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CHAPTER

Colorimetric Reactions
Diego A. Sampietro*, Melina A. Sgariglia, Jose R. Soberón,
Emma N. Quiroga and Marta A. Vattuone

1. INTRODUCTION
Colorimetry, the measurement of the absorption of visible light, allows the first
qualitative and quantitative estimation of the phytochemicals present in a plant,
soil or water sample. Colorimetric methods allow the assessment of an
unknown colour in reference to known colours (Thomas, 1996). In practice, they
provide qualitative or semiquantitative estimations rather than quantitative
determinations. Early colorimetric techniques consisted of the visual comparison
between the colour in a sample and that of several permanent colour standards,
which could be the same substance at known concentrations. The use of the
human eye as a colour detector, however, includes a subjective perception
reducing measurement reproducibility. The instrumental progress in visible
spectroscopic techniques allows the development of reliable and sensitive
instruments. Colorimeters and spectrophotometers, photoelectrically measure
the amount of coloured light absorbed by a coloured sample with respect to a
“blank” or colourless sample with reference. Modern spectrophotometers split
the incident light into different wavelengths and measure the intensity of that
radiation. Colorimetric measurements often involve the reaction of colourless
substances under analysis with specific reagents (Hagerman, 2002). This allows
the formation of coloured complexes with absorbance maxima at wavelengths
comprised in the visible light. In this chapter, general reactions of colour

Authors’ address: Instituto de Estudios Vegetales “Dr. A. R. Sampietro”, Facultad de

Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, España 2903,
4000, San Miguel de Tucumán, Tucumán, Argentina.
*Corresponding author: E-mail: dasampietro2006@[Link]
%" Isolation, Identification and Characterization of Allelochemicals/Natural Products

development are provided for the qualitative and quantitative determination of

natural products in a sample.

2. GENERAL COLORIMETRIC PRINCIPLES

2.1 Beer–Lambert Law
Electromagnetic radiation can be characterized as a wave with frequency (n) and
wavelength (l). This wave has energy (E) proportional to its frequency and is
given by the equation:
E = hn = hq/l
where, h is Plank’s constant and q is light velocity. When a wave of radiation
finds a substance and the energy is absorbed, a molecule of the substance is
promoted to an excited state (Thomas, 1996). The absorption of visible light
generally excites electrons of a molecule from a ground electronic state to an
excited one.
The amount of light absorbed by a substance in a solution is quantitatively
related to its concentration. This relationship is expressed by the Beer–Lambert
Law (Fig. 1):
Log10 (I0/I) = –log10 T = abc = A or I0/I = 1/T = 10 abc
where, I 0 is the intensity of incident light, I is the intensity of transmitted light, T
is the transmittance, a is the absorptivity or extinction coefficient, b is the path
length through an absorbing solution, c is the concentration of the absorbing
substance, and A is the absorbance (or optical density in old literature).
Absorptivity is constant for a given compound and is a function of the wavelength.
When c is expressed in moles per L and b is expressed in centimeters, the constant
is known as the molar absorptivity (e) and is expressed in L per mole per
centimeter (L mol–1 cm–1).
Beer–Lambert Law expresses a linear relationship between the absorbance
and the concentration of a given solution, if the length of the path and the

Figure 1 Absorption of radiation by a sample. I0: Intensity of incident light; I, Intensity of

transmitted light; b, path length of a sample; c, concentration of a sample.
 Colorimetric Reactions %#

wavelength of light are kept constant. This enables determination of the

concentration of a substance through the measure of the transmittance or
absorbance.

2.2 Deviations from the Beer–Lambert Law

Deviations from the linear relationship between concentration and absorbance
often occur and can lead to errors in the application of Beer–Lambert Law (Fig. 2).
High concentrations of substances in the solution, the use of radiant energy that
is not monochromatic or conditions within the solution causing shifts in the
chemical equilibrium, are common deviation sources (Brown, 1997). Linear
relationship can be evaluated by building a calibration curve, where the
absorbance of standard solutions is plotted as a function of their concentrations.
If the absorbance of an unknown sample is then measured, the concentration of
the absorbing component can be determined from this graph. In general,
transmittance should be in the range of 10-90% (absorbance 1.0-0.1) to avoid
high instrumental errors.

Figure 2 Deviation from Beer–Lambert Law

2.3 Details of Spectrophotometer Operation

Colorimetric analysis is often performed using a spectrophotometer (Brown,
1997). The cheapest spectrophotometers are single beam instruments (Fig. 3A).
They have a broadband lamp as the light source. A wavelength selector narrows
%$ Isolation, Identification and Characterization of Allelochemicals/Natural Products

the wavelength range to a typical band width of 20 nm. This light then passes
through one compartment with a typical path length of 1 cm and the transmittance
is measured by a photodetector. The instrument is calibrated to zero when a
shutter is lowered to 0% transmittance (or infinite absorbance). Then, a blank (a
reference solution) is placed in the cell and the instrument is adjusted to 0
absorbance (100% transmittance). A double beam instrument operates similarly,
but the beam is split to pass through a reference cell containing the blank and the
sample cell, simultaneously (Fig. 3B). A blank is kept in the reference cell for all
standardization and sample measurements, as the instrument reports the
difference in transmittance or absorbance measured from the two cells.

Figure 3 Typical designs of (A) single, and (B) dual beam spectrophotometers (Source:
Adapted from Brown, 1997).

Most modern instruments allow computer interfaces for data-logging or

automated operation. Also, flow-through cells may be used for continuous
measurements. Fibre optic units with modern instruments are computerized and
have sophisticated optical charged coupled device (CCD) arrays to monitor the
emission intensity at many wavelengths simultaneously. Statistical correlations
are used to generate highly selective methods and to reduce interference levels.
CCD detectors are becoming quite popular, as complete UV-Vis spectra can
be obtained in seconds, in situ sensing is available and computer interfacing is
enhanced by digital electronics.

2.4 Use of Chromogenic Reagents

Some natural products are coloured substances and can be directly analyzed
through colorimetry. Natural colourless or weakly coloured compounds,
however, must be converted to more coloured derivatives. The reaction for
 Colorimetric Reactions %%

colour formation may include enzymatic conversion, chemical modification of a

substance to produce coloured products or formation of complexes between a
substance and a colour-forming reagent. Several colour forming reagents are
commercially available for analysis of many substances.

2.5 General Clues for Colorimetric Reactions

I. Chromogenic reagents usually react with a mixture of compounds
belonging to the (correspond to the) same chemical class or many related
chemical classes. Colour reactions of these compounds are controlled by
the different kinetics producing variations in colour development (Fig. 4)
and reducing the accuracy of the colorimetric method for quantitative
analysis. Standard compounds should be selected according to past
knowledge of the main representative molecules that are known
components in a sample. Construction of several calibration curves with
different standard compounds help to validate the use of a colorimetric
method for quantitative analysis.

