Lab 2 Genetics Lab Medical club

Lab 2 Genetics Lab Medical club

Spectrophotometry and determination

of glucose concentration
Welcome to our second genetics lab, hope you enjoyed the first one and hope you enjoy this one as well.
Special thanks to doctor Aya Al-Smairat for editing it. Let us start!

 Introduction
- Radiation is a kind of energy propagating into a
space.
- This radiation oscillates periodically between a
minimum and a maximum as a function of time like
a wave.
- As you can see in the figure, the distance between
two maxima or two minima of a wave is defined as
the wavelength (λ) given in nanometer (nm).
- The different components of radiation are characterized by specific wave lengths.
- The sum of all component is called spectrum (Electromagnetic spectrum).
- We also defined spectrum as the range of wavelength characteristic of specific type of radiation.
- Electromagnetic spectrum includes gamma rays, X-rays, radio rays, infra-red, UV rays and the visible
light.
- Electromagnetic spectrum of visible light
range approximately from 400-700 nm,
and each color within this light has specific
wavelength.

 Spectrophotometry
- Spectroscopy is a general and broad term in chemistry. It is the study of electromagnetic radiation
emitted or absorbed by a given substance.
- There is a type of spectroscopy for virtually any type of electromagnetic radiation, for ex:
microwave, X-ray, and even gamma ray spectroscopy.
- Spectrophotometry is a very specific type of spectroscopy. It deals with visible light, near UV and
near infra-red. So, it is a quantitative study of these radiations. Also, it is analytical technique that
uses light to measure the chemical concentrations. In spectroscopy we measure how much chemical
substance absorbed light by measuring the intensity of light that passes through a sample solution.

 Spectrophotometry applications
- Within medical diagnostics and in molecular and biomedical applications, determination of the
concentration of substances and solutions is a very important step.
- For example, spectrophotometry can provide platform for diagnosing bilirubin, hemoglobin and
glucose in the serum of the blood, as it can be conducted easily and quickly and represents the most
cost-effective option.

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 Spectrophotometer
- It is an instrument that measures the amount of light passes through a sample.
- Its principle of work states that we have a light source that emits the light with range of wavelength
b/w 400-700 nm. The light will strike the monochromator, which will separate this light into its
component wavelength. The light will exit the wavelength selector with a single wavelength
(intensity I0). Some of the light will be absorbed by the sample and will exit the sample with
(intensity I).Then, the light will hit the detector which will analyze the data and display it on digital
display or a meter.
Wavelength selector

- In Spectrophotometer, the sample is contained in a cuvette, which is a

small tube of circular or square cross section used to hold the sample
and is made of glass, plastic or quartz.
- We use Quartz cuvettes for UV experiments (for any wavelength in the
UV spectrum) and glass or plastic cuvettes for visible light (if the
wavelength within the visible range).
- For visible light, we use glass or plastic cuvettes according to the type
of the experiment.
- If the speed is more important than accuracy in such experiment, we use
plastic cuvettes. And if the accuracy is more important, we use glass
cuvettes.
- This picture shows plastic cuvette in our lab.
- Things that should be taken into consideration are:
1- Path length (width of cuvette): is the distance that the light travels
through the solution.
2- Io: is the intensity of the incident light before hitting the sample.
3- I: is the intensity of the light after it has passed through the sample or the intensity of light
coming outside the sample.
Note:
Intensity: the number of
photons delivered at a
specific point in a given
1cm unit of time
Path length (b)

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 Transmittance
- It is the fraction of light that passes through the
sample at specific wavelength.
- It is the ratio between I and Io.
- Transmittance (T)= I/Io
- Percent transmittance %T= T*100%

 Absorbance
- Absorbance (A):is the amount of light absorbed by the sample.

- Absorbance and Transmittance are inversely related, as one goes up the other goes down.
- If too much light exits the solution, that means there is small absorbance and vice versa.
- The absorbance has a logarithmic relationship with transmittance.
- Absorbance of zero corresponds to a transmittance of 100%.
- Absorbance of 1 corresponds to 10%
transmittance.
- Absorbance of 2 corresponds to 1%
transmittance.

 Absorption Spectrum
- Is the plot of the wavelength versus the
absorbance.
- As the wavelength increases, the absorbance
increases until it reaches the maximum value,
where the wavelength equal (λmax) which also
corresponds to (Amax) which is the maximum
absorbance.

