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Anal yti ca Chi mi ca Acta, 87 (1976) 445-449

OEIsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Envi ronment Canada, Fi sheri es and Mari ne Servi ce, Hal i fax Laboratory, Hal i fax, hrova
Scoti a B3J 2R3 (Canada)
(Received 31st March 1976)
A simple and rapid method for estimating the content of wax esters in lubricating
oik has been developed through the use of isopropanol extraction to concentrate the wax
esters. The ratio of the absorption bands at 1749 and 1385 cm- can be used directly
for the determination of wax esters in lube oil samples. The method can be satisfactorily
applied at levels down to 1 %, with an overall deviation of less than 10 lo; for samples
containing as low as 0.5 %, the procedure can be used semi-quantitatively with an error
of about 20 %. This infrared method can also be used to determine marine and sulfurized
wax esters.
Many instrumental methods for lipid analysis [l] provide only non-specific
determinations of constituents such as triglycerides, free fatty acids,
phospholipids, and wax esters which may be found in complex mixtures.
However, the application of n.m.r. spectrometry to the determination of
wax esters in marine oils [2, 31 does not suffer from these difficulties and
a very simple rapid operation can give reliable results. Many studies also
indicate the potential of i.r. spectrometry in various lipid investigations [ 4-71;
the i-r. absorption of the carbonyl band has been used for monitoring
kinetic changes during the oxidation of various lipids [8, 91. Three com-
prehensive reports on the quantitative i-r. spectroscopy of lipids in solution
[lo--121 have also discussed the theoretical and practical aspects of i-r.
spectroscopy as an identification method.
Wax esters have a superior ability to cling to metal surfaces in the form of
very thin films [13] and are commonly used in the well-known Germ
process for improving lubricating oils [ 141. However, an effective, rapid
procedure for the determination of the relatively low content of wax esters
in complex lubricating oils has remained a problem. The analysis of
lubricating oil often involves complex mixtures and although i-r. techniques
can give information on major components they are not usually sufficiently
sensitive for most minor compounds. In this report, a simple rapid i.r. method
* FA 0 Trainee. Present Address: Instituto de Foment0 Pesquero, P-0. Box 1287,
Santiago, Chile.
is described for estimating wax esters in lubricating oils at concentrations
down to 1 9% after the wax esters have been concentrated by a single solvent
extraction. Quantitative integrations of the bands corresponding to r&O in
dialkyl esters (1740 cm-) and the CHs symmetric scissors (1385 cm-) are
used for calculating the content of wax esters.
Ma teriGk and apparatus
Commercial samples of spennaceti (BDH, Ltd.) and Duo-Seal pump oil
(Welch Scientific Co.) were used as model materials for wax esters (WE) and
pure lubricating oil (LO), respectively. The LO was shown to contain only
hydrocarbons without any WE additives. Sperm whale (Physeter catodon)
[2, 151 oil, which contained 78 % WE, and a sulfurized ester oil (Chrysler
Canada Ltd., Windsor, Ontario) were also used.
A UNICAM SP-1000 infrared spectrophotometer with a KBr window
(5 X 25 cm) taken from a Beckman IR cell was used.
A sample of LO (10 g) was weighed out in a 50-ml graduated centrifuge
tube (28 X 151 mm) with plastic screw cap. Reagent-grade isopropanol
(20 ml) was added to extract the WE from the LO; the tube was shaken
gently for about 1 min. Two phases formed within a few minutes after
stopping the agitation_ After the isopropanol (top) phase had been carefully
removed by a Mini-Pet syringe-type pipet, the isopropanol was removed
completely with a rotary evaporator. Three drops of the residual WE
concentrate were placed on the top part of the KBr window. After the
window had been replaced in the cell holder, a thin, uniform oil film was
established in the i-r. beam within minutes and the spectra were recorded
in triplicate. The ratio between the absorption bands at 1740 and 1385 cm-,
calibrated against WE and LO mixtures could be used directly for determining
the content of wax esters in the original lubricating oil. The ratio for the
range 0.05-0.4 showed a linear relationship with % WE in lube oil over the
range 0.5-4 % (or 5-35 % WE in the extracted oil).
Since the WE content in most lube oils is about 1% it is necessary to
increase the concentration of WE in the sample for i.r. spectrometry. Previous
experience with the extraction of WE by ternary solvent systems used for
the production of marine wax ester oils [IS], led to an investigation of the
most suitable solvent for extracting WE from hydrocarbon lubricating oils.
