Equations 2 to 6 were used to obtain estimated error for each component is on the accuracy with which overlap-
values for the concentrations of the five 2 to 3%. ping peaks can be separated. In most
components. These values were sub-
stituted in Equation 1. If the values
did not fit, the areas on the right side
of Equations 2 and 3 were readjusted,
keeping the sum the same. New values
were then calculated for [a] to [e] and
substituted in Equation 1. By re-
peating this procedure, five values for
the component concentrations were
obtained, which were consistent with the
six equations. With experience in the
use of the method, only one or two
trials are necessary.
The precision obtained in determining
the composition of a typical 1 to 1
butadiene-isoprene copolymer is shown
in Table II. The error is estimated to
be within 2 to 3%.
No reference polymer was available
for a check on the reliability of the
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
method. Since pure natural rubber con-
sists essentially of cis-1,4-polyisoprene,
known mixtures of natural rubber and
Downloaded via UNIV OF SAO PAULO on March 25, 2019 at 20:58:05 (UTC).
1 to 1 butadiene-isoprene copolymer
were prepared. The composition of
the copolymer was taken to be that
shown in Table II. The range of
mixtures was rather narrow, because of
obvious solubility limitations. The
calculated and experimentally deter-
mined compositions of three independent
mixtures are shown in Table III. The
Determination of Unsaturation and Average
Molecular Weight of Natural Fats by
Nuclear Magnetic Resonance
LEROY F. JOHNSON and JAMES N. SHOOLERY
Instrument Division, Varían Associates, Palo Alto, Calif.
The proton NMR· spectra of tri-
glycerides dissolved in CCI4 are charac-
terized by four sets of signals includ-
ing, respectively, the oleflnic protons,
the four glyceride methylene protons,
methylene groups attached to two
doubly-bonded carbon atoms, and
the remaining protons on saturated
carbon atoms. The area of the signals
produced by the Q and C3 glyceride
protons is measured using an electronic
integrator and a D.C. digital voltmeter.
With this measurement as an internal
standard, one can measure accurately
the number of oleflnic protons and the
total number of hydrogen atoms in like
manner. From these determinations
the average molecular weight can be
calculated. Finally, an iodine number
is calculated using the number of
1136 · ANALYTICAL CHEMISTRY
For quantitative analysis, proper
operation of the spectrometer is crit-
ical. This subject has been discussed
in detail {2-5). If the area of an absorp-
tion peak is to be proportional to the
concentration of protons contributing
to it, and independent of the relaxa-
tion times, rf power saturation must
be avoided. However, the signal has to
be strong enough to ensure accurate
peak area measurements. The rf
level chosen was a practical com-
promise between these two factors.
Signal strength can also be increased
by increasing the solution concentration
or the inside diameter of the sample
tube. For butadiene-isoprene copoly-
mers, solution concentration is limited
by solubility and viscosity. A 5%
solution of copolymer in CCh is barely
pourable. The maximum diameter of
the sample tube is determined by the
design of the probe. The use of non-
spinning tubes of up to 15-mm. outside
diameter is feasible with the insert
manufactured by Varían Associates for
the V-4331A probe.
Recently, the
Wilmad Glass Co., Buena, N. J., has
marketed a plastic spinner for use with
the Varían probe which will take 8-mm.
and 12-mm. tubes.
The accuracy of the method depends
oleflnic protons and the average
molecular weight. The agreement be-
tween the NMR and Wijs iodine num-
bers is remarkably good in all fats
studied except tung oil where con-
jugated bonds cause inaccuracy in the
Wijs method.
investigation of fatty acids and
The
their triglycerides (commonly called
natural fats) using high resolution nu-
clear magnetic resonance (NMR) has
been reported by Hopkins and Bern-
stein (S). Their spectra of natural
fats, taken at 40 Me., showed signals
assignable to oleflnic protons in the fatty-
acid chains, protons in the glyceryl
radical, and other groups of uniquely
distinct proton environments. Area
cases, visual judgment was good enough
for determining the areas to be cut out.
Too great an error in separating the
overlapping peaks produced incon-
sistencies in the six equations or nega-
tive values for some of the components.
The separation of peaks 4 and 5 was
the most critical and this is reflected in
the method of calculation described
previously.
ACKNOWLEDGMENT
The author thanks Harry Greenberg
for supplying the samples of copolymer
and natural rubber for this work.
LITERATURE CITED
(1) Bovey, F. A., Tiers, G. V. D., Filipo-
vich, G., J. Polymer Sci. 38, 73 (1959).
(2) Reilly, C. A., Anal. Chem. 32, 211R
(1960).
(3) Varían Associates, Instrument Div.,
Instruction Manual, V-3521 NMR
Integrator, Publ. 87-100-029.
(4) Varían Associates, "NMR and EPR
Spectroscopy,” Chap. 8, Pergamon
Press, New York, 1960.
(5) Williams, R. B., Ann. N. Y. Acad
Sci. 70, 890 (1958).
Received for review April 16, 1962.
Accepted June 11, 1962. Pittsburgh
Conference on Analytical Chemistry and
Applied Spectroscopy, Pittsburgh, Pa.,
March 1962.
measurements on the first two of these
allowed a calculation of degree of un-
saturation with an accuracy of 5%.
The present work was undertaken to
determine the accuracy of unsaturation
measurements using the latest instru-
mental equipment and techniques.
Measurement of the total number of
hydrogen atoms present in the average
fat molecule as well as the number of
olefinic protons allows a determination
of average molecular weight; this is
used to calculate an iodine number
which is compared to that obtained on
the same sample by the Wijs method.
EXPERIMENTAL
Quantitative information was ob-
tained using a Varían HR-60 spectrom-
eter equipped with a V-3521 NMR
integrator. Integrator output voltage
was measured with a Hewlett-Packard
405AR D.C. digital voltmeter. Re-
corded spectra were obtained with a
Varían A-60 spectrometer using 0.5-ml.
samples containing a trace of tetra-
methylsilane as an internal reference.
Signal positions were obtained directly
from the precalibrated charts with an
accuracy of ± 1 c.p.s.
Matheson Coleman & Bell spectro-
grade CCh used for all solutions was
checked for proton background and
had no detectable signals under condi-
tions used for obtaining quantitative
data. Fats used for this study are com-
mercially available products. One of
the fats, safflower seed oil, was dried
over anhydrous CaS04 for several days
and a subsequent solution produced an
identical determination of unsaturation
compared to that of the oil direct from The output level Total number of protons
the bottle. mately 50 volts. =

