Evaluation of Carbonyl Compounds

in Natural Products by o-Dianisidine

2008, 68, 447451

Mohamed Abou-Shoer&
Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt; E-Mail: aboushoerm@yahoo.com

Received: 25 February 2008 / Revised: 13 May 2008 / Accepted: 21 May 2008

Online publication: 15 July 2008

Abstract
A simple sensitive and rapid method is herein described for the qualitative and quantitative
chemical assessment of the fractional composition of carbonyl functionalities in natural
products. Several extracts, essential oils, fixed oils, oleoresins and pure natural isolates were
directly analyzed by applying a simple colorimetric procedure, by LC or DE-TLC using
o-dianisidine as a chromogenic reagent. The reagent is found to be very useful and could
also be used in the microscopic examination of powdered herbal products.

Keywords
Column liquid chromatography
Thin layer chromatography
o-Dianisidine
Carbonyl compounds assay
Essential oil and fixed oils

Introduction
Extracts, exudates and other natural
products or herbal materials like gums
and oils are intricate blends of many different chemical entities. Essential oils, in
particular, are naturally composed of unoxygenated or oxygenated derivatives of
terpenes (which include alcohols, aldehydes, esters, ketones, acids and oxides),
benzenoids and volatile phenylpropenoids such as vanillin and eugenol,

Full Short Communication

DOI: 10.1365/s10337-008-0708-1
0009-5893/08/09

C5-branched compounds, and various

nitrogen and sulfur containing compounds [1]. As it happens, the chemical
composition of the same essential oil,
especially if coming from dierent suppliers or dierent species, can noticeably
vary with changes in the growing and
harvesting conditions of the parent plant
(origin; location, soil and climatic conditions), extraction procedure, rening
techniques, and storage conditions.
Moreover, some encounters of poor-

quality oils are often experienced because

of adulteration [2]. Accordingly, quality
requirement poses as an inevitable need
for chemical analysis for tagging and
traceability of the oils as long as determination of the percent of a specic constituent might not be the sole parameter in
assessing quality in quality control labs.
Typically, assessing or verifying the
purity of essential oils includes measuring some physicochemical parameters
such as relative density, ester value or
optical rotation power. Simple chromatographic techniques are pretty helpful in many occasions, yet, there are a
few rigorous techniques for analyzing
essential oil composition and its purity.
Conventionally, GC, especially GC-MS,
is the most frequently used technique for
analyzing essential oil composition [3].
LC is another versatile technique that
has also found its way in this arena [4, 5].
The quality of essential oils was also
veried rapidly and non-destructively by
several vibrational spectroscopic methods such as ATR-IR, NIR and Raman
spectroscopy [6].
Carbonyl compounds, aldehydes or
ketones, are key chemical entities that
alter the aroma prole or the olfactory
properties of oils. The aldehydes geranial
and neral (citral) confer citrus oils its
lemony aroma and carvone gives caraway its distinct avor. The presence of a
nite amount of cinnamaldehyde in

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2008 Vieweg+Teubner | GWV Fachverlage GmbH

447

cinnamon oil labels and describes the

chemistry and the avor of such oil [7].
Analysis of essential oils for its content of carbonyl compounds, like aldehydes and ketones, still run into the
lengthy classical titremetric methods of
analysis or the advanced GC techniques.
The BP uses the general spray reagents,
anisaldehyde and phloroglucinol, for
revealing the spots of cinnamaldehyde
on TLC plates. Dill oil is assayed for its
content of carvone by hydroxylamine
hydrochloride [8].
The role and signicance of the presence carbonyl compounds in xed oils is
entirely dierent. Measuring the amount
of carbonyl compounds in oils and fat is
indicative of rancidity and progressive
breakdown. Frequently, p-anisidine was
earlier used for evaluating the degree of
rancidity in essential oils [9, 10].
Herein, o-dianisidine (ODA) is one of
several other reagents that were explored
and tested for their scope and aptness as
analytical reagents for assessing carbonyl compounds. Eventually, ODA has
opened up a variety of new capacities in
analysis.

Experimental
Apparatus
A Shimadzu UV 1601PC, UVvis double beam spectrophotometer (Kyoto,
Japan) was used equipped with a 1 cm
quartz cells and connected to an IBM
compatible computer. The software was
UVPC personal spectroscopy software
version 3.7 (Shimadzu). The spectral
width applied was set at 2 nm.
LC experiments were run on a Shimadzu LC system equipped with a model
LC-10 ADVP pump, DGU-12-A degasser, Rheodyne 7725i injector, a 20 lL
loop, SPD-10 AVP UVvis detector and
SCL-10 AVP system controller. Separations were by a 250 9 4.6 mm generation
RP column (5 lm particle size). Linomat
CAMAG TLC sample applicator was
used in TLC analysis and TLC photos
were captured by a digital camera, Fuji
Fie PIX S9600, 9 M Pixels and 10.7X
optical zoom. Powdered plant material
were investigated by a normal light
microscope.

