Gas Chromatographic Analysis of Some Alcohol

3,5-Dinitro benzoates with Application

to Analysis of Acetals
W. G. GALETTO, RICHARD

E. KEPNER,

and A. DINSMOOR WEBB

Departments of Viticulture and Enology and of Chemisfry, University of California, Davis, Calif.

The gas-liquid chromatography of

the 3,5-dinitrobenzoate derivatives of
a variety of alcohols has been demonstrated using SE-30 silicone fluid
as the liquid phase at a column temperature of 250" C. The effluent
material from the peaks has been
trapped and shown by infrared and
thin layer chromatography to be the
original derivative injected. Mixtures
of the derivatives can be separated
successfully without previous purification. The investigation of the structures of trace quantities of acetals by
gas chromatography of the 3,5dinitrobenzoate and 2,4-dinitrophenylhydrazone derivatives of the component parts is described.

OUKUP, SCARPELLINO,
AKD DANIELCZIK (5) have reported recently the

gas chromatographic separation of carbonyl 2,4-dinitrophenylhydrazones.Although the use of a flame ionization

detector precluded collection of unaltered effluent samples and proof that
intact 2,4-dinitrophenylhydrazoneswere
being chromatographed, the excellent
separations achieved suggested the
possibility of employing preparative
scale gas chromatographic techniques
to purify and identify trace quantities
of carbonyl derivative mixtures. In
turn, if successful gas chromatography
of unaltered carbonyl-2,4-dinitrophenylhydrazones were possible, it seemed
that there might be equal success in
gas chromatographic separation of 3,5dinitrobenzoates of alcohols.
Analyses of trace quantities of acetals
contained in complicated aroma isolates
have always been difficult. It was
observed (6) that higher boiling fractions of aroma material from Muscat
grapes yielded acetaldehyde-2,4dinitrophenylhydrazone upon treatment with
an acidic solution of 2,4-dinitrophenylhydrazine. As the high boiling point of
the sample precluded the presence of
free acetaldehyde, the evidence indicated that acetals were present.
The gas chromatographic separation
of alcohol 3,5-dinitrobenzoates and the
analysis of acetals through hydrolysis
and preparation of the carbonyl derivative followed by preparation of the 3,5dinitrobenzoate esters of the liberated
34

ANALYTICAL CHEMISTRY

alcohols and their identification by gas

chromatography is the subject of this
paper.
EXPERIMENTAL

Apparatus. Two gas chromatographs were used in this investigation.

The first was a Wilkens Instrument
and Research, Inc., Hy-Fi Model
600D flame detector instrument
operated isothermally a t 250" C.,
injector temperature 275' C., 19 ml./
minute Nz flow rate. The column
employed was a 6 foot long, 1/8-inch
o.d., stainless steel tube packed with
10% w./w. SE-30 silicone fluid on acidwashed chromosorb W. Detector signals were recorded with a MinneapolisHoneywell Electronik 16 recorder.
A Loenco Model 70 Hi-Flex dual
column, dual thermal conductivity detector instrument was used when it was
desired to collect effluent samples from
the instrument column. A l/*-inch
0.d. by 10 foot long stainless steel tube
packed with 10% w./w. SE-30 silicon
fluid on acid-washed Chromosorb W
was used. Signals were recorded on a
Minneapolis-Honeywell Electronik 16
recorder. The exit line from the sample side of the thermal conductivity
detector was provided with a special
heating coil which extended to the
termination of the line outside of the
instrument cabinet. Samples were collected in thin-walled glass capillary
tubes ll/n-mm. 0.d. by 12 inches in
length.
Thin layer chromatography, using
the techniques of Dhont and de Rooy
( I ) , and infrared spectra were used to
check the purity and identity of the
various derivatives before and after
gas chromatography.
Materials and Methods. The reagent from which 2arbonyl derivatives
were obtained was prepared by dissolving 1 gram of 2,4-dinitrophenylhydrazine in 5 ml. of concentrated H2SOa.
After dissolution, 6.7 ml. of HzO were
added slowly; the solution was allowed
to set overnight and filtered before use.
Derivatives of known alcohols were
prepared from 3,5-dinitrobenzoyl chloride by standard methods (4) and recrystallized to constant melting points.
The derivatives were dissolved in benzene a t low concentration and injected
into the Hy-Fi gas chromatograph
individually for determination of retention times and as mixtures for determination of the completeness of separation. The derivatives in benzene
were also injected into the Loenco

