NOTES
Reagents for the detection of antioxidants
239
on thin
layers of siliea
Antioxidants
are used in foodstuffs and plastic materials to prevent autoxiclation. Because only a few antioxidants
are permitted in foodstuffs and food packaging
materials, methods for their detection became necessary. Usually the antioxidants
are
extracted
from the food or plastic with su.itable solvents, followed by separation on
thin layers of silica gel l-s. The detection and identification
may be hindered by the
fact that the reagents used are not specific for antioxidants:
they may also produce
coloured spots with other extracted substances or they may not react with all antioxidants. Most investigators
therefore use a range of reagents,
In this paper some reagents are suggested and in Table I the colours obtained with reagents that produce different colours with different antioxidants
are
listed for a number of antioxidants.
In these cases 20 pg of antioxidant
were spotted
and the silica-covered
chromatoplate
was sprayed after elution. (For solvents see refs.
3 ancl 5.) The colours may be different when other quantities of antioxidant
are used.
Detection wage&s
Reagents which can be used for the detection of antioxidants
can be divided
into groups as follotis :
(A) Chemicals which procluce,colours when reduced; since antioxidants
are used
to prevent oxidation they are reducing substances.
(B) Substances
which can be coupled to phenols (many antioxidants
are substtiutecl phenols) : (I) diazo-compounds;
(2) aromatic
aldehydes;
(3) Gibbs reagent,
tihich forms indophenols that form coloured salts with alkali.
(C) Stable free radicals, which accept a hydrogen radical from the antioxidant.
(D) Substances that form coloured addition compounds.
(1) Potassium fwmnan~a~zate. Potassium permanganate
produces yellow spots on
a pink background with all oxidisable substances. It is therefore not specific for antioxidants.
It is prepared by dissolving a few grains of potassium permanganate
in IO ml
acetone.
(2) Fel~ric-fevricynlzide.
A mixture of ferric sulphate and ferricyanide
is turned
into Prussian blue by reducing agents. It is specific for antioxidants
but gives the
same blue colour with nearly all of them. .A disadvantage
is that some time after
spraying the whole chromatoplate
turns blue.
It is prepared by mixing one volume of an 0.5 y0 ferric sulphate (anhydrous)
solution in 1 N W,SO,, with one volume of an 0.2 o/o aqueous solution of potassium
ferricyanide.
(3) Di&&dyZ. BURTONO
suggested a modification
of type of reaction with the
ferric-ferricyanide
reagent by using dipyridyl, which gives a permanent white background.
The spots with all antioxidants
are red-brown.
One volume of an 0.5 y. ferric sulphate (anhydrous) solution is mixed with one
volume of an 0.5 O/~
solution of ti,a-dipyridyl in methanol.
(4) phc@hcmclybdic
acid. This reagent develops blue spots with reducing agents:
J. Chvontalog., 24 (IgGG) 239-243
v
N
2
WITH
ANTIOXIDANT’S
2
ochre
grey
brown
brown
light purple pink
yellow
blue
blue
blue
blue
purple
-
violet
brick red
orange
blue
brown
violet
buff
brown
rustbrown
brown
green
blue
green
rustbrown
blue
redbrown
green
ochre
violet
blue
yellow
bluish
violet
violet
pink
greY
greY
ycllo\v
yellow
bluish
brown
brown
brown
purple
brown
brown
greY
purple
brown
9
WITH
greY
red
purple
-
brown
-
rustbrown claret
rustbrown
brown
brown
ochre
-
pink
pink
pink
pink
pink
pink
pink
Purple
light purple purple
yellow
yellow
yellow
rustbrown
orange
blue
blue
blue
blue
blue
chocolate
orange
brick red
redbrown
5
4
RX1
S
AFTER SPRAYIKG
7
CHROMATOPLWES
6
ON SILICA COVERED
CoEorcvwith reagent mrrnber
DIFFERENT
Propyl gallate
blue
blue
Octyl gallate
blue
Dodecyi gallate
Nordihydroguaiaretic acid blue
Gum guaiacblue
a.l,s-Trihydrorybntbrophenone
blue
Stearoyl-$-aminophenol bble
4.4’-Butylene-bii-(6-tevt.butyl-3-methylphenol) blue
+q’-Thio-bis-( r64ert.butyl-3-methylphenol) blue
z,z?‘-Dihydroay-x,3’-dicyclohe2tyl_5’5_dime_yl-~‘~-dimethyldiphenylmethane
blue
Dilauryl thiodipropionate blue*
:
Butvlidene-z.a-bis(&tyllthio&ycolate)
blue”
Diphenyl-p-phenylenediaminc
blue
2-Naphthytp-phenylencdiamine
blue
z-lerf.-Butyl-q-hydroxyanisole
blue
q,q’-Cyclohexylidene-bis(2-cycl$exylphenol)
blue”
Anticxidaut
COLOURS OBTAINED
TABLE I
chpcolate
pink
-
grey
blue
grcy
-
black
-
broitn
violet
chocolate
ochre
brown
brown
brown
chocolate
chocolate
I2
brownish
ochre
yellow
yellow
yellow
rustbrown
pink
brown
-
.