Figure 4 Calibration curves for protocatechuic (PRO), p-hydroxybenzoic (POH), vanillic

(VAN), caffeic (CAF), syringic (SYR), p-coumaric (PCO), ferulic (FER), and
sinapic (SIN) acids determined after reaction with Folin Ciocalteu reagent.
Absorbance was measured at 750 nm (Source: Blum et al., 1991).
%& Isolation, Identification and Characterization of Allelochemicals/Natural Products

II. A sample composition should be evaluated using several colorimetric meth-

ods (different chemical, reactions). Although water is the solvent of many
colorimetric reactions, it interferes with some of them, hence is replaced by
organic solvents. For this reason, the solvent used in the samples must be
the same as that used to dissolve the standard compounds.
III. Colorimetric measures of a sample must be in the range of absorbances
comprised in the calibration curve. Extreme absorbance values (too high or
low) must be avoided. This ensures that the colorimetric measurements
will be as per norms of Beer–Lambert Law. Little changes in reagents used
in a colorimetric method can modify the slope of the calibration curve.
Hence, calibration curves should be built each time a colorimetric method
is applied for sample analysis.
IV. Sometimes components of the sample solution absorb at the wavelength
used for colorimetric measurements or react with the chromogenic reagent
increasing colour development. Partial isolation of the compounds of interest
before colorimetric measurements aid in overcoming or reducing this kind
of interference. Absorbance can also be read in the sample solution before
and after colour development at the same wavelength or at the wavelength
of maximum absorbance of interfering compounds. These values are further
used to correct the absorbance lectures of the colorimetric method. General
UV-Vis analysis can help to check if major interferences from other
compounds are detected in the sample.

3. COLORIMETRIC METHODS
3.1 Phenolic Compounds
Experiment 1: Total Phenolic Compounds (Folin Ciocalteu Reagent)
The Folin Ciocalteu reagent is used to quantify concentrations of easily oxidizable
compounds, such as phenols, by colour changes accompanying a redox reaction.
The hydroxylated compounds reduce Cu2+ to Cu+ in an alkaline medium, which
in turn reduces the phosphotungsten-phosphomolybdic acid of the Folin Ciocalteu
reagent to form an intensely blue complex (Singleton et al., 1999). This complex is
often quantified spectrophotometrically at 750 nm. A lithium sulfate salt is also a
component of the reagent added to reduce the amounts of precipitate that can
appear when high concentrations of reagent are used to increase the assay’s
reactivity. Folin Ciocalteu reagent has been widely used in phytochemistry and
chemical ecology for preliminary analysis of phenolic compounds (Blum et al.,
1991).

Materials and equipments required

Assay tubes, Erlenmeyer flasks and pipettes; analytical balance with 0.01 mg
sensitivity; spectrophotometer or filter photometer capable of measurement at
750 nm, equipped with a 1 cm path length microcuvette; vortex mixer.
 Colorimetric Reactions %'

Reagents
0.5 N NaOH (dissolve 20 g of NaOH in 500 mL of distilled water and make up to
1 L); 1% CuSO4 (dissolve 1 g of CuSO4 in 50 mL of distilled water and make up
to 100 mL); 2% Na2CO3 (dissolve 2 g of Na2CO3 in 50 mL of distilled water and
make up to 100 mL); 2.7% sodium and potassium tartrate (dissolve 2.7 g of
sodium and potassium tartrate in 50 mL of distilled water and make up to 100
mL); solution A (mix 10 mL of 2% Na2CO3 with 0.1 mL of CuSO4 and 0.1 mL of
sodium and potassium tartrate in an Erlenmeyer flask, prepare this solution
immediately before use); Folin Ciocalteu reagent (dilute the Folin Ciocalteu reagent
with distilled water in the ratio 1:1 v/v, just before use); 1 mM ferulic acid
standard (dissolve 1 mg of ferulic acid in 5.15 mL of distilled water, if ferulic acid
is not completely dissolved, warm the tube in water at less than 50ºC); aqueous
extract from a plant material.
Procedure
(i) Set up a calibration curve in five tubes: dilute 1 mM ferulic acid with distilled
water to prepare solutions 0.25, 0.50 and 0.75 mM. Label tubes from 1 to 5.
Then, add 0.2 mL of 0, 0.25, 0.50, 0.75 and 1 mM ferulic acid to tubes 1, 2,
3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes: prepare tubes 6 and 7 adding 0.1
and 0.2 mL of a plant extract, respectively. Complete volume of tube 6 to
0.2 mL with distilled water.
(iii) Add 2 mL of solution A in each tube and vortex. Then, add 0.4 mL of 0.5 N
NaOH. Stand for 10 minutes at room temperature.
(iv) Add 0.2 mL of the Folin Ciocalteu diluted in water (1:1) and vortex. Leave
to stand for 30 minutes at room temperature.
(v) Read absorbance at 750 nm using a 1 cm path length (1 mL) microcuvette.
Calculations
Step 1. Graph the calibration curve relating to absorbance at 750 nm to ferulic
acid concentration. Fit a line function to the measured data points applying
regression analysis (r 2 > 0.80). A calibration curve as shown in Fig. 5 should be
obtained.
Step 2. Calculate concentration of total phenolic compounds (C) in the plant
extract, expressed in equivalent concentrations of ferulic acid. For example, if
absorbance at 750 nm (A750) = 0.5 and extinction coefficient (e) obtained from
linear regression = 0.89, concentration (C) for tube 6 should be:
C (mM) = (A750 ¥ e) ¥ 2 hence, C = (0.5 ¥ 0.89) ¥ 2
C = 0.72 mM
In the example above, if calculations were on tube 7, concentration (C) should
be:
C (mM) = A750 ¥ e hence, C = 0.5 ¥ 0.89
C = 0.36 mM
& Isolation, Identification and Characterization of Allelochemicals/Natural Products

Figure 5 Absorbance at 750 nm vs ferulic acid concentration (Source: Adapted from

Singleton et al., 1999). e = extinction coefficient. A750 nm = Absorbance at
750 nm.

Observations
(i) Make at least three independent experiments (N = 3).
(ii) The Folin Ciocalteu reagent can react with many oxidizable compounds.
Although phenols are often the most abundant reactive compounds in a
sample, samples may also contain other reactive compounds such as
tyrosine, tryptophan, and ascorbic acid. To solve this problem, extraction
with solutions of acetone-trichloroacetic acid (TCA) can be used to precipitate
proteins eliminating tyrosine, tryptophan and other non-phenolic reactive
compounds (Hatch et al., 1993). Remotion of non-protein oxidizable
compounds is also achieved with polyvinylpolypyrrolidone (PVPP): phenolic
compounds bind to non-soluble PVPP under acidic conditions and are then
removed by filtration or centrifugation. Reaction with Folin Ciocalteu reagent
on sample aliquots occurs before and after precipitation with PVPP. Then,
absorbance of phenolic compounds (A750) is corrected as under:
A750 (corrected) = A 750 (before PVPP precipitation) – A750 (after PVPP
precipitation)
Concentration is determined with the corrected absorbance value as
previously indicated in Calculations.

Experiment 2: Total Flavonols and Flavones

Carbonyl and hydroxyl groups of flavonols and flavones form complexes with
Al3+ which absorb at 415 nm (Fig. 6).
Materials and equipments required
Assay tubes, Erlenmeyer flasks, pipettes; analytical balance with 0.01 mg sensitivity;
spectrophotometer or filter photometer capable of measurement at 415 nm,
equipped with a 1 cm path length microcuvette; vortex mixer.
 Colorimetric Reactions &

Figure 6 Reaction of Al3+ with a flavonoid molecule (Source: Adapted from Woisky and
Salatino, 1998).

Reagents
10% AlCl3 (dissolve 10 g of AlCl3 in 50 mL of 98% ethanol and make up to 100
mL); 1 M potassium acetate (dissolve 19.63 g of potassium acetate in 80 mL of
distilled water and adjust volume to 100 mL); 1 mg/mL stock quercetin solution
(dissolve 100 mg of quercetin in 80 mL of 98% ethanol and make up to 100 mL;
this solution can be kept in a freezer for at least one month); 0.1 mg/mL standard
quercetin solution (dilute 1 mL of quercetin stock solution in 9 mL of 98% ethanol;
this solution can be kept in the refrigerator for at least one week); an ethanolic
plant extract.

