Chem.

Educator 2001, 6, 7–9 7

Determination of the Iodine Value of Selected Oils: An Experiment

Combining FTIR Spectroscopy with Iodometric Titrations

E. Eugene Gooch

Department of Chemistry, Elon College, Elon College, NC 27244, [email protected]

Received July 14, 2000. Accepted October 17, 2000

Abstract: An experiment has been developed that combines the FTIR analysis of oils and fats with an
iodometric titration. While it is most appropriate for a sophomore or junior laboratory for majors, it also makes a
suitable group project or demonstration for nonscience majors. Integration of the experiment into an upper-level
biochemistry laboratory is described.

In recent years people have become increasingly aware of sample is determined by separate methods to be 825, a similar
the effects of diet on health. It is common knowledge that the calculation indicates the average number of double bonds
average person’s diet contains too high a percentage of fats, present per molecule:
especially saturated fats. This increases the risk factors for
cardiovascular disease. Lowering the amount of dietary 135 g I 2 × 825 g oil × 1 mol I 2 × 1 mol C=C
cholesterol and saturated fats has become a goal for more and =
100 g oil × 1 "mol" oil × 254 g I 2 × 1 mol I 2
more people, including a significant number of college
students. In science courses, students show substantial interest
in experiments involving the isolation and analysis of fats. It is 4.38 mol C=C per mole of oil or 4.38 C=C per molecule
appropriate to introduce into the undergraduate laboratory
curriculum analytical methods that can distinguish saturated Because elemental iodine is not sufficiently reactive with
from unsaturated fats. carbon–carbon double bonds, determination of iodine value is
typically done via the method developed by Wijs [4]. A
Background weighed sample of oil is dissolved in an organic solvent and a
measured amount of ICl is added. The solution is allowed to
Fats and oils are more correctly called triglycerides [1]. stand in the dark for 30 min. The excess ICl is converted to I2
Triglycerides contain three fatty acids esterified with glycerol. by addition of KI. The concentration of I2 is determined by
A representative structure is shown in Figure 1, which shows titration with standardized sodium thiosulfate. Synthesis of
one stearoyl and two palmitoyl groups esterified with glycerol Wijs reagent involves handling gaseous Cl2 [5], and many
to form a saturated triglyceride. In contrast, unsaturated chemists will wish to avoid this. An alternative reagent,
triglycerides contain a larger proportion of unsaturated fatty developed by Hanus, utilizes a solution of equimolar I2 and Br2
acids. The second structure in Figure 1 shows two molecules in glacial acetic acid [6]. The Hanus reagent gives iodine
of oleic acid and one molecule of linoleic acid esterified with values that are comparable to the Wijs reagent, but the Hanus
glycerol. reagent is easier to prepare and more stable during storage.
The degree of unsaturation of a triglyceride is expressed as Strong and Koch have developed an undergraduate laboratory
its iodine value (IV) [2], which is defined as the number of based on the Hanus method [7].
grams of iodine absorbed by one hundred grams of fat. This
definition exploits the ability of carbon–carbon double bonds
Instrumental Methods
to undergo addition of halogens. Consider the example
triglyceride structures in Figure 1. The first structure has no Instrumental methods for the determination of iodine values
double bonds, so its iodine value is zero. The second structure are well documented. Arnold used transmission IR to analyze
can add four moles of iodine per mole (882 g) of triglyceride. 25 different fats and oils with iodine values ranging from 31 to
Calculation of its iodine value is a stoichiometric exercise that 142 [8]. A transmission spectrum of a 10% solution of oil in
any freshman can do. CCl4 was run, and the olefinic absorbance was divided by the
aliphatic absorbance to produce a ratio. This ratio was found to
4 × 254 g I 2 × 100 g triglyceride be proportional to the iodine value. More recently, van der
iodine value = = 115
882 g triglyceride Voort’s group used FTIR to determine both iodine values and
saponification numbers (SN) [9]. This method used attenuated
Saturated fats and some tropical oils typically have iodine total reflectance (ATR) sampling, and a computer program was
values from 35 to 70, vegetable oils range from 80 to 150, and written to integrate sample handling, spectrum acquisition, and
more highly unsaturated oils have values from 160 to 195 [3]. numerical analysis of results.
Consider a sample of vegetable oil with an iodine value of Although ATR is the more modern technique, we choose
135. If the average molecular weight of triglycerides in the oil transmission IR for this undergraduate experiment. ATR

