ÇUKUROVA UNIVERSIY

SCIENCE AND LITERATURE FACULTY

CHEMISTRY DEPARTMENT.
BIOCHEMISTRY LABORATORY I

FOLİN-WU YÖNTEMİ İLE KAN ŞEKERİ TAYİNİ

(Folin-Wu Method for Blood Glucose Estimation)

Daniel Alejandro MALAVER RANGEL

2019144315

Porpoise of the experiment

Acquiring and obtaining a major knowledge of glucose and, most important, generating
a base in bioanalysis and bioanalytical techniques by carrying out a Folin-wu
methodology for calculating the quantity of glucose in a given blood sample.

Theorical information and experiment introduction.

The Folin-Wu method, also known as the Folin-Lowry method, is a laboratory technique used
for the quantification of glucose and other reducing sugars in a solution. This method was
developed in 1953 for measuring total blood sugar showing that is a test that is not normally
used in modern medicine. It involves adding a reagent containing pyruvic acid and phenol to a
sample of whole Blood. This method involves a colorimetric assay based on the reduction of a
compound known as Folin-Ciocalteu reagent (a mixture of phosphomolybdic and
phosphotungstic acids) by reducing sugars in an alkaline medium. The reduction of the
reagent leads to the formation of a blue color whose intensity is proportional to the amount of
reducing sugar present. During this experiment benedict reagent will be used with the purpose
to oxidate the glucose in the following way:

[1]
Due to the color that the complex adquire the analysis in the UV will be 500-1000cm^-1, this
is because the compound can give an expectrum in the visible region of the ligth expectrum.
The main logic behind such a study is seing how the intensity of the blue color fades away as
the concentration of glucouse changes. İn a broad point of view, the whole methodologyu can
be summarize as:

Preparation of Glucose Standard Curve

standard glucose solutions with known concentrations were prepared in order to calibrate the range
that will be worked with. The concentrations should cover the expected range of glucose
concentrations in the samples to be analyzed.

Preparation of Samples

A separation of the plasma and serum from the blood cells and proteins is necessary in order to
continue with the experiment, in this experiment, the blood sample that was separated from its protein
and blood cells will be called treated blood sample, Dilute the samples appropriately, especially if the
glucose concentration is expected to be high. Dilution might be necessary to fall within the range of
the standard curve.

Reaction

In test tubes or cuvettes, mix a volume of each sample (or standard) with an appropriate volume of
Folin-Ciocalteu reagent and Cu(II). Add sodium carbonate solution to each tube to create an alkaline
environment. This initiates the reduction reaction.-in this experiment benedic’s reagent was used as a
main source of CuSO4 Allow the reaction mixtures to incubate for a specified period. Usually, this
incubation period allows the color to develop, which is proportional to the glucose concentration.

Measurements

measure the absorbance of the blue color developed in each test tube or cuvette at a specific
wavelength (commonly around 750 nm) using a spectrophotometer. Plot a standard curve using the
absorbance values of the glucose standard solutions against their known concentrations.

Analysiz
Determine the glucose concentration of the sample by comparing their absorbance values to the
standard curve.
This method is sensitive to the presence of reducing substances other than glucose, which might
interfere with the assay. To minimize interference, additional treatments or specific filtration steps
might be employedthis is one of several methods used for glucose estimation in laboratories, and it's
important to ensure calibration, precision, and standardization while performing the assay to obtain
accurate and reliable results.

Calculations and information Tables

All the calculations were made using Excel.
Preparation of solutions
H2SO4 Sol.
Calculation M (solution neeeded)
Density (g/ml) 1,84 3,2693g from a 96% sol 3,40555556
Concentration 96
Vol (ml) Solution
Mw 98,08 needed
Concentration
wanted Den=1,84g/ml 1,85084541
0,666666667 N PREPARATION
0,333333333 M
100ml Mol q. 0,03333333
100ml g quan. 3,26933333 100ml Balon + 1,85ml 96%
 Glucose and Absorbance Calculations.
Four tubes were prepared with different volumes of a solution of glucose 0,1mg/ml for
calibrating the UV and obtaining a function of absorption and glucose concentration. The
following formula is used for calculating the dissolution of glucose.
0,1g
M Glucouse solution ( )∗V glucosesol
ml
M glucouse ∈the tubes =
V of the tubes. (6 , 25 ml)