Procedure
(i) Set up a calibration curve in five tubes: dilute 0.1 mg/mL quercetin standard
with 98% ethanol to obtain solutions with concentrations of 12, 24, 36 and
48 μM. Label tubes from 1 to 5. Then, add 0.5 mL of 0, 12, 24, 36 and 48 μM
quercetin to tubes 1, 2, 3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes: Prepare tubes 6 and 7 adding 0.25
and 0.5 mL of an ethanolic plant extract, respectively. Complete volume of
tube 6 to 0.5 mL with 98% ethanol.
(iii) Add 1.5 mL of 98% ethanol and swirl.
(iv) Add 0.1 mL of 10% AlCl3 and 0.1 mL of 1 M potassium acetate and swirl.
(v) Add 2.8 mL of distilled water. Let stand for 30 minutes at room temperature.
(vi) Read absorbance at 415 nm using a 1 cm path length (1 mL) microcuvette.

Calculations
Step 1. Graph the calibration curve relating to absorbance at 415 nm to quercetin
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate total concentration of flavonol and flavones (C) in the plant
extract expressed in equivalent concentrations of quercetin:
C (mM) = (A415 ¥ e) ¥ 2 (tube 6) or C (mM) = A415 ¥ e (tube 7)
& Isolation, Identification and Characterization of Allelochemicals/Natural Products

where, A415 is absorbance at 415 nm and e is extinction coefficient obtained from

linear regression.
Observations
(i) Make at least three independent experiments (N = 3).
(ii) 10% AlCl3 solution should be used within a week after preparation. The
solution must be stored at room temperature in a dark coloured bottle. If
precipitates are observed, solution must be decanted.
(iii) This method was originally developed to measure concentrations of
quercetin, the most common flavonol in plants (Woisky and Salatino, 1998).
It eliminates the interference of several phenolic compounds in the
spectrophotometric analysis. However, accuracy of the method is high
only when flavonols are the dominant flavonoids in a sample. Data will be
less representative in samples with high levels of flavones because Al3+-
flavones complexes have maximum absorbance at wavelengths less than
415 nm.

Experiment 3: Condensed Tannins (Proanthocyanidins)

Oxidative cleavage of proanthocyanidins leads to formation of coloured
anthocyanidins that are detected spectrophotometrically at 550 nm (Fig. 7).

Figure 7 Conversion of proanthocyanidins to coloured anthocyanidins in the butanol

acid assay (Source: Hagerman, 2002).

Materials and equipments required

Assay tubes, Erlenmeyer flasks, marbles, pipettes; analytical balance with 0.01 mg
sensitivity; water bath; spectrophotometer or filter photometer capable of
measurement at 550 nm, equipped with a 1 cm path length microcuvette; vortex
mixer.
 Colorimetric Reactions &!

Reagents
Butanol-HCl reagent, 95:5 v/v (add 950 mL n-butanol to 50 mL concentrated
HCl); 2% ferric reagent (make up 16.6 mL of concentrated HCl to 100 mL with
distilled water and add 2.0 g of ferric ammonium sulfate); 1 mg/mL stock catechin
solution (weigh 100 mg catechin and dissolve in 80 mL of 98% ethanol. Then,
make up the volume to 100 mL); 0.1 mg/mL standard catechin solution (dilute
1 mL of catechin stock solution in 9 mL of 98% ethanol); an ethanolic plant
extract.

Procedure
(i) Set up a calibration curve in five tubes: dilute 0.1 mg/mL catechin solution
with 98% ethanol to prepare solutions 17, 35, 50 and 70 μM. Label tubes
from 1 to 5. Then, add 1 mL of 0, 17, 35, 50 and 70 μM catechin to tubes 1,
2, 3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes: prepare tubes 6 and 7 adding 0.5
and 1 mL of an ethanolic plant extract, respectively. Complete volume of
tube 6 to 1 mL with 98% ethanol.
(iii) Add 6 mL of the Butanol-HCl reagent to each tube.
(iv) Add 0.2 mL of ferric reagent and vortex. Cover the mouth of each tube
with a glass marble and put the tubes in a boiling water bath for 50 minutes.
(v) Let the tubes cool at room temperature. Read absorbance at 550 nm using
a 1 cm path length (1 mL) microcuvette.

Calculations
Step 1. Graph the calibration curve relating absorbance at 550 nm to catechin
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate concentration of total condensed tannins (C), considering
absorbance at 550 nm (A550) of tubes 6 and 7 and extinction coefficient (e) obtained
from linear regression:
C (μM) = (A550 ¥ e) ¥ 2 (tube 6) or C (μM) = A550 ¥ e (tube 7)
Observations
(i) Make at least three independent experiments (N = 3).
(ii) Ferric reagent should be stored in a dark coloured bottle.
(iii) Store catechin solution in a freezer for not more than a month after
preparation.
(iv) Sometimes chemical characteristics of certain plant tannins, such as position
of the interflavan bond and oxygenation pattern, significantly affect the
colour development, underestimating tannin content of the sample.
(v) The presence of pigments, particularly chlorophyll, may interfere in this
method. An alternative is to read absorbance before heating and subtract
this value from the absorbance determined after heating. Chlorophyll may
also be eliminated by precipitation with Polyvinylpyrrolidone (PVP) or by
chlorophyll extraction of the dry sample with petroleum ether.
&" Isolation, Identification and Characterization of Allelochemicals/Natural Products

Experiment 4: Gallotannins
Hydrolyzable tannins, such as gallotannins, release gallic acid after hydrolysis.
Gallic acid reacts with rhodanine dye producing coloured complexes that are
detected spectrophotometrically at 520 nm. Plant samples often contain gallic
acid in free forms. Hence, not only total hydrolizable gallic acid (free and bound)
is measured, but also the free gallic acid found in the sample before hydrolysis
(Makkar, 2000).
Materials and equipments required
Assay tubes, Erlenmeyer flasks, pipettes, marbles; analytical balance with 0.01 mg
sensitivity; spectrophotometer or filter photometer capable of measurement at
520 nm, equipped with a 1 cm path length microcuvette; vortex mixer; rotary
evaporator; water bath; vacuum pump.

Reagents
Rhodanine solution 0.667% (weigh 667 mg rhodanine and dissolve it in 100 mL
methanol; this solution is stable for 2 weeks when stored in a refrigerator); 0.5 N
KOH (dissolve 2.8 g potassium hydroxide [KOH] in 100 mL distilled water);
H2SO4 solutions (prepare 0.2 and 22 N solutions diluting concentrated H2SO4);
1 mg/mL gallic acid stock solution (weigh 100 mg gallic acid and dissolve in
approximately 80 mL of 0.2 N H2SO4 and then make up the volume to 100 mL
with 0.2 N H2SO4; it can be kept frozen for at least one month); 0.1 mg/mL
standard gallic acid solution (dilute 1 mL of gallic acid stock solution with 9 mL of
0.2 N H2SO4; it can be stored in a refrigerator for at least two weeks); tannin
extract (place 200 g of finely ground plant material in a 25 mL glass beaker; add
10 mL of 70% aqueous acetone and shake for 20 minutes at room temperature;
transfer the liquid to centrifuge tubes and centrifugate for 10 minutes at 3,000 ¥ g
and 4°C; collect the supernatant and keep it on ice for tannin analysis).