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 Beer-Lambart’s law
- It states that the absorbance of a chemical substance is proportional to:
1- Concentration of the light absorbing molecules in the sample.
2- Path length of the beam through the sample.
A=ε×b×C
A: absorbance (has no unit).
ε: molar absorptivity (L/mol.cm).
b: path length (cm).
C: concentration of the absorbing chemical in the sample (mol/L).
- ε measures the amount of light absorbed per unit concentration. The compound with high molar
absorptivity absorbs more effectively. So, low concentrations of a compound with a high molar
absorptivity can easily be detected.
- The previous equation is similar to the mathematical expression Y=a × X
A= ε b C

Y a X
- Now we can plot the concentration vs absorbance. It will
be a linear relationship. As the concentration increases the
absorbance increases.
- Slope = a = εb

 How to Determine Unknown Concentration Using Spectrophotometric Method

- The concentration of an unknown sample can be
determined by comparing the absorbance data to
multiple standards of known concentration.
- In this method we have Multiple solutions with
known concentrations called Standards and we
have Sample solution with unknown
concentration.
- We must Measure the absorbance for all solutions
(standards and sample) by spectrophotometer.
Then construct a standard curve for the
concentration vs absorbance.
- In the standard curve, to find the unknown concentration, find the absorbance of your sample from
the Y-axis and then faced downward to see which concentration matches up with it.
- For our experiment we get multiple samples with known concentration (standards) by doing serial
dilution to a stock solution.
- Serial Dilution is the stepwise dilution of a substance in a solution that reduces its concentration.
- Stock solution is a concentrated solution that will be diluted to some lower concentrations for
actual use

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 How to prepare serial dilution from stock solution?

- Here we have stock solution with 300mg/dl, and we have 2 test tubes (test tube 1, test tube 2), each
one of them is filled by 1ml of distilled water. Then we are going to take 1ml from the stoke solution
and transfer it to test tube 1. Now we have a total volume of 2ml in test tube 1. we can say that we
did 1:2 dilution or we have done 2 times (2 folds) dilution.
- The dilution factor = final volume/initial volume.
- (final volume=total volume in test tube 1)
- (initial volume=the volume transferred from the stock
solution)
DF=Vf/Vi
DF=2/1
DF=2
- Now we can calculate the concentration of the solution in
test tube 1 by :
C1V1 (initial) =C2V2 (final)
C1=concentration of the stock solution
V1=the volume transferred from the stock solution
C2=the concentration of solution in test tube 1
V2=the volume of solution in test tube 1
So, we get 150mg/dl
- Then subsequently we will take 1ml from solution in test
tube 1 and transferred it to test tube 2. Again, in test tube 2
we have a total volume of 2ml and again we did 1:2 dilution
or 2 folds dilution.
- The concentration of solution in test tube 2 can be
calculated by the formula:
C1V1=C2V2 but here
C1= the concentration of solution in test tube 1
V1= the volume of solution transferred from test tube 1
C2= the concentration of solution in test tube 2
V2= the total volume of solution in test tube 2
So, we get 75mg/dl
- Now the solution in test tube 2 has much less concentration than the stock solution, each time we
diluted it 2 folds dilution. We can now multiply them to get the final dilution factor which is 4 folds
dilution.
- So, the dilution from the stock solution to the test tube 2 is the final dilution factor and it equals 4
(4x dilution).

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 Determination/measurement of glucose concentration

- We will use indirect method to get the glucose concentration, following the
instructions provided by the reagent manufacturing company.
- This reagent will work to give color (pink to red) to glucose solution. It contains two
enzymes (Glucose oxidase and peroxidase).
- This method is based on an enzymatic colorimetric assay.
- Glucose cannot absorb visible radiation that is why it is colorless compound.
- Glucose can be estimated indirectly by glucose oxidase - peroxidase method.
Glucose oxidase
Glucose + H2O2 + O2 Gluconic acid + H2O (oxidation reaction)
peroxidase
H2O2 + 4-aminoantipyrin + phenol Quinoneimine(pink to red) + 4H2O

- The intensity of quinoneimine color related to glucose concentration, that is why it is called indirect
method.
- Quinoneimine absorbs light at λ max equal 500nm.
- So, we have 2 steps:
1- The oxidation of glucose by glucose oxidase.
2- The oxidation of glucose reagent by peroxidase.
- Here is the leaflet attached to the reagent.

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Moving to the Lab work.

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 Equipment:
4
1- Glass pipette
2- Fixed volume micropipettes with 2 with
different sizes.
3- Tips for the pipettes.
4- Test tubes contain solution. 2

 Materials: 1
1- Distilled water for serial dilution.
2- Glucose standard solution with concentration of 300mg/dl of stock solution.
3- Glucose reagent.
4- Two samples with unknown glucose concentration (we are going to measure them).

3
1 2

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 Procedure:
- Serial dilution
First, we are going to perform serial dilution to get multiple glucose samples with known
concentrations.

- Using our stock solution

(glucose with concentration of 1 2
300mg/dl) and the two test
tubes (1,2).

- We are going to add 1ml of distilled water

for each test tube.

- Then, using the glass pipette we will take

1ml from glucose stock solution and we will
add it to test tube 1.