The graph of glucose vs absorption will look like

tup no 1 2 3 4 5 SAMPLE
GST 0 0,25 0,5 0,75 1 1
Su 1 0,75 0,5 0,25
Cu(II) 1 1 1 1 1 1
FM 1 1 1 1 1 1
Su(II) 3,25 3,25 3,25 3,25 3,25 3,25
6,25 6,25 6,25 6,25 6,25 6,25
GLU
conce. 0 0,004 0,008 0,012 0,016 0,00955
0,00002 0,15 0,294 0,434 0,57 0,345

glucose Absorbance vs Glucouse concentration

(mg/ml)
0.6
f(x) = 35.599 x + 0.00481199999999998
0.5

0.4

0.3

0.2

0.1

0
0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018
 Concentration in the plasma
The following terminology were used:
 Treated Blood: Solution of blood in which had a previous process of protein
extraction.
 Plasma: Blood taken from a person
 Sample’s glucose: glucose concentration calculated in the (UV) absorption
machine.
The calculation was planned in the following way:

glucose in the sample glucose in the treated blood glucose in the plasma

Treated Blood sample

blood Treated (ml) Glucouse of the blood sample treated
Plasma ml 1 1 (eq1) 0,0596875
Sample’s Volume
water ml 17 (ml) Glucouse founded in the plasma
H2SO4 ml 1 6,25 1,19375
Sample’s Glucose Glucouse founded in the plasma
Na2WO ml 1 Con. (mg/ml) (mg/dl)

Final
volume (ml) 20 0,00955 Equ(2) 119,375
 Conclusion and discussion of results.
The Folin-Wu method was developed approximately 70 years ago and it was used in a
prototype in 1917 for detecting glucose in urine due to the characteristic way that the benedict
reagent works, this method ,thus , can be considered and old method that is not suitable for
measuring the glucose in the blood.
Understanding the main logic of this test is understanding why this method is not a suitable
way to develop such an important study that can be life-depending as the quantity of sugar in
our circulative system. The oxidation of sugars in the presence of copper gives a color that is
easily observed by the human eye, because of this, the Folin-Wu test is also called a
colorimetric assay. Nevertheless, blood is a complex colloidal solution that can have other
oxidable compounds besides sugar, because of this the quantity of glucose will never be
accurate. Instead of the Folin-wu, enzymatic reactions as the hexokinase can be used giving
an exact and accurate analysis. Because of this, one can mention that the Folin-Wu method
lacks specificity.
Having a lot of interferences, this method is not useful to measure the sugar in blood due to
the complexity that blood might present. Requiring such a long and extensive work for
measuring just a single parameter is just a big warning for not using this method specially in
these times where a lot of analytical methods have been developed having. However, it is
factor what makes it an excellent educational experience due to the extensive work that one
must make to accomplish the objectives of the practice.
Since the need to separate the proteins from the blood to the fact that a calibration is
obligatory for managing the results makes it a full-filling experience creating a better
understanding of what bioanalysis can work.
 Reportages and Sources

 The benedict test, which is the main reagent used in the Folin-Wu method, was
precenting problems since 1917.

 Preparation, characterization, and antimicrobial activity of xyloglucan-chitosan film

from tamarind (tamarind indica L.) seed kernel,
Benedic’s reagent is used in the modern-day laboratory for calculating in an empirical way the
quantity of glucose in organic studies, a good example of this would be the flowing article.

volume 179,2023,107486,ISSN 0300-9440,https://doi.org/10.1016/j.porgcoat.2023.107486.

(https://www.sciencedirect.com/science/article/pii/S0300944023000826)
  An article showing the development of glucose blood tests.

References: Quagliarini, E., Caputo, D., Cammarata, R. et al. Coupling magnetic levitation of
graphene oxide–protein complexes with blood levels of glucose for early detection of
pancreatic adenocarcinoma. Cancer Nano 14, 16 (2023). https://doi.org/10.1186/s12645-023-
00170-1

Sources and Citations

 Auto-analyzer versus Folin-wu for blood glucose - JSTOR. (n.d.-b).

https://www.jstor.org/stable/3627183
 says:, C. (2023, April 14). Estimation of blood glucose level by Folin-Wu Method.
Biochemistry Den. https://biochemden.com/estimation-blood-glucose-level-folin-wu-
method/#:~:text=The%20folin%2DWu%20method%20was,form%20a%20pink
%20coloured%20complex.
 Wikimedia Foundation. (2023, February 19). Folin–CIOCALTEU reagent. Wikipedia.
https://en.wikipedia.org/wiki/Folin%E2%80%93Ciocalteu_reagent