Procedure
Calibration Curve
(i) Set up a calibration curve in five tubes: dilute 0.1 mg/mL gallic acid solution
with 0.2 N H2SO4 to prepare solutions 0.12, 0.25, 0.38 and 0.50 mM. Label
tubes from 1 to 5. Then, add 0.2 mL of 0, 0.12, 0.25, 0.38 and 0.50 mM gallic
acid to tubes 1, 2, 3, 4 and 5, respectively.
(ii) Add 0.3 mL of the rhodanine solution to each tube.
(iii) After 5 minutes, add 0.2 mL of 0.5 N KOH solution to each tube. Wait for
2.5 minutes and then add 4.3 mL of distilled water.
(iv) After 15 minutes, read absorbance at 520 nm using a 1cm path length
(1 mL) microcuvette.
Determination of Free Gallic Acid
(i) Set up four experimental assay tubes and add 0.2 mL of tannin extract to
each tube. Eliminate acetone drying in a rotary evaporator. Then, add
0.2 mL of 0.2 N H2SO4.
(ii) Proceed as previously indicated for calibration curve.
 Colorimetric Reactions &#

Determination of Gallic Acid Present in Free and in Gallotannin Forms

(i) Pipette 3.34 mL of tannin extract in a tube. Remove acetone by drying in a
rotary evaporator. Make up to 1 mL. Then add 0.1 mL of 22 N H2SO4, so
that the final H2SO4 concentration is up to 2 N. Freeze the contents and
remove air from these culture tubes by using a vacuum pump.
(ii) Cap each tube with a glass marble and put the tubes in a boiling water
bath for 4 hours to hydrolyze gallotannins to gallic acid. After hydrolysis,
make up the volume to 11 mL by adding 9.9 mL distilled water. H2SO4
concentration in this solution is 0.2 N. Prepare four tubes. Add 0.2 mL of
this solution in three of them. Add 0.2 ml of 0.2 N H2SO4 to the fourth
tube. This last tube will be used as the control.
(iii) Add 0.3 mL of the rhodanine solution to each tube.
(iv) Wait for 5 minutes and then add 0.2 mL of 0.5 N KOH solution to each
tube. Again wait for 2.5 minutes and add 4.3 mL distilled water. After
10 minutes, measure absorbance at 520 nm.
Calculations
Step 1. Graph the calibration curve relating absorbance at 520 nm to gallic acid
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate concentration of free gallic acid (Cfree) in the tannin extract as
under:
Cfree (mM) = A520 ¥ e
where, A520 is absorbance at 520 nm and e is extinction coefficient obtained from
linear regression.
Step 3. Calculate concentration of total gallic acid in free and gallotannin forms in
0.2 ml of the sample (Cs):
Cs (mM) = A520 ¥ e
However, original volume of tannin extract was 3.34 mL. Hence, total
concentration (CT) should be:
CT (mM) = Ctotal ¥ 0.2/3.34
Step 4. Calculate concentration of gallic acid in gallotannin forms (Cg form),
subtracting free gallic acid found in tannin extract:
C g form (mM) = CT – Cfree
Precautions
H2SO4 is highly corrosive. Rhodanine is toxic by inhalation, contact or swallowing.
Avoid exposure. Obtain special instructions before use.
Observations
(i) Ascorbic acid (generally added to prevent oxidation of phenols) interferes
in this assay and, therefore, should not be added to the solvent used for
tannin extraction.
&$ Isolation, Identification and Characterization of Allelochemicals/Natural Products

(ii) Rhodanine solution is stable for two weeks when stored in a refrigerator,
stock solution of gallic acid diluted in H2SO4 can be kept in the refrigerator
for one week and gallic acid dissolved in H2SO4 can be kept frozen for at
least one month.

Experiment 5: Lignin Content (Refer Chapter 14, Experiment 6)

3.2 Terpenoids
Experiment 6: Total Azadirachtin-like Limonoids
Triterpenoids form conjugated diene groupings with H2SO4. Addition of vanillin
transforms this complex to a blue chromophore that is detected at 577 nm (Dai
et al., 1999).
Materials and equipments required
Assay tubes, pipettes, Erlenmeyer flasks; spectrophotometer or filter photometer
capable of measurement at 577 nm, equipped with a 1 cm path length
microcuvette; analytical balance with 0.01 mg sensitivity; neem seeds or other
plant material containing limonoids.
Reagents
Dichloromethane; petroleum ether; methanol; concentrated H2SO4 (98%);
0.02 mg/mL vanillin reagent (dissolve 100 mg of vanillin in 50 mL methanol;
take 0.5 mL of this solution and make up to 50 mL with methanol); 0.1 mg/mL
standard azadirachtin solution (dissolve 10 mg of azadirachtin in 100 mL of
dichloromethane); neem seed extract (stir blended 2 g of neem seeds overnight
in 60 mL of petroleum ether at room temperature; filter and recuperate the
residues; stir the residue in 50 mL methanol at room temperature for 10
minutes; then, filter and evaporate the filtrate solution in a rotary evaporator;
an orange amorphous solid should be obtained; then, dissolve this material in
dichloromethane).
Procedure
(i) Set up a calibration curve in five tubes: dilute 0.1 mg/mL azadirachtin
solution with dichloromethane to prepare solutions 1.0, 2.6, 5.1 and 7.2 mM.
Label tubes from 1 to 5. Then add 0.7 mL of 0, 1.0, 2.6, 5.1 and 7.2 mM
azadirachtin to tubes 1, 2, 3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes and prepare tubes 6 and 7 adding
0.35 and 0.70 mL of the dichloromethane plant extract, respectively.
Complete volume of tube 6 to 0.7 mL with dichloromethane.
(iii) Add 0.2 mL of the vanillin solution to each tube and swirl. Stand for 2 min-
utes at room temperature.
(iv) Add 0.3 mL of concentrated H2SO4 to each tube in three portions (0.1 mL
each) and mix carefully.
(v) Add 0.7 mL of methanol. Stand for 5 minutes at room temperature.
(vi) Read absorbance at 577 nm using a glass (1 mL) microcuvette.
 Colorimetric Reactions &%

Calculations
Step 1. Graph the calibration curve relating absorbance at 577 nm to azadirachtin
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate content of azadirachtin like limonoids in equivalent azadirachtin
concentration (C), considering absorbance at 577 nm (A577) and extinction
coefficient (e) obtained from linear regression:
C (mM) = (A557 ¥ e) ¥ 2 (tube 6) or C (mM) = A557 ¥ e (tube 7)
Observations
Although this method was specifically described for azadirachtin, vanillin–H2SO4
also reacts with triterpenoid sapogenins, sterols and steroidal sapogenins
producing stable red-purple colours with l max in the region 515-545 nm.

Precautions
H2SO4 is highly corrosive. Methanol, petroleum ether and dichloromethane are
toxic by inhalation, contact or swallowing. Avoid exposure. Obtain special
instructions before use.

3.3 Alkaloids
Experiment 7: Precipitable Alkaloids (Dragendorff Reagent)
Dragendorff´s reagent form red precipitates with alkaloids in solution. The
precipitates are solubilized in NaI, being possible to read absorbance of the
coloured solution at 467 nm (Stumpf, 1984).
Materials and equipments required
Assay tubes, Erlenmeyer flasks, Pasteur pipettes, pipettes; analytical balance with
0.01 mg sensitivity; spectrophotometer or filter photometer capable of
measurement at 595 nm, equipped with a 1 cm path length glass microcuvette;
centrifuge capable to reach a speed of 7,000 ¥ g.