- Now concentration of solution in test

tube 1 = 150mg/dl.
1

- Subsequently we will take 1ml from

solution in test tube 1 and add it to test
tube 2.

- Now concentration of solution in test

tube 2 = 75mg/dl.

1
2

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- We have finished our serial dilution. In addition to the

glucose stock solution we have 2 glucose solutions with
known concentrations ( solution 1 with 150mg/dl and
solution 2 with 75mg/dl).

- Now we will bring new 6 tubes (empty test

tubes).
- We will label them as:
1- Blank
2- 300mg/dl
3- 150mg/dl
4- 75mg/dl
5- Sample A
6- Sample B Rack

- We will take 1ml of glucose reagent using the fixed volume

micropipette and we will add it to each test tube.
- We said in the previous slides that we use 2 different sizes
of micropipettes, for glucose reagent we use 1 ml
micropipette. (1ml=1000micro letter)
- The fixed volume micropipette, as its name refers, every
time it will pull a fixed volume of liquid and this allow us to
work faster.
- Here we use pipette require tips that come in contact with
liquid in solution. These tips are disposable, we must change
it each use to avoid contamination.

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- Now we have 1ml of glucose reagent in each test

tube.

- We will take 0.01 ml from

glucose stock solution and we
will add it to the test tube
labeled as (300mg/dl) , again we
will use fixed volume
micropipette but here it's
volume is 10 microliters or
0.01ml.

- Then, we will repeat the same step for the solution in

test tube 1. We will take 0.01ml or 10 microliters from
solution in test tube 1 and add it to the test tube labeled
as (150mg/dl).

- Again, we will repeat the same step for the solution in

test tube 2. We will take 0.01ml from solution in test
tube 2 and will add it to the test tube labeled as
(75mg/dl).

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- Then we will take 0.01ml from sample A

which has unknown concentration, and we
will add it to the test tube labeled as (sample
A).
- And finally, we will take 0.01ml from sample
B which also has unknown concentration,
B
and we will add it to the test tube labeled as
(sample B).
A

- Now Each test tube contains:

- Now, we must wait 15 min for the reaction to be completed. (If we are doing our experiment in the
room temperature). But our experiment was done in temperature higher than 35c°, it took about
only 10 min for the reaction to be completed, because it is an enzymatic reaction (enzymatic
colorimetric) affected by temperature. Then we get the color (pink to red). As you can see in the
picture. The intensity of this color is an indicator for glucose concentration in solutions.

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- Now glucose solutions are

ready to measure their
absorbance using the
spectrophotometer.

- This is the spectrophotometer we use in our

lab.
- To use this device, first we must adjust the
wavelength to the desired wavelength where
the absorbance is maximum. (for our
experiment, the desired wavelength is
500nm).

- To adjust it press "Gotoλ", then press

500 and we get 500nm on the screen
of the spectrophotometer. As you can
see in the figure.

- Now we are going to blank our instrument.

- We will transfer blank solution which contain glucose
reagent only to the cuvette. Here we use plastic cuvette
because we are working in the visible wavelength region.
Then we will place this cuvette in the sample compartment
of the spectrophotometer.

- After that, press "zero", the absorbance value

now is zero. (we did zero adjustment)
- My instrument (the spectrophotometer) is
ready to measure the absorbance of glucose
solutions

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- Now we will bring the test tube with glucose concentration 300mg/dl.

- We will transfer glucose solution to the plastic

cuvette, then we will place the cuvette in the
sample compartment of the spectrophotometer
and measure its absorbance. (It was A=0.769)

- The same steps will be repeated for test tube

with glucose concentration 150mg/dl and we
measure the absorbance. (A=0.389).

- For test tube with glucose concentration

75mg/dl the absorbance was (A=0.194).

- For sample A it was (A=0.336).

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- For sample B it was (A=0.179)

- Remember that absorption has no unit.

- We are going to use the previous data to construct the standard curve.

- When we plot the concentration versus the absorbance for our standard solutions, we get this
standard curve. Since we know that the absorbance of sample A is 0.336, we draw a line across the
graph till we get the line of best fit. When the line intercepts with the line of best fit we draw a line
to the x-axis (concentration axis). Then we can determine the concentration of sample A and the
same goes for the sample B.

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- Summary
 To measure glucose concentration for unknown sample (or samples), we need to draw a
standard curve for multiple glucose standard solutions (with known concentration of glucose).
 These solutions ( with known glucose concentrations) are obtained by doing serial dilution to a
stock solution of glucose.
 The reagent is added to all the solutions (standards and unknown samples) and the absorbance
is measured by spectrophotometer.
 Now we have data to plot the standard curve, we can find the concentration of the unknow
samples (absorbance is known).

If you have any question, please don’t hesitate to ask us!

2020 ‫مع تحيات فريق عمل النادي الطبي‬

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