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    AOCS Ce 1-62 FA COMP PDF

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    wil

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    of 4
    SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS: DEFINITION SCOPE acids that have been polymerized. AOCS Official Method Ce 1-62 Reapproned 1987 Fatty Acid Composition by Gas Chromatography ‘Methyl esters of fatty acids are separated and determined quantitatively by GLC using a packed column. ‘The method is applicable to methyl esters of fatty acids having 8-24 carbon atoms and to animal fas, vegetable oils, marine oils and fatty acids after their conversion to methyl esters. The method permits quantitative separation of mixtures containing saturated and unsaturated methyl estes. The condi tions specified inthis method are not suitable for determining epoxy or oxidized faty acids or fatty ‘APPARATUS 1. The gas chromatograph, which is commercially avail- able, should have as @ minimum the following charac teristics— (@) Column oven—capable of heating the column to at least 220°C and of maintaining the desired temperature to within + 1°C. (b) Sample inlet port—with minimum dead space which is independently heated to a temperature 20-50°C higher than column temperature. (©) Detectors—thermal conductivity (TC) or flame- ionization (FID), separately thermostated, which can be maintained at or above column temperature. 2. Recorder—If the recorder curve is to be used to caleu- late the composition of the mixture analyzed, an elec- tronic recorder of high precision is required. The char- acteristics ofthe recorder should be: (2)_ Rate of response—below 1.0 sec (the rate of response is the time taken for the recording pen to pass from O to 90% following the momentary introduction of a 100% signal). (b) Chart paper—width 25 em (10 in.) min. (©) Chart paper speed—25-100 cmv (10-40 in fr). 3. Integrator or calculator (optional)—rapid and accurate calculation can be performed with the help of an elec- tronic integrator or calculator. This must give a linear response with adequate sensitivity and baseline correc- tion should be consistent with good chromatographic practice. Horizontal, non-horizontal and tangential baseline correction must be controlled by selectable electronic peak logic. 4. Syringe—maximum capacity 10 pL, graduated in 0.1 pL. 5. Chromatographic column— (@) The column must be constructed of a material nert (0 the substances to be analyzed—glass, or failing that, stainless steel (see Notes, 1), with a length of 1-3 mand a 2-4 mm id, (b) Packing support—acid-washed and silanized diatomaceous earth, or other suitable inert support with a narrow range (25 um) of grain size between the limits of 60-120 mesh (125-250 um). (©) Stationary phase—polyester type of polar liquid (diethylene glycol polysuccinate, butancdial poly- succinate, ethylene glycol polyadipate), or any liquid (e-g., cyanositicones), meeting the require- ‘ments below. The stationary phase should amount to 5-20% of the packing. A non-polar stationary phase, such as methyl silicone, fluid or gum, can be used for separations of fully saturated materials REAGENTS 1. Gases— (a) Carrier gas for TC detector—helium, min purity 99,95 mol%; for FID, helium, nitrogen or argon, rin purty 99.95 mol% (©) FID—hydrogen, min purity 99.95 mols sir dry [ew point -59°C (75°F) maximum] ané hydro- carbon free (less than 2 ppm hydrocarbons equiva- lent CH) 2, Reference standards —a mixture of methyl estes, or the methyl estes of an oil of known composition, prefer ably similar to that of the fatty mater to be analyzed Reference mixtures simulating most fats and oils may be obtained from: Applied Science Laboratories, Inc., P.O. Box 440, State College, PA, USA. Supelco, Inc, Supelco Park, Bellefonte, PA, USA. Nu-Chek-Prep, inc, P. O. Box 172, Elysian, MN, USA. Analabs, Inc., 80 Republic Drive, North Haven, CT, USA. Alltech Associates, Inc, 2501 Waukegan Ra, Deerfield, IL, USA. PREPARATION OF METHYL ESTERS AOCS Official Method Ce 2-66 is recommended. PROCEDURE 1. Condition a new column while itis disconnected from detector by holding it about 10°C above its operating temperature with flow of inert gas at 20-60 mLimin for approximately 16 hr and then an additional 2 hr at 20°C above its operating temperature. In no case exceed the ‘manufacturer's recommended maximum temperature 2. Determining optimal operating conditions— (@) In selecting the test conditions, the following vari- ables must be taken into account: length and diam- eter of the column, temperature of the column, carrier gas flow, resolution required, size of the Page 1 of SAMPLING AND ANAIYSS OF COMERCIAL FATS AND OWS Ce 1-62 + Fatty Acid Composition by Gas Chromatography sample for analysis and time of analysis, The size ‘of the sample should be chosen so that the assem bly of detector and electrometer gives a lincar response. As a rule, the following figures will lead to the desired results, viz, at least 2,000 theoreti- cal plates for methyl stearate