Spectrophotometer

M.Sc. Zaid Nabeel

Lab 12
 Spectrophotometer

An instrument that measures the amount of light that

passes through (is transmitted through) a sample.
• Uses a type of light to detect molecules in a solution
• Light is a type of energy, and the energy is reported
as wavelengths, in nanometers (nm).
Two different types of Spectrophotometer:
 Ultraviolet (UV) Spectrophotometers.
Uses ultraviolet light of wave lengths from
200 nm to 350 nm.

 Visible (VIS) Light Spectrum

Spectrophotometers.
Uses visible light (white light) of wave
lengths from 350 nm to 700 nm.
• What do visible spectrophotometers measure?

• Measure amount of light absorbed by the dissolved solution

So concentration of substances will be obtained according to
Beer-Lambert law.
 Principle of spectrophotometry
• The spectrophotometer technique is to measure light intensity according to
wavelength of the light.
• It does by:
1. Diffracting the light beam into a spectrum of wavelengths
2. Direct it to an object ( Sample )
3. Receiving the light reflected or returned from the object
4. Detecting the intensities with a device detector.
5. Displaying the results as a graph on the detector and then the display device.

6
Introduction

7
The visible light spectrum

R O Y G B IV
• Light pathway Spectrophotometer:

9
Cuvettes :
• Cuvettes: designed to hold samples for spectroscopic
experiments. made of Plastic, glass or optical grade
quartz
• should be as clear as possible, without impurities that
might affect a spectroscopic reading.

10
 Cuvettes

Cuvettes are made from plastic, glass, or quartz.

a. Use quartz cuvettes for UV work.
b. Glass, plastic or quartz are acceptable visible work.
c. There are inexpensive plastic cuvettes that may be
suitable for some UV work.
Cuvettes
Cuvettes are expensive and fragile (except for “disposable”
plastic ones). Use them properly and carefully.
a. Do not scratch cuvettes or clean with brushes,it must
puttted in the cup of distil water after use.
b. Do not allow samples to sit in a cuvette for a long
period of time.
c. Wash cuvettes immediately after use.
Instruments
• Detectors: Convert radiant energy (photons) into an electrical signal.

13
Types of standred curve ;

1- Directly proportion
2- Inversely proportion

14
 Some Applications of a
spectrophotometer
• Determines the presence and concentrations of
samples.
• Determines the purity of a sample.
How to Ensure Accuracy?
• Repeat tests many times (2-3)and take average

• Run another sample that was tested before along with patient
samples and make sure its result is close to what it should be
Thank You