Lecture 4: Spectrophotometer

First Semester
2018-2019

Dr. Farah Anjum

Ms. Hanan Alrishi

Second Year: Nursing regular

Biochemistry: Practical Session 4
• Learning objectives:

– Determination of the wavelength of maximum absorbance of a

given solution

– Learn how to use spectrophotometer

– Show your result in graph

 Spectrophotometry
Is a technique used to measure how much a substance absorbs at
varying wavelengths of light.

2.
0
Absorbance

0.
0
20 25 30 35 40 45
0 0 0 0 0 0
Wavelength
 What is wavelength?
The distance between peaks (high points) is called wavelength.
Each wave has a certain shape and length.
• electromagnetic radiation is classified by
wavelength into radio wave, microwave,
infrared, visible light, UV, X ray, Gamma
rays.
• Longer wavelength (low frequencies) used
for radio, however gamma rays have short
wavelength (high frequency)
• Visible light is only a small portion of the
entire electromagnetic spectrum
• it includes the colors commonly observed
(red, yellow, green, blue and violet).
• The visible spectrum consists of electro-
magnetic radiation whose wavelengths
range from 380nm to nearly 760nm.
 Spectrophotometer

To measure the amount of light that a sample absorbs

Visible spectrophotometer: uses light over visible range (400 - 700 nm) of electromagnetic
radiation spectrum.
 Transmittance

Transmittance (T) is defined as the fraction of

incident light which is transmitted, ie, passes
through, a sample.
Thus,
 Transmittance
• Defined as the ratio of the intensity of light
emerging from the solution (I) to that of incident
light entering (Io)
 Transmittance

Transmittance is usually expressed as a percentage:

%T = (I/Io) x 100

100% transmittance means no light is absorbed by the

solution so that incident light is 100% transmitted
 Absorbance
Absorbance (A), or optical density, is a logarithmic
function of T and is expressed as:

A = 2 – log(%T)

Note that absorbance has no units.

How to use Spectrophotometer
 How to use Spectrophotometer
Step 1: Turn on the instrument to warm it up for 10 minutes.

Step 2: Prepare a BLANK cuvette by adding WATER and insert it

into cuvette holder.
Step 3: Prepare a SAMPLE cuvette, wipe off the
outside of the cuvette and insert it into the sample
cuvette holder.
Step 4: Close the lid and choose the proper
wavelength
 Step 5: Measuring Absorbance

Read absorbance for your sample.

 Method:
1) Prepare solution of a Dye
a) 1 ml of Dye into 9 ml of water (total 10 ml)

2) Determine spectrum:
a) Set up spectrophotometer
b) Choose the wavelength (400 – 700 nm)
c) Measure Absorbance
 Data Analysis
• Plot your data on a graph
– Wavelenght is plotted on the X-axis
– Absorbance is plotted on the Y-axis

• Put units on each axis

• Determine the wavelength of maximum absorbance
Absorbance, A, is directly proportional to concentration
A) Show your proper graphs and results
B) Determine the wavelength of maximum
absorbance

W 400 420 450 500 520 550 600 620 650 700

A 0.5 0.8 1.2 1.8 2.5 2.9 2.4 2.1 1.8 1.7