1. Spectrophotometry at a Glance
Presented by:
Nasir Nazeer

2. Introduction
Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet, and near-infrared, but does not cover time-resolved spectroscopic techniques.
Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement.
The spectrophotometer has well been called the workhorse of the modern laboratory. In particular, ultraviolet and visible spectrophotometry is the method of choice in most laboratories concerned with the identification and measurement of wide range of products and processes.
Modern spectrophotometers are quick, accurate and reliable and make only small demands on the time and skills of the operator.

3. The Spectrophotometer

4. Spectrophotometric Analysis
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.
The spectrophotometer can measure the amount of light or electromagnetic radiation (of certain frequency) transmitted or absorbed by the solution.
If there is too much or too little analyte, spectrophotometer cannot read the absorbance accurately.

5. Properties of Light
Electromagnetic radiation moves in waves

6. Regions of Electromagnetic Spectrum-the “colour” of light

7. Visible Light Colors & Wavelengths COLOR WAVELENGTH (λ in nm)
Ultraviolet
< 380
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

8. R O Y G B I V

9. The absorption process How does matter absorb radiation
When polychromatic light (white light), which contains the whole spectrum of wavelengths in visible region, is passed through an object will absorb certain of the wavelengths, leaving the unabsorbed wavelengths to be transmitted. These residual transmitted wavelengths will be seen as a color. This color is complementary to the absorbed colors.

10. Classes of Spectrophotometers
Single beam and double beam are the two major classes of spectrophotometer.
Single Beam:  In this type, all the light passes through the sample .To measure the intensity of the incident light the sample must be removed so that all the light can pass through. This type of spectrometer is usually less expensive and less complicated.
Double Beam: In this type, before reaches the sample the light source is split into two separate beams. From these one passes through the sample and second one is used for reference. This gives the advantageous because at the same time the reference reading and sample reading can take place.

11. Single Beam Spectrophotometer

12. Double Beam Spectrophotometer

13. Different types of Spectrophotometers
Visible Light: 
Visible spectrophotometers use incandescent, halogen, LED, or a combination of these sources and these spectrophotometers vary in accuracy. Plastic and glass  cuvettes  can  be used for visible light spectroscopy.
Ultraviolet Light: 
UV spectroscopy is used for fluids, and even for solids. Cuvettes, only made of quartz, are used for placing the samples.
Infrared Light:
 IR spectroscopy, which helps to study different structures of molecules and its vibrations. Different chemical structures vibrate in different ways due to variation of energy associated with each wave length. For example, mid-range and near infrared (higher energy) infrared tends to cause rotational vibrations and harmonic vibrations respectively.

14. Different types of Lamps used in Spectrophotometer
Visible spectrophotometer
Contains a tungsten lamp that produces white light. Tungsten lamp consists of a Tungsten filament, enclosed in a glass envelop with the wavelength range of 330 to 900nm, are used for visible region. They are generally useful for measuring moderately dilute solutions in which change in color intensity varies significantly with the change in solute. It has long life about 1200hr
Ultraviolet spectrophotometer
Contains a Deuterium/ Hydrogen lamp that produces light in the UV light part of the spectrum. It ranges about 200 to 450nm in wavelength. This lamp is generally more stable.

15. How a Spectrophotometer works?
Shines a beam of light on a sample.
The molecules in the sample interact with the light waves in 3 ways:
Absorb the energy
Reflect the energy
Transmit the energy between and through the atoms and molecules of the sample.
The spectrophotometer measures the amount of light transmitted through the sample (Transmittance).
By using an equation (Beers law), it converts the transmittance data to an absorbance value.

16. b. Do not allow samples to sit in a cuvette for a long period of time.
Cuvettes are made from plastic, glass, or quartz.
Use quartz cuvettes for UV work.
Glass, plastic or quartz are acceptable in visible work.
There are inexpensive plastic cuvettes that may be suitable for some UV work.
2. Cuvettes are expensive and fragile (except for “disposable” plastic ones). Use them properly and carefully.
a. Do not scratch cuvettes; do not store them in wire racks or clean with brushes or abrasives.
b. Do not allow samples to sit in a cuvette for a long period of time.
c. Wash cuvettes immediately after use.

18. Beer’s Law
The intensity of a ray of monochromatic light decreases exponentially as the concentration of the absorbing medium increases.
More dissolved substance = more absorption and less transmittance

19. Beer’s Law
cuvette
source
slit
detector

20. How absorbance is calculated?
Lambert's law is expressed by
I/Io = T
where I is the intensity of the transmitted light, Io is the intensity of the incident light, and T is the Transmittance. It is customary to express transmittance as a percentage:
%T = I/Io x100
A combination of the two laws (known jointly as the Beer-Lambert Law) defines the relationship between absorbance
(A) and transmittance (T).
A = log Io/I = log 100/T = ε c b
ε : is molar absorptivity ( L.mol-1. cm-1)
b : is path length (cm)
c : concentration (M)

21. After collecting data for your concentration an absorption spectrum graph is created.
These can be used when attempting to identify unknown substances
The absorbance spectrum is a graph of a sample’s absorbance at different wavelengths.
Measure the absorbance of standards containing known concentrations of the analyte
Plot a standard curve with absorbance on the X axis and analyte concentration on the Y axis
Measure the absorbance of the unknown(s).
Determine the concentration of material of interest in the unknowns based on the standard curve.

22. Standardization graph

23. Difference between Spectrophotometer and Colorimeter
A colorimeter measures the absorbance of a particular wavelength by a solution. It is usually used to determine the concentration of a known solute in a known solvent through the application of the Beer-Lambert law.
A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector.
(1) Wavelength selection, (2) Printer button, (3) Concentration factor adjustment, (4) UV mode selector (Deuterium lamp), (5) Readout, (6) Sample compartment, (7) Zero control (100% T), (8) Sensitivity switch, (9)ON/OFF switch