Indian Journal of Chemical Technology
Vol. 5, November 1998, pp. 383-386
Thin layer chromatographic separation and identification of some phenols
on calcium sulphate: Determination of phloroglucinol and resorcinol
Aditya K Misra··, Sri K Agarwalb, Subodh Kumarb
& Ram
P S Rajputb
·Chemistry Department, N.M.S.N. Dass (P.G.) College, Budaun 243 601, India
bChemistry Department, Bareilly College, Bareilly 243 005, India
Received 12 September 1997; accepted 25 August 1998
Thin layer chromatographic behaviour of thirty phenols on calcium sulphate has been studied in six
organic solvents. On the basis of Rfvalues, several importantbinary, ternary and quaternary separations of
phenols have also been achieved. A rapid, quantitative microgram separation of phloroglucinol and
resorcinol from synthetic mixtures containingmany phenols have been developed using chlorobenzene and
chloroform respectively.
Phenols have been known to impart resistance and
tolerance to plants against invasion by various
micro-organisms.
Soil amended with organic
matter is a usual practice for crop improvement
and control of diseases. As one of the mechanisms
of control by organic amendments, it has been
suggested that these phenols released during
.decomposition of organic matter are absorbed by
roots which might be imparting resistance or
tolerance. Some of the phenols are formed during
the decomposition of organic matter in soil\ and
may reduce plant growth possibly by inhibiting
nutrients uptake. They are also responsible for
inhibiting nitrification in soil. When they interact
with different components of the soil they might be
affecting their mobility. This is important when
organic matter is used for controlling diseases of
plants. A review was published by Roland2, on the
effect of phenols on human health, while
Brovillan~ and co-workers3 correlated the colour
of fruits and vegetables with molecular interactions
of phenolic compounds. Therefore, the separation,
identification and determination of phenols is of
great importance. On one hand, phenols were
separated on silica gel thin layer impregnated with
aluminium .and copper ions4 and ferric hexamine5
and on plain silica gel G6. The traces of phenols
and cresols have also been separated and identified
• Address for correspondence: 20, Saudagaran,
Bareilly 243 003 (UP), India
in contaminated
land leachates using planar
chromatography7. The phenols were also separated
quantitatively from the waste containing other
metals8 and from air samples9. Phenolic waste
water has been treated in RBC (rotating biological
contractor) reactorslO and the biodegradation of
phenolslJ has also been successfully carried out.
These studies have given us impetus to extend our
work on mechanism of movement of phenols in
soil in presence of organic waste. Because calcium
salts are also important components of various
soils and it was observed in earlier workl2 that
calcium soil is converted into ammonium soil with
the formation of calcium sulphate.
Rathore and coworkers
have successfully
utilized the calcium sulphate in two dimensional13,
sequential14,
reverse
phasel5,
thin
layer
chromatographic techniques for the separation of
carboxylic acids, herbicides16 and pesticides. The
steroids and lipids were also separated on calcium
sulphate thin layersl7•
In the present work, the movement of various
phenols of importance to conn-ol of diseases in
crop and fruit industry has been studied and
microquantities of phloroglucilJol and resorcinol
have been separated quantitatively.
Experimental Procedure
Apparatus
Thin layers of calcium sulphate were prepared
384
INDIAN ~. CHEM. TECHNOL., NOVEMBER
on glass plates of 15x3 cm size were subsequently
developed in various organic solvents 20x5 cm
glass jars.
Reagents
All chemicals and solvents used in this work
were of analytical grade (BD., Sarabhai, Merck or
E. Merck).
Test solution and detection reagents
0.01 M alcoholic solution of various phenols
were prepared and spotted on the TLC plates and
these spots were detected by spraying 2% FeCh,
20% titanium tetra chlorides in HCI and 4%
methanolic
thorium
diazotized sulphanilic
separately.
nitrate
(hexa
hydrate),
acid followed by NaOH
Solvent systems
The solvents used were: chloroform, acetone,
chlorobenzene, butanol, benzene, ethyl acetate, and
0.1 M ferric chloride.
Preparation of calcium sulphate thin layer plates
The calcium sulphate was sieved to obtain the
fine particles of uniform size and 70 mL of
deionised water was added to the 30 g of calcium
sulphate and slurried in a glass mortar, then spread
over the glass plates with the help of an applicator
to obtain 0.25 mm thick uniform layers and these
pfates were heated in an hot air oven at 110±5°C
for one hour to activate calcium sulphate. These
activated plates are spotted with phenols by a fine
glass capillary, and a paper strip about 2 cm wide,
moistened with corresponding organic solvent was
wrapped arou.nd the bottom of the plates. Plates
were developed in the different solvent systems.
