Exp 10_Determination of the degradants in caffeine solution using mass spectrometry

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Rutgers University *

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160:251

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Chemistry

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Feb 20, 2024

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pdf

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Uploaded by MinisterStarGoldfinch36

Sample analysis using mass spectrometry. Introduction The goal of this week lab is to identify the degradation products of caffeine using electrospray ionization (ESI)You make a solution of caffeine in water. Then a sample of caffeine would need to be subjected to degradation conditions, such as exposure to light, heat, or acidic or basic conditions. The resulting degraded sample would then be analyzed using ESI mass spectrometry. In general, ESI can provide information about the molecular weight and structure of the degradation products, allowing for their identification. For example, if caffeine is degraded under acidic conditions, it may form the degradation product theobromine, which has a molecular weight of 180.0803 g/mol. Similarly, under basic conditions, caffeine may degrade to form paraxanthine, which has a molecular weight of 182.0968 g/mol. It is important to note that the specific degradation products of caffeine will depend on the specific degradation conditions used and may also vary based on the duration and severity of the degradation. Therefore, careful experimental design and analysis are necessary to accurately identify the degradation products of caffeine using ESI mass spectrometry. For this experiment, you will be using a paper that describes the degradation of caffeine using a UV/H 2 O 2 system . Once you have degraded the caffeine sample using this system, you will inject the resulting degradation product into a mass spectrometer. The mass spectrum obtained from the sample can then be compared to the spectra in the paper to help identify the specific degradation product. Once the degradation product has been identified, you can use the degradation schemes in the paper to determine the specific pathway of degradation that occurred. Reagents and special equipment Caffeine solid 3.0 % Hydrogen Peroxide solution Mass spec grade ACN (acetonitrile) 50/50 ACN/water solution Automatic pipets Mass spectrometer single quad with ESI Procedure You need to make two solutions (1 ࠵? M caffeine and 1-0.75 ࠵? M degraded caffeine) and the inject them into mass spectrometer. Part 1. Preparation of the 1 ࠵? M caffeine solution All your calculations must be entered into the Table 1 on the excel file. 1. Weigh about 0.194 g of the caffeine powder and transfer it to 100 mL volumetric flask and bring to volume with deionized water, calculate the molarity and label the flask as stock solution 1. 2. Transfer 1.0 mL of the stock solution 1 to a new 100 mL volumetric flask and bring to volume with deionized water, calculate the molarity and label the flask as stock solution 2. 3. Use micropipettes to transfer 40 ࠵? L of the stock solution 2 to a 1.5 mL Eppendorf tube and add 1.5 mL of 50/50 ACN/water solution, calculate the concentration and label the

tube “ ms 2 ”, what means that this solution will be used for injection to mass spectrometer. You should have about 1 ࠵? M caffeine solution. Part 2 . Degradation experiment 1. You should use stock solution 1 from part 1 for the degradation studies. 2. Transfer 1.0 mL of the stock solution 1 to a new 100 mL volumetric flask and bring to volume with 3% hydrogen peroxide, calculate the molarity and label the flask as stock solution 3. 3. Transfer all or part of the solution 3 into a beaker of your choice, 50 mL, 100 mL, 150 mL or larger. 4. Put the solution under UV lamp for 30-90 min. 5. When you keep the solution under the UV light, think about how this solution need to be diluted to have the same concentration as your stock 3 from the Part 1. 6. Show your calculations to you TA for approval. 7. Dilute the degradation solution with 50/50 ACN/water to make it ready for injection into mass spec. Remember to label the Eppendorf tube with “ dms ”, what can be read as degraded for mass spec. Part 3: Using mass spectrometry to analyze the caffeine sample and degradants. When you come to the lab, mass spectrometer will be running, your TA will open gas flow, login to the instrument computer, and then discuss how to operate the instrument using MassLynx software. MassLynx software for waters Q and ESI+ • Sample analysis Use your 1.0 ࠵?࠵? caffeine solution for the first injection, calculated (195.0883 Da) m / z values for [ M + H] + of C 8 H 11 N 4 O 2 § Save the spectrum on your flash drive. § Assign the structure for each fragment on the spectrum. 2. Inject your degraded solution. § Save the structure on your flash drive. § Assign the structure for each fragment. 3. Look at the provided paper and make conclusions about the presence of any degradation products and assign the degradation pathway.

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