Spectrophotometry at a Glance

Presented by:

Nasir Nazeer

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.

The Spectrophotometer

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.

Properties of Light

Electromagnetic radiation moves in waves

Regions of Electromagnetic Spectrum-the “colour” of 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

Vis

ible

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ht

R O Y G B I V

The absorption processHow 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.

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.

Single Beam Spectrophotometer

Double Beam Spectrophotometer

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.

Different types of Lamps used in Spectrophotometer

Visible spectrophotometer Contains a tungsten lamp that produces wwhhiittee lliightght. 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.

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.

1. Cuvettes are made from plastic, glass, or quartz. a. Use quartz cuvettes for UV work.b. Glass, plastic or quartz are acceptable in visible work. c. 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.

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

Beer’s Lawcuvette

sourceslit

detector

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)

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.

Standardization graph

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