Reagents
Dragendorff reagent (Solution A: Prepare 0.35 M bismuth nitrate in 20% acetic
acid (v/v); Solution B: Prepare 2.45 M NaI in distilled water; prepare Dragendorff
reagent by mixing solutions A and B in a 1:1 (v/v) relation)); 0.1 mg/mL standard
papaverine hydrochloride solution (dissolve 1 mg of papaverine hydrochloride
in 100 mL distilled water); a plant extract.
Procedure
(i) Set up a calibration curve in four tubes: dilute 0.1 mg/mL papaverine
solution with distilled water to prepare solutions 0.1, 0.2 and 0.3 mM. Label
tubes from 1 to 4 Then, add 0.2 mL of 0, 0.1, 0.2 and 0.3 mM papaverine to
tubes 1, 2, 3 and 4, respectively.
&& Isolation, Identification and Characterization of Allelochemicals/Natural Products

(ii) Set up your experimental assay tubes. Prepare tubes 5 and 6 adding 0.15
and 0.3 mL of a plant extract, respectively. Complete volume of tube 6 to
0.3 mL with distilled water.
(iii) Add 0.1 mL of Dragendorff reagent to each tube.
(iv) Centrifugate the tubes for 1 minute at 7,000 ¥ g. Carefully remove the
supernatant with a Pasteur pipette.
(v) Dissolve each pellet in 1mL of 2.45 M NaI. Add 10 μL aliquots of each tube
to 1 mL of 0.49 M NaI and swirl.
(vi) Read absorbance at 467 nm using the glass microcuvette.

Calculations
Step 1. Graph the calibration curve relating absorbance at 467 nm to papaverine
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate content of total alkaloids in equivalent papaverine concentration
(C), considering absorbance at 467 nm (A467) and extinction coefficient (e) obtained
from linear regression:
C (mM) = (A467 ¥ e) ¥ 2 (tube 5) or C (mM) = A467 ¥ e (tube 6)

Observations
After centrifugation, the supernatant must be completely removed or the
remaining bismuth nitrate will cause an increased background. Proteins can
precipitate in the presence of Dragendorff reagent. It is recommended to remove
the proteins from the samples before addition of the reagent.

Experiment 8: Pyrrolizidine Alkaloids (Methyl Orange Method)

Protonation of pyrrolizidine alkaloids in chloroform solution with acetic acid and
their stoichiometric reaction with aqueous methyl orange lead to the formation
of yellow complex that can be detected at 525 nm (Birecka et al., 1981).

Materials and equipments required

Assay tubes, Erlenmeyer flasks; analytical balance with 0.01 mg sensitivity,
centrifuge capable to reach a speed of 400 ¥ g, Whatman # 1 paper,
spectrophotometer or filter photometer capable of measurement at 525 nm,
equipped with a 1 cm path length glass microcuvette.

Reagents
Methyl orange reagent (dissolve 500 mg powdered methyl orange in 100 mL
distilled water at 40º C for 20 minutes; let cool at room temperature and then
filter with Whatman # 1 paper); 1.25% acetic acid in distilled water; 2% H2SO4 in
ethanol (dissolve 2 mL H2SO4 in 100 mL ethanol); 0.25 M H2SO4 (dissolve 1.40
mL H2SO4 in 100 mL ethanol); Plant Extract (Soak 1 kg of dried milled plant
material in methanol for 24 hours; filter and evaporate the solution to dryness;
dissolve the residue in 0.25 M H2SO4; extract chlorophyll with petroleum ether;
add Zn dust to reduce N oxide forms of alkaloids to tertiary alkaloids; filter and
 Colorimetric Reactions &'

add NH3 to reach pH > 10. Add chloroform and vortex. Aspirate the chloroform
phase with a Pasteur pipette and use as the plant extract.); standard monocrotaline
solution, 0.1 mg/mL (dissolve 10 mg of monocrotaline in 100 mL of chloroform).
Procedure
(i) Set up a calibration curve in five tubes: dilute 0.1 mg/mL monocrotaline
solution with chloroform to prepare solutions 6, 12, 25 and 50 μM. Label
tubes from 1 to 5. Then, add 5 mL of 0, 6, 12, 25 and 50 μM monocrotaline
to tube 1, 2, 3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes: prepare tubes 6 and 7 adding 2.5
and 5 mL of the chloroform plant extract, respectively. Complete volume
of tube 6 to 5 mL with chloroform.
(iii) Add 10 μl of 1.25% acetic acid and vortex.
(iv) Add 25 μl of methyl orange solution and vortex.
(v) Stand for 5 minutes. Transfer the chloroform phase to glass centrifuge
tubes.
(vi) Centrifugate at 400 ¥ g for 2 minutes.
(vii) Transfer 1.5 mL of each supernatant to a tube.
(viii) Add 0.1 mL of 2% ethanolic H2SO4 to each tube and vortex.
(ix) Read absorbance at 525 nm using the glass microcuvette.

Calculations
Step 1. Graph the calibration curve relating to absorbance at 525 nm to
monocrotaline concentration. Fit a line function to the measured data points
applying regression analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate total pyrrolizidine alkaloids in equivalent monocrotaline
concentration (C), considering absorbance at 525 nm (A525) and extinction
coefficient (e) obtained from linear regression:
C (μM) = (A525 ¥ e) ¥ 2 (tube 6) or C (μM) = A525 ¥ e (tube 7)

Precautions
Chloroform and methyl orange are toxic by inhalation, contact or swallowing.
Avoid exposure. Obtain special instructions before use.

3.4 Lipids
Experiment 9: Total Sterol Content (Lieberman–Buchard Method)
Free sterols and sterol esters react with acetic anhydride and concentrated H2SO4,
forming a blue-green complex which absorbance can be read at 550 nm.
Materials and equipments required
Assay tubes, pipettes, Erlenmeyer flasks; analytical balance with 0.01 mg sensitivity,
water bath; Vortex mixer, spectrophotometer or filter photometer capable of
measurement at 550 nm, equipped with a 1 cm path length glass microcuvette.
' Isolation, Identification and Characterization of Allelochemicals/Natural Products

Reagents
Chloroform–methanol mixture: Mix chloroform with methanol in 2:1 ratio;
Lieberman–Buchard reagent [add slowly, while stirring continuously, 10 mL of
concentrated H2SO4 to 60 mL of acetic anhydride in an Erlenmeyer flask. As the
reaction is strongly exothermic, cooling the Erlenmeyer flask in an ice bath is
recommendable. Then, add 30 mL of acetic acid and 0.6 g of anhydrous sodium
sulfate]; Standard cholesterol solution (0.4 mg/mL): Dissolve 40 mg of cholesterol
in 100 mL of chloroform:methanol (2:1); walnut extract: Grind 1 g of walnut
thoroughly in a mortar with 5 mL of chloroform:methanol mixture. Transfer to
a test tube washing the mortar with 5 mL of the same solvent into the same
tube; cap the tube and place it in a water bath at 50ºC for 10 minutes; filter using
Whatman # 1 paper. Measure the volume of filtrate and add enough CaCl2 to
prepare a 0.02% solution; vortex; leave the tube until there are two separate
phases; remove the upper phase carefully with a Pasteur pipette; double the total
volume of the lower phase by the addition of chloroform:methanol:water ([Link]);
vortex and leave until two phases separate; remove the upper phase with a
Pasteur pipette and measure the volume of the lower phase, containing the
lipids; measure 0.5 mL of the obtained extract and transfer to another tube for
sterol determination; the remaining lipid extract is placed in a water bath at 80ºC
and evaporated to dryness; weigh the dry lipid residue.

