Gas Chromatography: Determination of Fatty Acid composition in Fats and Oils

Transcription

1 Chem 3410 Experiment 4 Determination of fatty acids by GC 16 Gas Chromatography: Determination of Fatty Acid composition in Fats and Oils Introduction The following procedure for the determination of fatty acid composition is an official method of the American Oil Chemists' Society. The composition of fatty acids in different fat/oil samples (personal samples are recommended), will be determined. Gas chromatography is a technique for carrying out the separation and measurement of mixtures of materials that can be volatilized. These materials may be gases, liquids, or solids that have appreciable vapor pressures at temperatures up to a few hundred degrees. In capillary gas chromatography a stationary phase, generally a stable non-volatile liquid, is spread in a thin film on a wall of column. A carrier gas acts as an inert moving phase to transport the sample components from an injection port at the head of the column through the column to a detector. Sample injection is an arrangement by which a solid, liquid, or gaseous sample is transmitted as a short pulse into the carrier-gas stream before it enters the column. The sample should be vaporized and carried to the leading end of the column in negligible time. The detector, commonly flame ionization, monitors the composition of the carrier-gas stream as it leaves the column. Simple, sensitive, and stable, the flame ionization detector has contributed in a major way to the explosive growth of gas chromatography. A significant advantage is that it provides a recorder response proportional to concentration of substance in the effluent from a column. (Suggested Reading: Chapters 23 & 24 in Quantitative Chemical Analysis by Harris and Chapter 27 in Principles of Instrumental Analysis by Skoog, Holler and Neiman) Preparation of reagents and apparatus (a) Prepare 100 ml of an esterification reagent M sodium methoxide solution (check as this maybe prepared for you by your instructor). (b) Prepare 100 ml of saturated salt solution in water. (c) Prepare 35 mg sample of lipids (fat or oil) in threaded test tube. Ensure that the top edge of the tube is not broken. Add 1 ml of iso-octane and dissolve the sample in it. Add 10 ml of esterification solution (0.50 M sodium methoxide), close the tube, vortex for 2 minutes and place in a hot heating block for 10 minutes and remove the tubes and allow the contents to cool undisturbed for 30 minutes.

2 Chem 3410 Experiment 4 Determination of fatty acids by GC 17 (d) Add 6 ml of iso-octane and top with ~5 ml of the salt solution. Close tube and mix by inverting only, vigorous mixing causes emulsion formation and the 2 phases will not separate. Place this tube in a test tube stand and wait until phases separate and is clear. A centrifuge can be applied to speed up separation and clarification. Note: Always place an equal counterweight in the centrifuge. (e) Transfer the upper clear phase into an auto sampler vial up to neck of the vial, using a Pasteur pipet. (f) Set carrier gas at the total flow of 75 ml/min; injector and detector temperatures set at 235 C, column temperature at programming: 125 C for 2 minutes, program to 220 C at 5 C/min, held for 5 minutes. Program the running sequence on ChemStation, program controlling GC to do injection and data acquisition. (g) Place vials into autosampler carousel, set injection to 1µL. With the samples, add at least two standard mixtures to verify elution order of components. Standard samples have to be placed at the beginning and close to end of set of samples. When all parameters are set start your run with the ChemStation and not on the GC. Figure 4-1. Schematic drawing of a gas chromatograph Data presentation

3 Chem 3410 Experiment 4 Determination of fatty acids by GC 18 Retrieve your sample data from the ChemStation; analyze them to check if integration was done properly. Check how the baseline was drawn and correct it if not as expected by changing integration parameters. Identify peaks on chromatogram base on retention data from analyzed standard samples. Print report and chromatogram for each sample analyzed. Prepare a report which should have printed identification of peaks and data on the composition of fatty acids in your samples. Calculate the weight % of trans fatty acids and weight % of saturated fatty acid in your sample.