REAGENTS
brown
!PY
WY
WY
fPY
II
DIFFERENT
-
$
2
-&
ti
w
2
_
g
a
i
‘Iy
cl
*
blue
blue
blue
blue
violet
buff
buff
blue
blue
blue
yellow
Purple
bluish purple
ochre
yellow
pink
claret
orange
pink
yellow
yellow
blue
blue
yellow
yellow
-
purple
blue
i
brick red
yellow
rustbrown
yellow
blue
blue
blue
green
blue
ochre
blue
blue
brown
yellow
rustbrown
blue
blue
violet
violet
ochre
-
blue
orange
purple
yellow
-
blue
brown
violet
orange
blue
blue*
l Colour develops only after heating the chromatoplate.
*l Mixed colour of green, brown and pink.
u-Octadecyl /I-(4’-hydroxy-3,3-di-ferYt.-(butylbenzene)-propionate
Tris-(nonyl-phenyl)phosphite
Di-octylphenylphosphite
z,z’-Methylene-bis-[6-(zmethylcyclohexyl)-4methylphenol]
tyl-m-cresol)-r,3,3crotonaldehyde
z-Hydroxy-q-alkylbenzophenones
2-(z’-Hydroxy-5’-alkylphenyl)-benzotriazoles
Phenyl?x-naphthylamine
Phenyl$-naphthylamine
z,z’-Methylene-bis-(qmethyl-6-M.-butylphenol)
z,z’-Thio-bis-(+-methyl6-tert.-butylphenol)
2.6Di-buf.-butylphenol
a,6-Di-tevt.-butylq-methylphenol
a,a’-Propylene-bis(+4’,6.6’-fert.-butylphenol)
r,3,5-Trimethyl-z,4,6tris-(3.5-di-tert.-butyl;hYdrYbenzYl)-
4,4’, 4”-7rk.c(6-ht.-bu-
purple
pink
pink
violet
violet
buti
purple
brown
Pm@
green
yellow
yellow
yellow
yellom
violet
buff
pink
pink
brown
brown
brown
brown
purple’
blue
-
red
Pm-+
brown
-
-
WY
brown
brown
brown
grey
grev
brownish violet -
buff
blue
blue
bluish grey
grey *l
bluish
grey
pink
violet
brown
violet
brown
brown
red
violet greenish
blue
yellow
green
rustbrown
yellow
light orange
yellow
ochre with blue
blue
rim
brown
greenish
yellow
pink
yellow
gre
Y
brown
brown
violet
*. .
I-
E
242
NOTES
it is not specific for antioxidants
and though the colour may differ from purple to
green-blue for various substances it generally gives dark blue spots.
zo g of phosphomolybdic
acid are dissolved in go 0/Oethanol. The plate is heated
for 10 min at 105O.
Groau$ B(r)
(5) Dinzo-reage?zt. Diazotized $-nitroaniline has been used for a long time for the
detection of aromatic compounds; with antioxidants it forms yellow to brown spots.
Soo mg $-nitroaniline
are dissolved in a mixture of 250 ml water and 20 ml
hydrochloric
acid (25 “/o). A 5 % sodium nitrite solution is added dropwise until the
solution is colourless.
(6) Red snlt. Some typical colours are obtained with some types of antioxidant
when red salt is used, e.g. orange spots are given by benzophenone derivatives that are
used as U.V. absorbers’ in plastics and claret spots by phosphites.
It is prepared by dissolving successively in IO ml water zoo mg sodium acetate
and 200 mg red salt A.L. (C.I. 37275).
B (2)
Coupling of antioxidants with aromatic aldehydes has the disadvantage that the
colour is not very reproducible, but it may be useful because different bright colours
can be obtained.
(7) A&saZdehyde. This is prepared by dissolving 500 mg p-methoxybenzaldehyde
(anisaldehyde) in a mixture of IO ml glacial acetic acid and 55 ml methanol and adding
5 ml concentrated sulphuric acid.
