Comparison of attenuated total reflection infrared spectroscopy to capillary gas chromatography for trans fatty acid determination

Gas chromatography (GC) has been a standard analytical tool in lipid chemistry. The rapid attenuated total reflection (ATR) infrared (IR) American Oil Chemists’ Society (AOCS) Recommended Practice (Cd 14d-97) was compared to the capillary GC AOCS Recommended Practice (Ce 1f-97) that was optimized to accurately determine total trans fatty acids on highly polar stationary phases. This comparative evaluation was validated in an independent laboratory. These procedures were used to quantitate the total trans fatty acid levels in partially hydrogenated vegetable oils, measured as neat (without solvent) triacylglycerols (TAG) by ATR and as fatty acid methyl ester (FAME) derivatives by capillary GC. Unlike FAME, TAG determination by ATR required no derivatization, but samples had to be melted prior to measurement. Five blind replicates for each of three accuracy standards and three test samples were analyzed by each technique. The GC and ATR determinations were in good agreement. Accuracy was generally high. The ratios of ATR mean trans values (reported as percentage of total TAG) to the true values (based on the amount of trielaidin added gravimetrically) were 0.89, 0.98, and 1.02 for accuracy standards having about 1, 10, and 40% trans levels. The corresponding GC values, determined as percentage of total FAME, were 0.98, 0.99 and 1.04. The ratios of mean trans values determined by these techniques were ATR/GC 0.85, 1.04, and 1.01 for test samples having trans levels of about 0.7, 8, and 38%, respectively. The optimized GC procedure also minimzed the expected low bias in trans values due to GC peak overlap found with the GC Official Method Ce 1c-89. Satisfactory repeatability and reproducibility were obtained by both ATR and GC.     

Simultaneous determination of lodine value and trans content of fats and oils by single-bounce horizontal attenuated total reflectance fourier transform infrared spectroscopy

A method for the simultaneous determination of iodine value (IV) and trans content from the Fourier transform infrared (FTIR) spectra of neat fats and oils recorded with the use of a heated single-bounce horizontal attenuated total reflectance (SB-HATR) sampling accessory was developed. Partial least squares (PLS) regression was employed for the development of the calibration models, and a set of nine pure triacylglycerols served as the calibration standards. Regression of the FTIR/PLS-predicted IV and trans contents for ten partially hydrogenated oil samples against reference values obtained by gas chromatography yielded slopes close to unity and SD of <1. Good agreement (SD<0.35) also was obtained between the trans predictions from the PLS calibration model and trans determinations performed by the recently adopted AOCS FTIR/SBHART method for the determination of isolated trans isomers in fats and oils.     

A rapid method for the estimation oftrans unsaturation in hydrogenated oils and fats

Glycerides and methyl esters of fatty acids containingtrans unsaturation both show peaks at 10,38 μ. The absorptivities of the glycerides (a_10.38 μ) have a straight line relationship with the concentration of thetrans unsaturation, calculated as methyl elaidate. This relationship has been utilized as a quick method for estimating thetrans unsaturation in hydrogenated fats such as Vanaspati. The infrared spectrum of a given hydrogenated fat is taken in the region of 9–11 μ, the absorptivity at thetrans-peak is calculated, and the corresponding methyl elaidate content is read from a graph.     

A rapid method for the determination oftrans unsaturation in fats and derivatives

Thetrans unsaturation present in samples of unsaturated fats and derivatives may be determined from measurement of the infrared absorbance at two wavelengths, one due totrans configuration of a double bond and the other due to the ester or acid group. The ratio of the two absorbance values has a linear relationship with thetrans unsaturation content. Thus, a drop of sample is diluted with CS₂ and the infrared absorbance measured at 10.36 μ and at 8.5–8.6 μ if esters and 10.7 μ if acids. To calculate the per cent isolatedtrans double bonds in the sample, the ratio of the two absorbance values is substituted into the linear equation which was developed from known samples. The method is rapid since the sample need not be weighed nor made up to a known volume and thus may be applied to samples such as those collected from a gas chromatograph or after thin layer chromatography. Presented at the AOCS Meeting, New York, October 1968.     