448

Reagents
o-Dianisidine (3,3-dimethyoxybenzidine,
E. Merck Darmstadt, Germany, oil of
citron, caraway, anise, cumin, parsley,
fennel, rose were from Grasse-Argenteuil
(France), eucalyptus and lavender, cinnamon, clove, nutmeg and rosemary
from Johs Thoms (Hamburg, Germany).
Olive oil, nigella seed oil and cotton seed
oil are commercial grade from the local
market, TLC plates are silica gel
(E. Merck) 0.2 mm in thickness.

methylene chloride (ca 20 mg) were

treated as the standard solutions.
Commercial samples of vitamin A
(ca. 50 mg) were dissolved in 5 mL of
hexane and added to 5 mL of 0.5% ODA
solution in 80% formic acid in a separating funnel. The red-colored acidic
layer is measured against retinal reference solution at 530 nm while the organic
layer containing retinol was also evaluated against reference retinol at 400 nm.

Calibration Graphs
Colorimetric Analysis
Determination of the
Aldehydes or Ketones Content
Standard Aldehyde Solutions

An accurately weighed quantity (2530

mg) of vanillin, cinnamaldehyde, salicylaldehyde, benzaldehyde, p-dimethylaminobenzaldehyde, retinal and anisaldehyde
were placed in 25 mL volumetric asks
and dissolved to volume in methylene
chloride.
ODA Reagent Solution

0.125 g of ODA was dissolved in methylene chloride in 25 mL volumetric ask.

Reaction and Conditions

Volumes of 0.53 mL were sampled

from each solution into 250 mL measuring asks. To each ask, 1 mL of
acetic acid and 1 mL of ODA solution
were added. The mixture was allowed to
stand for 1530 min with occasional
shaking and then completed to volume
with methanol. Careful warming of the
solutions was found to accelerate the
color development and completion of
the reaction. The intensity of developed
color was measured at its recorded kmax.

Several aliquots from the standard

solutions (0.53 mL) were transferred to
50 mL volumetric asks, 1 mL of ODA
reagent solution and 1 mL acetic acid
were added with occasional shaking
during the specied time. The reaction
asks are completed to volume with
methanol. One millilitre from the colored solution is diluted to 10 mL with
methanol and the solution is measured
spectrophotometrically at the kmax nm
(Fig. 1).

LC
The elution was carried out with acetonitrilewater (40/60 v/v). The ow rate
was 1 mL min-1. All measurements
were performed at room temperature
and the injection volume was 20 lL.
Reference and commercial samples
(ca. 1 mg of cinnamon oil, cinnamaldehyde, benzaldehyde and bitter almond
oil) were dissolved in 10 mL of the mobile phase then the solution was used for
injection. Alternatively, 2 mg of ODA
solution were added to 1 mg of the oil in
methylene chloride. The solution was
acidied with 0.5 mL glacial acetic acid
and the CH2Cl2 was carefully vaporized
and the developed colored solution is
completed to volume as the reference
and used for injection.

Sample Preparation

An accurately weighed quantity of cinnamon, clove, bitter almond oils, commercial samples of vitamin A and benzyl
alcohol together with articial solutions
of vanillin, salicylaldehyde, anisaldehyde
and p-dimethylaminobenzaldehyde in

LC Applications
Detection of Benzaldehyde
in Benzyl Alcohol

Benzyl alcohol was used for the LC

measurements. Ten milligrams of each of

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the sample and reference benzyl alcohol

were transferred into a 100 mL volumetric ask. One millilitre of ODA reagent is added to each solution. The
mixture was acidied by 2 mL glacial
acetic acid. The solution is allowed to
stand for 15 min with occasional shaking
then completed to volume with the mobile phase, mixed well and used for
injection.
Similarly, the same techniques were
used to monitor dierent dill oil samples
for its content of carvone and commercial guggulsterones from guggul samples.

