Analysis of Triglycerides

8/2/2019 Analysis of Triglycerides

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Analysis of Triglycerides via LC and FTIUsing the LC-Transform InterfaceAN-19

NTRODUCTION:

iglycerides are esters of various fatty acids with the trihydric alcohol, glycerol. This work deals wi

e measurement of unsaturation in materials which contain fatty acids notably, vegetable oils.easurement of the degree of unsaturation is particularly important because organoleptic and texture

operties of foods such as baked goods are influenced by the degree of unsaturation. Stability in coo

ocess and rancidity are also influenced by unsaturation. Finally, recent health issues associated witturated fats have assumed a high level of importance.

frared spectroscopy has numerous applications in the analysis of fats and oils. This includes the

termination of unsaturation, measurement of hydrogenation, and measurement of cis/trans content

ouble bonds. Oxidation and/or polymerization during cooking has been measured by infrared metho

nalysis of the various natural triglyceride fats and oils shows that they are mixtures of various fatty

ters, rather than simple mono-acid esters. Traditionally measurements of unsaturation have employet chemical methods such as saponification and iodination of the double bonds. Infrared techniques

ve been shown to provide fast and labor efficient determinations of unsaturation. Arnold and Hartu

1971, demonstrated a good correlation of iodine value and infrared absorbance ratios for a broad r

vegetable oils. Afran and Newbery2 in 1991extended the approach using FTIR combined with AT

this work, using mixtures of triglycerides, Afran and Newbery demonstrated a direct correlationtween C=C/C-C stretch ratio and the number of double bonds in triglycerides and natural oils.

y use of a chilled sample stage, De Ruig3 demonstrated that spectra of the crystalline forms of

glycerides yielded information about the chain lengh of the fatty acid moiety, isomeric forms, cis-r

aracterization, 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.

hile these measurements demonstrate the utility of FTIR in the determination of unsaturation, they

ot address the complex mixture of triglycerides that characterize the natural oil.

quid chromatography is often the separation of choice for many food analyses because many of theixed materials are relatively non volatile or thermally labile and are; therefore, difficult to analyze

ing gas chromatography. Liquid chromatography does an adequate job of separating many of thesebstances although the use of spectroscopy allows one to gain additional structural information. Wit

ditional sample preparation, retention times are often not enough for identification due to the numb

components in many of the food matrices.

frared spectroscopy is especially useful in this area because, it allows for the differentiation betwee

turated and unsaturated fats via observation of the C=CH absorbance region at 3000 cm-1. The deg

saturation/unsaturation can also be determined for unknowns by generating a response curve for

andards based on the total carbon number versus the ratio of the intensities of the antisymmetric CHd ester C=O peaks. Similar response curves can be generated from other peak ratios as well. FTIR

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rther advantages in that the technique can identify free fatty acids and fatty acid esters in a complex

atrix. The following is a brief study on triglycerides using the LC-Transform as the interface betwe

quid chromatography and IR spectroscopy. Based on the aforementioned reasons this hyphenated

chnique can serve as a powerful analytical tool.

EXPERIMENTAL

iglyceride standards were used initially to determine the feasibility of depositing the analytes onto sk as well as optimizing the deposition conditions. A standard mixture containing 1 mg/mL each of

glycerides listed in Table 1 was prepared in a solution of 50/50 acetonitrile and acetone. The soluti

as separated on a Waters HPLC system with the eluent running directly into the flow divider on a

odel 102 LC-Transform. A secondary detector (light scattering - Alltech Varex) was connected to tutput of the splitter in order to make a comparison with the IR reconstruction. Following the

romatographic run the germanium disk on which the analytes were deposited was transferred to the

ptics in the sample compartment of a Nicolet Magna Series FTIR system. The IR run was collected

ing OMNIC Series software. A Gram-Schmidt reconstruction was automatically generated and the

dividual spectrum for each peak was then acquired. Following the analysis of the standard mixture

lution of 1 mg/mL olive oil was analyzed under the same conditions.

XPERIMENTAL CONDITIONS

Chromatography (Waters 600-MS)q

Column: 250 x 4.6 mm Hypersil - ODS (Keystone Scientific)q

Mobile Phase: 50/50 ACN/Acetone for 16 min.

Linear ramp to 10/90 in 24 min.q

Column Flow: 1 mL/minq

Injection Volume: 50 Lq

Concentration: 1 mg/mLq

ight Scattering (Varian)

Drift tube: 85Cq

Gas Flow: 2.00 SLPMq

C-Transform Model 102 (Lab Connections)Sheath Temperature: 55Cq

Sheath Gas Flow: 17q

Nebulizer Flow: 0q

Nozzle Height: 5 mmq

Disc Rotation Rate: 10/minq

Flow to Disk: 30 Lq

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Magna 550 Spectrometer (Nicolet)

Detector: DTGSq

Scan Velocity: 0.6329q

Scan Number: 8 scans per Spectrumq

Sampling Interval: 8.12 sec.q

Resolution: 8 cm-1q

Disc Rotation: 10/minq

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

Results

he standard solution was injected under the conditions listed previously. The light scattering trace i

ven 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.

ll four components look very similar because they are all unsaturated compounds. The areas of spemportance include the ester C-O stretch at 1161 cm-1, symmetric CH2 deformation at 1462 cm-1, an

e ester C=O stretch at 1745 cm-1.

Figure 5. C-H Stretch Region of Tripalmitolein.

gure 5 shows an expanded x-scaling of the 3300 - 2500 cm-1 region of tripalmitolein. The CH2

metric stretch region is identified at 2855 cm-1

and the CH2 antisymetric stretech region at 2925 cmhe antisymmetric CH3 stretch can be seen as a shoulder on the 2925 region. The determination of a

nsaturated triglyceride is made by the identification of a stretch between 3000 and 3020 cm-1. In or

judge the efficiency of this FTIR method, the ratio of the peak heights for the =CH and symmetric

H2 were plotted vs. the number of double bonds (Figure 6). The outlying point on the graph is for t

palmitolein. The deviation is due to the fact that there are 6 fewer carbons present in this species th

each of the other three.

fter it was determined that the deposition parameters were optimized and gave deposits with good

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ectral resolution, the olive oil sample was injected. Initially an injection of a lower concentration w

ade to get a clear light scattering trace (upper trace, Figure 7). A full 50 L injection was then mad

d analyzed on the IR (lower trace, Figure 7).

ll seven peaks showed triglyceride type IR fingerprints. Figure 8, the spectrum of the third peak, is

presentative of these spectra.

gure 9 shows spectra for the 3300 - 2500 cm-1 region for all seven peaks. When viewing the 3000

m-1 region it was evident that there was a varying degree of unsaturation. Peaks 2, 5, and 7 seem to

e 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

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

he study presented here adequately demonstrated the ability of this LC/FTIR method to characteriz

gree of unsaturation/saturation in a natural oil. The ability of this LC-IR method to effectively anal

e triglycerides in olive oil shows that this could be a powerful technique for the determination of fa

variety of food products. Conventional LC methods can be used and the IR analysis performed veryuickly thereafter. The structural information necessary to make conclusions on the nature of the oil

easily obtained without additional sample preparation methods.

ontributing Authors Sheri L. Jordan, Larry T. Taylor, Department of Chemistry, Virginia Polytech

stitute and State

Arnold, R.G., and Hartung, T.E., J. Food Sci., V36, pp166-168, (1971).

Afran, A., and Newbery, J.E., Spec. Int., V3, pp39-42, (1991).

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Analysis of Triglycerides

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