Evaluation of the CP-Sil 88 and SP-2560 GC columns used in the recently approved AOCS official method Ce 1h-05: Determination of cis-, trans-, saturated, monounsaturated, and polyunsaturated fatty acids in vegetable or non-ruminant animal oils and fats by capillary GLC method

The AOCS Official Method Ce 1h-05 was recently approved at the 96th AOCS Annual Meeting (2005) by the Uniform Methods Committee as the official method for determining cis and trans FA in vegetable or non-ruminant fats and oils. A series of experiments was undertaken using a margarine (hydrogenated soybean oil) sample containing approximately 34% total trans FA (28% 18∶1 trans, 6% 18∶2 trans, and 0.2% 18∶3 trans), a low-trans oil (ca. 7% total trans FA), and a proposed system suitability mixture (12∶0, 9c−18∶1, 11c−18;1, 9c,12c,15c−18∶3, 11c−20∶1, and 21∶0) in an effort to evaluate and optimize the separation on the 100-m SP-2560 and CP-Sil 88 flexible fused-silica capillary GC columns recommended for the analysis. Different carrier gases and flow rates were used during the evaluation, which eventually lead to the final conditions to be used for AOCS Official Method Ce 1h-05.     

High performance liquid chromatography and glass capillary gas chromatography of geometric and positional isomers of long chain monounsaturated fatty acids

Positional and geometrical isomers of monounsaturated long chain fatty acids were analyzed by the combination of high performance liquid chromatography (HPLC) and glass capillary gas chromatography (GC). A preparative group separation ofcis andtrans isomers of the monounsaturated fatty acid methyl esters was achieved according to chain length by reversed-phase HPLC, and using a highly sensitive interference refractive index detector. After collection of the different fractions containingcis andtrans forms of the monounsaturated fatty acid methyl esters, the fractions were analyzed for their content of positional isomers using glass capillary GC with Silar-5 CP as stationary phase. The preparative step in the HPLC was also used analytically for the determination of the ratio between thecis andtrans monounsaturated fatty acids. A comparison was made between the results obtained with the HPLC technique and the results of a GLC technique with a packed OV-275 column. There was a good correlation between the 2 techniques with a tendency to highertrans values with the HPLC technique (4%). It was shown with reference substances that 18∶1ω6-cis to ω11-cis and 18∶1ω5-trans to ω12-trans, the most common monounsaturated fatty acid isomers in partially hydrogenated vegetable oils, could be almost quantitatively recovered in the HPLC step. Most of the individual positional isomers of monounsaturated fatty acids of varying chain length could be separated and determined in the glass capillary GC step with the exception of those isomers containing the double bond in a relatively high ω-position. The relative standard deviation of the technique as determined with reference substances was better than 4%. The described technique was applied to the analysis of the isomeric monounsaturated fatty acid content in partially hydrogenated vegetable and marine oils, and about 5 samples a day could be executed. Part of this work has been presented at the ISF/AOCS World Congress, New York (1980)JAOCS 58, (4), 1981, abstr. no. 184.     

Methyl esters from a partially hydrogenated vegetable oil is a better infrared external standard than methyl elaidate for the measurement of totaltrans content

An infrared spectrophotometric procedure, based on the fatty acid methyl ester mixture derived from a partially hydrogenated vegetable oil as the calibration standard, has been developed for accurate analysis of the totaltrans content of hydrogenated fats. This procedure produces more accurate results than the current official methods of Association of Official Analytical Chemists and American Oil Chemists’ Society, both of which use methyl elaidate as the external standard. The results obtained with this procedure were in close agreement to those determined by the combined procedure of silver-nitrate thin-layer chromatography and capillary gas-liquid chromatography. The improved results, obtained with the partially hydrogenated vegetable oil methyl esters as the calibration standard, may be attributable to its assortment oftrans isomers, which may have different absorptivities relative to methyl elaidate.     

Industrial validation of fourier transform infrared trans and lodine value analyses of fats and oils

A Fourier transform infrared (FTIR) edible oil analysis package designed to simultaneously analyze for trans content, cis content, iodine value (IV), and saponification number (SN) of neat fats and oils by using calibrations based on pure triglycerides and derived by application of partial-least-squares (PLS) regression was assessed and validated. More than 100 hydrogenated rapeseed and soybean samples were analyzed by using the edible oil analysis package as well as the newly proposed modification of the AOCS IR trans method with trielaidin in a trans-free oil as a basis for calibration. In addition, ∼1/3 of the samples were subsequently reanalyzed by gas chromatography (GC) for IV and trans content. The PLS approach predicted somewhat higher trans values than the modified AOCS IR method, which was traced to a combination of the inclusion of trilinolelaidin in the calibration set and the effects of baseline fluctuations. Eliminating trilinolelaidin from the triglyceride standards and the use of second-derivative spectra to remove baseline fluctuations produced excellent concurrence between the PLS and modified AOCS IR methods (mean difference of 0.61% trans). Excellent internal consistency was obtained between the IV and cis and trans data provided by the edible oil analysis package, and the relationship was close to that theoretically expected [IV=0.86 (cis + trans)]. The IV data calculated for the GC-analyzed samples matched the PLS IV predictions within 1 IV unit. The trans results obtained by both IR methods were linearly related to the GC data; however, as is commonly observed, the GC values were significantly lower than the IR values, the GC and IR data being related by a slope factor of ∼0.88, with an SD of ∼0.80. The concurrence between the trans data obtained by the two FTIR methods, and between the FTIR and GC-IV data, as well as the internal consistency of the IV, cis and trans FTIR predictions, provides strong experimental evidence that the edible oil analytical package measures all three variables accurately. Co-Director, McGill IR Group.     

