Drylab : Quantifying Nicotine in a Complex Sample Using the Multipoint Internal Standard Method

ANSWER THESE QUESTIONS TO COMPLETE THIS DRY LAB: ALL data has been given below.

Clearly identify the weight percent of nicotine in each cigarette. Clearly report the total nicotine (in milligrams) in each cigarette.

The purpose of this experiment is to quantify nicotine in cigarette tobacco using a GC equipped with a flame ionization detector (FID).

This experiment employs the multipoint internal standard method. Three standards are separated by the GC, then one unknown sample is separated by the GC, and a control sample is separated by the GC. Each standard contains a known amount of internal standard (cotinine) and a known amount of the analyte of interest (nicotine). The chromatographic data resulting from these standards will be used to construct an internal standard curve. For this calibration curve, the peak area ratio (area standard/area internal standard) will be plotted as a function of the concentration ratio of the two compounds. This calibration curve will allow you to calculate the concentration of nicotine in your unknown sample. You will also use the calibration curve to check that your control sample yields the expected amount of nicotine.

You will have one control cigarette and one unknown cigarette in this exercise. You will first extract the nicotine from tobacco using methanol solvent extraction. Then, using the internal standard method, you will analyze the methanol solution for nicotine. From the resulting chromatograms, calculate the percent nicotine (% w/w) contained in each cigarette.

MATERIALS

Methanol

Nicotine stock in methanol (prepared to be 2.5 mg/mL)

Cotinine stock in methanol (prepared to be 2.5 mg/mL)

20 mL vials

GC vials

Micropipettes

The control cigarette (according to the control chart, this should contain 12.0 0.6 mg nicotine)

SOLVENT EXTRACTION OF NICOTINE

1. Obtain one unknown and one control cigarette from your instructor.
2. For each cigarette, remove the paper cover and weigh the total tobacco content of the cigarette.

Mass of total tobacco in control cigarette: 676 mg

Mass of total tobacco in unknown cigarette: 687 mg

3. Now weigh an exact amount of the tobacco (~400 mg) into a 20 mL vial. Next, add 8 mL methanol to the vial. Close the lid tightly and shake it for a few minutes. Store the vial in a cool place until time for analysis (preferably over 1 day).

Mass of tobacco aliquot from control cigarette: 389 mg

Mass tobacco aliquot from unknown cigarette: 425 mg

A Class A 10 mL graduated cylinder was used to deliver 8.00 0.08 mL of methanol

SOLUTION PREPARATION

Step 1. Be sure to do this section only on the day of analysis, not beforehand. Prepare three standard solutions in three GC vials. Each standard solution will be a dilution of the nicotine stock solution. The three standards should be: a 2-fold dilution (400 L methanol and 400 L of the 2.5 mg/mL nicotine stock), a 4-fold dilution (600 L methanol and 200 L of the 2.5 mg/mL nicotine stock), and 800 uL of a full strength nicotine stock (2.5 mg/mL nicotine stock). For ease in preparing these samples, use a 200 L micropipette and GC vials.

Step 2. Further prepare the standard solutions for GC injection by mixing exactly 200 L of solution from Step 1 with exactly 200 L of internal standard stock (2.5 mg/mL cotinine stock) into a new GC vial. Do this for all three standards.

Step 3. Prepare control cigarette and unknown cigarette solutions by mixing exactly 200 L of 2.5 mg/mL cotinine stock with exactly 200 L of the cigarette extracted solutions into a GC vial. Do this for both cigarettes.

Step 4. Finally, prepare a qualitative solution of cotinine by mixing approximately 200 L of cotinine stock with about 200 L of methanol into a GC vial. This will allow you to figure out the exact retention time for cotinine.

Step 5. Run each prepared solution from steps 2, 3, and 4 through the GC-FID using the GC method* below. Use 1.0 L for each manual injection. Each run will take about 11 minutes to finish, so you will need about one hour and thirty minutes to collect all the data.

Step 4 qualitative solution = Data file: test42b82.CHR

Step 2 4-fold dilution = Data file:test42b83.CHR

Step 2 2-fold dilution = Data file:test42b84.CHR

Step 2 full strength = Data file:test42b85.CHR

Step 3 control = Data file: test42b86.CHR

Step 3 unknown = Data file: test42b87.CHR

Step 6. After you have collected all the data, calculate the total amount of nicotine in each cigarette and the percent nicotine (by weight) in each of the two cigarettes.

ANSWER THESE QUESTIONS TO COMPLETE THIS DRY LAB: data below

Clearly identify the weight percent of nicotine in each cigarette. Clearly report the total nicotine (in milligrams) in each cigarette.

*GC Method

Column: EC-WAX 30 m x 0.32 mm x 0.25 um

60:1 split injection

Injector 250C

FID detector 280C

Isothermal oven 220C

Labe Red A OVVT 13.013 Merge Doch Detector Dette test2 CHO Co AS Temper Init temp Hold Ramp Final temp 10120 2,59 11.700 Retention Area Height 0.150 322.9260 2. 160 5177.4940 11.700 2284.3500 11.740 928.755 269.038 Lab A OG IN EP DON Ova RING Init temp Hold Ramp Final tomp 2. 1. Area Retention 2.168 2.950 11.710 5021.1770 428.6820 2144,7230 Height 930. 105 100.345 259.184 A 141 e CORO OS Terce Init temp Hold Ramp Final temp 2.300 Retention Area Height 2.2004731.1580 930.540 12.003 795.1640 189.803 11.716 1700.7510 227.234 Les VOC 1: M De CRM Ta thu H - LH: 0 Open RVS Tere Init temp Hold Ramp Final temp 2.300 23 11.15 Retention Area Haight 2.2004667.8220 020.641 2.933 1364.6320 323.634 11.716 1676.7810 217.037 Are we NO Gesch D CRD VAS Yerrer Init temp Hold Ramp Firul temp 10.00 21 43 + Retention Area 0.150 1 120, 1315 0.783 546.1435 0.900 434.7905 1.300 786,3020 1.550 491.8045 2.183 6362.2300 2.946 B12.1030 4.300 522.1410 11.716 1030.0320 Height 54.025 49.428 48.011 45.875 44.205 931.420 136.7041 84.930 241.416 UND AVOT 14-20 De Chic Die C OVAS Tower Init temp Hold Ramp Final tomp 0.000 2.13 13. Retention Area Height 0.000 353.6040 55.436 0.266 1370.2540 43.758 0.800 432.3020 26.559 2.183 4335.3425 917.093 2.986 551.3160 123.943 11.716 1746.4880 222 241