1. Introduction to Instrumental Analysis - Spectrophotometry
Clinical Analytical Chemistry
CLS 231
Introduction to Instrumental Analysis - Spectrophotometry
Lecture 11
Done by Lecturer : Amal Abu-Mostafa

2. Session Objectives: Introduction to Spectrophotometry
Properties of Light
Colors & Wavelengths
What are Spectroscopy and Spectrophotometry
Instruments of Measurement
Absorption of Light
The Spectrophotometer
Definitions & Symbols
Beer’s Law
Spectrophotometric techniques
Applications of a spectrophotometer
Overview of Quantitative Spectrophotometry

3. Introduction to Spectrophotometry
Properties of Light:
Electromagnetic radiation moves in waves
Light (called electromagnetic radiation) moves in waves.
Wavelength = different types of light have different wavelengths. Some are longer than others. For instance, in the visible light spectrum, red light waves are longer than blue light waves.
Wavelengths are commonly given in ????
Lambda λ

4. Electromagnetic Spectrum

5. Colors & Wavelengths Visible Light COLOR WAVELENGTH (λ in nm)
Ultraviolet
< 380, UV region ( nm)
Violet
380 – 435
Blue
436 – 480
Greenish-blue
481 – 490
Bluish-green
491 – 500
Green
501 – 560
Yellowish-green
561 – 580
Yellow
581 – 595
Orange
596 – 650
Red
651 – 780
Near Infrared
> 780
Visible Light

6. What are Spectroscopy and Spectrophotometry??
Light can either be transmitted or absorbed by dissolved substances.
Presence & concentration of dissolved substances is analyzed by passing light through the sample.
Spectroscopes measure electromagnetic emission
Spectrophotometers measure electromagnetic absorption

7. Instruments of Measurement
Two most common:
Visible Spectrophotometer
Atomic-Absorption Spectrophotometer

8. Instruments of Measurement
What do visible spectrophotometers measure?
Amount of light absorbed by the dissolved substance
Qualitative
Quantitative
The absorption of light indicates the presence of the substance. This is a qualitative measurement.
The amount of light absorbed measures the concentration of the dissolved substance. This is a quantitative measurement.

9. Absorption of Light White light All colors Polychromatic light
When white (polychromatic) light passes through a coloured solution some of the light is absorbed by the substances in the solution, and the rest passes through.
For Example: Green solution absorbs light other than green.

10. Absorption of Light Monochromatic light Light of one color
For example: If white light is made to pass through a red filter, all light except red is filtered out and absorbed. Therefore, only red light hits the solution.
Red light is absorbed
by the green solution

11. The Spectrophotometer

12. The Spectrophotometer
a) Single-beam
b) Double-beam

13. The Spectrophotometer
Contains:
Light source (Lamp)
Optical filters or prism
Tube or cuvette
photoelectric cell, or detector, or Photomultiplier tube.

14. The Spectrophotometer
Light source (Lamp)
UV light from 200 to below 380 nm = deuterium or hydrogen lamp.
Visible region from 380 nm to 700 nm = tungsten or tungsten-halogen.

15. The Spectrophotometer
Optical filters/prisms:
To limit light to a certain wavelength
Monochromator can isolate a specific wavelength of white light and allow it to pass through the solution being analyzed.
Tubes or cuvettes:
Visible range = glass cuvette
UV range = quartz cuvette
Photocell: To detect transmitted light,
Or Detector: Convert radiant energy (photons) into an electrical signal.
Or Photomultiplier tube: very sensitive detector

16. Definitions & Symbols:
Spectrophotometry
Definitions & Symbols:
Radiation Intensity (I)
It : is the radiation transmitted by the solution.
Io : is the radiation transmitted by the pure solvent (blank).
Transmittance (T)
It’s also referred to as %T or T x 100
%T = It x 100 Io
Io It

17. Definitions & Symbols:
Spectrophotometry
Definitions & Symbols:
ABSORBANCE (A)
A = log(1/T) = -log(T)
A = log Io = log Io – log It It
Absorbance is what is generally recorded from a spectrophotometer.

18. Beer’s Law
More dissolved substance = more absorption and less transmittance.
Beer-Lambert’s Law is:
A =  l C
Log Io =  l C It
A= Absorbance (no units)
Io = intensity of incident light
It = intensity of transmitted light
 = molar extinction coefficient, molar absorptivity
c = concentration of the absorbing species (mol/L)
l = path length of the light-absorbing sample (cm)

19. Sample Problem
Cytosine has a molar extinction coefficient of 6 x 103 mol-1 cm-1 at 270 nm at pH 7. Calculate absorbance of 1 x 10-3 M cytosine solution in 1mm cell at 270 nm.
Solution:
A = Log Io =  l C It
 = 6 x 103 mol-1 .cm-1
l = 1mm = 0.1 cm
C = 1 x 10-3 M
A =  l c = (6 x 103)x (0.1) x (1 x 10-3)
= 6 x 10-1
= 0.6

20. Spectrophotometric techniques
Are used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer.
Spectrophotometry: Measures light absorbed by solution at a specific wavelength.
One of the simplest and most widely used methods to determine the amount of protein or nucleic acid present in a given solution

21. Applications of a spectrophotometer
Determines the presence and concentrations of samples.
Determines the purity of a sample.
Look at the change of samples over time.

22. Overview of Quantitative Spectrophotometry
A. Measure the absorbance of standards containing known concentrations of the analyte
B. Plot a standard curve with absorbance on the Y axis and analyte concentration on the X axis
C. Measure the absorbance of the unknown(s)
D. Determine the concentration of material of interest in the unknowns based on the standard curve

24. LINEAR RANGE:
If there is too much or too little analyte, spectrophotometer cannot read the absorbance accurately.

25. Thank you