MWROCHEMICAL JOURNAL 17, 655-660 (1972)

Spectrophotometric Microdetermination of Some

Anthraquinone Derivatives Separated by Thin Layer
Chromatography
G. S. VASILIKIOTIS AND H. ALEXAKI-TZIVANIDOU
Laboratory of Analytical Chemistry,
University of Thessaloniki,
Thessaloniki, Greece.
Received July 18, 1972

INTRODUCTION

In connection with a research project on the photo-oxidation of an-

thracene absorbed on aluminum oxide, a new procedure for the:
separation and detection of some anthracene derivatives has been1
developed (6).
Thin layer chromatography in conjunction with spectrophotometric
microanalysis was investigated in order to develop a quantitative deter-
mination of these derivatives for a further study of the anthracene
photo-oxidation.
In the present work anthracene, anthraquinone, ahzarin (1,2dihy-
droxyanthraquinone) , quinizarin ( 1,4-dihydroxyanthraquinotoe] and
chrysazin ( 1,8-dihydroxyanthraquinone) were separated by ti w
chromatography. Good separation results were obtained and eati a
was scraped off from the plate. Quantitative evaluation of in-
components was carried out by a simple extraction-spectrophotormetric
technique.
MATERIALS AND METHODS
Materials
Anthracene, anthraquinone, quinizarin, and chrysazin were from
Fluca A. G. Alizarin was from E. Merck A. G. The purity of these
compounds was tested by their melting point and by chromatographic
tests. Chrysazin was recrystallized twice from absolute ethanol and
quinizarin was recrystallized from ethanol: water (1: 1) and sublimed at
120” C and 15 mm Hg. The rest of the above compounds were used
without any other purification. Standard solutions were prepared by
dissolving these compounds separately in a mixture of benzenechloro-
form-methanol (90: 5 : 5) (solvent system A). Their concentrations were
1 mg/ml for quinizarin, 0.8 mg/ml for chrysazin and anthraquinone,
655
Copyright 0 1972 by Academic Press, Inc.
All rights of reproduction in any fortq reserved.
656 VASILIKIOTIS AND ALEXAKI-TZIVANIDOU

and 0.6 mg/ml for alizarin, and they were kept in darkness. Solvents
were analytical grade reagents. Silica Gel GF 254, according to Stahl,
was from E. Merck A. G. Water used was deionized and distilled.
Apparatus
A Unicam SP 500 spectrophotometer was used for the spectrophoto-
metric measurements.
Thin Layer Chromatography
Thin layer plates (0.25 mm thick) were prepared by the following
procedure: 40 g of silica gel were mixed with 80 ml of 0.1 M potassium
dihydrogen phosphate solution (pH + 4.4) by constant stirring until the
slurry had a uniform consistency and was free from air bubbles. Then
glass plates 20 X 20 cm were coated by means of a Shandon spreader.
The plates were allowed to dry for 30 min and activated at 110’ C for
l- hr. After activation they were stored over a silica gel desiccant, A
solvent system of benzene-carbon tetrachloride-acetic acid (50:75:0.8)
was used (6).
Quantitative Analysis
A quantity of 0.1 ml of the sample solution was spotted zonally on
the plate by means of a microsyringe. Plates were developed in a
saturated Shandon chromatographic tank until the solvent front
ascended 12 cm from the spotting line. All developments were run in
a dark room and at a temperature of 23 + lo C.
Zones were located in short-wave (254 nm) uv light after the solvent
had evaporated and were removed separately and quantitatively with
the surrounding support into lo-ml stoppered ccntrifugc tubes. The re-
moved material was extracted in the following manner. After adding
exactly 4 ml of the appropriate solvent, the samples were mixed for 2
mitt, then centrifuged on a clinical centrifuge at 2,000 rpm for 10 min
and decanted into lo-mm cells. In all cases these extracts were optically
clear and they were measured spcctrophotometrically against their
solvents as a blank. By this procedure the final concentration of the
compound to be analyzed was equal to the spotted quantity dissolved
in 4 ml of the solvent. So, if x was the quantity spotted in micrograms,
then the final concentration was x/4 ppm.
RESULTS AND DISCUSSION
Quantitative Analysis of Quinizarin and Chrysazjn
The absorption spectra of a quinizarin solution, in solvent system A,
showed a we&defined peak at 484 nm with a molar extinction coefficient
 ANTHRAQUINONE DERIVATIVE ANALYSIS 657