After development, the spots were detected by
spraying the detection reagents and Rfvalues were
measured. In case of impregnated plates, the slurry
was made in 0.01 M ferric chloride solution in
place of demineralised water.
Procedure
For quantitative separation of resorcinol and
phloroglucinol, a stock solution of phloroglucinol
12.1 mg/mL and a stock solution of resorcinol 11.0
mg/mL was prepared in absolute alcohol. A known
amount of synthetic mixtures containing resorcinol
0.055 to 0.110 mg and phloroglucinol 0.0605 to
0.1210 mg was applied with the help of
d
I
'h
'I
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1998
micropipette on the line of application separately
and the plates were developed in chloroform and
chlorohenzene respectively. Pilot plates were run
simultaneously to locate the position of spots by
detecting with a suitable spraying reagent. The
areas corresponding to the location of resorcinol
and phl<;)foglucinol were scrapped out from the
TLC plates and the phloroglucinol and resorcinol
were eluted with small amounts of butanol,
suspended particles were filtered off and solutions
were diluted to 100 mL with butanol. Phloroglucinol
and
resorcinol
were
determined
spectrophotometrically
by dimethylaminobenzaldehyde method18, 2 mL of the sample solution, 2
mL of the reagent containing 0.5 mg of pdimethylaminobenzaldeh}'de
in 17 mL of 1:2
sulfuric acid. After 30 min read at 518 mil for
resorcinol and after 30 min at 550 mil for phloroglucinol.
Results and Discussion
The TLC results presented in this study
illustrate several important features. Table 1 gives
an account of Rf values of thirty phenol on
different coating in various solvent system while
the Tables 2 and 3 give an account of quantitative
work. These results depict the various possible
important and analytically
difficult
phenolie
separations
and separations
of phenols
of
agricultural importance, which make the crop
resistant to plant diseases but have harmful effect
on human health.
When calcium sulphate was used as an
adsorbent the mobility of phenols depends upon
the solvents used and their nature, i.e., metahydroxy function such as orcinol, phloroglucinol,
resorcinol etc. show gre~ter mobility than phenols
having ortho-dihydroxy function (catechol, gallic
acid, pyrogallol). Orcinol, phloroglucinol
and
resorcinol show higher movement in general which
could be due to the higher polarity and
intermolecular H-bonding. In rest of the cases
adsorption,
H-bonding and steric effect are
responsible.
Adsorption
on calcium su Iphate
dihydrate occurs mainly by dipole interactions and
by H-bonding to the calcium sulphate dihydrate
active sites on the surface, m-nitrophenol for
instance can interact with calcium sulphate with
either of two polar functional groups. The other
factors which are responsible for migration are
·"1'11
1"1"
,
"'1'1"
II
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II
1+II I
MISRA et al.: THIN LAYER CHROMATOGRAPHIC
Table I-Rfvalues
of 30 phenols on calcium sulphate thin layers
Phenols
3,4-diaminotoluol
o-aminophenol
Xylenol orange
p-nitrophenol
p-bromophenol
Phloroglucinol .