4 Chem 3410 Experiment 5 Determination of ASA by fluorometry 19 Introduction The Fluorometric Determination of Acetylsalicylic Acid in an Aspirin Tablet Acetylsalicylic acid is an analgesic (pain reliever) which is found in aspirin tablets. In addition to acetylsalicylic acid, aspirin tablets contain other ingredients such as binders and buffering agents. In this experiment a portion of an aspirin tablet is dissolved in water and converted to salicylate ion by the addition of sodium hydroxide. COOH + 2 OH - O-C-CH 3 O Acetylsalicylic acid (MW ) COO - OH Salicylate ion + CH 3 COO - + H 2 O The salicylate ion strongly fluoresces at about 400 nm after it has been excited at about 310 nm. A series of standard solutions of salicylate ion are prepared; the fluorescence of the standards and the samples are measured; and the working curve method is used to determine the concentrations of salicylate ion in the sample solutions. The concentration is used to calculate the percentage of acetylsalicylic acid in the aspirin tablet. (Suggested reading: Chapters in Quantitative Chemical Analysis by Harris and Chapter 15 in Principles of Instrumental Analysis by Skoog, Holler and Neiman) Apparatus 2 L beaker 100 ml beaker mortar and pestle Filter paper (medium porosity) Glass funnel buret 2-1 L volumetric flasks ml volumetric flasks wash bottle 100 ml graduated cylinder hot plate or Bunsen burner Chemicals Aspirin tablet Sodium hydroxide solution (4 M) Salicylic acid (reagent grade)

5 Chem 3410 Experiment 5 Determination of ASA by fluorometry 20 Procedure 1. Obtain an aspirin tablet from the instructor. Record sample number on the tablet or the brand and/or manufacturer s name if it is available. 2. Place the tablet in a clean, dry mortar. Use a clean pestle to grind the tablet into a powder. Weigh 0.1 g of the powder to the nearest 0.1 mg into a 100 ml beaker. 3. Place about 1 L of distilled or deionized water in a 2 L beaker. Heat the water to just below boiling. 4. Fold a piece of filter paper and place it in a glass funnel. Place the funnel in the top of a 1 liter volumetric flask. Use a spray of distilled or deionized water from a wash bottle to rinse the powder in the 100 ml beaker into the funnel. Allow the solution which flows through the funnel to drain into the volumetric flask. 5. Slowly pour the hot water which is in the 2-L beaker over the solid and through the funnel. The acetylsalicylic acid, which is in powder form, slowly dissolves in water and drains into the funnel. Some tablets contain binders which will not dissolve. The insoluble binders are separated from the acetylsalicylic acid during this step. After the solid has completely dissolved, or after no further solid appears to dissolve, allow the solution in the flask to cool to room temperature. Dilute the solution to the mark with room-temperature water. Pour at least 500 ml of the hot water through the funnel before concluding that the solid will not dissolve further. 6. Weigh g of salicylic acid to the nearest 0.1 mg. Place the weighed acid in a labeled, 1-L volumetric flask. Add about 500 ml of distilled and deionized water and swirl the contents of the flask until the solid has dissolved. Dilute the solution to the mark with water. The result is a stock solution of salicylic acid. 7. Respectively label nine, 100 ml volumetric flasks with B, U1, U2, U3, 1,2,3,4, and 5. Use a pipet to deliver 2 ml of 4 M sodium hydroxide solution to each of the nine flasks. Use a buret to add 2, 4, 6, 8, and 10 ml, respectively, of the salicylic acid stock solution to the 100 ml volumetric flasks which are labeled 1, 2, 3, 4, and 5. Use a pipet to place 10 ml of the 1-L solution of the tablet into each of the flasks which are labeled U1, U2, and U3. Fill each flask to the mark with distilled or deionized water.

6 Chem 3410 Experiment 5 Determination of ASA by fluorometry Measure the excitation and emission spectrum of salicylic acid using standard 5. Get the exact excitation and emission wavelengths and adjust the monochromator which controls the excitation wavelength and emission wavelength appropriately. 9. Fill a cuvet with the well-stirred solution from one of the nine 100 ml volumetric flasks. Place the cuvet in the fluorometer. Measure and record the relative fluorescence of the solution. The instructor will provide the operating instructions for the fluorometer. 10. Similarly measure and record the relative fluorescence of each of the remaining eight solutions. Calculations 1. Use the mass of the salicylic acid (MW ) to calculate the concentration of salicylic acid in the stock solution. 2. Use the volumes of the stock solution which were added to the 100 ml volumetric flasks to calculate the concentrations of salicylate ion which are in flasks 1, 2, 3, 4, and Prepare a working curve 4. From the working curve, determine the concentration of salicylate ion which is in flasks U1, U2, and U3. 5. Use the dilution factor to calculate three values for the concentration of acetylsalicylic acid in the 1-L solution. 6. Use the three acetylsalicylic acid concentrations and the mass of the tablet which was used to prepare the solution to calculate three values of the percentage of acetylsalicylic acid in the tablet. 7. Determine the mean and standard deviation of the results.