The plate is heated for IO min at 105’.
(8) V~CWX%Z.
400mg vanillin are dissolved in a mixture of 95 ml methanol and
5 ml concentrated sulphuric acid. The plate is heated for IO min at 105~.
Grou$
Groq?~ B (3)
(9) Gibbs reagent. Though it is not specific for antioxidants it develops very typical colours, particularly with some antioxidants which do not give specific colours with
other reagents (e.g. butylhydroxytoluene).
It consists of IOO mg z,G-dichloro-+benzoquinone-+chlorimine
in IOO ml ethanol. Spraying of the chromatoplate with this solution is followed_by a spray of a 2 O/~
borax solution in 50 yO ethanol.
Groz@ C
(IO) ~,&-Di~1aelayZ-~-Ihicryllzydya~~vZ.WOGGON et al. 4 described this reagent for the
quantitative
determination
of antioxidants
on chromatoplates:
the violet-coloured
stable free radical a,a-diphenyl-/?-picrylhydrazyl
is decolorised stoichiometrically
by
antioxidants. All the spots are yellow against a deep violet background.
It is prepared by dissolving IOO mg a,cc-diphenyl-P-picrylhydrazyl
in IOO ml
g6 % ethanol.
Groau$ D
(XI) Palladizts~z chloride. Palladium
chloride forms addition
many aromatic compounds, the colours of which may differ.
J. Chromalog.,
24 (IgGG) 239-243
compounds
with
243
NOTES
ISO mg palladium chloride are dissolved in 100 ml 0.2 .iV hydrochloric
acid.
(12) RmSvzomy &mhzcJzZoride. 20 ml antimony pentachloride are mixed with 80 ml
carbon tetrachloride. The plate is heated for IO min at 105’.
Dcj5nmaent
Uk.wsity
of Food Claemistry,
of Utrecht (TJze Netlaerlands)
R. P. VAN
DER
%hDE*
Ii. 1-I.CAMPBELL
AND IX. TN. \VISI~:,
J. Chromalog.,
12 (1963) 17s.
R. F. VAN DER I-IEIDE, Thesis, UlxXChC, 1964, pp. IoI-III.
E-1.
WOCGON,0. KORN AND D. JENLE, Nnlzyc~?~g.
g (1965) 4g5.
R. P. VAN DER HIZIDE, A. C. MAAGDIZNRIZRG
AND J. I-I. VAN DIZRNIUJT, Cl&er?z.
Weclcblad,
(196.9 440.
6 G. M. BARTON, J. Chromatog..
20 (x965) ISg.
7 E. KNAPPE.
D. PETERI AND I. ROI-IDE.WALD.Z. Anal. Ch.wt., 197 (1943) 3G4.
S J. I-I. VAN DER NEUT AND A. C. MAAGDENBIZRG.
Plnslics (Lo&ogz). 31 (1966) 66,
2
3
4
5
Received
Or
March ~st, 1966
* Present: acldrcss: Esso Rcscarcl~ SR., Zavcntem,
BcQium.
1. C?UOH%atO~. 24 (IgG6)
The
cytogenetics
XII.
Thin-layer
compounds
239-243
of Lotus
chromatography
in the
separation
of secondary
phenolic
in Lotus (Leguminosae)
The successful separation of secondary phenolic compounds in Lo&s through the
use of the Shandon thin-layer chromatographic
equipment and silica gel G as the
coating material has recently been reported by GRANT AND WHIZTTER’. The availability of commercially prepared coated plates would eliminate the initial time needed to
learn the technique required in order to ‘obtain a satisfactory coating on the plates as
well as the time required for the messy preparation of the silica gel coating on the
plates. This note reports the results obtained in the separation of secondary phenolic
compounds in Lotzts using the techniques reported in the earlier paper by GRANT AND
WWETTER~ but using prepared Eastman Chromagram sheets, Type K301R with a
fluorescent indicator, and the Eastman Chromagram Developing Apparatus (Eastman
Organic Chemicals, Distillation Products Industries, Rochester, New York).
tests
Samples of fresh leaves of Lotzbs were prepared by weighing out 0.08 g and
leaving them in 0.5 ml of I y0 hydrochloric acid in methanol at room temperature, in
“T’ the dark, overnight. The plates were prepared for development by applying an approximately
7 ~1 spot of sample solution with a micropipette at a distance of 2.0 cm
from the base. Two spots were run for each sample. Ascending development was carried
Preliminary
J. ~b’ol?tatog.,
24 (IgGG) 243-244