© 2001 Springer-Verlag New York, Inc., S1430-4171(01)01438-8, Published on Web 11/30/2000, 10.1007/s00897000438a, 610004eg.pdf
8 Chem. Educator, Vol. 6, No. 1, 2001 Gooch

O
O yielded the formula below for iodine value; the formula from
H3C (CH2 )16
C
O CH2
C CH3 Arnold’s group is provided for comparison [8]
H2C O (CH2) 7 (CH2 )7
O O

C CH CH C CH3 IV = 740 (Aolefin/Aalkane ) – 2

H3C (CH2) 14 O O (CH2) 7 CH2 (CH2 )4
O O
CH2
H2C C CH3 R = 0.91
C (CH2) 7
O (CH2) 7
H3C (CH2) 14 O

IV = 495 (Aolefin/Aalkane ) – 31
Figure 1. A saturated fat (left) and an unsaturated oil.
R = 0.98 (ref 8)
Table 1. Typical Iodine Values for Selected Vegetable Oils
Oil Analyized Iodine values determined by
where Aolefin is the absorbance of the olefinic (alkene) C–H
IR Hanus method
stretch above 3000 cm–1 and Aalkane is the absorbance of the
saturated (alkane) C–H stretch near 2950 cm–1.
Almond 117 + 5 97 + 6
Corn 150 + 18 112 + 6
Olive 86 + 19 93 + 10 Experimental Procedure

