method, a series of standards with known concentrations of the component to be analyzed is prepared. The standards and the unknowns are injected using the same chromatographic conditions and the peak areas are measured. 6 A plot of the peak area versus the concentration is made and the line equation is used to determine the concentration of the caffeine in the different coffee types. Caffeine is a stimulant that increases the activity of the central nervous system. Caffeine when taken in moderation has little to no effect on health; however, the FDA estimates

Onukogu 3 toxic effects, like seizures, can be observed with rapid consumption of around 1,200 milligrams of caffeine, or 0.15 tablespoons of pure caffeine. 7 As a results regulations were put in place by the FDA and the recommended/tolerated amount of caffeine per day for healthy adults is 400 mg. 8 Experimental Materials used: 1000 ppm standard, 10 mL volumetric flasks, autosampler vials, light roast coffee, dark roast coffee, espresso, hot plate, weighing balance, paper towel, 150 mL Erlenmeyer flask, DI water, 50 mL beaker, 20 mL dichloromethane, stirring rod, 100-1000 μl micropipette, GC- FID Preparation of Standards: The 1000 ppm standard was obtained from the fume hood. The volumes of the stock solution needed to make the 50 ppm, 100 ppm, 150 ppm, 250 ppm, and 500 ppm in a 10 mL volumetric flask were calculated using the following formula: C 1 V 1 = C 2 V 2 50 ppm : V 1 = 50 ppm × 0.1 L 1000 ppm = 0.5 mL 100 ppm : V 1 = 100 ppm × 0.1 L 1000 ppm = 1 mL 150 ppm: V 1 = 150 ppm × 0.1 L 1000 ppm = 1.5 mL 250 ppm: V 1 = 250 ppm × 0.1 L 1000 ppm = 2.5 mL 500 ppm: V 1 = 500 ppm × 0.1 L 1000 ppm = 5 mL The 5 solutions were made in 5 separate 10 mL volumetric flasks and diluted to the 10 mL mark with dichloromethane. Then 2 mL of each solution was added into individually labelled autosampler vials with a micropipette.

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Onukogu 4 Preparation of the Coffees: 500 mg of the light roast coffee was weighed using the weighing by difference technique on the scale. The coffee was placed on a piece of paper and crushed till it was relatively homogenized. Then it was added into a 150 mL Erlenmeyer flask and 25 mL of deionized water was added to it. The flask was placed on a hot plate and was left to boil for five minutes. After this was done the flask was placed on a paper towel for a few minutes and then transferred into a 50 mL beaker and left to cool for a while. After it was cooled 20 mL of dichloromethane (DCM) was added to the coffee water and a stirring rod was used to mix the mixture for five minutes. Then 2 mL of the DCM layer which was the bottom layer was transferred into a labelled autosampler vial with a micropipette, and it was ensured that no coffee particles was transferred into the vial. This process was repeated for the dark roast coffee and espresso. Data Collection: All the autosampler vials were placed into the auto sampler tray of the GC-FID in the order 50 ppm,100 ppm, 150 ppm, 250 ppm, 500 ppm, light roast coffee, dark roast coffee, and espresso. The GC-FID settings were as follows: -1 μl injections, the split mode was set to 50:1 i.e., 1 part enters the GC and 50 does not enter the GC. -The injector temperature - 280 °C -The carrier gas, He, flowed at a rate of 1.8 mL/min -The detector temperature - 300 °C -Initial oven temperature - 100 °C. This was held for 2 minutes and then after it was increased at a rate of 16 °C per minute until the temperature was up to 180 °C. Then after that it was increased to 20 °C per minute until the temperature was up to 250 °C. The sequence was set up with 101 as the first sample and 108 as the last sample. The GC-FID ran the sequence and automatically recorded and integrated the data.

Onukogu 5 Results and Discussion A calibration curve of the peak areas versus concentrations for the standards was generated. The trendline equation for the graph was y = 0.1099x – 0.243, and this was used to determine the caffeine concentration in the standards and the different coffee types by substituting the peak areas for ‘y’ in the equation. The concentration of caffeine was seen to increase in the order 50 ppm, 100 ppm, 150 ppm, 250 ppm, 500 ppm, espresso, light roast coffee, and dark roast coffee. 50-ppm standard had the lowest caffeine concentration of 52.7 ppm and the dark roast coffee had the highest caffeine concentration of 709 ppm as seen in Table 1. The 100-ppm standard had a caffeine concentration of 100 ppm, the 150-ppm standard had a caffeine concentration of 149 ppm, the 250-ppm standard had a 246 ppm caffeine concentration, the 500-ppm standard had a 502 caffeine concentration, the espresso had a 345 ppm caffeine concentration and the light roast coffee had a caffeine concentration of 420 ppm. The concentrations of the 500-ppm standard, the light roast and dark roast coffee were significantly higher than the FDA recommended amount of caffeine (400 ppm). The data for the dark roast coffee is out of range and could be a result of random/experimental error. Table 1: The different standard solutions and the different types of coffee with their retention times, peak areas, and caffeine concentrations Sample Retention Time (min) Peak Area (pA*s) Caffeine Concentration (ppm) 50 ppm 9.561 5.54667 52.7 100 ppm 9.561 10.79312 100 150 ppm 9.563 16.13172 149 250 ppm 9.563 26.80567 246 500 ppm 9.566 54.93758 502 Espresso 9.564 37.62313 345 Light Roast Coffee 9.565 45.86778 420 Dark Roast Coffee 9.568 77.66491 709

