INTRODUCTION:

Glycerides:

Fatty acids often occur as esters of the trihydric alcohol, glycerol. The fully esterified triglycerides are typical of plant seed-oils, the properties of which depend on the fatty acids present, their relative amounts and their position in the molecule. If one of the acids is a hydroxy acid, further acylation of this hydroxy group can give rise to a tetra -acid triglyceride, but this occurs only rarely.Triglycerides are used as a source of energy, they also provide flavour and palatability. Naturally occurring unsaturated fatty acids are predominantly in cis form and their double bonds are not conjugated [1]. Triglycerides rarely exist in glycerol ester of single fatty acid but contain mixed fatty acids. Food triglycerides have fatty acid carbon chain length ranging from (4 to 24) [2]. Spectroscopic methods are very important in determining the structure of triglycerides. This review helps in understanding how various spectroscopic methods help in establishing the structure of triglycerides.

Triglyceride

Spectral Techniques:

1. IR Spectra:

Triglycerides are transparent to UV-visible region but possess many infrared absorption bands that are characteristic of their general structure. Most long chain triglycerides have very similar infrared spectra, although the presence of functional groups in the fatty acid chain will add additional bands. Infra red spectroscopy is a good method for quantitating total triglyceride. The strong band at 1742 cm^-1 due to carbonyl stretching can be used to measure total moles of triglyceride, provided no other ester molecule is present. Neither fatty acid chain length nor degree of unsaturation significantly affects absorptivity at 1742 cm^-1. Acetotriglycerides have a prominent band at 1235 cm^-1 due to the acetate group [3]. Saturated triglycerides of different chain lengths can be distinguished by measuring the ratio of the C-H (2926 cm^-1) and the ester carbonyl (1742 cm^-1) absorption bands after suitable calibration. Band at 967 cm^-1is characteristic of isolated transdouble bond.

2. Mass Spectra:

A mass spectrum is very important in the identification of triglycerides.Molecular weights up to C₆₆ can be examined using mass spectra.Pure triglycerides can be identified from their degradation pattern. The presence of certain fragment ions is very helpful in identifying the triglyceride. Triglycerides are characterized by the presence of following type of fragment ions:

i) Acyl ion [RCO]⁺, Loss of acyloxy ion [RCOO]⁺, Loss of acyloxymethyl ion [RCOOCH₂]⁺

ii) Mass spectra of glycerides also shows characteristics peaks for [RCO +74]⁺ and [RCO + 128]⁺ ions[4].

iii)

[RCO + 128]⁺

[RCO +74]⁺

In a glyceride molecule, the fatty acid composition can be easily deduced from the large (M-RCOO)⁺ and (RCO)⁺peaks. In the mass spectrum of 2-stearo-1, 3-myristopalmitin (b-MStP), there are three peaks at 523, 551 and 579 (m/z) corresponding to the loss of three different acyloxy groups from the molecular ion (806). The three (RCO)⁺ peaks at 211, 239 and 267 confirm this identification. The fatty acid esterified at the 2-position can be determined since it produces little or no (M-RCOOCH₂)⁺ fragmentation compared with the acids at 1- and 3- positions. Thus comparison of the 509,537 and 565 peaks from b-MStP shows 509 to be much smaller than 537 and 565, indicating 18:0 is esterified at the secondary hydroxyl.

In case of diglyceride, 1,3-diglyceride isomer is readily distinguished by the appearance of a prominent (M-RCOOCH₂)⁺fragment while 1,2-diglyceride are characterised by the preferential loss of the acyloxy group from the 2-position[3].

Mass Spectra of 1-Palmitoyl-2-linoleoyl-3-oleoyl—glycerol[5]

M⁺ = 856; M-18 = 838

[M - R₁COO]⁺ = 601; [M – R₂COO ]⁺ = 577; [M – R₃COO ]⁺ = 575

[M - R₁COOCH₂ ]⁺ = 587; [M – R₂COOCH₂ ]⁺ = 563; [M – R₃COOCH₂ ]⁺ = 561

[R₁CO + 128]⁺ = 367; [R₂CO + 128]⁺ = 391; [R₃CO + 128]⁺ = 393

[R₁CO + 74]⁺ = 313; [R₂CO + 74]⁺ = 337; [R₃CO + 74]⁺ = 339

[R₁CO]⁺ = 239; [R₂CO]⁺ = 263; [R₃CO]⁺ = 265

NMR Spectra:

The nuclear magnetic resonance spectra of trigycerides exhibit characteristic peaks, each produced by protons of different character. Differences between the NMR spectra of individual triglyceride are strictly a function of fatty acid composition (double bonds, chain length, hydroxyl groups, etc.). Acetotriglycerides are clearly distinguished by a sharp signal from the OOCCH₃ protons at d 2.05. NMR can also distinguish between the various crystalline forms of triglycerides. It is possible to determine the total glyceride content of a sample by comparing the area of the d 4.20 NMR signals (glyceride-CH₂O-protons) with suitable calibration standard. Signals at d 5.2 – 5.4 corresponds to olefinic protons of unsaturated fatty acid and H-2 proton of glycerol. Remaining protons of glycerol (H-1 and H-3) resonate at d 4.1 – 4.3 as two sets of double doublets (J = 6Hz). Peak area in the region d 5.2 – 5.4 is helpful in determining the degree of unsaturation. Allylic protons of unsaturated fatty acids exhibit peak at d 2.05 as multiplet¹.Total unsaturation can also be determined by compairing peaks of allylicmethylenes (d 2.05) and terminal methyls (d 0.8 – 1.0). Polyunsaturated fatty acids shows bisallylic peak at ~ d 2.7 arising from methylene protons sadwitched between two double bonds. Terminal methyl group exhibit peaks near d 0.9, which is inturn depends upon its nearness to double bonds [2,6,7].

¹H NMR of compound 1,2,3-Propanetriyl tri-(E)-9-octadec-enoate showed a triplet at d 0.89, representing nine protons, which was due to terminal methyl groups. A signal at d 1.29, integrating to sixty protons, representing twenty-six methylene groups. A multiplet at d 1.60, representing six protons, was due to methylene group b to carbonyl group. Signals at d 2.01 and d 2.32, representing twelve and six protons, were due to methylene groups adjacent to double bond and carbonyl group respectively. A multiplet in the range d 4.00 - 4.43, integrating to four protons, was due to methylene groups of glycerol molecule. A multiplet in range d 5.15 - 5.55, integrating to seven protons, was due to olefinic protons and methine group of glycerol [8].

1,2,3-Propanetriyl tri-(E)-9-octadec-enoate

Comparison of peaks of mono, di and tri glycerides[9]

Monoglyceride:

H_a = d 4.18 – 4.25; H_b= d 3.97, H_c = d 3.64 & 3.73, H_d,e,f = d 2.4; H_h = d 1.25; H_g = d 1.64;

H_i = d 0.88

Diglyceride:

H_a = d4.20; H_b= d4.13, H_c = d2.48 ,H_d, = d 2.4; H_e= d 1.64; H_f = d 1.25; H_g = d0.88

Triglyceride:

H_a = d 4.17 & 4.33 (dd); H_b= d 5.3 (m), H_c, = d 2.35; H_d= d 1.64; H_e = d 1.25; H_f= d 0.88

Comparison of signal of glyceride moiety at H^b, distinction between mono-, di-and triglycerides can be made easily. H^b signal of tri, -di- and monoglycerides appears at d 5.13, d 4.13 and d 3.97 respectively, showing that as more and more hydroxyl group of glycerol moiety get esterified with fatty acid, signal for H^b shifts downfield.

CONCLUSION:

Spectral techniques are very important in determining the structure of the molecules. Careful study of the spectra helps in precise determination of structures. Mass spectra and NMR spectra are found to be very important in establishing the structure of glycerides. With the help of NMR and mass spectra distinction between mono, di and triglycerides can be done easily.

REFERENCES:

1. Shiao TY and Shiao MS. Determination of fatty acid composition of triglycerides by high resolution NMR spectroscopy. Botanical Bulletinof Academia Sinica. 1989; 30(3): 191-199.

2. Gerdova A, Defernez M, Jakes W, Limer E, McCallum C, Nott K, Parker T, Rigby N, Sagidullin A, Watson AD, Williamson D and Kemsley EK. Quantitative NMR. Magnetic Resonance in Food Science: Defining Food by Magnetic Resonance. Royal Society of Chemistry. 2015; 17- 30.

3. LitchfieldC. Analysis of Triglycerides. Academic Press, New York and London. 1972. pp. 206-224.

4. Kinghorn AD, Falk H and Kobayashi J. Progress in the Chemistry of Organic Natural Products 100. Springer. 2014.

5. Miyazawa T, TazawaH andFujino Y.Molecular Species of Triglyceride in Rice Bran. Cereal Chemistry. 1978; 55(2): 138-145.

6. Salinero C, Feas X, Mansilla JP, Seijas JA, Tato MPV, Vela P and Sainz MJ.¹H Nuclear Magnetic Resonance Analysis of triglycerides composition of cold-pressed oil from Camellia japonica. Molecules. 2012; 17: 6716-6727.

7. KnotheG and Kenar JA. Determination of the fatty acid profile by ¹H-NMR spectroscopy. European Journal of Lipid Science and Technology. 2004; 106: 88 – 96.

8. Sasaki S. Handbook of Proton-NMR Spectra and Data. Vol. 10. Academic Press, Tokyo. 1986; p. 401.

9. Lie Ken JieMSF and Lam CC.¹H-Nuclear magnetic resonance spectroscopic studies of saturated, acetylenic and ethylenictriacylglycerols. Chemistryand Physics of Lipids.1995; 77: 155-171.

Received on 04.09.2017 Modified on 20.09.2017

Asian J. Research Chem. 2017; 10(5): 708-710.

DOI: 10.5958/0974-4150.2017.00120.1