FTIR estimation of free fatty acid content in crude oils extracted from damaged soybeans

A user-interactive computer-assisted Fourier transform infrared (FTIR) method has been developed for estimation of free fatty acids (FFA) in vegetable oil samples by deconvolution of the infrared (IR) absorbances corresponding to the triglyceride ester and FFA carbonyl bonds. Peak areas were used to determine FFA as a percentage of the total carbonyl areas in weighed standards of refined, bleached, deodorized soybean oil containing from 0 to 5% added oleic acid. These data for percent FFA by FTIR were compared to corresponding FFA data obtained by two titration methods-the AOCS Official Method Ca 5a-40 and the Official Method with a slight modification. Correlation coefficients were 0.999 for the Ca 5a-40, 0.999 for the modified and 0.989 for the FTIR methods. FFA in samples of crude soybean oils extracted from damaged beans (0.5 to 2.1% FFA) were measured by FTIR and compared to data obtained by titration of the same samples (correlation coefficient, 0.869). To whom correspondence should be addressed at National Center for Agricultural Utilization Research, Agriculture Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604. ¹The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

The determination ofTrans isomers in GLC fractions of unsaturated esters

The determination of the amount oftrans unsaturated isomers present in collected fractions of unsaturated fatty acid methyl esters separated by gas liquid chromatography is accomplished by measuring the infrared absorbances of the collected peak at 10.36 and 8.55 μ. The ratio of these two absorbances is proportional to thetrans unsaturation. The interfering polyester column bleed is removed from the collected peaks by collection on alumina and elution into the spectrophotometer cell with CS₂.     

Application of a Novel,Heated, Nine-Reflection ATR Crystal and a Portable FTIR Spectrometer to the Rapid Determination of Total <Emphasis Type="Italic">Trans</Emphasis> Fat

Since trans fat labeling requirements became mandatory in the US and many other countries, there has been a need for rapid and accurate analytical methodologies that can facilitate compliance with the various regulations. The determination of total trans fatty acids by mid-infrared (IR) spectroscopy is a widely used procedure that was standardized and validated as AOCS Official Method Cd 14e-09 (negative second derivative infrared spectroscopic method for the rapid (5 min) determination of total isolated trans fat) in 2009. The C–H out-of-plane deformation mid-IR band observed at 966 cm⁻¹ is uniquely characteristic of isolated (non-conjugated) double bonds with trans configuration. AOCS Official Method Cd 14e-09, the most recent attenuated total reflection-Fourier transform IR (ATR-FTIR) official method, entails the measurement of the height of the negative second derivative of the trans absorption band. In the present study, the performance of a novel, portable FTIR system equipped with a heated 9-bounce diamond ATR crystal was evaluated and compared to that of a conventional benchtop single-bounce ATR-FTIR spectrometer. The introduction of the 9-bounce diamond ATR crystal resulted in the lowering of the limit of quantification of trans fat, as a percentage of the total fat, from approximately 2 to 0.34%. The data collected from accurately weighed gravimetric standards and 28 unknown test samples ranging in trans fat content from about 0.5 to 54%, as a percentage of the total fat, indicated that this IR official method and the use of the new 9-bounce portable ATR-FTIR instrumentation could lead to a five-fold enhancement in sensitivity relative to single-bounce systems. Implementing these changes would facilitate regulatory compliance and verification of fat and oil samples for trans fat content in the US and other countries, since all of the published regulations (e.g., “0 g trans fat per serving”) have levels of trans fat, as percentage of total fat, that exceed 0.34%.     