Onukogu 6 Figure 1. Calibration curve of the peak areas (pA*s) versus the standard concentrations (ppm) Calculations: Line equation – y =0.1099 x – 0.243 Caffeine concentrations in espresso y = 37.62313 x = y+0.243 0.1099 x = 37.62313 + 0.243 0.1099 ≅ 345 ppm Caffeine concentration light roast coffee y = 45.86778 x = 45.86778 + 0.243 0.1099 ≅ 420 ppm Caffeine concentration dark roast coffee y = 77.66491 x = 77.66491 + 0.243 0.1099 ≅ 709 ppm y = 0.1099x - 0.243 R² = 0.9998 0 10 20 30 40 50 60 0 100 200 300 400 500 600 Pear Area (pA*s) Concentration (ppm) Peak Areas vs Standard Concentrations

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Onukogu 7 50 ppm y = 5.54667 x = 5.54667 + 0.243 0.1099 ≅ 52.7 ppm 100 ppm y = 10.79312 x = 10.79312 + 0.243 0.1099 ≅ 100 ppm 150 ppm y = 16.13172 x = 16.13172 + 0.243 0.1099 ≅ 149 ppm 250 ppm y = 26.80567 x = 26.80567 + 0.243 0.1099 ≅ 246 ppm 500 ppm y = 54.93758 x = 54.93758 + 0.243 0.1099 ≅ 502 ppm

Onukogu 8 Conclusion The purpose of this experiment was to use gas chromatography to determine the concentration of caffeine in 3 different types of coffee (espresso, light roast, and dark roast) and standard solutions. The GC-FID was used to run the sequence and integrate the data. The peak areas and the standard concentrations were used to generate a calibration curve and trendline equation. The equation was used to determine the concentration of caffeine in the standard solutions and the 3 different coffee types. The 150 ppm had the lowest caffeine concentration of 52.7 ppm and the dark roast coffee had the highest caffeine concentration for 709 ppm. The 50 ppm, 100 ppm, 150 ppm, and the 250 ppm caffeine concentrations were within the FDA recommended caffeine concentration of 400 ppm; however, the caffeine concentrations of the 500 ppm, espresso, light roast and dark roast coffees had caffeine concentrations higher than the FDA recommendation. The data of the dark roast coffee was out of range and could be a result of random or experimental error such as the method the solution was prepared.

Onukogu 9 References 1. Thet, Kyaw; Woo, Nancy, Gas Chromatography , Aug 29, 2023, https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Analytical_Che mistry)/Instrumentation_and_Analysis/Chromatography/Gas_Chromatography 2. Jacobs, A. Gas Chromatography: Analysis of Caffeine in Coffee; University of Florida/Department of Chemistry: Gainesville, FL, 2023 3. Jacobs, A. Gas Chromatography: Analysis of Caffeine in Coffee; University of Florida/Department of Chemistry: Gainesville, FL, 2023 4. Jacobs, A. Separations- Gas Chromatography; University of Florida/Department of Chemistry: Gainesville, FL, 2023 (PowerPoint) 5. Jacobs, A. Gas Chromatography: Analysis of Caffeine in Coffee; University of Florida/Department of Chemistry: Gainesville, FL, 2023 6. Jacobs, A. Gas Chromatography: Analysis of Caffeine in Coffee; University of Florida/Department of Chemistry: Gainesville, FL, 2023 7. Food and Drug Administration: Spilling the Beans: How Much Caffeine is Too Much; Aug. 07, 2023 https://www.fda.gov/consumers/consumer-updates/spilling-beans-how-much-caffeine-too-much 8. Food and Drug Administration: Spilling the Beans: How Much Caffeine is Too Much; Aug. 07, 2023 https://www.fda.gov/consumers/consumer-updates/spilling-beans-how-much-caffeine-too-much

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