TLC Analysis

Fig. 1. Calibration curves, colorimetric, for cinnamon oil (cinam), cinnamaldehyde (cinamald),
vanillin (van), dill oil (dill) and benzalldehyde (benz)

Table 1. Natural products tested with ODA

ODA Spray Reagent

Pure compounds

The TLC spray reagent is prepared by

dissolving 1 g of ODA in 95 mL MeOH,
2 mL concentrated HCl and 1 mL glacial acetic acid. The reagent is stable in
dark bottles and cool plate for several
days.

Essential oils

Volatile Oils and Samples

About 10 mg of the standard or reference oil and sample to be analyzed are
dissolved in 5 mL of CH2Cl2. In parallel,
10 mg of the sample oil are mixed with
20 mg of ODA and dissolved in 2 mL
CH2Cl2 and the solution is acidied with
two drops acetic acid. Both solutions
were analyzed by TLC, injection volume
510 lL volume using toluene: ethyl
acetate (9:1) as the mobile phase. The
plates were then sprayed by ODA spray
reagent and heated in an oven at 100 C
for 2 min. The developed orange-red
colored spots on the chromatograms
were evaluated by DE TLC Videodensitometry (Sorbpolymer JSC, Krasnodar,
Russian Federation.) and quantitatively
analyzed [11].
Microscopy

Powdered cinnamon bark were mounted

in chloral hydrate solution and analyzed
by the ordinary light microscope. The
mount was then stained with the ODA
solution with one drop of HCl reexamined under the microscope. Secretory

Full Short Communication

Oleoresins
Phenols and
miscellaneous
Fixed oils

Anisaldehyde, benzaldehyde, cinnamaldehyde,

p-aminobenzaldehyde, salicyladehyde, vanillin,
geranial, neral (citral), carvone, santonin,
khellin, orcinol, phloroglycinol, retinal,
phytomenadione
Anise, black cumin (Nigella), caraway, cardamom,
chamomile, cloves, coriander, cinnamon, cumin,
dill, fennel, ginger, lavender, rosemary, rose,
lemon, lavender, orange, marjoram, wintergreen,
nutmeg, parsley, thyme, sassafras, oil of citron
Olibanum, ginger oleoresin, guggul gum
Some avones, methyl salicylate, eugenol,
silymarin, thymol, salicin
Nigella, cotton seed oil, cardamom seed oil, olive oils

tissues containing the oil were stained

deeply red and easily identied.

Results
ODA is found to be very reactive to
many of the tested essential oils and
other natural products producing yellow
to intense cherry reddish coloration.
Some other oils were neutral to the reagent or may develop very faint or dark
coloration by time. Experiments carried
out on over 50 chemical items, covering
single compounds and complex natural
mixtures of natural origin like essential
oils, have revealed that the chromogenic
reaction with ODA varies depending on
the nature of the involved carbonyl
(Tables 1, 2). The reaction with ODA is
more instant and intense with di-unsaturated aldehydes than with monounsaturated aldehydes, which are themselves more sensitive than saturated
aldehydes. Cinnamaldehyde and salicylaldehyde were found to be very reactive

Table 2. Absorption maxima for the colors

developed with ODA
Sample

Product (color) kmax

Anisaldehyde
Anthraquinone
Benzaldehyde
Caraway oil
Cardamom
Cinnamaldehyde
Cloves
Coriander oil
Cotton oil
Dill oil
Eugenol
Ginger
Khellin
Nutmeg oil
Oleobanum
Reserpine
Salicylaldehyde
Santonin
Sassafras oil
Vanillin
Vitamin A
(retinal)

470
No change
480
470
440480
510
470
406
466488
392
470
480
No change
440477
430456
400477
533
No change
452
450470
530

as compared to citral and carvone. The

concentrations chosen ranged from 1 to
8 mg mL-1 for benzaldehyde, cinna-

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449

Discussion

Fig. 2. DE-TLC analysis of ginger, cinnamon and clove oils

maldehyde, bitter almond oil, cinnamon

oil, vanillin and anisaldehyde. Furthermore, ODA was found to produce a
deep reddish color with xed oils like
virgin olive oil and cotton oil; which is
most probably due to the presence of
aromatic carbonyl compounds (decarboxymethyl oleuropein dialdehyde and
gossypol respectively).
Under the presented experimental
conditions, the plots of absorbances for
the colored products developed from the
reaction of ODA with vanillin, cinnamaldehyde, anisaldehyde, benzaldehyde
and salicylaldehyde, versus dierent
sample concentrations showed a linear
relationship. The linearity of the data
was evaluated by measuring a series of
dierent concentrations of the analyte. A
minimum of six concentrations were
analyzed and each concentration was
repeated three times.
Very distinctive intense reddish
orange spots were obtained on TLC
plates developed for eugenol, retinal,
salicylaldehyde, ginger oleoresin and
olibanum. Oil of citron, cardamom oil
and caraway oil have developed bright
colored spots but not as intense as those
produced with cinnamaldehyde. In
addition, unexpectedly, clove oil and
eugenol was found to be also very
reactive with ODA with the instant
development of deep cherry-red color.
Samples of cinnamon oil, ginger and
dill oil were quantitatively analyzed by
TLC, using calibration plots prepared