of 7.8 x 10:’ liter mole-’ cm-*. A similar solution of chrysazin has an

absorption maximum at 433 nm with a molar extinction coefficient of
1.0 X IO’ liter molt’ cm ‘. These wavelengths were used for direct
absorbance measurements of quinizarin and chrysazin (separately).
Both systems conform to Beer’s law over the range studied. The
concentration limits for the final measurement in lo-mm cells, were
5-25 ppm for qunizarin and 4-20 ppm for chrysazin.
In the case of quinizarin and chrysazin the removed material from
the plates was extracted with the solvent system A [i.e., benzcne-chloro-
form-methanol (90:5:5)]. Solutions were measured against a solvent A
blank.
The concentration of each compound was calculated by means of a
calibration curve, constructed in the following way to increase the pre-
cision of the method. Two coated plates (20 X 20 cm) were divided in
three sections each. In one section, a zone of 0.1 ml of the unknown
solution which contained quinizarin or chrysazin or both, was spotted.
In this case the concentration of the unknown solution must be between
200 and 1000 ppm for quinizarin or 160 and 800 ppm for chrysazin.
In the rest of the five sections, zones of 0.1 ml quinizarin or chrysazin
refcrcncc solutions wcrc spotted. The concentrations of reference
solutions lay in the ranges of the above given limits. Both plates were
developed under the same exact conditions and after development the
absorbance measurements were pcrformcd as described above. In this
way a reference curve of five points was constructed and the concen-
tration value of the unknown was determined separately for quinizarin
and chrysazin.
Quantitative Analysis of Alizarin
The method described below is based on the information that ahzarin
gives a color reaction with alcoholic solution of 0.5 N potassium hy-
droxide (5). Actually an aqueous solution of 1 N sodium hydroxide
was used to produce the color reaction. Absorption spectra of the
formed blue-violet solution has an absorption maximum at 566 nm
with a molar extinction coefficient of 1.3 X 10’ liter molc-l’cm-l.
It was reported (3, I) that the stability of the color reaction of quini-
zarin with sodium hydroxide increases with increasing concentration of
sodium hydroxide. In *this case, it was found that a minimum of 2 N
concentration in sodium hydroxide is necessary to keep the color stable.
Beer’s law is obeyed from 3 to 15 ppm in 2 N sodiuin hydroxide
solution. Maximum color formation occurs after I min of agitation. A
reference curve was drawn by using solutions of alizarin from 120 .to
600 ppm and by spotting 0.1 ml on the plates as described above.
 658 VASILIKIOTIS AND ALEXAKI-TZPJANIDOU