Procedure
(i) Set up a calibration curve in five tubes: dilute 0.4 mg/mL cholesterol solu-
tion with chloroform:methanol (2:1) to prepare solutions 0.2, 0.4, 0.6 and
0.8 μM. Label tubes from 1 to 5. Then, add 0.5 mL of 0, 0.2, 0.4, 0.6 and
0.8 μM cholesterol to tubes 1, 2, 3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes and prepare tubes 6 and 7 adding
0.25 and 0.50 mL of a plant extract, respectively. Complete volume of tube
6 to 0.5 mL with chloroform:methanol (2:1).
(iii) Add 5 mL of Lieberman–Buchard reagent to each tube.
(iv) Cap the test tubes, swirl and place in a water bath at 35ºC for 10 minutes.
(v) Measure the absorbance at 550 nm.
Calculations
Step 1. Graph the calibration curve relating absorbance at 550 nm to cholesterol
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate concentration of total sterols (C) in the plant extract expressed
as cholesterol equivalent concentration. Consider absorbance at 550 nm (A550)
from experimental tubes and extinction coefficient (e) obtained from linear
regression as under:
C (μM) = (A550 ¥ e) ¥ 2 (tube 6) or C (μM) = A550 ¥ e (tube 7)

Observations
The Lieberman–Buchard test can be done at room temperature. However, placing
the test tubes in a water bath at 35ºC speed up the reaction.
 Colorimetric Reactions '

3.5 Carbohydrates
Experiment 10: Total Neutral Carbohydrates (Phenol Sulfuric Method)
H2SO4 in hot conditions dehydrates the carbohydrates to form either furfural
(from pentoses, Fig. 8A) or 5-hydroxymethyl furfural (from hexoses, Fig. 8B).
These products of carbohydrate dehydration can react with phenol to form a
coloured complex that can be detected spectrophotometrically at 490 nm (Dubois
et al., 1956).

Figure 8 Hot acid dehydration of (A) pentoses, and (B) hexoses.

' Isolation, Identification and Characterization of Allelochemicals/Natural Products

Materials and equipments required

Assay tubes, marbles, Erlenmeyer flasks, pipettes; analytical balance with 0.01 mg
sensitivity, water bath, spectrophotometer or filter photometer capable of
measurement at 490 nm, equipped with a 1 cm path length microcuvette.
Reagents
H2SO4 concentrated; 80% phenol, w/v (Dissolve 0.8 g phenol in a boiling water
bath. Then cool and dilute with distilled water to reach 1 L. This solution should
be prepared with caution because phenol is hydroscopic and toxic.); 1 mg/mL
stock glucose solution (Weigh 90 mg glucose and dissolve in 25 mL of distilled
water; make up to 50 mL. It can be kept frozen for at least six months.);
0.18 mg/mL standard glucose solution (dilute 1 mL the glucose stock solution in
9 mL of distilled water); an aqueous plant extract.
Procedure
(i) Set up a calibration curve in five tubes: dilute 0.18 mg/mL glucose solution
with distilled water to prepare solutions 60, 120, 190 and 250 μM. Label
tubes from 1 to 5. Then, add 0.8 mL of 0, 60, 120, 190 and 250 μM glucose
to tubes 1, 2, 3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes and prepare tubes 6 and 7 adding 0.4
and 0.8 mL of the plant extract, respectively. Complete volume of tube 6 to
0.8 mL with distilled water.
(iii) Add 0.04 mL of 80% phenol to each tube and swirl. Add 2 mL of H2SO4
concentrated. Cover the mouth of each tube with a glass marble and put
the tubes in a boiling water bath for 20 minutes. Let the tubes cool to room
temperature.
(iv) Read absorbance at 490 nm using a 1 cm path length (1 mL) microcuvette.
Calculations
Step 1. Graph the calibration curve relating absorbance at 490 nm to glucose
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate concentration of total sugars (C) in the plant extract expressed
as glucose equivalent concentration. Consider absorbance at 490 nm (A490) from
experimental tubes and extinction coefficient (e) obtained from linear regression:
C (μM) = (A490 ¥ e) ¥ 2 (tube 6) or C (μM) = A490 ¥ e (tube 7)
Precautions
H2SO4 is extremely corrosive and can cause immediate and serious skin or eye
damage. It can also cause damage to clothing and equipment. Avoid exposure.
Obtain special instructions before use.

Experiment 11: Total Reducing Carbohydrates (Somogyi–Nelson Method)

Carbohydrates with free aldehyde or keto groups, in alkaline conditions, reduce
Cu2+ to Cu+ which in turn reduces the arseno-molybdate complex of Nelson
reagent to molybdenum blue which absorbs at 520 nm (Fig. 9).
 Colorimetric Reactions '!

Figure 9 Somogyi–Nelson reaction (Source: Adapted from Somogyi, 1952).

Materials and equipments required

Assay tubes, marbles, pipettes, Erlenmeyer flasks; analytical balance with 0.01 mg
sensitivity; water bath; spectrophotometer or filter photometer capable of
measurement at 520 nm, equipped with a 1 cm path length microcuvette.
Reagents
Somogyi reagent [Solution A: 10% CuSO4; Solution B: Dissolve 28 g Na2HPO4
(anhydrous) in 700 mL of distilled water. Add 40 g of tetra hydrated sodium and
potassium tartrate; then, add 100 mL 1 N NaOH and 120 g Na2SO4 (anhydrous);
reach to 900 mL with distilled water, stand 48 hours at room temperature and
filter through Whatman # 1 paper; mix solution A with solution B in the ratio 1:9
just before use.]; Nelson reagent (dissolve 25 g of (NH4)6Mo7O24 ◊ 4 H2O in 450
mL of distilled water; add 21 mL of concentrated H2SO4; after mixing, add 3 g
Na2HAsO4 ◊ 7 H2O previously dissolved in 25 mL distilled water; keep at 37ºC
for 2 days and store in a brown bottle at room temperature); 1 mg/mL stock
glucose solution (weigh 100 mg glucose and dissolve in 80 mL of distilled water;
then, make up the volume to 100 mL. It can be kept frozen for at least six
months); 0.1 mg/mL standard glucose solution (dilute 1 mL of glucose stock
solution in 10 mL of distilled water); an aqueous plant extract.
Procedure
(i) Set up a calibration curve in five tubes: dilute 0.18 mg/mL glucose solution
with distilled water to prepare solutions 0, 0.1, 0.2, 0.3 and 0.4 mM. Label
tubes from 1 to 5. Then, add 0.5 mL of 0, 0.1, 0.2, 0.3 and 0.4 mM glucose
to tube 1, 2, 3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes and prepare tubes 6 and 7 adding
0.25 and 0.50 mL of a plant extract, respectively. Complete volume of tube
6 to 0.5 mL with distilled water.
(iii) Add 0.5 mL of Somogyi reagent to each tube. Cover the mouth of each
tube with a glass marble and put the tubes in a boiling water bath for
'" Isolation, Identification and Characterization of Allelochemicals/Natural Products

15 minutes. Let cool to room temperature. Add 0.5 mL of Nelson reagent

to each tube and swirl.
(iv) Add 1 mL of distilled water to each tube and vortex.
(v) Read absorbance at 520 nm using a 1 cm path length (1 mL) microcuvette.
Calculations
Step 1. Graph the calibration curve relating absorbance at 520 nm to glucose
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate concentration of total reducing sugars (C) in the plant extract
expressed as glucose equivalent concentration. Consider absorbance at 520 nm
(A520) from experimental tubes and extinction coefficient (e) obtained from linear
regression:
C (mM) = (A520 ¥ e) ¥ 2 (tube 6) or C (mM) = A520 ¥ e (tube 7)
Observations
Compare total carbohydrate and total reducing carbohydrate contents in the
extract and calculate the relative participation of reducing carbohydrates referred
to total carbohydrate content.