instrument and the effluent samples

collected for investigation.
Ethyl isoamyl acetal was prepared
by reacting 0.05 mole each of anhydrous
ethyl alcohol and isoamyl alcohol with
0.05 mole of redistilled acetaldehyde.
Dry HC1 bubbled into the alcohol solution before addition of the aldehyde
served as catalyst. After addition of
the acetaldehyde, the flask was mechanically stirred for 1 hour a t room
temperature. At this time the lower
water phase was removed with a pipet
and anhydrous K&03 was added.
After filtration, the desired ethyl isoamyl
acetal was separated from the other two
acetals and the two alcohols by preparative scale gas chromatography on a
polyneopentylglycoladipate column.
Analysis of the ethyl isoamyl acetal
was accomplished as follows: to 4 drops
of the 2,4-dinitrophenylhydrazinereagent solution contained in a 3-ml.
centrifuge tube was added with mixing
1 p1. of the ethyl isoamyl acetal.
After 10 minutes the precipitate (2,4dinitrophenylhydrazone of the carbonyl
portion of the acetal) was centrifuged
to the bottom of the tube. The supernatant liquid was transferred by pipet
to a 5-ml. pear-shaped flask, 1.5 mi. of
H20 were added, the flask was fitted
to a simple distillation head and condenser, and 0.8-1.0 ml. of the sample
was distilled.
To the distillate. cooled to 0" C..
was added 1.0 ml. of benzene containing
0.9 gram of 3,bdinitrobenzoyl chloride
(9). The mixture was shaken and kept
a t 0" C. for approximately 10 minutes.
Two-tenths of a microliter of the benzene phase was injected directly into
the Hy-Fi gas chromatograph for
analysis.
The 2,4-dinitrophenylhydrazone precinitate in the centrifuge tube was
taken to dryness and &en dissolved
in a few drops of benzene or methylene
chloride. Five- to lo-& samples of
the solution of the carbonyl derivative from the hydrolyzed acetal and of
selected known carbonyl 2,4-dinitrophenylhydrazones were injected into
the Hy-Fi gas chromatograph using
the same column as was used for the
3,5-dinitrobenzoates, but operated isothermally a t 270" C.
RESULTS AND DISCUSSION

The gas chromatographic retention

times for 23 alcohol-3,5-dinitrobenzoates investigated are listed in Table I.
A typical chromatogram showing the

$4

z
8
0

m
0
Y)

s
0

20.0

Figure 1.
alcohols

15.0

7.5

Time, min.

5.0

2.5

Gas chromatogram of 3,5-dinitrobenzoate derivatives of known

separation of the 3,Bdinitrobenzoates

of 12 different alcohols is presented in
Figure 1. Investigation of single derivatives, recrystallized to constant melting
point, showed that a single sharp peak
with relatively little tailing was obtained for each derivative. When the
Loenco gas chromatograph with the
thermal conductivity detectors is used,
it is found that separated peaks can be
collected easily in capillary collection
tubes. Rechromatography of a collected peak yields a single peak with the
same retention time, indicating either
that decomposition is insignificant or
that the initial compound reacts cleanly
under the heat of the injector to give a
second substance which is stable. The
infrared spectra determined on a number
of the collected fractions were identical
with the spectra for the corresponding
alcohol-3.5-dinitrobenzoatesin the Sadtler (3) tables indicating that unaltered
alcohol derivatives emerged from the
column. Further confirmation of the
identity of the material collected from
the gas chromatograph was obtained by
running thin layer chromatograms ( I ) ,
in which case it was found that the component before and after gas chromatography behaved identically. The minimum detectable quantity of derivative
was not determined. However, only a
very small fraction of the total derivative

obtained from the hydrolysis of 1 pl.

of ethyl isoamyl acetal was required
to give easily visible peaks on the
chromatogram.
The boiling points of the alcohols used
to prepare the 3,bdinitrobenzoate deTable 1.