Catechol violet
Orcin monohydrate
Thymol blue
2,4-dinitrophenol
Resorcinol
Bromocresol green
a-naphthol
l3-naphthol
Bromopyrogallol red
Picric acid
1-(pyridyl-2-azo )naphthol-2
Quinol
Tannic acid
Thymol phthalein
Naphthalene
aminothiazole
Bromophenol
Quinhydrone
Gallic acid
blue
Pyrogallic acid
2,4,6-tribromophenol
m-aminophenol
VanilIine
a-nitroso l3-naphthol
p-aminophenol
Ethyl
acetate
0.86
0.94
0.91
0.92
0.98
0.97
0.92
0.95
0.94
0.96
0.96
0.91
0.95
0.96
0.96
0.00
0.97
0.67
0.65
0.79
0.95
0.88
0.15
0.06
0.17T
0.85
0.82
0.68
0.03
0.96
0.90
0.14
0.87
0.96
0.98
0.97
0.98
0.89
0.89
0.77
0.98
0.90
0.90
0.76
0.27T
0.85
0.97
0.97
0.87
0.97
0.87
0.95
0.17T
0.30
0.84
ND
0.47
0.46T
0.80
0.87
0.96
0.51
0.89
0.88
0.93
0.50LT
0.98
0.50LT
0.78
0.57
0.88
0.50LT
0.50LT
0.92
0.96
0.87
0.92
0.50LT
0.50LT
0.93
0.91
0.78
0.95
0.82
0.97
0.66
0.73
Chloroform
Coating "A"
Acetone
Ferric
chloride
0.78
0.93T
0.84
0.90
0.77
0.83
0.32
0.47
0.75
385
BEHAVIOUR OF PHENOLS
0.05
0.07
0.68
0.14
0.17T
0.85
0.44
0.90
0.83
0.87
Coating"B"
Chlorobenzene
Chlorobenzene
Butanol
Benzene
0.23
0.24
0.55
0.85
0.98
0.00
0.05
0.78
0.26
0.95
0.75
0.42
0.11
0.97
ND
ND
0.30
0.93
0.98
0.00
0.92
0.97
0.96
0.94
0.98
0.99
0.90
0.90
0.95
0.88
0.95
0.87
0.86
0.95
0.97
0.98
0.92
0.00
0.00
0.76
0.00
0.92
0.66
0.00
0.85
0.83
0.00
0.91
0.88
0.71
0.74
0.78
0.90
0.90
0.69
0.35T
0.84
0.00
0.66
0.93
0.94
0.82
0.89
0.99
0.77
0.05
0.75
0.94
0.96
0.96
0.99
0.33
0.86
0.00
0.77
0.14
0.90
0.00
0.80
0.87
0.36
0.909
0.93
0.85
0.99
0.94
0.95
0.85
0.90
0.46T
0.14
0.06
0.00
0.07
0.96
0.58
0.92
0.00
0.94
0.98
0.90
0.96
0.93
0.97
0.98
0.95
0.95
0.96
0.94
0.97
0.06
0.82
0.00
0.14
0.91
0.98
0.93
0.00
0.80
0.56T
0.87
0.85
0.00
0.00
0.93
0.92
0.66
0.00
0.00
0.50LT
0.88
0.08
0.75
0.78
ND - not detected; LT - Long tailing; T - Tailing
Coating A - Pure calcium sulphate; Coating B - Calcium sulphate impregnated with 0.1 M ferric chloride
Table 2--Quantitative separation of resorcinol on calcium sulphate thin layers
Mixture
0.1 mL of 10-2 solution of each of the
following phenols
Gallic acid, pyrogallic acid
m-aminophenol, p-bromophenol
thymol blue, phloroglucinol,
a-napthol, l3-naphthol, m-amino
phenol mixed with the resorcinol
(0.055 to 0.110 mg)
acidity or basicity of the phenols and solvation
effect. In butanol, all the phenols have higher Rf
values due to their high solubility in butanol and
cannot be· separated, in this solvent system,
although the separation mechanism is unknown.
On the basis of Rf values a large number of
Amount
Amount
found
Percentage
error
applied
mgxlO-2
mgxlO-2
5.50
5.50
5.50
11.00
11.00
5.48
5.54
5.27
10.70
10.70
-2.10
+0.90
11.00
11.29
+2.65
-4.10
-2.67
-1.90
interesting separations were achieved and some of
them are as follows .. Separation of catechol,
bromocresol green, bromophenol blue, bromopyrogallol
red from
18 phenols
including
resorcinol, p-bromophenol, 2,4,6-tribromophenol,
in chloroform. The o-aminophenol,
m-amino
386
INDIAN 1. CHEM. TECHNOL., NOVEMBER
Table ~uantitative
separation of phloroglucinol
Amount
Mixture
1998
on calcium sulphate thin layers
Amount
11.93
6.14
11.81
found
6.02
mgx10·2
Percentage
error
6.05
12.10
applied
mgxlO·2
12.10
6.05
0.1 mL of 10.2 solution of each of the
+1.62
-5.32
-2.32
following phenols
Orcinol, a-napthol, p-naphthol
tribromophenol, venilline
2,4-dinitrophenol were mixed with
phloroglucinol (0.0605 to 0.1210 mg)
phenol and p-aminophenol can be separated from
each other in chloroform. Resorcinol can be
-1.35
separations of analytical interest like m-aminophenol from p-aminophenol, o-aminophenol from
m-aminophenol, picric acid from 2,4-dinitrophenol
in chlorobenzene
solvent system. The unique
feature of these studies \\;'ere the quantitative
separation of resorcinol (55 to 110 J.lg) and
phloroglucinol (60.50 to 121 J.lg)in simple solvents
like chloroform and chlorobenzene within short
period.
separated from 18 pi1enols in chloroform. Bromophenol blue and bromocresol green can be
separated from remaining 28 phenols in acetone
solvent system. Some phenols of agricultural
importance such as phloroglucinol, catechol and
pyrogallol can be separated from other phenols in
chlorobenzene.
