Lab 2: Quantification of Total Phenolic Content using the Follin-Ciocalteu

Method
Introduction
Phenolics are the largest group of phytochemicals that account for most of the
antioxidant activity in plants or plant products. Flavonoids, tannins and
proanthocyanidins are some of the common phenolic antioxidants found in plants (John
et al. 2014). These compounds may exhibit many biological effects including
antioxidant activity. Antioxidants are able to prevent a number of diseases due to their
ability to scavenge free radicals (Baba and Malik 2015). Antioxidant activities of most
plant samples are well-correlated with their total phenolic content.

In this study, the total phenolic acid content was quantified

spectrophotometrically by using Folin-Ciocalteu reagent. Folin-Ciocalteu assay is
based on transfer of electrons in alkaline medium from phenolics compounds
tophosphotungstic or phosphomolybdenum complexes of Folin-Ciocalteu reagent,
resulting in the formation of a blue complex (Varma and Sharma 2017). Sodium
carbonate as a buffer for pH adjustment and the end-point of the reaction was attained
after 30 min (Bioquochem.com n.d.). The reduced Folin-Ciocalteu reagent is detectable
with a spectrophotometer in 765nm. Total phenolics content was expressed as mg
Gallic acid Equivalents (GAE) because gallic acid was used as reference standard in
this experiment.

Objective
- To evaluate the absorbance of phenols at different concentrations
- To determine the concentration of total phenolic content in unknown solution

Apparatus & Materials

4x volumetric flask (10ml), 4x unwrapped test tubes, 10x test tubes wrapped with
aluminium foil, Gallic acid standard solution (1000ppm), Follin-Ciocalteus reagent
(10 times dilution), Sodium carbonate (7.5% w/v), Unknown gallic acid solution

Procedure
As refer to lab manual.
Results
Before Data Handling:
Group Standard 1 Standard 2 Standard 3 Unknown
(25ppm) (50ppm) (100ppm)
1 0.272 0.511 1.090 0.852
2 0.325 0.677 1.149 0.980
3 0.422 0.776 1.028 0.684
4 0.238 0.308 0.456 0.048
5 0.463 0.695 0.992 0.860
6 0.324 0.609 1.144 0.836
7 0.239 0.527 1.562 1.107
8 0.214 0.430 0.915 0.845
9 0.614 0.988 1.672 1.141
Table 1.0: Absorbance of gallic acid at different concentration before data
handling

Standards Mean Standard Deviation Percentage Relative Standard

Deviation/ % (RSD)
1 (25ppm) 0.346 0.124 35.84%
2 (50ppm) 0.613 0.189 30.83%
3 (100ppm) 1.112 0.335 30.13%
Unknown 0.817 0.303 37.09%
Table 2.0: Mean, standard deviation and percentage relative standard deviation
of standards before data handling
 Absorbance (Abs) at 765nm
1.2
against Concentration (ppm) 1.112

Absorbance (Abs) at 765nm 1

0.8
0.613
0.6
y = 0.0102x + 0.0965
0.346 R = 0.9997
0.4

0.2

0
0 20 40 60 80 100 120
Concentration (ppm)
Absorbance Linear (Absorbance)

Graph 1.0: Absorbance against concentration of standards and unknown before

data handling

After Data Handling:

Group Standard 1 Standard 2 Standard 3 Unknown
(25ppm) (50ppm) (100ppm)
1 0.272 0.511 1.090 0.852
2 0.325 0.677 1.149 0.980
3 0.422 0.776 1.028 0.684
4 0.238
5 0.463 0.695 0.992 0.860
6 0.324 0.609 1.144 0.836
7 0.239 0.527 1.107
8 0.214 0.430 0.915 0.845
9 1.141
Table 3.0: Absorbance of gallic acid at different concentration after data
handling
 Standards Mean Standard Deviation Percentage Relative Standard
Deviation/ % (RSD)
1 (25ppm) 0.312 0.084 26.92%
2 (50ppm) 0.604 0.112 18.54%
3 (100ppm) 1.053 0.084 7.98%
Unknown 0.913 0.143 15.66%
Table 4.0: Mean, standard deviation and percentage relative standard deviation
of standards before data handling

Absorbance (Abs) at 765nm

against Concentration (ppm)
1.2
1.053
Absorbance (Abs) at 765nm

0.8
y = 0.0098x + 0.0875
0.604
R = 0.9953
0.6

0.4 0.312

0.2

0
0 20 40 60 80 100 120
Concentration (ppm)
Absorbance Linear (Absorbance)

Graph 2.0: Absorbance against concentration of standards and unknown after

data handling

Calculation
Concentration of total phenolic content in unknown (Before Data Handling):
y = 0.0102x + 0.0965
0.817 = 0.0102x + 0.0965
x = 70.7 ppm GAE