Gas Chromatographic Retention Times of Some Alcohol-3,5-Dinitrobenzoates

Alcohol
Methyl
Ethyl

?g"$

sec-Butyl
Isobutyl

rivatives and the melting points of the

derivatives used in this investigation
are also listed in Table I. Since the
column oven temperature (250' C.) is
well above the melting points of all of
the compounds studied, it is not surprising that there is 'no correlation
between the melting points and the
retention times. The nearly perfect
correlation between alcohol boiling
points and retention times is interesting,
however, as it signifies that conversion
of alcohols to 3,5-dinitrobenzoate esters
does not alter their relative positions in
series arranged according to vapor
pressures.
Analysis of 1 11. of the acetal, l-ethoxy1-isopentoxyethane, was accomplished
by hydrolysis and identification of the
products through gas chromatography of
the derivatives. The acidic 2,sl-dinitrophenylhydrazine solution hydrolyzed
the acetal very rapidly giving a copious
precipitate of the carbonyl derivative
almost instantaneously after the addition of the acetal to the reagent. A tenminute period of shaking and waiting is
suggested simply to assure that all of
the acetal is hydrolyzed. Simple distillation of 60-S0~0of the volume of the
diluted hydrolysis mixture serves to
separate most alcohols from the carbonyl derivative. Water-soluble alcohols with boiling points lower than that
of water would be distilled over because
of the low boiling points, while most
higher boiling, less water-soluble alcohols
would be carried across by steam
distillation. It is t'rue, of course, that
extremely ~at~er-soluble,very high
boiling alcohols and glycols would not be
recovered by this technique. Although
no detailed study was made of the
completeness of the reaction between

%pentyl
3-Methyl-2-butyl
n-Butyl
Active amy1
Isoamyl
2-Hexyl
n-Amyl
cis-3-Hexenyl
2-Heptyl
n-Hexyl
Isoheptyl
n-Hept 1
n-Oct y?'
Benzyl
n-Nonyl
2-Phenethyl
n-Decyl

B.p., "C.
64.7
78.5
82.3
97.2
99.5
108.4
119.3
114

117.7
128
130.5

140
138
156-157
160.4
157.2
168
176
195

205.2
213
220

231

3,5-DvB
m.p., C.
106.5-106.8
93.0- 93.2
121.5-121.8
73.0
75.2- 75.5
85.5- 86.0
60.7- 60.9
76.0- 76.4
63.0- 63.2
84.0- 84.9
60.0- 61.0
37.5- 38.2
44.0- 45.0
48.5- 50.0
49.5- 50.0
57.9- 58.7
56.5- 57.0
46.0- 46.5
61 - 62
112 -113
52 - 53

107.3-107.8
56.5- 57.0

3,5-DNB
retention
time, min.
3.4
3.6

3.7
4.3

4.4

4.7
5.2
5.2

5.4
6.0
6.1
6.5

6.9
8.1
8.2
8.3

9.6
10.4
13.3

14.0

16.6
17.1
21.5

VOL. 38, NO. 1, JANUARY 1 9 6 6

35

Table II. Gas Chromatographic Retention Times of Alcohol-3,5-Dinitrobenzoates and Carbonyl-2,4-Dinitrophenylhydrazone from Hydrolyzed
Acetal

Compound
3,5-DNB from acetal
(Peak 1)
3,5-DNB from acetal
(Peak 2)
2,PDNH from acetal
Ethyl-3,5-DNB
Isoamyl-3,5-DNB
Acetaldehyde-2,4DNH

Retention
time,
min.
3.6
6.1
4.1
3.6
6.1
4.1

Table 111. Gas Chromatographic Retention Times of Certain Carbonyl-2,4Dinitrophenylhydrazones

Compound
Acetaldehyde
Propionaldehyde
Acetone
Isovaleraldehyde

Retention
time,
min.
4.1

4.9

5.1

6.6

the 3,5-dinitrobeneoyl chloride and the

alcohols under the conditions used, it
was observed that the conversion was
complete enough to give two large
peaks, in addition to the benzene peak,
on gas-liquid chromatography of a
portion of the reaction products from
1 111. of acetal. These peaks had
retention times corresponding to