In butanol, no separation was
achieved due to higher Rf values of phenol.
phloroglucinol,
catechol violet, thymol blue, References
bromopyrogallol red, bromocresol green, tannic
I Strack D, in Plant Biochemistry, edited by Dey P M;
acid, galiic acid, a-nitroso, (3-naphthol show no
Harbomee J B (Academic Press, London, UK), 1997,387.
2 Roland B, Naturwiss Rundsch, 49(1996) 47.
movements perhaps due to the nonpolarity of
3 Brovillard R, Figueiredo P, Elthakiri H & Dangles 0,
benzene or due to the adsorption on calcium
Proc. Phytochem Soc Evr, 41(1997) 29.
sulphate in benzene. Bromopyrogallol red from
4 Petrovic M, Kastelan-Macan
M & Horvat A J M, J
remaining 26 phenols can be separated in ethyl
Chromatogr, 607 (1992) 163.
acetate. Amino phenols are weakly acidic than
5 Singh D K & Misra A J Ltq Chromatogr, 15 (1992) 369.
6 Ferry J & Larson R A, J Chromatogr Sci, 29 (1991) 476.
phenol owing the inhibited resonance of the
7 Fulter J E & Wall P. J Planar Chromatog Mod TLC,
hydroxyl group with the benzene ring caused by
6(1993),372.
the amino group. An amino group in the ortho or
8 Schewedt G, Ger D E 19, 520, 289 (C. A62D3/00) 6 Feb.
para position has a much greater inductive effect
1997, Appl. 19,520,289,2
June 1995,4.
than one in the meta position with the exception to
9 Bartulewicz J, Bartulewicz E, Gawlowski J & Niedzielski
J, Chem Anal (Warsaw). 41 (1996) 939.
this the meta and para amino phenols can be
10 Banerjee G, Water Res, 31 (1997), 705.
successfully separated in aqueous ferric chloride
11 Rao P S, Mohan V, Murthy D V S, indian J Chem
solvent system on calcium sulphate thin layers.
Technol. 4(1997) 45.
While para amino phenol shows lowest Rf values 12 Misra A K, Synthesis and ion exchange applications of
some new inorganic ion exchangers.
Ph.D. Thesis.
in this solvent system. Orcin monohydrate, thymol
Rohilkhand University (UP), India, 1988, p.26.
blue, bromocresol green, a-naphthol, 1-(pyridyl-2
13 Rathore, H S & Gupta S, J Ltq Chromatogr, 10 (1987)
azo) naphthol-2, quinhydron gallic acid and meta
3619.
amino phenol shows decrease in Rf values in this 14 Rathore H S & Saxena S K, J Ltq Chromatogr, 10 (1987)
3623.
solvent system and can be separated from other
15 Rathore H S & Saxena S K, int J Environ Anal Chem, 33
phenols. The coating 'B' gives good results in
(1988) 209.
chlorobenzene
solvent system and orthoamino
16 Rathore H S, Ali I, Gupta S & Begum T, J Planar
phenol, bromopyrogallol red, gallic acid, a-nitroso
Chromatogr ModTLC, 2 (1989) 119.
(Reinhold
(3-naphthol-2 and tannic acid shows no movement 17 80bbit J M, Thin layer chromatography
Publishing
Corporation,
Chapman
Hall
Ltd,
London),
due to the formation of complexes with ferric ion.
1963.
In general, the Rf values are lower on the coating 18 Snell F D & Snell C T, Colorimetric method of analysis.
'B' in comparison to the Rfvalues on pure calcium
Vol. 3A (D Van, Nostrand Company Inc., Princiton, New
Jersey), 1961, 109 .
sulphate which causes a large number of
.,
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II
1"1" , I'I!I 'II'!I
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