7 Chem 3410 Experiment 6 Determination of iron by AA 22 Determination of Iron in Cereal by Atomic Absorption Spectrophotometry Introduction The nutritional value of trace amount of certain metals, such as iron and manganese, is well known. In this experiment, the amount of iron present in a dry, breakfast cereal is determined. The powdered cereal is digested with a nitric acid-perchloric acid solution. Atomic absorption spectrophotometry is used to determine the concentration of iron in the resulting solution. The effect of potentially interfering substances in the cereal is minimized by use of the standard-addition technique. (Suggested reading: Chapters 5 (5-3) & 21 in Quantitative Chemical Analysis by Harris and Chapter 9 in Principles of Instrumental Analysis by Skoog, Holler and Neiman) Apparatus 50 ml beaker Iron Hollow Cathode Lamp 10 ml graduated cylinder Mortar and Pestle 1 ml graduated pipet 4 ml pipet Hot plate mm test tube 25 ml volumetric flask 250 ml volumetric flask Chemicals Acetylene Compressed Air Breakfast Cereal Iron (II) Ammonium sulfate hexahydrate (reagent grade) Nitric acid-perchloric acid solution (1:1 volume) Procedure 1. Weigh appropriate amount of iron(ii) ammonium sulfate hexahydrate to the nearest 0.1 mg to prepare 100 ml of ~100 mg/l aqueous iron standard solution. 2. Grind about 2 g of the cereal using a mortar and pestle into a powder. Weigh to the nearest 0.1 mg about 0.5 g of the powdered cereal and transfer it into a 50 ml beaker.

8 Chem 3410 Experiment 6 Determination of iron by AA 23 Caution: The remainder of the experiment could be hazardous. The use of safety glasses is required at all times. 3. Place the beaker on a hot plate in the hood. Cautiously add 10 ml of the nitric acidperchloric acid solution. Gently warm the beaker until the sample is colorless. The acid helps to dissolve iron present in the sample during this step. 4. After digestion is complete, transfer the solution to a 25 ml volumetric flask. Rinse the beaker twice with 5 ml portions of distilled or deionized water. Pour the rinsing into the volumetric flask. Dilute the solution to the mark with distilled or deionized water. 6. Label five mm test tubes as S, 1, 2, 3, and 4. Use a pipet to add 4 ml of the 25 ml of the sample solution to each of the five test tubes. Use the 1 ml graduated pipet to transfer 0.20 ml of the 100 mg/l iron solution to tube 1, 0.30 ml to tube 2, 0.40 ml to tube 3, and 0.60 ml to tube Use a graduated 1 ml pipet to add 1.00 ml of distilled or deionized water to tube S, 0.80 ml to tube 1, 0.70 ml to tube 2, 0.60 ml to tube 3, and 0.40 ml to tube 4. Each tube should contain a total of 5.00 ml of solution. 8. Insert the iron hollow cathode lamp into the atomic absorption spectrophotometer. Refer to the supplied instructions and optimize the signal and prepare for the analysis. 9. Successively aspirate the solutions in the five test tubes into the flame. Record the instrumental reading from each solution. 10. Close the acetylene quick-shut-off valve and allow the flame to extinguish. After the flame has extinguished, close the valve on the acetylene tank, open the quick-shut-off valve and allow the acetylene to drain from the acetylene line. Shut off the air supply and turn off the instrument. Turn off the hood. Calculations 1. Calculate the exact concentration (mg/l) of iron (AW ) in the standard solution using the mass of the iron(ii) ammonium sulfate hexahydrate (MW ) and the volume of the solution (100.0 ml).

9 Chem 3410 Experiment 6 Determination of iron by AA Use the concentration of the standard solution, the volume of the solution which was added to each of test tubes 1 through 4, and the final solution volumes (5.00 ml) to calculate the concentration (mg/l) of iron which was in each of the tubes. 3. Prepare a plot of absorbance (or instrumental reading) (y axis) as a function of the added iron concentration for the solutions that are in tubes S, 1, 2, 3, and 4. Draw a straight line through the data points and extrapolate it to intersection with the concentration axis. The distance on the concentration axis between the origin and the intersection with the extrapolated line corresponds to the concentration of iron which is in tube S. (Refer to section 5-3 in your textbook on constructing a calibration curve for standard addition experiments and obtaining the concentration of the analyte in the unknown sample). 4. Use the dilution factor and the iron concentration that is in tube S to calculate the concentration in the undiluted sample solution. Use that concentration and the total sample-solution volume (25.0 ml) to calculate the mass (mg) of iron in the sample. Express your result as mg of iron/g of cereal.