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Analysis of Triglycerides via LC and FTIR Using the LC-Transform Interface AN-19
INTRODUCTION:
Triglycerides are esters of various fatty acids with the trihydric alcohol, glycerol. This work deals with the measurement of unsaturation in materials which contain fatty acids notably, vegetable oils. Measurement of the degree of unsaturation is particularly important because organoleptic and texture properties of foods such as baked goods are influenced by the degree of unsaturation. Stability in cooking process and rancidity are also influenced by unsaturation. Finally, recent health issues associated with saturated fats have assumed a high level of importance. Infrared spectroscopy has numerous applications in the analysis of fats and oils. This includes the determination of unsaturation, measurement of hydrogenation, and measurement of cis/trans content of double bonds. Oxidation and/or polymerization during cooking has been measured by infrared methods. Analysis of the various natural triglyceride fats and oils shows that they are mixtures of various fatty acid esters, rather than simple mono-acid esters. Traditionally measurements of unsaturation have employed wet chemical methods such as saponification and iodination of the double bonds. Infrared techniques have been shown to provide fast and labor efficient determinations of unsaturation. Arnold and Hartung1, in 1971, demonstrated a good correlation of iodine value and infrared absorbance ratios for a broad range of vegetable oils. Afran and Newbery2 in 1991extended the approach using FTIR combined with ATR. In this work, using mixtures of triglycerides, Afran and Newbery demonstrated a direct correlation between C=C/C-C stretch ratio and the number of double bonds in triglycerides and natural oils. By use of a chilled sample stage, De Ruig3 demonstrated that spectra of the crystalline forms of triglycerides yielded information about the chain lengh of the fatty acid moiety, isomeric forms, cis-rans characterization, and position of double bonds in the chain.

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Figure 1. Correlation of C-H stretch ratios and iodine values for vegetable oils.

Figure 2. Correlation of double bond peak ratios and double bond frequency in triglycerides. While these measurements demonstrate the utility of FTIR in the determination of unsaturation, they do not address the complex mixture of triglycerides that characterize the natural oil. Liquid chromatography is often the separation of choice for many food analyses because many of these mixed materials are relatively non volatile or thermally labile and are; therefore, difficult to analyze using gas chromatography. Liquid chromatography does an adequate job of separating many of these substances although the use of spectroscopy allows one to gain additional structural information. Without additional sample preparation, retention times are often not enough for identification due to the number of components in many of the food matrices. Infrared spectroscopy is especially useful in this area because, it allows for the differentiation between saturated and unsaturated fats via observation of the C=CH absorbance region at 3000 cm-1. The degree of saturation/unsaturation can also be determined for unknowns by generating a response curve for standards based on the total carbon number versus the ratio of the intensities of the antisymmetric CH2 and ester C=O peaks. Similar response curves can be generated from other peak ratios as well. FTIR has
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further advantages in that the technique can identify free fatty acids and fatty acid esters in a complex matrix. The following is a brief study on triglycerides using the LC-Transform as the interface between liquid chromatography and IR spectroscopy. Based on the aforementioned reasons this hyphenated technique can serve as a powerful analytical tool.

EXPERIMENTAL
Triglyceride standards were used initially to determine the feasibility of depositing the analytes onto the disk as well as optimizing the deposition conditions. A standard mixture containing 1 mg/mL each of the triglycerides listed in Table 1 was prepared in a solution of 50/50 acetonitrile and acetone. The solution was separated on a Waters HPLC system with the eluent running directly into the flow divider on a Model 102 LC-Transform. A secondary detector (light scattering - Alltech Varex) was connected to the output of the splitter in order to make a comparison with the IR reconstruction. Following the chromatographic run the germanium disk on which the analytes were deposited was transferred to the optics in the sample compartment of a Nicolet Magna Series FTIR system. The IR run was collected using OMNIC Series software. A Gram-Schmidt reconstruction was automatically generated and the individual spectrum for each peak was then acquired. Following the analysis of the standard mixture a solution of 1 mg/mL olive oil was analyzed under the same conditions.

