CH 4200, Fall 2004 Prof.

Greenlief

Fatty Acid Determination Using Gas Chromatography

Objectives This experiment introduces a procedure that is used routinely for fat analysis in which nonvolatile fatty acids are chemically converted to the corresponding volatile methyl esters. The resulting volatile mixture can be analyzed by gas chromatography. Background Fatty Acids Fats consist of glycerol esters and long chain aliphatic acids (fatty acids). The general glycerol ester structure is shown below: CH2-O-CO-R1 | CH2-O-CO-R2 | CH2-O-CO-R3 The backbone of these compounds contains from 4 to more than 20 carbon atoms. Most natural sources of these compounds have an even number of carbon atoms because the biosynthetic pathway builds the backbone two carbons at a time. Fatty acid chains may contain one or more double bonds at specific positions (unsaturated and polyunsaturated), or they may be fully saturated. The physical and chemical properties of a fat depends on the composition of the fatty acid mixture. Animal fats tend to have a larger proportion of long chain saturated acids and are solids at room temperature. Fats from plant sources contain a higher proportion of unsaturated acids and are often liquids at room temperature due to hydrogen bonding. Polyunsaturated fats are usually of vegetable origin. Crisco is an example of a vegetable-derived, unsaturated fatty acid that has been hydrogenated to form a solid material. Fats are used in cooking because they are very high boiling compounds. Their high boiling points therefore make this class of compounds ill suited for analysis by gas chromatography. However, the glycerol esters can be chemically decomposed into methyl esters of each individual fatty acid. In this lab, your samples will be inter-esterified in methanol using a BF3 catalyst. The corresponding mixture of methyl esters will be extracted into hexane and chromatographically separated. Methyl pentadecanoic acid (C15) will be added as an internal standard. This compound is ideal as an internal standard because it should behave similarly to the analytes of interest, but it is unlikely to be present in your natural fat sample since it has an odd number of carbon atoms on the backbone.

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CH 4200, Fall 2004 Prof. Greenlief

Experimental I. Preparation and analysis of fatty acid methyl ester (FAME) samples from fat samples Weigh out about two grams of oil or fat in a small beaker and record the exact weight. Dissolve the sample in 50 ml of chloroform and transferred to a 100 ml volumetric flask and dilute to the mark. Transfer 1 ml of the unknown sample to a 10 ml screwtop culture tube with Teflon liner. Now add exactly 1.00 ml of a standard solution of 0.814 mg/ml pentadecanoic acid. When you esterify the glycerides in the fat sample, you will also esterify the pentadecanoic acid standard. If we assume i.) the efficiency for esterification of the standard is the same as that of the glycerides, and ii.) the response of the detector to each of the FAMES including the C15 internal standard is the same, then we can quantify the amount of each ester in the fat by comparison of the integrated areas with the known concentration of the standard. Evaporate most of the chloroform under a stream of nitrogen until ~100 l of the solution remains. If the solution is dried completely, it will be hard to re-dissolve with the esterification reagent. Next, add 1 ml of interesterification reagent [25 vol% of a 12% BF3-methanol solution, 20 vol% benzene and 55 vol% methanol]. Flush the tube with nitrogen, seal it, and heat it in a 100C water bath for 30 minutes. After interesterification, extract the methyl esters with hexane and H2O so that the final mixture of the reagent, hexane and water, is in a proportion of 1:1:1 (i.e., add 1 ml each of hexane and water to the reaction mixture). Shake the mixture vigorously by hand for 2 min. If a stable emulsion is formed, break it by centrifugation. Transfer about half of the top hexane phase to a small test tube for injection. Be careful to remove only the organic layer. Do not inject directly from the reaction vial because of the risk of injecting water. Water can ruin the GC column. NOTE: You may want to run your calibration standards while the esterification reaction is in progress.