PROCEDURE
control was adjusted to attenuate the Z
50-volt charge to approximately 14 (O',
( 1
The spectrum of a typical fat, volts. The voltmeter can be fixed to (Y -

safflower seed oil, 50 vol. % in CC14,
is shown in Figure 1. The sharp
signal at the extreme right side of the
spectrum (high applied field) is from a
trace of tetramethylsilane added to the
solution to serve as an internal reference
standard. The spectrum is divided
into seven groups of signals labelled A
through G. These groups of signals are
assigned as follows: A, hydrogen
directly attached to doubly-bonded
carbon (olefinic protons) and the
methine proton in the glyceryl moiety;
B, the two methylene groups in the
glyceryl moiety [in each group the
individual protons are magnetically
nonequivalent because of hindered rota-
tion of the C—C bond; hence these
signals are characteristic of the AB
portion of an ABX group (5)]; C, CHj
groups attached to two doubly-bonded
carbon atom's; D, the three CH2 groups
alpha to carboxyl; E, CH2 groups
attached to saturated carbon and
doubly bonded carbon; F, CH2 groups
bonded to two saturated carbon atoms;
read 0.00-9.99 volts and still perform
measurements up to about 15 volts.
Fixing the decimal point eliminates
interruptions due to voltmeter scaling
and, in this case, gives a better indica-
tion of the readings to be taken between
A and B and between B and C.
The first voltmeter reading is a
measure of the number of olefinic
protons and the methine proton in the
glyceryl moiety. Because the integral
voltage is cumulative, the second read-
ing includes the signals contributing to
area A as well as those from the four
glycerine methylene protons producing
area B. The integral voltage per
proton is obtained from one fourth of
area B and this number subtracted from
area A leaves a number which is propor-
tional to the number of olefinic protons.
The total number of protons is simply
obtained by dividing the total integral
by the number corresponding to one
fourth of area B.
Given three digital voltmeter read-
A + B, Z total
ings, X —
X)/4
These relationships are close approxi-
mations of the actual values which can
only be obtained by correcting for C13
satellite signals. Carbon-13 is natur-
ally abundant to the extent of 1.108%
and has a nuclear spin of Vs· The spin
coupling constant between C13 and
bonded protons depends upon the
hybridization of the carbon orbitals (6).
Olefinic proton signals have C13 satellite
signals approximately 80 c.p.s. each
side of the C12—H signals, while protons
on saturated carbon atoms have satel-
lites ± ^60 c.p.s. from the main signals.
Consequently, the high field C13 satel-
lites of signals in area A would be
expected to fall in area B and the low
field satellites of B in A. Likewise, the
high field satellites of C are included
with B. In all cases the low field and
the high field satellites each amount to
0.0055% of the corresponding main
band. Corrections for the satellite
signals modify Equations 1, 2, and 3

