Fresenius J Anal Chem (1994) 349:451-453
Fresenius' Journal of
Determination of glycols by HPLC with refractive index detection
L. Nitschke, L. Huber
I Bayerische Landesanstalt ffir Wasserforschung, Kaulbachstrasse 37, D-80539 Mfinchen, Germany
Received: 6 September 1993/Accepted: 1 December 1993
Abstract. A simple and fast HPLC method for the deter-
mination of glycols is described. It is characterized by a
reversed-phase separation using water as eluent and a
refractive index detection. The method was applied to in-
vestigate the biodegradation of glycols in a laboratory ac-
tivated sludge plant and to determine the content of gly-
cols or alcohols in detergents. The detection limit is
4 rag/1 ethylene glycol or propylene glycol in an aqueous
sample.
Introduction
The determination of glycols, especially diethylene glycol
(DEG), has become very popular in the eighties when
DEG had been illegally added to wine. In most cases, gas
chromatography was used to determine its content in
wine [ 1 - 5 ] . Also thin-layer chromatography [6], 13C-
NMR [7] and HPLC (with derivatization to make an UV
or fluorescence detection possible [8]) were applied. Nor-
mally, glycols are mainly used as intermediate products in
the chemical industry, as solvents, and antifreezes. Large
amounts of antifreezes containing diethylene glycol and
propylene glycol (PG) are used at airports during the win-
ter period for de-icing airplanes and runways. A large
part of the formed mixture of water and glycols is recy-
cled by distillation at modern airports, especially aque-
ous solutions with a high content of glycols. The rest of
the mixture is treated by the activated sludge process in
waste water treatment plants. Although these glycols are
easily biodegradable by adapted activated sludge, there
are sometimes problems caused by low waste water tem-
peratures in winter and by the load, especially shock-
loads. Therefore laboratory activated sludge units were
tested by us to find out the optimal conditions for the
biodegradation of glycols, or its restrictions. The deter-
mination of diethylene glycol and propylene glycol in the
influent and especially in the effluent of a laboratory ac-
Correspondence to." L. Nitschke
Springer-Verlag 1994
tivated sludge plant was additionally of special interest
for the control of more common chemical parameters
such as COD, TOC, ammonia etc. A simple and rapid
HPLC method has been developed for this purpose, re-
quiring no special sample pretreatment. The method is
characterized by using a reversed-phase column, water as
eluent, and refractive index detection.
Experimental
Chemicals
All glycols were obtained from Merck, Darmstadt. Sol-
vents for HPLC (water, methanol) were of LiChrosolv
quality and obtained from Merck, too.
HPLC procedure
The HPLC equipment consisted of the data system (Kon-
tron 450) and two pumps (Kontron 420). A refractive in-
dex detector (Erma ERC-7512) was used for the measure-
ments. The filtered samples were injected by a valve fitted
with a 100 gl loop. A Hypersil ODS column (particle size
5 ~m, 250 4.6 ram) was used for the reversed-phase sep-
aration of the glycols. The mobile phase was water and
the flow rate 2 ml/min. The separation was carried out at
room temperature. The temperature control of the refrac-
tive index detector was set to 30C.
Results and discussion
Reversed-phase chromatograms of an influent and an ef-
fluent of a laboratory activated sludge plant are shown in
Fig. 1. The influent is an aqueous solution containing a
prescribed content of nutrients (peptone, beef extract)
and mineral salts (for details see DIN 38412, Teil 26). A
certain amount of the antifreeze about 88% DEG and
270 PG has been added to the solution. The added
amount corresponds to 400 mg/1 DEG and 9 rag/1 PG,
respectively. As can be seen from Fig. 1, PG and DEG can
be detected in the influent and effluent of a laboratory
452

(D
O)
03 C
C DEG 0
0 C1
g1 (.0

[_

--A

B
] I = I ]
0 3 6 5 iO
time [mini time [min]
Fig. 1. Chromatograms of (A) influent (400 mg/1 diethylene glycol and Fig. 3. Chromatogram of a mixture of (/) 100mg/1 glycerol, (2)
9 mg/1 propylene glycol are added to the nutrient solution), (B) effluent 150 mg/1 propylene glycol, (3) 200 rag/1 diethylene glycol, (4) 400 mg/1
of a laboratory activated sludge plant. H P L C conditions: column: propan-2-ol, (5) 250 mg/1 triethylene glycol in water. For HPLC condi-
Hypersil ODS (2504.6 mm I.D., particle size 5 gm); injection: 100 gl; tions see Fig. 1
eluent: water; flow rate: 2 ml/min; detection: refractive index

biodegradation) from the stationary phase. The frequen-

activated sludge plant with the described HPLC method.
There are no interferences from other substances. The
broad peak at the beginning of the chromatogram has
been ascribed to the content of inorganic ions in the
aqueous solutions. There ions very weakly interact with
the column material, i.e. they are transported more or less
with the solvent front. On the other hand, organic sub-
stances added as nutrients are adsorbed on the C~8 col-
umn material. By using water as eluent these substances
have a long retention time and pass the column very slow-
ly. For this reason, glycols can be detected in the influent
and effluent of laboratory activated sludge plants with-
out interferences from other substances. After recording
several chromatograms, the column has to be rinsed with
methanol or another appropriate solvent to elute the ad-
sorbed organic substances (widely unknown products of
cy of the cleaning procedure depends on the composition
of the injected samples. In our case it is necessary to rinse
the column with methanol for I h (flow rate 2 ml/min)
after 10 to 15 chromatograms have been recorded.
The calibration plot of propylene glycol is shown in
Fig. 2. The correlation coefficient and the 3a detection
limit were calculated to 0.9996 and 4 mg/1, respectively. A
similar calibration was obtained from DEG. The repro-
ducibility was determined with an aqueous solution con-
taining 100mg/1 PG. A relative standard deviation of
0.6% was obtained. The concentration of DEG was de-
termined to be 7.3 mg/1 in the effluent as shown in Fig. 1.
This means that the biodegradation of DEG in the labo-
ratory activated sludge plant amounts to over 98%. The

(D PG
0
peak area [mV*min] C
6 0
CI
(,9

C.

propan-2-ol

[ I [
O 3 6 9
0 50 100 150 200 250 300 350 tme [mini
concentration [mg/I]
Fig. 4. Chromatogram of a detergent containing 570 propylene glycol
Fig. 2. Calibration plot of propylene glycol in water and propan-2-ol each. For HPLC conditions see Fig. 1

concentration of PG in the same sample is below the de-
tection limit. It should be pointed out that the H P L C
method described here is also applicable to the determi-
nation of other glycols or alcohols without interferences
by other organic substances. An important requirement,
however, is a limited length of the alkyl chain of the gly-
cols and alcohols (maximum 4 up to 6 - C H 2 - groups),
depending also on the number of polar groups in the
molecules. A chromatogram of a sample containing 4 dif-
ferent glycols and one alcohol is shown in Fig. 3. The
peaks are well resolved.
Based on the German detergents law, our institute and
certain other authorities have to check the declared for-
mulations of detergents. The above H P L C method is sat-
isfactorily applicable to determine glycols and alcohols in
the presence of surfactants. A chromatogram of a deter-
gent containing propylene glycol and propan-2-ol is
shown in Fig. 4.
In summary, it may be emphasized that the described
H P L C method is a fast and simple procedure to deter-
453
mine glycols and alcohols with a limited length of the
alkyl chain in the presence of other organic substances
such as nutrients of a laboratory activated sludge plant or
surfactants.
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