Rapid determination of the totaltrans content of neat hydrogenated oils by attenuated total reflection spectroscopy

A Fourier transform infrared spectroscopy procedure is described for quantitating the levels of totalrans triglycerides or their fatty acid methyl ester derivatives in neat fats and oils. It requires either warming or no preparation of the laboratory sample, and about 5 min for spectroscopic measurement, band area integration, and calculation of thetrans content from a linear regression equation. To eliminate the strongly sloping background of the 966-cm⁻¹ trans band, the single-beam spectrum of thetrans-containing fat is “ratioed” against that of an unhydrogenated oil or a reference material that contains onlycis double bonds. Thus, a symmetric absorption band on a horizontal background is obtained. The area under thetrans band can then be accurately integrated between the same limits, 990 and 945 cm⁻¹, for alltrans levels investigated. To speed up the analysis, an attenuated total reflection liquid cell was used, into which oils, melted fats or their methyl esters were poured without weighing or quantitative dilution with the toxic and volatile carbon disulfide solvent. Thetrans levels determined by attenuated total reflection were closer to those found by capillary gas chromatography when the hydrogenated fat was measured against the corresponding unhydrogenated oil than when it was measured against acis reference material. Small differences were found betweentrans levels in hydrogenated fat test samples and the corresponding methyl ester derivatives (9.3 and 2.2% at about 2 and 41%trans, respectively). The lower limits of identification and quantitation were 0.2 and 1%, respectively.     

Determination ofcis unsaturation in oils by near infrared spectroscopy

An analytical method has been developed capable of giving reproducible and accurate determinations of thecis unsaturation present in refined oils, hydrogenated oils, and finished shortenings. Molar absorptivity measurements indicate that similar methods are applicable to the analyses of mixed esters or fatty acids. The method depends upon the measurement of a band in the near infrared at 2.143 μ that is caused bycis unsaturation. The following pertinent conclusions are drawn from data obtained in this investigation.

1. The areas under the absorption bands forcis polyenes are integral multiples of the areas under the bands ofcis monoenes.
2. Thecis content of oils containing less than 10%trans triglycerides can be determined accurately by measuring the area under the absorption band.
3. Accurate results can be obtained for samples containing quantities oftrans fatty acids in excess of 10% if a point baseline is used and the peak absorbance measured.

     

Reduction with zinc of triple bonds tocis double bonds in long chain conjugated fatty acids

The methyl ester of santalbic acid (trans-11-octadecen-9-ynoic acid) was hydrogenated with powdered zinc in 50% propanol to yield 90% of thecis,trans-diene ester; similarly, 7,9-octadecadiynoic methyl ester was converted tocis,cis-diene ester but yields were no better than 75%. Presented in part at the AOCS Meeting, Chicago, October 1967. East. Market. Nutr. Res. Div., ARS, USDA.     

Study on the effect of antioxidants in the autoxidation of methyl nonconjugated octadecadienoates

Many studies have been published on the effect of antioxidants on unsaturated fatty acid esters but the differences of the effects of antioxidants on geometric isomers have never been investigated. In this study, methylcis-9,cis-12-octadecadienoate and itstrans isomer methyltrans-9,trans-12-octadecadienoate were used as methyl nonconjugated dienoates, and BHA, BHT, PG, NDGA, 4,4′-dihydroxy-3,5,3′,5′-tetratert-butyl diphenyl methane, L-thyroxine sodium salt,a-tocopherol and sesamol were used, as antioxidants. The differences of the effects of antioxidants on both geometric isomers were investigated by determining the induction period using the weighing method. Also determined were the infrared and ultraviolet spectra, peroxide values, conjugated diene contents, isolatedtrans double bond contents and molecular weights for the controls and the samples containing antioxidants. Thecis,cis isomer was more easily autoxidized and had a shorter induction period than thetrans,trans form. By the end of the induction period, no isolatedtrans double bond forms in thecis,cis isomer, but a considerable amount of isolatedtrans double bond decreased in thetrans,trans isomer. In general, the effects of antioxidants, except NDGA, on thecis,cis isomer were larger than thetrans,trans form.     

Determination of trans fatty acids in hydrogenated vegetable oils by attenuated total reflection infrared spectroscopy: Two limited collaborative studies

