Please show calculations for how to prepare the standards, unknown, and control.

ANALYZING NICOTINE IN E-LIQUIDS USING GC-FID PRE-LAB READING Read Harris pages 609 - 612 general chromatography, you may skip the portion on the different types of chromatography) Read Harris pages 634 - 651 (gas chromatography, you may skip the Retention Index, Splitless Injection. On-Column, and Thermal Conductivity Detector sections) Read Harris pages 109 - 112 (principles of internal standard calibrations) Refer to your calibrations primer for plotting multi-point internal standard calibration data LEARNING OBJECTIVES Learn how to perform chromatographic analyses Learn how to plot internal standard calibration data Learn how to determine unknown concentrations using internal standard calibrations Learn how to create professional quality tables, figures, and equations EXPERIMENT OVERVIEW To quantify nicotine in e-cigarette liquids, we will use a gas chromatograph (GC) equipped with a flame ionization detector (FID). In this GC experiment, we will employ the multi-point internal standard method. We use this method because it is the most accurate method when there may be small differences in injection volume from run to run. We will run a set of five nicotine standards. Each standard contains a known amount of internal standard (quinoline) and a known amount of the analyte of interest (nicotine). The chromatographic data resulting from these standards will be used to construct a calibration curve. For this, the peak area ratio (standard internal standard) will be plotted against the concentration ratio of the two compounds. This curve will allow us to calculate the concentration of our unknowns. You will have one control e-liquid and one unknown e-liquid in this exercise. The first step in the analysis is to weigh out a portion of each e-liquid and to dilute them with methanol. You will then prepare a set of standard solutions and further dilute the control and unknown sample (while also adding a known concentration of internal standard). Next, you will analyze all of the standards and samples on the GC-FID. From the resulting chromatograms, you will be able to calculate the concentration of nicotine in each e-liquid. MATERIALS Methanol 200. ppm w/v nicotine stock in methanol 250. ppm wiv quinoline stock in methanol An unknown e-liquid A control e-liquid EXTRACTION OF NICOTINE 1. Obtain one unknown and one control e-liquid and nicotine and quinoline stocks from your instructor or TA. Keep the nicotine and quinoline stocks in your drawer, but retum the control and unknown dropper bottles to the refrigerator (or to the TA) once you have made your control and unknown samples. 2. For each e-liquid carefully mass-200. mg of e-liquid into a 10 mL volumetric flask. Record the exact mass. The density of the e-liquids used in this lab is 1.3 g/mL. Fill to volume with methanol. Cap and shake it for a few minutes and transfer to a vial for storage. SOLUTION PREPARATION 1. Prepare standards using methanol as the dilution solvent. These standards should be prepared directly in GC vials assuming additivity of all solutions (use micropipets). The final volume of each should be 1.00 mL. The nicotine concentration should vary as follows: 20.0, 30.0, 40.0,50.0, and 60.0 ppm w/v. The quinoline concentration should be 25.0 ppm w/v for each standard. Create a table in your notebook that clearly shows what volume of each stock and methanol needs to be added to create these standards. 2. Prepare control and unknown e-liquid samples for analysis. In a GC vial, add 100. uL of your control and 100. uL of quinoline stock and dilute to a total volume of 1.00 mL by adding 800. uL MeOH (the quinoline concentration in this sample should thus be 25.0 ppm w/v). Repeat this scheme for the unknown. 4. After consulting with your instructor or TA about how to properly set up the instrument, add your samples to the sequence queue. Watch the first run of the sequence and look at the acquired chromatogram. If the expected analytes are present, you may leave. The data will be ready for you to analyze next lab period. 5. After you have collected all the data, calculate the concentration (in ppt w/v; t = thousand) of nicotine in each of your e-liquids. Use nicotine and quinoline peak areas (not area %) in your calculations WASTE All of your waste generated in this lab should be collected and disposed in the appropriate waste container in the hood