II. Instrumental Set-Up Refer to the Chromatographic Instrumentation: Gas Chromatography procedure for a detailed description of the instrumental adjustments required for this analysis. Fixed Settings: Generally the operator must adjust gas flows to the columns, the inlets, the detectors, and the split ratio. In addition, the injector and detector temperatures must be set. The detectors are generally held at the high end of the oven temperature range to minimize the risk of analyte precipitation. All of these parameters should have been set to the correct values, but double check all the parameters to ensure that the values are correct. Detector Temperature Detector A: 250C Detector B: 300C

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CH 4200, Fall 2004 Prof. Greenlief

Injector temperature Both injectors 220C Integrator chart speed: 2 cm/min

III. Separation of Fatty Acid Methyl Esters (FAMES) The following experiments will be run on column A using a standard sample containing the following FAMES in approximately the following concentrations. (Get the exact concentrations from your TA.) FAME Methyl myristate Methyl pentadecanoate Methyl palmitate Methyl palmitoleate Methyl stearate Methyl linoleate Methyl oleate Methyl linolenate Approximate Concentration 0.10 mg/ml 0.80 mg/ml 1.50 mg/ml 0.15 mg/ml (* 16 carbon backbone, 1 double bond) 0.70 mg/ml 0.35 mg/ml 2.00 mg/ml 0.15 mg/ml

C14:0 C15:0 C16:0 C16:1* C18:0 C18:2 C18:1 C18:3

Note: You can often assign each peak of the chromatogram of each standard sample by considering the melting point and the interactions of the analyte with the stationary phase (column). The assigned peaks can be cross-checked by relating the concentration and the area of the peak measured. The above list is given in the order of elution, but you will want to confirm this by checking the concentrations against the relative areas of the peaks. A. Isothermal Chromatogram of FAME Standard Set the OVEN TEMP to 180C and allow the GC to warm up. While its warming, set: SIG 1 A FINAL VALUE 181C INIT VALUE 180C FINAL TIME 1 minutes. INIT TIME 15 minute RATE 0C/min When the instrument is ready, the NOT READY light will turn off, and you can begin your run. Inject a 1 microliter sample onto column A using proper injection technique as described in the Chromatographic Instrumentation: Gas Chromatography document. Collect the chromatogram and answer the following question. Question 1: Do you get good separation of all peaks with the isothermal program? Why? B. Temperature Program for FAME Standard
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CH 4200, Fall 2004 Prof. Greenlief

Next you will analyze the same sample with a temperature program. You will run the temperature program listed below on column A of the instrument to compare the effect of isothermal to temperature programmed methods. The effect should be noticeable by the change in the retention times of the esterfied compound peaks. The program is as follows: INIT VALUE INIT TIME RATE 15C /min FINAL VALUE FINAL TIME 120C 1 min 210C 7 min

(To get rid of tick marks on the chromatogram, press INTG, the number 8, and then ENTER on the integrator keypad.) Question 2: How do the retention times of the sample components differ in the two chromatograms? Explain this result. C. Temperature Program for FAME Sample Using the same temperature program as in part III.B above, inject 1 l of the dry esterification product for analysis. Assign the peaks according to the results of the standard chromatograms collected in section III B. IV. Quantitative Analysis of FAMES From your chromatograms: A. Identify the esters present in your sample qualitatively by retention times. You will notice a relationship between carbon number and retention time, which can be used to identify FAMES having chain lengths longer than C18. B. Quantify the amount of each of the FAMES in your sample, but only do this for the seven components (not including the C15 internal standard) which were present in your standard sample if they appear. This requires several steps. You will need to compare the sample peak areas with the standard peak areas. Since the concentrations of FAMEs responsible for the standard peaks are known, this comparison will allow you to calculate a concentration from the sample peak area. However, because the sample injection is different from the standard injection (e.g., small differences in volume, split ratio, dilutions, etc.) you cannot make a direct comparison. This is where the internal standard comes in. You can correct for variations in sample volume by taking the ratio of all peak areas to the internal standard in both the sample and the standard. Since the internal standard has a known concentration in both sample and standard, it can be used to correct for sample variations. Ax denotes the area of a peak due to compound x in the sample. In order to relate this area to the area of compound x in the standard you must first correct for sample variations to arrive at a corrected area denoted Ac,x . You can find Ac,x for each peak in the chromatogram using the equation