Extraction of the removed material was done with 2 N sodium

hydroxide solution.
Quantitative Analysis of Anthraquinow
This determintaion was based on the information from the literature
(1,2) that when a solution of anthraquinone is heated with a reducing
agent, 9,lOdihydroxyanthracene @HA) is formed. The aqueous SOIU-
n.‘on of an alkali salt of DHA is slightly red but gives a deep red color
afte ‘t heating. Under these conditions it is easily reoxidized to anthra-
quino ‘Tie in contact with air. But if an excess of sodium dithionite
(Na,sg?J is added to this solution, oxidation of DHA is prevented.
Absort’tiOn spectra of a sobrtion of sodium salt of DHA has a maxi-
mum abs+cVtiOn at 418 IW with a molar extinction coefficient of
8.3 X lo3 hliter mole-l cm-‘.
Beer’s law El obeyed from 4 to 20 ppm in anthraquinone. A reference
curve was cons,‘ructed by using solutions of anthraquinone from 160
to 800 ppm and . by spotting zones with 9.1 ml. Extraction of rgmQved
material was done with a freshly prepared aqueous sofution of 10%
Na,S,O, in 1 N Na,QH. The extract was heated on a steam bath for
10 min. Maximum c *olor development occurred after 39 min and
spectrophotometric mea. surements were then taken,
In the course of this i. WeStigatiOn a great number of determinattions
have been carried out and thus considerable experience with the method
has been achieved. It was 1‘ound that the total amount of the applied
compound should vary between the given values. For smaller amounts
the error of determination is l’arge, and larger quantities cause tailing,
All operations must be performted with great care and therefore bright
illumination conditions, especidll,v direct sunlight, should be avoided.
Owing to the volatility of the solvent system A, it is necessary to work
as much as possible at temperatures ,?ot exceeding 20’ C.
In Table 1, results of five deterr.rinations are summarized. In this
case, an artificial mixture of aliza_*in, chrysazine, quinizarin, and
anthraqumone was used. One advanl’age of this procedure is that
all compounds are detectable under uv light and it is unnecessary to
use any other means of detection of zones.
Application of this procedure to irradiated samples of anthracenc
absorbed on aluminum oxide was performed as previously reported (6),
Dublicate samples of the so&urn eontaining the four possible phctto-
products were spotted. A prelinninaq q&native study of these samples
is recommended to adjust &besp&ted Wlume, in order to obtain wcll-
defined zones. Results were quite satisf&ory and work on photo-
oxidation conditions is in progress..
 ANTHRAQUINONE DERIVATIVE ANALYSIS 659

TABLE 1
DETERMINATION
OFANTHRAQUINONE DERIVATNESMIXTURE.YSEPARATED
BY
THIN LAYER CHROMATOGRAPHY a
Determination no. 1 2 3 4 5

Quantity in mirrograms spotted

Compound
(Xrnax,4 * Rj X 100 Fbwid

Alizarin 7 10.0‘
(566; 1.3 x 10%) -
10.2 10.3 9.1 IO,1 10.11

Anthraquinone 34 12.0
(418;8.3 x 103) -~-
13.0 11.8 11.5 12.4 12.6

Chrysazin 43 12.0
(433; 1.0 X 104)
12.1 11,4 12.3 12.1 11.9

Quinizarin 54 15.0
(484; 7.8 x I@) 7-
15.2 15.3 14.9 15.1 14.8

a Artificial mixtures.
bWavelength in nm; molar extinction coefficient (e) in liter mole-* cm-l.

SUMMARY

A method for the spectrophotometric determination of anthraquinone deriva-

tives, after separation on thin layers of silica gel, has been developed. These
derivatives are photo-oxidation products of anthracene and they may occur sepa-
rately, together, or in combination with other substances. Thus anthraquinone,
alizarin, chrysazin, and quinizarin, at microgram levels, can be analyzed quanti-
tatively.

ACKNOWLEDGMENT

The authors wish to thank Prof. E. Voyatzakis for his interest in this work and
for his suggestions.

REFERENCES

I. Feigl, F., and Jungreis, E., Detection of anthraquinone and anthracene in spot
test analysis. Microchem. J. 3, 213-214 (1959).
2. Feigl., F., Goldstein, D., and Haguenauer-Castro, D., Contributions to Organic
spot test analysis. Z. Anal. Chem. 178, 419-428 (1960).
3, Field, K., and Godly, E. W., The determination of quinizarin in hydrocarbon
oil. Analyst 91, ‘287-289 (1966).
660 VASILIKIOTIS AND ALEXAKI-TZIVANIDOU

4. Harrison, R. B., and Heysmau, L. T., Methods for the detection, determination
and identification of quinizarin in hydrocarbon oil. Analyst 86, 566569
(1961).
5. Masschelein-Kleiuer, L., Microanalysis of hydroxyquinones in red lakes.
Microchim. Acta. 1080-1085 (1967).
6. Voyatzakis, E., Vasilikiotis, G., and Alexaki-Tzivanidou, H., Separation of
some anthracene derivatives by thin layer chromatography. Anal. Lett. 5,,
445-449 (1972).