ISSN: 0975-8585

Research Journal of Pharmaceutical, Biological and Chemical

Sciences
Flow Injection Spectrophotometric System for Determination of Flavonoids in
Tea Using Modified Dowd Assay
Piyanete Chantiratikul* and Kwanyuen Leamsomrong
Department of Chemistry and Center of Excellence for Innovation in Chemistry (PERCH-CIC), Faculty of Science,
Mahasarakham University, Kantarawichai, Maha Sarakham,44150 Thailand

ABSTRACT
A simple, fast and reliable FI-system for the determination of flavonoids in tea was investigated. The
proposed method was based on the reaction of sodium nitrite and aluminum chloride in alkaline solution, with
spectrophotometric detection at 500 nm. The experimental parameters were optimized. Linear calibration graph
-1
was obtained in the range of 0.5-50.0 mg L (as catechin). The relative standard deviations (n=20) were in the
-1
-1
range 1.41-2.19%, with detection limit (S/N=3) was 0.03 mg L . The sample throughput was 32 samples h . The
results revealed no significant interference. According to paired sample t-test, there was no significant difference
between Dowds standard spectrophotometric method and the proposed method. The method was successfully
applied to flavonoids analysis in tea samples.
Keywords: Flow injection; Spectrophotometry; Flavonoids; Dowd assay; Tea

*Corresponding author

October December

2012

RJPBCS

Volume 3 Issue 4

Page No. 1322

ISSN: 0975-8585
INTRODUCTION
Teas are used as beverages worldwide and widely consumed in China and Japan. The
two main types of tea are green and black. Most phenolic compounds found in tea are
polyphenols which consisting of more than one benzene ring with each containing at least one
hydroxyl group (-OH). Health benefits of tea are believed to be widely due to the presence of
high levels of polyphenols, mainly flavonoids. Flavonoids are phenol derivatives that are
distributed in plants and known to exhibit higher antioxidative activities. The main polyphenols
present in tea are the flavonoids. The most subclass of flavonoids in tea is the flavanols
(primarily catechins) [1]. The flavonoids are largely responsible for the distinctive taste and
color of tea [2]. A number of studies suggest that tea consumption may reduce the risk for
cardiovascular disease [3], decrease carotid atherosclerosis [4], blood pressure [5] and
chloresterol [6].
There are several methods for determining flavonoids in tea such as LC-MS [7], RP-HPLC
with UV detection [8-9], NIR reflectance spectroscopy [10], capillary electrophoresis [11,12] and
flow injection with adsorptive stripping voltammetry [13,14] and amperometry [15-16].
This study was aimed to establish a simple, fast and reliable flow injection system based
on the reaction of sodium nitrite and aluminum chloride in alkaline solution with visible
detection at 500 nm for the determination of flavonoids contents in tea infusion. The
optimizations of experimental parameters and validation of the proposed method were also
investigated.
MATERIALS AND METHODS
Reagents
All reagents used were of analytical reagent grade and all solutions were prepared in
deionized water (18.2 M cm) from a Milli-Q water purification system (Millipore Co., USA).
AlCl3, NaNO2, NaOH, fructose, sucrose were purchased from BDH, England. Catechin and NaCl
was purchased from Fluka, Switzerland. The calibration curve was prepared by diluting catechin
solutions from 0.5 to 50.0 mg L-1 from stock solutions.
Preparation of Samples
Tea sample solutions were prepared by infusing 1 g of tea powder samples (purchased
from a local market in Maha Sarakham province) in hot double-distilled water for about 10 min.
Then, the tea solutions were filtrated and adjusted to a 100 ml volumetric flask prior to
injection into the proposed FI-system.

October December

2012

RJPBCS

Volume 3 Issue 4

Page No. 1323

ISSN: 0975-8585
Analytical Procedure
The content of flavonoids was determined by spectrophotometric method described
previously [17] and modified in our laboratory. Briefly, aliquots of standard catechin solution or
appropriate dilution of samples solution were reacted with 5.0% w/v NaNO 2 solution. Then, a
flavonoid-aluminum complex was occurred using aluminum trichloride. The pink color product
was measured at 500 nm after standing at room temperature for 5 min and compared to that
of catechin standard.
A schematic diagram for the proposed flow injection analysis system is presented in
Fig.1. A peristaltic pump (Perkin Elmer, FIAS-300, U.S.A.) was used for propelling the carrier
solution (CS) and reagent (RS1, RS2). Solinoid injection valve, I, with a sample loop (200 l) was
used for introducing catechin as standard solutions, as well as sample solutions into carrier
stream. PTFE tubing (i.d.= 0.89 mm) was used for flow lines. The absorbance was measured
with UV-Visible spectrophometer (Perkin Elmer Lambda Bio-40, U.S.A.). A personal computer
with FIA monitor data processing software (Perkin Elmer, U.S.A.) was used for controlling the
apparatus and recording data.
P

I
MC1

MC2

CS
R1
R2

DD

Figure 1: Schematic manifold of FIA spectrophotometric system for determination of flavonoid. CS: carrier
(deionized water); R1: AlCl3 solution mixed with NaNO2 solution; R2: NaOH solution; P: peristaltic pump; I:
injection valve; MC1 and MC2: mixing coil 1 and 2; D: spectrophotometric detector; W: waste.