those for ethyl-3,5-dinitrobenzoate and

isoamyl-3,5-dinitrobenzoate (Table 11).
Injection of a benzene solution of 3,5dinitrobenzoyl chloride or 3,5-dinitrobenzoic acid gave no discernible peaks
other than that of the benzene solvent.
The retention time of the 2,4-dinitrophenylhydrazone derivative of the carbonyl portion of the hydrolyzed acetal
is also given in Table 11. In Table I11
are listed, for comparison purposes, the
retention times of four known carbonyl2,4 - dinitrophenylhydrazones which
were run gas chromatographically under
conditions identical to those used for
the hydrolysis product. It is evident
that the hydrolysis products from an
acetal can be determined successfully in
the form of the derivatives by means of
gas chromatography.
In order to confirm that 2,4-dinitrophenylhydrazone derivatives do pass
through the gas chromatograph without
decomposition, samples of known isovaleraldehyde - 2,4 - dinitrophenylhydrazone were injected into the Loenco
instrument and collected after passage
through the gas chromatograph. The
derivative before and after gas chromatography showed identical behavior
on a thin layer chromatogram.
The fact that ~arbonyl-2~4-dinitrophenylhydrazones and alcohol-3,5-dinitrobenzoates can be separated easily
by gas chromatography without decomposition suggests that a number of
other derivative compounds also might
be separated and identified in this
manner. Perhaps of more significance,

however, is the fact that gas chromatography can be used as a method to

obtain the derivative in a pure state
when the quantity is too small to permit
purification by recrystallization techniques. A small sample of a mixture of
derivatives thus could be resolved into
its components and the components
tentatively identified through retention
times. After collection of the separated
components melting points can be
determined using a micro hot stage and
the identifications thus be further
confirmed. Although this report is
limited to a discussion of the successful
application of these techniques to the
analysis of trace quantities of complex
acetals, it appears that functional group
separations and analyses of very small
samples of complex aroma or flavor
extracts should be facilitated.
LITERATURE CITED

(1) Dhont, J. H., de Rooy, C., Analyst

86, 74 (1961).
(2) Lipscomb, W. N., Baker, R. H., J.
Am. Chem. SOC.64, 179 (1942).
(3) Sadtler, S. P., Catalog of Infrared

Spectrograms, Philadelphia, Pa.

(4) Shriner, R. L., Fuson, R. C., Curtin,
D. Y., The Systematic Identification
of Organic Compounds 4th ed., p.
212, Wiley, Kew York, G..Y., 1956.
( 5 ) Soukup, R. J., Scarpellino, R. J.,
Danielczik., E.., ANAL.CHEM.36. 2255
(1964).
(6) Webb, -4.D., Kepner, R. E., Food
Res. 22, 384 (1957).
RECEIVED
for review September 7, 1965.
Accepted November 15, 1965.

Gas Chromatographic Determination of Campesterol,

P-Sitosterol and Stigmasterol
ANDREW ROZANSKI
Research Laboratories, The Upjohn Co., Kalamazoo, Mich.
An accurate gas chromatographic
method for the determination of campesterol, p-sitosterol, and stigmasterol is
described. The sterols are separated
as trimethylsilyl ethers on a stainless
steel column, packed with 0.75-1 .O%
silicone gum rubber on an acid-washed,
silanized support, having about 4 0 0 0
theoretical plates. The quantification
is carried out by measuring ratios of
peak areas before and after addition
of a standard amount of one of the
sterols to the sample, or by using
cholesterol as an internal standard.
For the first time, the relative responses
in the flame ionization detector of the
trimethylsilyl ethers of these three
sterols and cholesterol have been
accurately determined and found to be
significantly different. The method is

36

ANALYTICAL CHEMISTRY

easily used on a routine basis with

precision and accuracy hitherto unattainable, and has been applied to
the analysis of soybean sterols.

of the commonly occurring phytosterolscampesterol (24a -methyl - cholest - 5 en-3B-o1),

0-sitosterol
(240-ethylcholest-5en-3P-o1), and stigmasterol
(24p-ethyl-cholest-5,22-dien-3~-ol) - is
important in a number of studies and
Complete
applications (7, 8, 19).
analysis of a mixture of such closely related sterols had been very difficult
before the successful application (25) of
gas-liquid chromatography (GLC) to
steroids. Their separation by paper
(18) or thin-layer (4) chromatography
XACT DETERMINATION

is difficult and a t best semiquantitative

results might be expected. It is possible to determine stigmasterol in soybean sterols by two specific methods
based on its higher degree of unsaturation-e.g., by an infrared (10) and by a
radioactive isotope dilution technique
(6).
GLC is capable of separating campesterol, @-sitosterol, and stigmasterol and
has been frequently reported (1, 2 , 7,
8, 11, IS, 15, 20, 21, 23). Derivatives such as esters (12-14, 22) and
ethers (S), including trimethylsilyl
(TMS) ethers (12, 24) have also been
separated by GLC. Although this
informative and inherently precise
method has been often used for the
separation and identification, attempts
a t quantitative analysis (1, 7 , 8, 11, 14,