450

from serial concentrations, after spraying the plates with ODA. ODA produced chromatograms with clear and
discrete spots compared to those produced by UV-quenching or through
spraying the plates with anisaldehydesulfuric SP (Fig. 1). Calibration curves
obtained by ODA are much steeper and
better intercepts.
The slope of calibration curves is
expected to vary according to the chromophoric nature of the analyte; yet, the
intensities of the produced colors are
evidently in the range applicable to many
pharmaceutical products and raw materials. The real gain was reected on as a
very much lower sample size was needed
for the analysis when compared to other
chemical methods like the hydroxyl
amine method and still also practical to
other sensitive methods like GC and LC.
The S.D. deviation and of the measurements and the slope were the guide for
evaluating the detection and quantitation limits for each analyte.
The ODA sensitivity and selectivity
values are evident when very small
quantities of benzaldehyde in benzyl
alcohol was easily measured colorimetrically by ODA without resorting to LC.
Nevertheless, LC chromatograms for the
analyzed sample of benzyl alcohol for its
content of benzaldehyde after derivatization with ODA retained the alcohol
peak with complete absence of the aldehyde peak as it has been transformed to
the ODA derivative.

The reaction of ODA with saturated or

unsaturated aldehydes and ketones has
instantly produced very bright and intense colors. This has triggered developing ODA as a powerful derivatizing
reagent for boosting photometric absorbance and selectivity in the analysis of
carbonyl compounds in natural products. The developed reaction brings a
substantial improvement in the sensitivity in detecting carbonyl functionalities
in an economic fashion in routine quality
assurance exercises.
The high value of the correlation
coecient and the intercept value were
used to evaluate the linearity of the calibration curves. Regression analysis of
these plots using the method of least
squares had produced correlation coefcients (r) equal to 0.99930.9999 indicating a good linearity (Fig. 2). The
sensitivity of the method to dierent
analytes is evident from the slope and
intercept values in the calibration curves.
Extended chromophores, like in cinnamaldehyde, possessed a steeper line.
The enhanced sensitivity associated
with the use of ODA as a derivatizing
agent, qualitative and quantitative
assessment of the purity of some essential oils, was evident through using only
a few milligrams of cinnamon oil to assess the percentage of cinnamaldehyde
colorimetrically.
As a TLC spray agent, ODA proved
to be very successful in identifying carbonyl compounds in natural products
extracts. ODA is found to be sensitive
and economical as analytical tool or
technique. Scarcely conjugated carbonyls like guggulsterones and carvone are
easily detected in LC. Aromatic or conjugated aldehydes and ketones, and
some phenolics were among the tested
natural products and were found to be
strikingly reactive with ODA.
ODA reacted also well with cotton
oil, olive oil and with the unusual
essential and the xed oils of Nigella
sativa seed (Ranunculaceae family) and
produced deep colors. TLC chromatograms for the xed and essential oils of
Nigella, using ODA and anisaldehyde
sulfuric as spray reagents, were selec-

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tively dierent in identifying their composition. The dark red color produced
by the reaction of ODA with retinal was
very useful in assessing the fraction of
retinal produced by time while storing
retinol (vitamin A). Accordingly, the
reaction appears extremely useful in
stability studies to measure shelf-life
decomposition or oxidation products of
phytopharmaceuticals.

expensive instruments or lengthy techniques at early stages of analysis is

easily performed with ODA. The reagent smoothly adds extra chromophore for feasible selective colorimetric
assessment of these entities as colored
derivatives. In the light of the performed measurements, the reagent is
also qualied to be used as an LC precolumn derivatizing reagent and as pre
or post-solvent-developing TLC visualizing reagent.

Conclusions
The use of ODA for analysis of carbonyl compounds proers several
advantages. ODA introduces a simple
direct colorimetric technique to analyze
or assess the quality of essential oils,
xed oils or crude natural extracts for
its content of carbonyl compounds. It
serves as valuable tool for quick and
routine screening. Rapid preliminary
sorting out, without the need for

Full Short Communication

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