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Application of Transmission FTApplication of Transmission FT--IR IR Spectroscopy for the Spectroscopy for the transtrans Fat Fat

Determination in Edible OilsDetermination in Edible Oils

M.I. Bhanger & S.T.H. SheraziM.I. Bhanger & S.T.H. SheraziNational Center of Excellence in Analytical National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, Chemistry, University of Sindh, Jamshoro,

PakistanPakistanwww.ceacsu.edu.pkwww.ceacsu.edu.pk

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Fats and oilsFats and oils

Edible oils are composed of triglycerides, which are the ester of one molecule of glycerol and three molecules of fatty acids

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transtrans Fatty AcidsFatty AcidsAccording to Food and Drug Administration (FDA), trans FA are defined as the sum of all unsaturated fatty acids that contain one or more isolated (i.e. non-conjugated) double bonds in a trans configuration.

(trans FA present in foods / dietary supplements, mandatory for nutritional label since January 2006)

TransTrans--Unsaturated ChainUnsaturated ChainCisCis--Unsaturated ChainUnsaturated Chain

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Health risks ofHealth risks of trans trans FAFA

Numerous research and clinical studies determine the impact of trans fatty acids on cholesterol levels and coronary heart diseases (CHD). (2 % - 23% risk)

A correlation exist between the intake of trans isomers and LDL-cholesterol increase and HDL cholesterol decrease.

Some studies also indicated that TFA raise lipoprotein level, an independent inherited factor of coronary heart diseases.

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Analytical MethodsAnalytical Methods

� Gas chromatography

� 13C NMR spectroscopy

� Infrared spectroscopy

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At NCEACAt NCEACwww.ceacsu.edu.pkwww.ceacsu.edu.pk

� Focus on the application of FTIR spectroscopy to food and biological matrices.

� Development of rapid instrumental methods for the quantitative analysis of edible oils (FFA, tFA, PV, FAC) by FTIR spectroscopy

� The rich functional group information available in the mid-infrared portion of the oil spectrum provide a basis for developing a method.

� The challenge has been to take a traditionally qualitative procedure quantitative

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FTFT--IR spectrum of edible oilIR spectrum of edible oil

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MEASURE FUNCTIONAL GROUP(S)

CHEMICAL METHOD

DEGREE OF UNSATURATION

C=C IODINE VALUE

TYPE OF UNSATURATION

cis-C=C, trans-C=C TRANS ANALYSISFATTY ACID PROFILE (GC)

CHAIN LENGTH CH SAPONIFICATION NUMBER

HYDROPEROXIDES OOH PEROXIDE VALUE

CARBONYL COMPOUNDS

HC=O ANISIDINE VALUE

CARBOXYLIC ACIDS COOH FREE FATTY ACIDS

HYDROXYL GROUPS (MONO/DIGLYCERIDES)

OH HYDROXYL NUMBER

MOISTURE OH MOISTURE CONTENT

SOLIDS CONTENT CH, C=C, O-C=O SOLID FAT INDEX

IR QUALITY FACTOR INFORMATIONIR QUALITY FACTOR INFORMATION

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FTFT--IR spectrometersIR spectrometersMany advantages over traditional dispersive instruments :

� complete scan in seconds.

� multiple scans provide high S/N ratio by signal averaging.

� Thus recent advances have made possible to determine not only trans FA but also its FA composition without any chemical treatment.

� Result � FT-IR spectroscopy now more amenable to development as a quantitative tool.

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Determination of IsolatedDetermination of Isolated transtrans Isomers Isomers by Infrared (IR) Spectroscopyby Infrared (IR) Spectroscopy

Method adopted by the American Oil Chemists� Society (AOCS) and AOAC.

Based on the CH out-of-plane deformation band at 966 cm-1 � a unique characteristic of isolated trans isolated double bond.

Widely employed method, particularly in the analysis of hydrogenated oils in industries.

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Factors Limiting Accuracy of Factors Limiting Accuracy of Traditional AOCS MethodTraditional AOCS Method

All triglycerides exhibit a weak absorption band that underlie the trans absorption band

Intensity of these underlying absorptions varies with triglyceride composition of the oil; can contribute 3-5 percent to the measured trans values

Baseline variability due to intense triacylglycerol absorptions in proximity to the isolated trans band near 1000 cm-1

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Soybean oil2

Ab

s

HydrogenatedSoybean oil

2

Ab

s

Wavenumbers (cm-1)

trans

10004000 3000 2000

1

1

966 cm-1

In IR spectroscopy the high characteristic trans absorption at 966 cm occurs on an elevated and sloping baseline due to acylTG as seen here. Thus measurement of its height and area becomes less accurate especially when trans level goes < 2 %.

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Recent modes of FTIR Recent modes of FTIR spectroscopy spectroscopy

� Internal reflection or ATR

� Transmission FTIR

� 2 D procedures

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Internal reflection (ATR) infrared spectroscopy

Limitations

SensitivityLess sensitivity for trace level tFA due to short path length (~ 4 um)

Accuracy

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� Fats and oils : High viscosity

The limitation of sensitivity / accuracy was overcome by :

� Transmission cell - 200 um path-length

� Diluting the oil/fat with odorless mineral spirits (OMS) for transcontent (< 1%).

Transmission FTIR � an accurate quantitative method for the low level of determination of

trans FA.

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Present WorkPresent Work

In the present study, using transmission FT-IR we have developed a long range of calibration (0.01~37.87%) for the determination of low to high transvalues in a series of industrially hydrogenated and deodorized oils.