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CH 4200, Fall 2004 Prof. Greenlief

A c,x = A x

A C15 ,standard A C15 ,sample

CONC C15 ,sample CONC C15 ,standard

where AC15 ,standard and AC15 ,sample denote the internal standard areas in the standard solution and the sample, respectively. Note that the C15 signal area is proportional to the mass of analyte passing through the detector. Since the mass is equal to the concentration of C15 times the volume, the ratio of Area/conc. is equal to a volume times a proportionality constant. The above equation results in a cancellation of the proportionality constant, and so this equation can be rewritten as
A c,x = A x InjectedVolume C15 ,standard InjectedVolume C15 ,sample

to emphasize the importance of the internal standard in correcting for sample volume. The resulting Ac,x is an area that has been corrected to give a volume equivalent to what would be expected if the internal standard peaks has identical areas in sample and standard chromatograms. From the corrected areas, the concentration of each component of the mixture can be calculated using the expression
CONC x = A c, x CONC x ,standard

A x , standard This expression reflects the fact that the properly corrected area of compound x can be compared with the area of compound x on the standard chromatogram, whose concentration is known. Thus the ratio of the areas gives the ratio of concentrations, and the product of this ratio and the known concentration of compound x in the standard gives the concentration of compound x in the sample.

Use the results of this quantitative analysis to calculate the percentage composition of each fatty acid in grams of compound per gram of oil. (Decide if C15 should be included in this calculation.) The development of an exact formula for this calculation is left to you. Additional Questions 3. Some FAME sample chromatograms may contain additional peaks. What might these be due to assuming they are not simply contamination of the sample? 4. Suppose we found out that the sensitivity scale used in the run of the standard was different from that of the sample. Should we consider the change of the sensitivity? If not, why (Hint: C15)?

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CH 4200, Fall 2004 Prof. Greenlief

5. What is the resolution (R) for the C16:0 and C16:1 peaks in the standard chromatogram?

Lab Write-up Format for Gas Chromatography Lab The Gas Chromatography Lab report will be a formal report. The report must be typed, and must include the following sections
Title of Analysis, Name of Analyst, Date of Analysis General methods (a page and a half maximum) Raw Data, chromatograms Data Analysis Questions Conclusions For General Methods give a brief description of the techniques used in the analysis. This should include, for example, the temperature program, flow rates, pressures, the column type, detector type, esterification procedure, etc. It should include enough detail so that another analyst can duplicate the experiment if necessary. This section must be written in the third person (do not write I/We prepared a sample), and it must be written in the past tense. This is standard writing practice. This section CANNOT be a list of steps; it must be in sentence form. No credit will be given for procedural checklists. For Raw Data, tables of retention times and integrations of relevant peaks must be included. These tables should be labeled sequentially (Table 1, Table 2, Table 3, etc) and should be referred to in the text of the Data Analysis. These should be Microsoft Excel Spreadsheet tables (or a similar program like Sigmaplot, etc). This also MUST include copies of the chromatograms. They can be reduced, if necessary. For the Data Analysis section should include any graphs and tables of calculated numbers that you might feel necessary. (This lab does require many graphs.) There is no need to show all of the calculations performed (mostly the results of those calculations will be fine), but representative calculations and/or formulas might provide some clarity to the section. You might include sample calculations in an Appendix, or you may choose to include them in the Data Analysis section. This is your choice, however, make it completely clear where each of the numbers comes from for your data analysis.

Questions should be answered in this section of the report. The answers should be written in essay style using complete sentences and coherent paragraphs. Conclusions: This section will contain the final thoughts and recap of any important points made in data analysis. This can be a paragraph, or a bulleted (or numbered) list would also be acceptable. Any important results should be briefly noted in the conclusion.

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CH 4200, Fall 2004 Prof. Greenlief

The TAs will not ask to see any photocopies of the lab notebooks for grading at the time that the labs reports are turned in, however, they may spot check lab notebooks to check for completeness during some of the lab periods. Therefore, it would be to everyones advantage to keep all notes and observations in the lab notebooks.

FAMEs by GC-7