The extraction with single solvents such as benzene, chloroform, acetone,
hexane, diethyl ether, isopropanol, cyclohexane, tolueneqrethanol and
carbon tetrachloride was evaluated. Isopropanol and acetone showed
considerable discrimination in their dissolving power for wax esters and
lube oils. The partition coefficients of WE between LO and isopropanol or
acetone at room temperature are such that the single-stage recovery of WE
is approximately 80 % of that originally present, and the upper solvent phase
contains less than 4 % of LO. Because the absorption of t.he carbonyl group
is proposed for the determination of WE by i-r., acetone would have to be
removed completely from the extract; high-purity isopropanol is the best
solvent for extracting WE from LO in one step. Multiple extractions were
also evaluated; the WE content obtained by multiple extractions was only
4 times higher than that in the original sample, and this enrichment did not
meet the requirements for a simple determination by i-r.
In the procedure recommended, 20 ml of isopropanol was used to extract
the wax esters from a sample (10 g) of LO. By shaking gently for 1 mm,
separation of the two phases can usually be obtained rapidly without
foaming problems_ After the isopropanol has been evaporated, about
0.8-0.9 g of WE concentrate, including some LO can be removed from a
sample of LO (10 g) which originally contained 1% WE. The recovery of
WE decreases from 84 to 69 70 when the WE content is increased from 0.5
to 4 %. Correspondingly, the percentage of WE in the isopropanol concen-
trate is increased from 6 to 39 %. The overall deviation in this extraction
step is less than 5 %I for a sample of LO containing 1 70 WE. The amounts
of WE thus concentrated can be satisfactorily analyzed by i-r. spectro-
metxy (Table 1). The use of 10 ml of isopropanol for the extraction of the
WE from a LO sample (10 g) was also investigated. As expected, the
concentration of WE in the extract was higher than that obtained with 20 ml
of isopropanol, but the percentage recovery and the overall operating
errors were much worse.
The i-r. spectra for a pure lube oil, wax esters and mixed reference LO
samples which contain 10 or 30 % added WE, are compared in Fig. 1. There
is no absorption from 1600-1800 cm- for a pure lube oil (Fig. 1, left).
The band B at 1385 cm- is the symmetric scissors of -CH3 (63 and
-C(CHJ)7 (6 ,) groups [ 171. The combination absorption bands from the
asymmetric scissors of CH2 and CHS and the symmetric scissors of -CH2-O-
(ester) [17, 181 near 1470 cm- are not specific enough to be used for WE
analysis. The carbonyl absorption band A (YCO) is clearly recorded in Fig. 1
(center) for model WE at 1740 cm -*_ For sulfurized WE, band A shifts
about 10 cm- to a lower frequency; this may result from the overlap
between carbonyl electrons and the 3d orbitals of sulfur [19]. The carbonyl
frequencies and intensities of a large number of esters of all kinds have been
given by Flett [20] ; the carbonyl intensities of esters are remarkably constant,
change little with conjugation, and can be used for the determination of
WE in lube oils as shown in Fig. 1 (right).
The integrated absorbance of band A is substantial; the ratio of the band
heights A/B is even more specific and reproducible, and shows a linear
calibration for the WE content whether expressed in terms of isopropanol
Reproducibility of the determination of wax esters in lube oils by i-r. spectrometrya
Sample No. of
WE content, Wt %
Added Found + s
LO l Sperm whale oil
LO + Sulfurized esters
LO + Sperm whale oil
LO c Sperm whale oil
Sample Ab
Sample Bb
2.0 :
2.0 i
- i
0.43 2 0.09
1.11 r 0.09
0.94 5 0.08
1.04 * 0.10
2.13 * 0.14
1.88 f 0.08
3.91 ? 0.21
0.81 t 0.06
1.20 + 0.11
=WE, wax esters; LO, pure lube oil; s, standard deviation. bCommercial lubricating oils.
= no
z Q

Fig. 1. 1-r. spectra for lube oil (left); wax esters (center,- for Model WE and ----- for
sulfurized esters oil); and mixed oil (right, - sample containing 10 % WE and --- sample
containing 30 % WE)_ Bands A (1740 cm-) and B (1385 cm-) were used.
extracts (6-34 % WE) or in terms of the original LO samples (O-5-4 % WE),
respectively; After measurement of the i-r. bands A and B, the original WE
content of the lubricating oil can be determined directly from the calibration
The reproducibility and standard error for various LO samples are
presented in Table 1. This method can be satisfactorily applied for estimating
WE in LO at a level of 1 %, with an overall deviation of less than 10 %; for a
sample containing 0.5 % WE the method can be used semi-quantitatively
with an error of about 20 %. As shown in Table 1, the procedure can also be
used for determining natural marine wax esters and sulfurized wax esters,
etc., in LO samples. Extraction with isopropanol followed by i-r. spectrometry
is superior to other chemical or chromatographic methods for determination
of wax esters in various lubricating oils.
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