Experiment 12: Starch

Addition of an adequate quantity of KI to a starch solution (weakly acid) and
dilute hydrogen peroxide leads to the formation of a blue starch-iodide complex.
This complex can be detected at 620 nm (Chinoy, 1939).
Materials and equipments required
Assay tubes, Erlenmeyer flasks, pipettes; analytical balance with 0.01 mg sensitivity;
water bath; spectrophotometer or filter photometer capable of measurement at
620 nm, equipped with a 1 cm path length microcuvette.

Reagents
Chloroform; H2O2 of 20 volumes; 0.1 N KI (Dissolve 8.3 g in 300 mL of distilled
water. Make up to a final volume of 500 mL); 2.5 mg/mL standard starch solution
(dissolve 1 g of soluble starch in 400 mL of distilled water by heating for 15 minutes
in a water bath; cool to room temperature and make up to 500 mL in a flask).
Procedure
(i) Set up a calibration curve in five tubes: dilute 2.5 mg/mL starch solution
with distilled water to prepare solutions 0.3, 0.6, 0.9 and 1.3 mg/mL. Label
tubes from 1 to 5. Then, add 2 mL of 0, 0.3, 0.6, 0.9 and 1.3 mg/mL starch
to tubes 1, 2, 3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes and prepare tubes 6 and 7 adding 1
and 2 mL of a plant extract, respectively. Complete volume of tube 6 to
2 mL with distilled water.
(iii) Add 2 mL of 0.1 N KI to each tube and swirl.
(iv) Add 2 mL of H2O2 to each tube and swirl.
 Colorimetric Reactions '#

(v) Stand tubes for 1 hour at room temperature.

(vi) Add 5 mL of chloroform to each tube and swirl.
(vii) Remove the chloroform phase of each tube and discard the chloroform
phase.
(viii) Repeat steps 6 and 7 two times to eliminate the iodine excess from the
aqueous phase.
(ix) Read absorbance of the aqueous phase at 620 nm.

Calculations
Step 1. Graph the calibration curve relating absorbance at 620 nm to starch
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate concentration of total starch (C) in the plant extract expressed as
starch equivalent concentration. Consider absorbance at 620 nm (A620) from
experimental tubes and extinction coefficient (e) obtained from linear regression:
C (mM) = (A620 ¥ e) ¥ 2 (tube 6) or C (mM) = A620 ¥ e (tube 7)

Observations
After chloroform addition, it is advisable to shake the tubes gently avoiding
emulsion of the organic solvent with the aqueous solution. Turning tubes from
end to end ensures efficient shaking and removal of iodine from the aqueous
solution. Three or four changes of chloroform are adequate to remove all the
iodine from the solution. This is easily noticed because the last volume of
chloroform remains colourless.

3.6 Proteins and Amino Acids

Experiment 13: Protein Content (Bradford Method)
The absorbance maximum of an acidic solution of the dye Coomassie® Brilliant
Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. The
dye has been assumed to bind via electrostatic attraction of the dye’s sulfonic
groups to the arginine residues of the proteins. Formation of the coloured
complexes is measured spectrophotometrically at 595 nm (Bonjoch and Tamayo,
2001).
Materials and equipments required
Assay tubes, Erlenmeyer flasks, pipettes; analytical balance with 0.01 mg sensitivity,
a spectrophotometer or filter photometer capable of measurement at 595 nm,
equipped with a 1 cm path length microcuvette.
Reagents
Bradford reagent: dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 mL 95%
ethanol. Add 100 mL 85% (w/v) phosphoric acid; dilute to 1 L with distilled water
when the dye has completely dissolved and filter through Whatman #1 paper
just before use; stock solution of commercial bovine serum albumin, BSA (dissolve
'$ Isolation, Identification and Characterization of Allelochemicals/Natural Products

100 mg of BSA in 100 mL of the same buffer used to prepare the plant extract);
0.1 mg/mL standard BSA solution (dilute 1 mL of BSA stock solution in 9 mL of
buffer); this solution can be stored at –20°C and used whenever needed); plant
extract (proteins are often extracted from plant tissues in buffer solutions; literature
can be selected according to the tissue and protein to be extracted).

Procedure
(i) Set up a calibration curve in five tubes: dilute 0.1 mg/mL BSA solution with
buffer to prepare solutions 25, 50 and 75 μg/mL. Label tubes from 1 to 5.
Then, add 2 mL of 0, 25, 50, 75 and 100 μg/mL BSA to tubes 1, 2, 3, 4 and
5, respectively.
(ii) Set up your experimental assay tubes. Prepare tubes 6 and 7 adding 0.10
and 0.20 mL of a plant extract, respectively. Complete volume of tube 6 to
0.20 mL with buffer solution.
(iii) Add 1 mL Coomassie Brilliant Blue solution and swirl. Allow to stand
5 minutes at room temperature.
(iv) Read absorbance at 595 nm using a 1 cm path length (1 mL) microcuvette.

Calculations
Step 1. Graph the calibration curve relating absorbance at 595 nm to BSA
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate total protein concentration (C) in the plant extract expressed as
BSA equivalent concentration. Consider absorbance at 595 nm (A595) from
experimental tubes and extinction coefficient (e) obtained from linear regression:
C (μg/mL) = (A595 ¥ e) ¥ 2 (tube 6) or C (μg/mL) = A595 ¥ e (tube 7)

Observations
If the unknown protein concentration is too high, dilute the protein, assay a
smaller aliquot of the unknown, or generate another calibration curve in a higher
concentration range (e.g. 10 to 100 μg).

Experiment 14: Amino Acid Content (Acid-Ninhydrin Method)

Ninhydrin produces the oxidative deamination of the amino groups of amino
acids releasing ammonium, the corresponding aldehyde and ninhydrin in reduced
form. Released ammonium reacts with an additional mol of ninhydrin and with
reduced ninhydrin, thus producing a complex (Ruhemann’s purple)
spectrophotometrically detected at 570 nm (Fig. 10).

Materials and equipments required

Assay tubes, marbles, Pasteur pipettes, pipettes; analytical balance with 0.01 mg
sensitivity; water bath; spectrophotometer or filter photometer capable of
measurement at 520 nm, equipped with a 1 cm path length glass microcuvette.
 Colorimetric Reactions '%

Figure 10 Reaction with Ninhydrin (Source: Adapted from Bates et al., 1973).