A, Y = =
to the following:
G, the three terminal CH3 groups. (Figure 1), taken as described, the
The weak shoulder 65 c.p.s. from following relationships apply:
SiMe4 is the low field line of a triplet Area per proton =

signal from CH3CH2CH=CH— as in Area per proton =

(F —

X)/4 (1)
linolenic acid. The other peaks of this [1.0112 (F -
X) -

Number of olefinic protons =

triplet are 58 and 51 c.p.s. from SiMe4. 0.0055 -0.0055(C)]/4 =

Quantitative measure of the amount of X-{Y -

X)/i
w
this grouping is not possible because of (F -

X)/4 0.2528F -
0.2542X -

0.0014(C) (4)
overlapping signals; however, semi-
quantitative information is available
from the height of the peak at 65 c.p.s.
This signal is more apparent in the
spectrum of linseed oil, Figure 2.
Integration of the spectra was ac-
complished using a sweep rate of about
25 c.p.s./sec. and a radio frequency
power level of approximately 100 Mgauss.
Under these conditions no satura-
tion effects are expected even for nuclei
with long relaxation times (8). The
charge on the integrating capacitor
is most conveniently read with a D.C.
digital voltmeter. Readings were taken
midway between areas A and B, between
B and C, and after accumulating the
total integral which occurred about 60
c.p.s. beyond area G. The integrator
input level control -was adjusted to
provide for a total integral of approxi-

VOL 34, NO. 9, AUGUST 1962 · 1137

Number of olefinic protons =

1.00556* 0.0055(7 *) - - -

[1.0112(7 *) 0.0055* - - -

0.0055(C)]/4
[1.011(7 *) 0.0055* 0.0055(01/4
- - -

1.2653* -
0.25837 + 0.0014(0 ,

0.25287 -
0.2542* -

0.0014(0
Total number of protons =

Z
[1.011(7 *) 0.0055* - - -

0.0055(01/4
^/[0.25287 0.2542* 0.0014(0] (6) - -

The quantity C, which depends on

the number of =C—CH¡¡—CH2—C=
groups, can be estimated from the
spectrum in terms of area B—e.g., in
safflower seed oil C is approximately
equal to 1.0 B and hence 1.0(7 *). —

A general formula for natural fats The number of olefinic protons, V, is
may be written in the following manner: simply:
7 2(x + y + e) =
(9)
CHj0C0(CHj)o(CH=CH)iCH8
Equation 8 may be solved for (a +
CH—OCO(CH2)t( CH=CH)„CH8 6 + c) in terms of T and V.
¿H2OCO(CH2)c(CH=CH),CH, (a + b + c) =
(T
conjugated double bonds produce signals
which are shifted toward lower field by
0.4-1.0 p.p.m. The total unsaturation
is still readily measured by including
these signals with those associated with
the unconjugated double bonds. Thus,
-
V -

14)/2 (10) the NMR method provides an indication

of total unsaturation, expressed as a
The molecular weight using this Likewise Equation 7 may be expressed calculated iodine number. It is inter-
formula is: in terms of T and V using Equations esting to note that the theoretical iodine
9 and 10. number for tung oil, calculated from gas
Mol. wt. = 173.1 + 45.1 +
Mol. wt. = 218.2 + chromatographic data (/), is 229.
14.027(a + b + c) + 26.038(* + y + z) While not from the same sample of tung
(7) 7.013(7 -
7 -
14) + 13.0197
oil, this value does support the correct-
Mol. wt. = 120.0 + 7.0137 + ness of the NMR value of 225.
The first term, 173.1, is the formula 6.0067 (11) On a percentage basis there is a fairly
weight of the CeH6Oe glyceryl triester large discrepancy between the NMR
radical, while the second term, 45.1, Thus, the quantitative data obtained and Wijs iodine numbers for coconut
is the formula weight of three methyl with the digital voltmeter may be con-
groups. verted to average molecular weight oil. Here again, calculations from gas
The total number of protons, 7, in the through Equations 5, 6, and 11. chromatographic data reveal a theo-
above formula is Finally, the degree of unsaturation retical value, 12.1, which is in better
may be expressed in a calculated iodine agreement with the NMR iodine num-
= 5 + 9 + 2(o + 6 + c) number. ber. The reason for the discrepancy
2 (x + y+ z) (8) 126.91
in this case is not fully understood; how-
_,·
Iodine No. =
s—:— ,, X ever, it does appear to be real since the
Equiv. wt. (fat)
.