Hanus solution is a corrosive mixture of iodine and bromine in

analysis required 128 scans versus only 4 scans for concentrated acetic acid. It should be kept in the hood and dispensed
transmission spectra; fewer samples could be analyzed by ATR from an automatic-delivery pipet if possible. Spills should be
in a single laboratory period. Also, switching between neutralized immediately with 10% sodium bisulfite solution. Carbon
transmission and ATR accessories involves significant effort, tetrachloride is a suspected carcinogen and should be handled only in
and optical alignment changes in the sample compartment can small amounts under the hood. Analytical reagents used were
make it difficult to obtain reproducible spectra. Still, the effort purchased from Fisher Scientific or Aldrich Chemical and were of
(and the expense of the ATR accessory) should be justified if reagent grade purity. Methyl ester standards were purchased from
Sigma Chemical, refrigerated during storage, and used without further
both IV and SN values are to be determined in a single period.
purification. Vegetable oils were obtained from local retail outlets or
existing chemical stock. FTIR analysis was performed on a Nicolet
Results Magna 550 optical bench; spectra were acquired, annotated, and
analyzed using OMNIC version 3.0 software.
We have combined the estimation of iodine values via FTIR For FTIR analysis, about 50 mg (recorded to 0.001 g) of oil was
with the classical iodometric determination described in weighed into a clean, dry vial. Sufficient CCl4 was added to bring the
reference 7. The experiment provides a straightforward mass of the solution to 10 to 20 times the mass of the sample [10].
introduction to sampling and transmission IR spectroscopy. It The vial was capped and the solution swirled until the oil dissolved
connects general, analytical, and organic chemistry via and the solution was drawn into a 1-mL Luer-tip glass syringe. The
identification of simple (alkene and ester) functional groups syringe was used to fill a Perkin-Elmer demountable IR cell with a
0.5-mm spacer between two 25 mm × 4-mm rectangular NaCl
and stoichiometric calculations. When commercial samples of
windows. A transmission spectrum (4 scans, 4000 to 400 cm-1) was
vegetable oils and fats were provided to the students, a single obtained and stored on disk. The maximum absorbance of any olefinic
period was sufficient for both the Hanus method and FTIR C–H absorption bands (between 3000 and 3050 cm–1) was recorded
analysis as described in the experimental procedure. along with the aliphatic C–H band (between 2900 and 2075 cm–1).
Iodine values obtained by the Hanus method were compared Hanus iodine solution was prepared by dissolving 26.4 g of I2 in
to values predicted from the FTIR analyses. The agreement 1200 mL of glacial acetic acid with warming, then cooling, adding
between Hanus iodine values and IR estimates was good, with 17.1 g Br2 and diluting to 2.000 L in a volumetric flask [7]. Sodium
the more competent students reporting relative errors within thiosulfate (25.00 g) was dissolved in 1.000 L of water and
10%. These results compare favorably with van der Voort’s standardized [11] against KIO3. A 25.00-mL aliquot of Hanus
solution was treated with 20 mL of 3 M HCl and 10 g of solid KI to
results [9].
convert unreacted IBr to I2. The iodine content of the Hanus solution
In a worst-case scenario (Table 1), we ran the experiment was determined by titration with thiosulfate using 2% starch indicator.
during a sophomore organic laboratory where students had not For best results, classical analysis of the oils were done in
yet had qualitative analysis. Magnetic stirring was not used. duplicate. Approximately 250 to 300 mg of oil was weighed into a
Tolerable results were obtained; the larger differences between clean, dry Erlenmeyer flask. The mass was recorded to the nearest
IR and Hanus iodine values were traceable to two artifacts. milligram. The oil was dissolved in 15 to 20 mL of CHCl3 and 20.00
First, titrant is added to a two-phase solution and good mixing to 25.00 mL of Hanus solution was added via volumetric pipette. The
is essential (magnetic stirring is recommended); otherwise, flask was stoppered and allowed to stand in the dark for 45 min. After
iodine remaining in the CHCl3 layer is not immediately reaction, each oil sample was treated with 20 mL of 3 M HCl and 10 g
neutralized by thiosulfate. Second, if the oils have a substantial of solid KI and titrated with thiosulfate. A magnetic stirrer was used
to mix the solutions during the titration; the optimal speed was as high
color, students will overshoot the endpoint when they mistake
as possible without causing splashing. Rapid addition of titrant is
the color for excess iodine. possible early in the titration, as the color of excess iodine fades from
Estimating iodine values from IR absorbances was brown to orange to yellow. Addition of 1 mL of 2% starch indicator
straightforward. A calibration curve was prepared by the just before the endpoint in reached ensures a sharp endpoint. The
instructor using methyl stearate (IV = 0), methyl oleate (IV = relationship below was used to calculate the iodine value from
83), methyl linoleate (IV = 173), and mixtures of 50% methyl titration of the oil (equivalent to the equation in reference 6):
stearate/methyl oleate (IV =41) and 50% methyl oleate/methyl IV = [Vf – Vi] (for the Hanus solution) – [Vf – Vi] (for the Hanus
linoleate (IV = 129) for the standards. Linear regression solution plus lipid) × M × 126.9 m–1, where M is the molarity of

© 2001 Springer-Verlag New York, Inc., S1430-4171(01)01438-8, Published on Web 11/30/2000, 10.1007/s00897000438a, 610004eg.pdf
Determination of the Iodine Value of Selected Oils Chem. Educator, Vol. 6, No. 1, 2001 9