Reagents
Sulfosalicylic acid 3% (w/v); phosphoric acid 6 M; glacial acetic acid; acid-ninhydrin
solution (Warm 1.25 g ninhydrin in 30 mL glacial acetic acid and add 20 mL of
6 M phosphoric acid. Acid ninhydrin will keep stable only for 24 hours, at 4°C);
toluene; 1 mg/mL stock proline solution (weigh 100 mg of proline and dissolve
in 80 mL of 3% sulfosalicylic acid; then, make up the volume to 100 mL with 3%
sulfosalicylic acid); 0.1 mg/mL standard proline solution (dilute 1 mL of proline
stock solution in 9 mL 3% sulfosalicylic acid); plant extract (0.5 g of plant material
was homogeinzed in 10 mL of 3% aqueous sulfosalicylic acid and the homogenate
filtered through Whatman # 2 filter paper).
Procedure
(i) Set up a calibration curve in five tubes: dilute 0.1 mg/mL proline solution
with 3% sulfosalicylic acid to prepare solutions 10, 20, 40 and 60 μg/mL.
Label tubes from 1 to 5. Then, add 0.50 mL of 0, 10, 20, 40 and 60 μg/mL
proline to tubes 1, 2, 3, 4 and 5, respectively.
(ii) Set up your experimental assay tubes and prepare tubes 6 and 7 adding
0.25 and 0.50 mL of a test plant extract, respectively. Complete volume of
tube 6 to 0.50 mL with buffer solution.
(iii) Add 0.5 mL acid ninhydrin to each tube.
(iv) Add 0.5 mL glacial acetic acid to each tube.
(v) Cover the mouth of each tube with a glass marble and put the tubes in a
boiling water bath for 1 hour. Let cool in an ice bath.
(vi) Add 1 mL toluene to each tube and vortex. Stand the tubes for 1 minute.
(vii) Aspirate the toluene phase containing the chromophore with a glass Pas-
teur pipette.
(viii) Read absorbance of the toluene phase at 520 nm using the glass
microcuvette.
'& Isolation, Identification and Characterization of Allelochemicals/Natural Products

Calculations
Step 1. Graph the calibration curve relating absorbance at 520 nm to proline
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate total protein concentration (C) in the plant extract expressed
as proline equivalent concentration. Consider absorbance at 520 nm (A520)
from experimental tubes and extinction coefficient (e) obtained from linear
regression:
C (μg/mL) = (A520 ¥ e) ¥ 2 (tube 6) or C (μg/mL) = A520 ¥ e (tube 7)

Observations
It is advisable to express free proline concentration relative to dry weight of
plant material.

3.7 Glycosides
Experiment 15: Cyanide Production (Konig Reaction)
Total cyanide reaction is usually used to determine the release of cyanide from
plant cyanogenic glycosides (Goldstein and Spencer, 1985). Cyanide subjected to
an oxidant is transformed into cyanogen halide, CN+ (Fig. 11). The cyanogen
halide reacts with pyridine to produce an intermediate which hydrolizes to a
conjugated dialdehyde, the glutaconic aldehyde. This compound is then coupled
with a primary amine or a compound containing reactive methylene hydrogens
to form coloured compounds that can be read at 580 nm.

Figure 11 Reactions involved in detection of cyanide production.

 Colorimetric Reactions ''

Materials and equipments required

Assay tubes, marbles, pipettes; analytical balance with 0.01 mg sensitivity;
spectrophotometer or filter photometer capable of measurement at 580 nm,
equipped with a 1 cm path length glass microcuvette.

Reagents
1 N NaOH (Dissolve 40 g of NaOH in 400 mL of distilled water. Make up to a
final volume of 1 L); acetic acid 2 N (dilute 57.2 mL of acetic acid to a final volume
of 1 L); oxidant reagent (dissolve 2.5 g of succinimide in 300 mL of distilled water;
add 0.25 g N-chlorosuccinimide and stir to dissolve; dilute the solution to 1 L);
coupling compound reagent (dissolve 12 g of barbituric acid in 120 mL of pyridine;
make up to a final volume of 400 mL with distilled water); 0.5 mg/mL stock
NaCN solution (dissolve 50 mg of NaCN in 100 mL of 1 N NaOH); standard
NaCN solution (dilute 1 mL of stock NaCN solution in 49 mL of 1 N NaOH);
cassava extract (collect root peels of cassava; grind 1 g of root peel in a mortar;
stand for 2 hours. Add 7 mL of 1 N NaOH; grind again in the mortar; filter and
use for the assay).

Procedure
(i) Set up a calibration curve in five tubes: dilute standard NaCN solution with
1 N NaOH to prepare solutions 20, 40, 60 and 80 μM. Label tubes from 1 to
5. Then, add 1 mL of 0, 20, 40, 60 and 80 μM NaCN to tubes 1, 2, 3, 4 and
5, respectively.
(ii) Set up your experimental assay tubes and prepare tubes 6 and 7 adding
0.50 and 1 mL of cassava extract, respectively. Complete volume of tube 6
to 1 mL with 1 N NaOH.
(iii) Add 0.5 mL of acetic acid 2 N and 5 mL of the oxidant reagent to each tube.
(iv) Add 1 mL of the coupling compound reagent and swirl. Stand tubes for
10 minutes.
(v) Read absorbance at 580 nm.

Precautions
NaCN is extremely poisonous, eating or inhaling it is harmful, or even fatal.

Calculations
Step 1. Graph the calibration curve relating absorbance at 580 nm to NaCN
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate cyanide concentration (C) produced from cassava extract
expressed as NaCN equivalent concentration. Consider absorbance at 580 nm
(A580) from experimental tubes and extinction coefficient (e) obtained from linear
regression:
C (μM) = (A580 ¥ e) ¥ 2 (tube 6) or C (μM) = A580 ¥ e (tube 7)
 Isolation, Identification and Characterization of Allelochemicals/Natural Products

Experiment 16: Total Cardenolide Content (Cardiac Glycosides)

In alkaline conditions, cardenolides are formed with 2,2‘,4,4‘-tetranitrobiphenyl
(TNBP) reagent from a stable coloured complex, whose absorbance is measured
at 620 nm (Genkina and Eídler, 1975).
Materials and equipments required
Assay tubes, marbles, Erlenmeyer flasks, pipettes; analytical balance with 0.01 mg
sensitivity, water bath, spectrophotometer or filter photometer capable of
measurement at 620 nm, equipped with a 1 cm path length glass microcuvette.
Reagents
2,2‘,4,4‘-tetranitrobiphenyl (TNBP) reagent (dissolve 150 mg of TNBP in 100 mL
of absolute ethanol); stock digitoxin solution (dissolve 100 mg of digitoxin in
10 mL ethanol); 50 μg/mL standard digitoxin solution (dilute 0.5 mL of the stock
digitoxin solution to 100 mL with absolute ethanol); Asclepias leaf extract (add 5 g
of fresh young leaves of Asclepias glaucescens [or other Asclepias spp] to 50 mL of
95% ethanol; boil for 5 minutes; after cooling, filter the extract).

Procedure
(i) Set up a calibration curve in five tubes: dilute 50 μg/mL digitoxin solution
with ethanol to prepare solutions 1.8, 3.5, 5.3 and 7 μM. Label tubes from 1
to 5. Then, add 3.7 mL of 0, 1.8, 3.5, 5.3 and 7 μM digitoxin to tubes 1, 2, 3,
4 and 5, respectively.
(ii) Set up your experimental assay tubes and prepare tubes 6 and 7 adding
1.85 and 3.7 mL of Asclepias leaf extract, respectively. Complete volume of
tube 6 to 3.7 mL with ethanol.
(iii) Add 1 mL of TNBP solution and 1 mL of 0.1 N NaOH to each tube.
(iv) Stand tubes for 40 minutes.
(v) Read the absorbance at 620 nm.

Calculations
Step 1. Graph the calibration curve relating absorbance at 620 nm to digitoxin
concentration. Fit a line function to the measured data points applying regression
analysis with r 2 > 0.80 (refer Calculations in Experiment 1).
Step 2. Calculate total cardenolide concentration (C) in plant extract expressed as
digitoxin equivalent concentration. Consider absorbance at 620 nm (A620) from
experimental tubes and extinction coefficient (e) obtained from linear regression:
C (μM) = (A620 ¥ e) ¥ 2 (tube 6) or C (μM) = A620 ¥ e (tube 7)
Observations
The epigeal plant parts contain large amounts of substances (i.e. pigments,
chlorophyll and resins) that are soluble in ethanol, which can interfere with this
colorimetric method. Chromatographic methods can overcome this kind of
interference.
 Colorimetric Reactions 

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