A standard/sample solution was injected into a carrier stream of deionized water and
flowed to merged with a stream of RS1 (AlCl3 solution mixed with NaNO2 solution) and RS2
(NaOH solution), respectively. The complex formation was take place inline in mixing coil (MC1
and MC2) and was then monitored by measuring absorbance change at wavelength 500 nm. A
calibration graph was plotted between absorbance and catechin concentration in standard
solution. It was employed for the determination of flavonoids in tea samples.
RESULTS AND DISCUSSION
Selection of detection wavelength
Catechin solution as standard solution was prepared according to the standard
procedure and the absorption spectra was obtained in the range from 400 to 800 by a

October December

2012

RJPBCS

Volume 3 Issue 4

Page No. 1324

ISSN: 0975-8585
spectrophotometer. The maximum absorption wavelength of the pink color product was 500
nm (Fig. 2).

Figure 2: The UV-vis absorption spectra of complexes at different catechin concentrations, (a) blank; (b) 5 mg L-1 catechin; (c)
10 mg L-1 catechin; (d) 25 mg L-1 catechin; (e) 50 mg L-1 catechin.

Optimization of experimental variable for FI-system

The effect of a sample injection volume in the range of 50 to 500 l was investigated. It
could be seen that the absorbance increased with an increase of sample volume up to 200 l,
beyond which the highest absorption remained constant. A sample injection volume of more
than 200 l caused the broad peak and used analysis time. Thus, a sample injection volume of
200 l was selected for further study.
The effect of flow rate of carrier (double-distilled water, CS) and reagent solutions,
which was AlCl3 solution mixed with NaNO2 solution (RS1) and NaOH solution (RS2), was
studied in the range of 0.8 to 2.4 ml min-1. The results indicated that the sensitivity slightly
increased with an increase of flow rate. However, too low flow rates caused board peak and
could lead to poor reproducibility and sample throughput. Thus, the signals decreased when
the flow rate increased. Therefore, 1.7, 1.4 and 1.1 ml min -1 were selected as the optimum flow
rate of carrier, RS1 and RS2, respectively.
The effect of reaction coil length (RC1) was examined in the range of 100 to 1600 mm.
The results showed that the absorbance was almost identical when the 750-1300 mm reaction
coil length was used. Longer reaction coils gave a longer residence time, but the dispersion of
the sample zone became larger, and the output peaks were broadened. The reaction coil length
of 800 mm was chosen for rapid analysis measurement.
The effect of reaction coil length, RC2 was investigated in the range of 200 to 1000 m. It
can be seen that the absorbance increased with an increase of the length of the reaction coil up
to 300 mm. At longer than 300 nm, too large dispersion was occurred. Therefore, 300 mm was
used for further study.

October December

2012

RJPBCS

Volume 3 Issue 4

Page No. 1325

ISSN: 0975-8585
The effect of concentration of sodium nitrite solution was examined in a range from 0.1
to 1.0% w/v. It was found that the sensitivity increased with increasing of sodium nitrite
concentration. At the concentration of sodium nitrite solution was more than 0.5%, air bubble
was occurred. In further experiments, 0.5% sodium nitrite solution was selected.
The effect of concentration of aluminum chloride solution was studied from the range of
0.5 to 2.0% w/v. The sensitivity increased with increasing concentration of aluminum chloride
solution. However, a concentration of aluminum chloride was higher than 1% caused
precipitation and high baseline. The suitable concentration of aluminum chloride solution was
1% for optimum FIA system.
The effect of concentration of sodium hydroxide solution was studied from the range of
0.1 to 4.0% w/v. The sensitivity increased with increasing concentration of aluminum chloride
solution and remained constant when sodium hydroxide concentration was higher than 1.0%.
In order to reduce reagent consumption, the suitable concentration of sodium hydroxide
solution was 1% for further study.
The studied parameters for flavonoids determination using down assay and their
optimum value were summarized in Table 1.
Table 1: The studied parameters and their optimum value of FI system for determination of flavonoids

Analytical characteristics
Wavelength (nm)
Sample injection volume (l)
-1
Flow rate of carrier (ml min )
-1
Flow rate of sodium nitrite (R1) (ml min )
-1
Flow rate of sodium hydroxide (R2) (ml min )
Length of mixing coil 1 (mm)
Length of mixing coil 2 (mm)
Concentration of sodium hydroxide (M)
Concentration of sodium nitrite (%w/v)
Concentration of aluminium trichloride (%w/v)

Studied range
400-800
50-500
0.8-5.4
0.8-5.4
0.8-5.4
100-1600
100-1000
0.1-4.0
0.1-1.0
0.5-2.0