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FTFT--IR Spectral measurementsIR Spectral measurements

Trielaidine (t-elad.) standards and partially hydrogenated oil are solids at room temperature. The viscosity is reduced by adding OMS in ratio 1:2.

Standards are prepared by spiking the trielaidine in canola oil for Standards are prepared by spiking the trielaidine in canola oil for measuring measuring transtrans band at band at 967 967 cmcm--1 1 ..

Prior to the loading of 200 µm KCl cell, all standards and samples were heated to 50 0C to avoid any crystallization during the analysis.

The transmission FT-IR spectra of all standards and hydrogenated samples were recorded under the same parameters

The FT-IR spectra of before deodorization and hydrogenation were subtracted from sample spectra that were recorded after deodorization and hydrogenation process to obtain accurate results.

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The absorbance of The absorbance of transtrans band at band at 967 967 cmcm--1 1 of prepared of prepared standards spiking the trielaidine in canola oil ranging standards spiking the trielaidine in canola oil ranging

from from 00..011 011 to to 3737..874 874 %.%.

23B_Corr(37.874%TRANS)22B_Corr(23.980%TRANS)21B_Corr(21.252%TRANS)20B_Corr16.436%TRANS)19B_Corr(14.666%TRANS)18B_Corr(13.300%TRANS)17B_Corr(10.442%TRANS)16B_Corr(7.821% TRANS)15B_Corr(4.751%TRANS)14B_Corr(3.526%TRANS)13B_Corr(1.268%TRANS)12B_Corr(O.938%TRANS)11B_Corr(0.677%TRANS)10B_Corr(0.534%TRANS)9B_Corr(O.352%TRRANS)8B_Corr(0.17%TRANS)7B_Corr(0.14%)6B_Corr(0.11%)5B_Corr(0.089%)4B_Corr(0.055%)3B_Corr(0.041%)2B_Corr(0.026%)1B_Corr(0.011)PURE CANOLA B_Corr(0.000%TRANS)

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

Ab

sorb

ance

960 980 1000 Wavenumbers (cm-1)

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Plot ofPlot of transtrans peak area versus addedpeak area versus addedtranstrans to canola oilto canola oil

0 5 10 15 200

5

10

15

20

25

30

35

40

Linear Regression for Data1_Trans:Y = A + B * X

Parameter Value Error------------------------------------------------------------A -0.15776 0.05362B 1.84724 0.00817------------------------------------------------------------

R SD N P------------------------------------------------------------0.99979 0.20926 23 <0.0001------------------------------------------------------------

Per

cen

tag

e o

f ad

ded

Tra

ns

Trans Peak Area 990-945cm-1

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TQ Analyst calibration of the TQ Analyst calibration of the transtrans standards standards

(trielaidine added in canola oil)(trielaidine added in canola oil)

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0.0 0.2 0.4 0.6 0.8

0

5

10

15

20

25

30

%T

rans

Area 945-990 cm-1

A SB-ATR

0 4 8 12 16

0

5

10

15

20

25

30

%T

rans

Area 945-990 cm-1

B SR-Transmission

Sherazi et al. Talanta 80, 600-606, 2009

Comparison of FTIR responses of two techniques to increasing level of trans hydrogenated oil added to pure canola oil

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0.00 0.02 0.04 0.06

0.0

0.3

0.6

0.9

1.2

1.5

% T

rans

Add

ed

Area 945-990 cm-1

Almond Grapeseed Safflower Sesame SunflowerA

A

0.0 0.2 0.4 0.6

0.0

0.3

0.6

0.9

1.2

1.5

% T

rans

Add

ed

Area 945-990 cm-1

Almond Grapeseed Safflower Sesame Sunflower

B

SB- ATR T - SR

SB-ATR (A) and T-SR (B) calibration plots obtained from the spectra of five different oils spiked with trielaidin (0 to ~1.2% w/w) ratioed against the spectrum of the unspiked oil. (lower regression SD, more accurate det.)

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transtrans fatty acids (%) in partially hydrogenated fatty acids (%) in partially hydrogenated oils and pure cooking oils by GC and FToils and pure cooking oils by GC and FT--IRIR

Samples GC FT-IR

1. PHO-1 9.12 ± 0.23 8.06 ± 0.022. PHO-2 26.51 ± 0.55 25.74 ± 0.053. PHO-3 10.72 ± 0.64 9.61 ± 0.034. PHO-4 10.69 ± 0.22 8.58 ± 0.015. PHO-5 16.32 ± 0.44 15.17 ± 0.046. PHO-6 12.55 ± 0.41 12.21 ± 0.047. PHO-7 20.21 ± 0.61 19.19 ± 0.03

8. CO-8 0.52 ± 0.05 0.60 ± 0.019. CO-9 0.45 ± 0.16 0.51 ± 0.0110. CO-10 0.52 ± 0.12 0.46 ± 0.0111. CO-11 1.33 ± 0.21 1.76 ± 0.0112. CO-12 1.63 ± 0.23 1.83 ± 0.0113. CO-13 1.17 ± 0.16 0.83 ± 0.0114. CO-14 1.14 ± 0.23 1.65 ± 0.01

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ConclusionConclusion

FTIR spectroscopy, in conjunction with :

developments in sample-handling techniques, chemometrics and detailed functional-group information provided by mid-IR region

is exploited to develop instrumental methods for trans Fat in edible oils & fats at very low level.

Accuracy and sensitivity limitations of the FTIR official methods are overcome using transmission FTIR procedure that is currently being validated.

The results of transmission FT-IR spectroscopy were found in good agreement with the GC results and have shown better sensitivity for low trans (< 1 %) values in the analyzed edible oil samples.

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