no. of olefinic H’s X 100 range in iodine number for coconut oil is
reported to be within 7.5-10.5 (1,2).
Table Iodine No. Values of Various 126917 Molecular weights calculated from
1.
Iodine No. (12) NMR data using Equation 11 can be
Fats Mol. wt.
related to those obtained from chemical
NMR
WIJS data through the saponification value.
Oil No. No. RESULTS AND DISCUSSION
Table II shows the res1'’Is of such meas-
Coconut 10.5 ± 1.3 8.0-8.7 Table I shows the results of our study urements on three of the oils. The
Olive 80.8 ± 0.9 83.0-85.3
Peanut 94.5 ± 0.6 95.0-97.2 of nine natural fats. The errors listed agreement between the two methods
Soybean 127.1 ± 1.6 125.0-126.1 for each NMR-iodine number are stand- is as good as in the corresponding iodine
Sunflower ard deviations derived from at least 10 values.
seed 135.0 ± 0.9 136.0-137.7 The total time spent in obtaining the
Safflower measurements on each fat. Iodine
seed 141.2 ± 1.0 140.0-143.5 numbers determined by the Wijs method NMR data is about 20 minutes per
Whale 150.2 ± 1.0 149.0-151.6 are given as a total range which is taken sample. Calculation time can un-
Linseed 176.2 ± 1.2 179.0-181.0 from duplicate runs done by two differ- doubtedly be made quite short by using
Tung 225.2 ± 1.2 146.0-163.5 ent analytical laboratories. suitable nomographs prepared with the
The agreement between the two meth- aid of Equations 5, 6, 11, and 12.
Table ods of determining iodine number is
II. Average Molecular Weights
of Fats from Saponification Values and remarkably good for all fats except ACKNOWLEDGMENT
NMR tung oil. The high concentration of
eleostearic ester, containing conjugated The authors are indebted to DeSoto
Sap. NMR Mol. double bonds, has been shown to inter- Chemical Coatings, Inc., Berkeley,
Oil Value Mol. Wt.
Wt.
fere with the true measurement of un- Calif., for supplying samples of linseed
Olive 189.3 887.1 873.7 ± 5.3 saturation by the Wijs method (4, 7, 9). oil and tung oil. Iodine numbers by the
Peanut 188.8 891.5 882.3 ± 7.4
Safflower The NMR spectrum of this fat, Figure Wijs method were determined by Curtis
seed 191.5 879.0 874.9 ± 9.3 3, differs from that of the other oils and Tompkins, Ltd., and Pacific Chem-
since the olefinic protons associated with ical Laboratories, both of San Francisco.