An attenuated total reflection infrared spectroscopy procedure was collaboratively studied among two sets of five laboratories for quantitating the total trans fatty acid levels in neat (without solvent) hydrogenated vegetable oils, measured as triacylglycerols in one study, and as fatty acid methyl ester derivatives in the other. Unlike the fatty acid methyl esters, the triacylglycerols required no derivatization but had to be melted prior to measurement. To obtain a symmetric absorption band at 966 cm⁻¹ on a horizontal background, the single-beam spectrum of the trans-containing fat was "ratioed" against that of a refined oil or a reference material that contained only cis double bonds. A single-bounce horizontal attenuated total reflection cell that requires 50 μL of undiluted test samples was used for oils, melted fats, or their methyl esters. For fatty acid methyl esters, the reproducibility relative standard deviations were in the range of 0.9 to 18.46% for 39.08 to 3.41% trans, determined as methyl elaidate per total fatty acid methyl esters. For five pairs of triacylglycerol blind duplicates, the reproducibility and repeatability relative standard deviations were in the ranges of 1.62 to 18.97%, and 1.52 to 13.26%, respectively, for 39.12 to 1.95% trans, determined as trielaidin per total triacylglycerols. Six pairs of spiked triacylglycerol blind duplicates (quality assurance standards) exhibited high accuracy in the range of 0.53 to 40.69% trans and averaged a low bias of 1.3%. These statistical analysis results were compared to those collaboratively obtained by the recently adopted AOCS Cd14-95 and AOAC 994.34 Infrared Official Methods.     

The deposition of polyunsaturated fatty acids in the rat fed partially hydrogenated vegetable oil

The composition of deposited polyenoic fatty acids in rats fed liquid or partially hydrogenated corn oil was determined by gas chromatography, which did not distinguish isomeric forms, and by lipoxidase which reacted with thecis, cis-methylene-interrupted acids. The two methods gave similar results for the liver fatty acids of rats fed either the unhydrogenated or partially hydrogenated oil. Of the fatty acids from the epididymal fat pads of rats fed the hydrogenated product, an appreciable quantity of linoleate isomers did not react with lipoxidase. The total amount of linoleic acid deposited was related to the total fatty acid pattern of the dietary oil. It appeared that thetrans-acids were mostly metab-olized and that the originalcis,cis-linoleic acid remaining in the partially hydrogenated product was preferentially incorporated into tissues. One of 10 papers to be published from the Symposium “Hydrogenation” presented at the AOCS Meeting, New Orleans, April 1970.     

Raman spectroscopic analysis of thecis/trans isomer composition of edible vegetable oils

A new method is presented for determining thecis/trans isomer content of edible vegetable oils. The intensities of Raman lines near 1656 and 1670 cm⁻¹ are associated with thecis andtrans configuration, respectively. A precision of ca. 1% can be obtained in thecis/trans isomer analysis of binary mixtures of methyl esters and triglycerides of monoenes and dienes and of hydrogenated vegetable oils. The spectroscopic data also provide the iodine value of vegetable oils or isolated fractions with precision for a single determination of ca. 1%. Presented at the AOCS meeting, Houston, May 1971. W. Market. Nutr. Res. Div., ARS, USDA.     

Improved methods for the isolation and study of the C18, C20 and C22 monoethylenic fatty acid isomers of biological samples: Hg adducts,HPLC, AgNO3-TLC/FID,and ozonolysis

The monoethylenic isomers of C₁₈, C₂₀ and C₂₂ chain lengths of the depot fat of a nonhominid primate (cynomolgus monkeys,Macaca fascicularis), fed a partially hydrogenated herring oil (IV=76.0) for 30 months, were examined by 2 different approaches. The first isolation method involved preparative gas liquid chromatography and argentation thin layer chromatography (TLC). The second sequence involved a chain-length fractionation system based on the TLC of the methoxy-bromomercuri quence involved a chain-length fractionation system based on the TLC of the methoxy-bromomercuri adducts of the total methyl esters to isolate groups of acids of common degrees of unsaturation, and then high performance liquid chromatography on a reverse-phase column. In both cases, the monoethylenic isomer distribution was determined by ozonolysis in BF₃/MeOH. Comparable results were obtained with the 2 methods. The second approach is recommended for small biological samples, especially for those containing a relatively high proportion of di- and other polyethylenic isomers which might interfere. Presented in part at the AOCS annual meeting, New Orleans, May 1981.     

Automated quantitative analysis of isolated (nonconjugated) trans isomers using fourier transform infrared spectroscopy incorporating improvements in the procedure

A method for determining the isolated (nonconjugated)trans isomer content of fats and oils was developed using Fourier Transform Infrared Spectroscopy (FTIR), which permits automatic sample analysis and calculation of percenttrans content. Integrated band areas were used as a measure of the intensity of the band associated with thetrans C=C double bond. Measured band areas for samples with known percenttrans content were fit with a second order polynomial, resulting in a correlation coefficient of 0.99998 and a standard error of estimate of 0.11 over the range of 0 to 50%trans content. This technique also allows the analysis of neat samples, a significant improvement over current procedures in that it eliminates the need for dilution by volatile solvents and any attendant errors. Presented at the AOCS meeting in New Orleans, LA in May 1987.