Optimum value
500
200
1.7
1.4
1.1
800
300
1.0
0.5
1.0

Validation of the proposed FI-system

In order to investigate the linearity response, the catechin standard solutions were used
for preparing the calibration curve at the concentration range from 0.5 to 50.0 mg L -1. The
equation of calibration graph was expressed as Y = 0.0065X + 0.0089, where Y and X were
absorbance and catechin concentration, respectively, with a correlation coefficient of 0.9913.
The relative standard deviation (RSD) was 2.14% for 12.55 mg L-1 (n=20). The limit of detection
(LOD) was 0.0288 mg L-1 (calculated from 3S/N). Thirty-two samples per hour were obtained for
sample throughput. The investigation interference species was conducted with regard to
possible chemical interferences. The tolerable concentration is defined as the concentration of
species causing less than 5% relative error. The results revealed no significant interference
from citric acid, fructose, sucrose and sodium chloride. These data demonstrated that the
October December

2012

RJPBCS

Volume 3 Issue 4

Page No. 1326

ISSN: 0975-8585
proposed FI-system is sensitive and adequate to determine flavonoids contents at low
concentrations.
Application of the proposed FI-system to real samples
The proposed FI-system was applied to the determination of flavonoids in 9 tea powder
samples and compared with the original spectrophotometric method (Table 2). It was found
that the proposed and original spectrophotometric assay [17] were in good agreement with a
correlation coefficient of 0.999 as shown in Fig. 3. Due to the ability to operate continuously, it
is possible to analyze about 32 samples h-1, making the method for fast determination of for
flavonoids contents in tea.
Table 2: Flavonoids contents in tea infusion samples obtained by FI system and spectrophotometric method

Sample No
1
2
3
4
5
6
7
8
9

-1

FI system (mg g )
3.350.08
0.320.01
3.960.10
13.690.32
8.060.20
0.45 0.01
0.0340.02
5.930.06
13.400.21

-1

Dowds method (mg g )

3.390.06
0.320.01
4.390.33
14.980.33
8.710.13
0.46 0.01
0.0420.00
7.060.13
14.630.01

FIA/Dowd
0.98
1.00
0.90
0.91
0.92
0.97
0.80
0.83
0.91

Figure 3: Correlation relation between the proposed FI system and original spectrophotometric method.

CONCLUSION
A simple, fast and reliable FI-system based on the reaction of NaNO2 and AlCl3 in alkaline
solution for the determination of flavonoids in tea was developed. The complex product has
maximum absorption wavelength of 500 nm. This system could be used for the determination
of flavonoids in the range 0.5-50.0 mg L-1 (as catechin). It showed no significant difference with
the original spectrophotometric method. This proposed method is sensitive, fast, reliable and
October December

2012

RJPBCS

Volume 3 Issue 4

Page No. 1327

ISSN: 0975-8585
adequate to determine flavonoids contents and can be directly applied to the determination of
flavonoids in tea samples.
ACKNOWLEDGEMENTS
The authors are grateful for the financial support from Mahasarakham University
Budget fiscal year 2011 and Center of Excellence for Innovation in Chemistry (PERCH-CIC),
Commission on Higher Education, Ministry of Education.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]

Hodgson JM, Croft KD. Mol Aspects Med 2010; 31: 495-502.
Peterson J, Dwyer J, Bhagwat S, Haytowitz D, Holden J, Eldridge AL, Beecher G,
Aladesanmi J. J Food Comp Anal 2005; 18: 487-501.
Iwai N, Ohshiro H, Kurozawa Y, Hosoda T, Morita H, Fumakawa K, Okamoto M, Nose T. J
Epidemiol 2002; 12: 191-198.
Mursu J, Nurmi T, Tuomainen TP, Ruusunen A, Salonen JT, Voutilainen S. J Nutr 2007; 98:
814-818.
Hodgson JM, Croft KD. J Sci Food Agric 2006; 86: 2492-2498.
Hodgson JM. Asia Pacific J Clin Nutr 2008; 17: 288-290.
Justesen U, Knuthsen P, Leth T. Cancer Lett 1997; 114: 165-167.
Hertog MGL, Hollman PCH, van de Putte B. J Agric Food Chem 1993; 41: 1242-1246.
Bramati L, Aquilano F, Pietta P. J Agric Food Chem 2003; 51: 7472-7474.
Schulz H, Engelhardt UH, Wegent A, Drews H, Lapczynski S. J Agric Food Chem 1999; 47:
5064-5067.
Kreft S, Knapp M, Kreft I. J Agric Food Chem 1999; 47: 4649-4652.
Ozyurt D, Demirata B, Apak R. Talanta 2007; 71: 1155-1165.
Volikakis GJ, Efstathiou CE, Talanta 2000; 51: 775-785.
Volikakis GJ, Efstathiou CE. Anal Chim Acta 2005; 551: 124-131.
Pedrosa VA, Malagutti AR, Mazo LH, Avaca LA. Anal Lett 2006; 39: 2737.
Yashin AY. Russ J Gen Chem 2008; 78: 2566-2571.
Adom KK, Liu RH. J Agric Food Chem 2002; 50: 6182-6187.

October December

2012

RJPBCS

Volume 3 Issue 4

Page No. 1328