1138 . ANALYTICAL CHEMISTRY

 LITERATURE CITED
(1) Archer-Daniels-Midland Co., Minne-
apolis, Minn., “Composition and Con-
stants of Natural Fats and Oils.”
(2) Fieser, L. F., Fieser, M., “Organic
Chemistry,” p. 408, Reinhold, New
York, 1956.
(3) Hopkins, C. Y., Bernstein, H. J.,
Can. J. Chem. 37, 775 (1959).
Purification of Acetonitrile
for Exact Measurements
J. F. COETZEE, G. P. CUNNINGHAM, D. K. McGUIRE, and G.
Department of Chemistry, University of Pittsburgh, Pittsburgh 13, Pa.
Acetonitrile is attracting increasing
attention as a solvent for electro-
chemical and other reactions. Since it
is a comparatively inert solvent, several
possible impurities are sufficiently reac-
tive to modify its properties significantly,
even in very low concentrations. The
conventional method for the purifica-
tion of acetonitrile (repeated dis-
tillation from phosphorus pentoxide)
is not entirely satisfactory. Two alter-
native methods that give superior
results are described. The results of
polarographic and gas chromato-
graphic tests for logical impurities are
given. The persistent polarographic
wave at —2.2 volts vs. the saturated
calomel electrode is not caused by
acetic acid, as previously assumed, but
by unsaturated nitriles.
the last several years there
During
has been a rapidly increasing
interest in the use of acetonitrile as a
solvent for electrochemical and other
reactions. Various theoretical studies
of a quantitative nature have been
carried out in acetonitrile.
Typical Examples. Polarography
of inorganic substances (11, 12, 16)
and various classes of organic com-
pounds (18), as well as solid electrode
voltammetry of inorganic substances
(11,16).
Measurement of electrode potentials
of common inorganic couples (IS).
Conductometry of salts, particularly
substituted ammonium salts (4, 17).
Quantitative studies of dissociation
equilibria of acids (6, 10) and amine-
type bases (14), using conductometric
and spectrophotometric methods.
Measurement of the autoprotolysis
constant of acetonitrile (7).
Electron spin resonance studies of
electrolytically generated transient free
radicals (9).
Aromatic chlorination rate studies
(*).
(4) Mikasch, J. D. von, Frazier, C., Ind.
Eng. Chem., Anal. Ed. 13, 782
(1941).
(5) Pack, F. C., Planck, R. W., Dollear,
F. G., J. Am. Oil Chemists’ Soc. 29, 227
(1952).
(6) Pople, J. A., Schneider, W. G., Bern-
stein, H. J., “High Resolution Nuclear
Magnetic Resonance,” p. 98, McGraw-
Hill, New York, 1959.
(7) Shoolery, J. N., J. Chem. Phys. 31,
1427 (1959).
as a Solvent
R. PADMANABHAN
In addition to these quantitative
theoretical studies, numerous empirical
and semiempirical investigations (par-
ticularly acid-base titrations) have pro-
vided results that are useful for prac-
tical and comparative purposes (14).
Since acetonitrile is a comparatively
inert solvent, the presence of many
possible impurities, even in very low
concentrations, would make it un-
suitable for exact studies. Several
authors have commented on difficulties
encountered in the purification of aceto-
nitrile for such purposes.
EFFECT OF IMPURITIES
Numerous methods are available for
the preparation of nitriles (S, 13). We
were unable to obtain information from
the manufacturers about the methods
actually used in the commercial
preparation of acetonitrile and other
nitriles. Technical data reports are
available from the Eastman Kodak
Co. for n- and isobutyronitrile (8),
but not for acetonitrile.
Polarographic and other tests have
been carried out for a number of sub-
stances that logically may be present as
impurities in acetonitrile, particularly
isonitrile, water, and the various hy-
drolysis products of acetonitrile—
namely, acetamide, ammonium acetate,
ammonia, and acetic acid.
Acetonitrile is a relatively weak base,
much weaker than water (11,12), and
an extremely weak acid (7,14). Several
possible impurities are acids or bases
sufficiently strong to modify the proper-
ties of the solvent significantly, even
when present in very low concentrations.
The presence of acetic acid would be
particularly objectionable in exact
studies of bases in unbuffered solutions,
and that of ammonia in similar studies
of acids, even if the concentrations of
these impurities are as low as 10-5M.
Although the effect of water is less
(8) Stáhli, H., Mitt. Oebiete Lebensm.
Hyg. 46, 121 (1955).
(9) Varían Associates, Palo Alto, Calif.,
Tech. Info. Bull. Vol. 3, No. 1 (1960).
Received for review March 5, 1962.
Accepted May 21, 1962. Pittsburgh
Conference on Analytical Chemistry and
Applied Spectroscopy, Pittsburgh, Pa.,
March 5, 1962.
marked, the presence of relatively low
concentrations of this persistent im-
purity can cause large errors in cer-
tain measurements.
Examples. A. The polarographic
half-wave potential of the solvated
proton (as 1 X 10 ~*M perchloric acid)
in acetonitrile as solvent becomes 0.15
volt more negative on adding 1 X
10-2M water (11). This large shift is
mainly the result of the pronounced
increase in the solvation energy of the
proton which occurs when the relatively
strong base water converts the species
CH3CNH+ into H2OH+ (11,12).
B. The dissociation constant of the
protonated form of the Hammett in-
dicator 4-chloro-2-nitro-iV-methylaniline
increases by a factor of 20 when 2 X
10 ~*M water is added (as shown in
unpublished results, this laboratory).
The cause of this large effect is basically
the same as for example A.
C. The conductance of 0.1M n-
butylamine is doubled by adding 8 X
10-2Af water (14), because the acid
properties of water (proton donor
strength, as well as solvation of anions
by hydrogen bonding) are much stronger
than those of acetonitrile.
CONVENTIONAL METHOD FOR PURIFYING
ACETONITRILE
The conventional method for the
purification of acetonitrile generally
involved some kind of pretreatment to
remove acetic acid—for example, shak-
ing with aqueous potassium hydroxide,
solid potassium carbonate, or alumina—
followed by repeated distillations from
phosphorus pentoxide until the residue
was no longer colored (orange or black).
Certain authors included an additional
preliminary drying operation (shaking
with calcium chloride, magnesium sul-
fate, etc.) and an additional (final)
distillation of the product, either alone
or with added potassium carbonate or
barium oxide.
Repeated distillation of acetonitrile
from phosphorus pentoxide has a major
VOL 34, NO. 9, AUGUST 1962 · 1139