EXPERIMENTAL CONDITIONS
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Chromatography (Waters 600-MS) Column: 250 x 4.6 mm Hypersil - ODS (Keystone Scientific) Mobile Phase: 50/50 ACN/Acetone for 16 min. Linear ramp to 10/90 in 24 min. Column Flow: 1 mL/min Injection Volume: 50 L Concentration: 1 mg/mL

Light Scattering (Varian)

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Drift tube: 85C Gas Flow: 2.00 SLPM

LC-Transform Model 102 (Lab Connections)

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Sheath Temperature: 55C Sheath Gas Flow: 17 Nebulizer Flow: 0 Nozzle Height: 5 mm Disc Rotation Rate: 10/min Flow to Disk: 30 L

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Magna 550 Spectrometer (Nicolet)

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Detector: DTGS Scan Velocity: 0.6329 Scan Number: 8 scans per Spectrum Sampling Interval: 8.12 sec. Resolution: 8 cm-1 Disc Rotation: 10/min

Figure 3. Light scattering detector signal from chromatography of triglyceride standards.

Results
The standard solution was injected under the conditions listed previously. The light scattering trace is given in Figure 3. A reconstruction was generated on the IR and from this the spectra.

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Figure 4. Spectra of Triglyceride Standards. All four components look very similar because they are all unsaturated compounds. The areas of spectral importance include the ester C-O stretch at 1161 cm-1, symmetric CH2 deformation at 1462 cm-1, and the ester C=O stretch at 1745 cm-1.

Figure 5. C-H Stretch Region of Tripalmitolein. Figure 5 shows an expanded x-scaling of the 3300 - 2500 cm-1 region of tripalmitolein. The CH2 symetric stretch region is identified at 2855 cm-1 and the CH2 antisymetric stretech region at 2925 cm-1. The antisymmetric CH3 stretch can be seen as a shoulder on the 2925 region. The determination of an unsaturated triglyceride is made by the identification of a stretch between 3000 and 3020 cm-1. In order to judge the efficiency of this FTIR method, the ratio of the peak heights for the =CH and symmetric CH2 were plotted vs. the number of double bonds (Figure 6). The outlying point on the graph is for the tripalmitolein. The deviation is due to the fact that there are 6 fewer carbons present in this species than in each of the other three. After it was determined that the deposition parameters were optimized and gave deposits with good

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spectral resolution, the olive oil sample was injected. Initially an injection of a lower concentration was made to get a clear light scattering trace (upper trace, Figure 7). A full 50 L injection was then made and analyzed on the IR (lower trace, Figure 7). All seven peaks showed triglyceride type IR fingerprints. Figure 8, the spectrum of the third peak, is representative of these spectra. Figure 9 shows spectra for the 3300 - 2500 cm-1 region for all seven peaks. When viewing the 3000 cm-1 region it was evident that there was a varying degree of unsaturation. Peaks 2, 5, and 7 seem to be the most saturated species while all others show some higher degree of unsaturation.

Figure 6. C=C/CH2 Determination by Peak Ratios for the Triglyceride Standards.

Figure 7. Olive Oil Chromatography: light scattering detector and the Gram-Schmidt reconstruction IR chromatogram.

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Figure 8. Olive oil: spectrum of third eluted peak.

Figure 9. Olive oil: C-H stretch region spectra of 7 peaks.

CONCLUSIONS
The study presented here adequately demonstrated the ability of this LC/FTIR method to characterize the degree of unsaturation/saturation in a natural oil. The ability of this LC-IR method to effectively analyze the triglycerides in olive oil shows that this could be a powerful technique for the determination of fats in a variety of food products. Conventional LC methods can be used and the IR analysis performed very quickly thereafter. The structural information necessary to make conclusions on the nature of the oil can be easily obtained without additional sample preparation methods. Contributing Authors Sheri L. Jordan, Larry T. Taylor, Department of Chemistry, Virginia Polytechnic Institute and State
1Arnold,

R.G., and Hartung, T.E., J. Food Sci., V36, pp166-168, (1971). 2Afran, A., and Newbery, J.E., Spec. Int., V3, pp39-42, (1991).

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