and its elution within about 15 min Temperature 175 180 Iss 185 (b) Where the apparatus allows, the injection port should be at a temperature of about 250-275°C and the detector at a temperature equal to, oF higher than, that ofthe column, (c) The flow of hydrogen to the flame ionization detector is, as a tule, about 0.5-1 times that of the cartier gas, and the flow of air about 5-10 times that ofthe hydrogen 3, Determining the efficiency and the resolution — (a) Carry out the analysis of a mixture of methyl Stearate and oleate in about equivalent proportions (e.g. methyl esters from cocoa butter). Choose the size of the sample, the temperature of the column and the carrier gas flow so that the maximum of the methyl stearate peak is recorded about 15 min after the solvent peak and rises to three-quarters of the full scale. Calculate the number of theoretical plates n (efficiency) by the formula: n= 16(€R Jw, and the resolution, R, by the formula— R=2Mw, +05) Where— aR, = the retention distance, measured in mm, from the start to the maximum peak of methyl stearate , = width in mm of the peak for methyl stearate, measured between the points of intersection of ‘the tangents of the inflection points of the curve ith the baseline w)=Wwidth in mm of the peak for methyl oleate measured between the points of intersection of the tangents of the inflection points of the curve with the baseline ‘A= distance between the respective peak maxima for methyl stearate and oleate () Operating conditions to be selected are those which will afford at least 2,000 theoretical plates Page 2 of 4 4. Analys for methyl stearate and a resolution of at least 1.25. Additionally, linolenic acid (Cy4.,) ester should be separable from arachidic acid (Coy) and gadoleic acid (Cy, estes (c) Asa rule, the operating conditions will be those defined above. Nevertheless, i is possible to work with a lower column temperature where the deter- mination of acids below C,, is required, or at a higher temperature when determining fatty acids above Coy (2) On occasion, itis possible to employ temperature programming in both the previous cases. For example, ifthe sample contains the methyl esters of fatty acids below C) , inject the sample ata column temperature of 100°C and immediately raise the temperature at arate of 4-8°C/min to the optimum temperature. In some cases, the two procedures can be combined. After the Programmed heating, continue the elution at a Constant temperature until all the components have been eluted. If the instrument does not employ programmed heating, work at two fixed tempe tures between 100°C and 195°C. Liquid phase characteristics will determine the starting tempera- ture or the upper temperature if the analysis is performed isothermally. (a) The sample for examination should be 0.1-2 pL. of the solution of methyl esters obtained according. to AOCS Official Method Ce 2-66. In the case of esters not in solution, prepare an approximate 1=10% solution and inject 0.11 pl of this solution (b) If the object is to determine constituents present only in trace amounts, the sample size may be increased (up to tenfold), CALCULATIONS Identification of peaks— (a) Analyze the reference standard mixture of known composition under the same operating conditions as those employed for the sample, and measure the retention distances (or retention times) for the constituent esters. 2. Quantitative analysis— 2) Apart from exceptional cases, assume that the whole of the components of the sample are repre- sented on the chromatogram, so that the total of the areas under the peaks represents 100% of the constituents (total elution). (b) If the equipment includes an integrator, use the figures obtained therefrom. If not, determine the area under cach peak by multiplying the height by the breadth at mid-height and, where necessary, faking into account the various attenuations used during the recording. (©) For the general case, in which significant amounts ‘of components below C,, are absent, calculate the content of a particular constituent (expressed as percent of methyl esters) by determining the Percentage represented by the area of the corre a" Ce 1-62 * Fatty Acid Composition by Gas Chromatography “SAMPLING AND ANALNSIS OF COMMERCIAL FATS AND OWS sponding peak relative to the sum of the areas of all the peaks. ‘Area percent of the component i expressed as ‘methyl esters = A, = x 100 TA; Where— ‘A, = area of the peak corresponding to component i ‘SA, = sum of the areas under all the peaks (4) Correction factors, particularly in the presence of acids below C,, of acids with secondary groups, or when using a TC detector, must be used to convert the percentages of peak areas into mass percentages of the components. Determine the correction factors with the help of a chromatogram derived from the analysis of a reference mixture of methyl esters of known composition under operating conditions identical to those used for the sample. For this reference mixture: B

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