Concentration of total phenolic content in unknown (After Data Handling):

y = 0.0098x + 0.0875
0.913 = 0.0098x + 0.0875
x = 84.3 ppm GAE
Concentration = 3.2603 x 10
= 32.6 ppm GAE

Discussion
Gallic acid act as a reference standard of phenolic content in this experiment. According
to Agbor et al. 2014, 725nm to 765nm are the wavelength for maximal absorbance of
the polyphenols produced in gallic acid. In this experiment, absorbance of gallic acid
solutions of different concentration including 25ppm, 50ppm and 100ppm was
measured at 765nm. It is important to measure at the wavelength of maximum
absorbance because absorption of maximum shows characteristic of each compound.
Measuring at wavelength of maximum absorbance provides information on the
electronic structure of gallic acid, ensure the highest sensitivity and minimize the
deviation form Beers law (Kenkel 2014).

The mean and standard deviation was calculated based on nine groups of data. Before
data handling, the mean of the absorbance for 25ppm, 50ppm and 100ppm gallic acid
are 0.346, 0.613 and 1.112 respectively. This shows that absorbance of TPC increased
in gallic acid when the its concentration increased. The standard deviation for
25ppm,50ppm and 100ppm of gallic acid before data handling are 0.124, 0.189 and
0.335 respectively. Comparing with data before handling, standard deviation after data
handling was relatively low which 25ppm is 0.084, 50ppm is 0.112 and 100ppm is
0.084. This is because there are many outliers contributed from different groups before
data handling. Relative standard deviation(RSD) was also calculated by dividing
standard deviation of a series of values from the average of the values to measure the
precision in data analysis (Helmenstine 2014). The RSD of absorbance for 25ppm of
gallic acid before data handling is 35.84% despite after data handling is 26.92%. As for
RSD of 50ppm before data handling is 30.83% and after data handling its RSD becomes
18.54%. This large value of relative standard deviation means the absorbance readings
from each group were largely different. However, the absorbance value for 100ppm
before data handling is the least precise, 30.13% when comparing with the data after
handling which is 7.98%. The big difference in relative standard deviation for 50 ppm
is because many of the readings were contributed to outliers.
Besides, a standard curve was created based on the absorbance of TPC in gallic acid on
different concentration of gallic acid. From the standard curve, the concentration of
total phenolic content in unknown was calculated as 70.7ppm GAE. Yet, the total
phenolic content in unknown was calculated as 32.6ppm GAE after data handling.
Comparing with the RSD percentage before handling which is 37.09%, the RSD
percentage after handling shows a more precise result which is only 15.66%. The
difference in absorbance value and concentration of unknown samples between the data
might be due to human error. Some of the groups may not have measure accurate
volumes needed by using the micropipette incorrectly or the samples were not placed
in dark places. It could also be due to insufficient time places in dark places for
complete reaction leading to a difference in values. Besides, using the wrong blank may
affect the absorbance reading too.

It is important that to ensure there is no precipitation when measuring the absorbance.

The presence precipitate or materials can cause the reading to be distorted by light
scattering effect.

Conclusion
In conclusion, the absorbance of phenols increased when increasing the concentration.
To ensure the reliability of result, we should apply data handling method. The
absorbance of the concentration of total phenolic content in unknown solution measure
from standard curve before handling is 70.7ppm but after handling is 32.6ppm. Its RSD
shows that the data after handling is more precise.
References
Agbor, G.A., Vinson, J.A., and Donnelly, P.E., 2014. Folin-Ciocalteau Reagent for
Polyphenolic Assay. International Journal of Food Science, Nutrition and
Dietetics, 3(8), 147-156.

Baba, S.A., and Malik, S.A., 2015. Determination of total phenolic and flavonoid
content, antimicrobial and antioxidant activity of a root extract of Arisaema
jacquemontii Blume. Journal of Taibah University for Science, 9(4), 449-454.

Bioquochem.com, n.d. Folin Ciocalteau Phenolic Content Quantification Assay Kit

[Online]. Available at:
http://www.filgen.jp/Product/Bioscience4/BioQuoChem/KB-03-
006%20Folin%20Ciocalteau%20Book.pdf [Accessed 27th May 2017].

Helmenstine, A.M., 2014. What Is Relative Standard Deviation? [Online]. About.com

Education. Available from:
http://chemistry.about.com/od/chemistryglossary/g/Relative-Standard-Deviation-
Definition.htm [Accessed 27h May 2017].

Kenkel, J., 2014. Analytical chemistry for technicians. 4th ed. Boca Raton: Lewis
Publishers.

John, B., Sulaiman, C.T., George, S., and Reddy, V.R.K., 2014. Total Phenolics and
Flavonoids in Selected Medicinal Plants from Kerala. International Journal of
Pharmacy and Pharmaceutical Sciences, 6(1), 406-408.

Varma, A., and Sharma, A.K., 2017. Modern Tools and Techniques to Understand
Microbes. Switzerland: Springer, 422-423.