thiosulfate titrant, [Vf – Vi] is the final burette reading minus the initial Solomons, T. W. G. Organic Chemistry, 7th ed.; Wiley & Sons:
burette reading for the iodometric titration, m is the mass in New York, 1999.
milligrams of the oil analyzed, and IV is the iodine value of the fat or 2. Oddly enough, the simple stoichiometry of iodine values is not
oil. covered in commonly used general or organic chemistry textbooks.
3. Lide, D. R., Ed. CRC Handbook of Chemistry and Physics, 74th ed.;
Conclusion CRC Press: Boca Raton, FL, 1994; pp 7–29.
4. Cox, H. F. The Chemical Analysis of Foods, 2nd ed.; P. Blaikiston’s
This experiment is quite adaptable to several undergraduate Son & Co.: Philadelphia, PA, 1938; pp 293–295.
chemistry courses. It would work quite well in a combined 5. Tyree, S. Y., Ed. Inorg Synthesis, Coll. Vol. IX; McGraw-Hill: New
quantitative and instrumental analysis laboratory. Students York, 1967; pp 131–133.
could make and standardize their own thiosulfate solutions, 6. Jacobs, M. B. The Chemical Analysis of Food and Food Products,
perform the iodometric titrations on the oils and finish the 3rd ed.; Van Nostrand: New York, 1958; pp 373–375. Both Wijs
FTIR analysis in four- five hours. and Hanus iodine solutions are now commercially available through
When we originally developed this experiment for our Fisher Scientific Co.
biochemistry laboratory, we ran a three-week sequence of 7. Strong, F. M.; Koch, G. H. Biochemistry Laboratory Manual, 3rd
experiments. During the first week, students isolated lipids ed.; W. C. Brown: Dubuque, IA, 1981; pp 139–146.
from a variety of nuts and grains, then characterized the oils 8. Arnold, R. G.; Hartung, T. E., J. Am. Oil Chem. Soc. 1971, 36, 166–
via TLC [12]. During the second week, students determined 168. Instructors are encouraged to determine a unique calibration
curve for their instrument and sample cell(s).
SN and IV via FTIR; they also saponified their triglycerides
and converted them to fatty-acid methyl esters (FAMEs). A 9. van der Voort, F. R.; Sedman, J.; Emo, G.; Ismail, A. A. J. Am. Oil
Chem. Soc. 1992, 69 (11), 1118–1122.
strong finish to the sequence of experiments resulted during
the third week when students analyzed the FAMEs via GCMS 10. The concentration of lipid in CCl4 should be 5 to 10%. Care should
be taken to ensure that absorption bands fall within the linear
as described by Rubinson [13]. dynamic range of the instrument.
The FTIR estimation of iodine value (as a group experiment
11. Skoog, D. A.; West, D. M.; Holler, F. J. Fundamentals of Analytical
or a demonstration) is an excellent addition either to a basic Chemistry, 6th ed.; Saunders: New York, 1992; pp 861–864.
chemistry laboratory for students majoring outside the sciences
12. Boyer, R. F. Modern Experimental Biochemistry, 3rd ed.; Benjamin
or to a sophomore organic laboratory. We have modified and Cummings: San Francisco, CA, 2000; pp 303–319.
expanded an experiment for nonscience majors from the text 13. Rubinson, J. F.; Neyer-Hilvert, J. J. Chem. Educ. 1997, 74 (9),
by Richardson [14]. In one laboratory period, a sample of meat 1106–1108.
is dehydrated and defatted with organic solvent. The second 14. Richardson, B. C.; Chasteen, T. G. Experience the Extraordinary
period begins with the FTIR analysis of the residual fat, and Chemistry of Ordinary Things, 2nd ed.; Wiley & Sons: New York,
the exercise concludes with an analysis of the students’ results 1995; pp. 149–160, modified as indicated in boldface. A 100-mL
and a discussion of the differences between saturated and round-bottom flask with 19/22 joint is charged with 35 mL of
unsaturated fats. The experiment generated a substantial toluene and ten grams of ground meat. It is fitted with a distillation
amount of enthusiasm among nonscience majors. On several head, thermometer adapter, and condenser and heated under a hood
with an electric mantle. A 25-mL graduate is used as the receiver.
occasions students have decided to change their eating habits The azeotropic distillation is stopped when 25 mL of toluene is
as a result of their observations in this laboratory! collected. The volume of the water layer in the graduate is
determined. The residual toluene is decanted from the pot and
Acknowledgment. Appreciation is expressed to the National evaporated in the hood. The remaining fat is weighed during the next
Science Foundation (NSF-ILI grant # 96-50908), to Elon College for laboratory period. The defatted, dehydrated meat is allowed to dry in
the matching funds, and to the reviewers for their comments. the hood, is weighed, and is assumed to be residual protein and
insoluble material.

References and Notes

1. Information on fats and oils can be found in many texts, for example,
Stryer, L., Biochemistry, 4th ed.; Freeman: New York, 1995 and

© 2001 Springer-Verlag New York, Inc., S1430-4171(01)01438-8, Published on Web 11/30/2000, 10.1007/s00897000438a, 610004eg.pdf