Spectrophotometry

Spectrophotometry

Spectrophotometry is one of the most useful tools available to the biochemist.

It offers a high degree of precision, sensitivity, and accuracy. In addition, it is inexpensive and applicable to the measurement of a variety of substances.

Principles of Spectrophotometry There is interaction between

electromagnetic radiation (light) and matter

A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light.

Principles of Spectrophotometry The amount of light passing through

the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, The signal changes as the amount of light absorbed by the liquid changes.

Electromagnetic Radiation (EMR)

•10-200nm vacuum UV region•200-380nm near

UV•380-780nm visible

region

Type of Radiation

Frequency Range (Hz)

Wavelength Range

Type of Transition

gamma-rays

1020-1024 <1 pm nuclear

X-rays 1017-1020 1 nm-1 pm inner electron

ultraviolet 1015-1017 400 nm-1 nm

outer electron

visible 4-7.5x1014 750 nm-400 nm

outer electron

near-infrared

1x1014-4x1014

2.5 µm-750 nm

outer electron molecular vibrations

infrared 1013-1014 25 µm-2.5 µm

molecular vibrations

microwave 3x1011-1013 1 mm-25 µm

molecular rotations, electron spin flips*

radio waves

<3x1011 >1 mm nuclear spin flips*

Electromagnetic Spectrum

Ultra-Violet/VisibleSpectrophotometry

UV/VIS Spectrophotometry is used to determine the absorption or transmission of UV/VIS light (180 to 820 nm) by a sample.

It can also be used to measure concentrations of absorbing materials based on developed calibration curves of the material.

Spectrophotometer

Device which measures the amount of light absorbed by a substance.

The amount of light absorbed is directly related to the concentration of the analyte in the solution (A= a b c ) Beer’s law.

Each substance has maximum absorption of light at certain wavelength (λmax)

Quantitative

Qualitative

Components of spectrometry Lamp: light source(s) Monochromator: a means of isolating

a particular wavelength band of the light source, provide Monochromatic light which is light in which all photons have the same wavelength

Cuvette or cell: a sample holder Detector: a device to measure light

intensity.

Instrument

Light source

•Tungsten (visible)

•Deuterium or hydrogen

(UV)

1. Prism2. Diffraction

grating3. Optical filtration

Cell•Glass (visible)

•Quartz( UV)

Single Beam Spectrometer

The light source shines onto or through the sample.

The sample transmits or reflects light. The detector detects how much light

was reflected from or transmitted through the sample.

The amplifier then converts how much light the sample transmitted or reflected into a number.

A schematic showing that a single-color light is selected from continuous spectrum light source by a monochromator. The monochromator usually includes a grating which separates different color light at different angle. A specific color (angle) light passes through a slit and exits the monochromator.

Types:

A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample.

A single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted.

Single Beam Spectrometer

Double Beam Spectrometer

Experiment

Objective: Determine the absorbance spectrum

of a sample solution. Determine the wavelength of

maximum absorbance.

Approach: Measure the intensity of transmitted light

for various wavelengths of light. For each wavelength, calculate the

absorbance of the solution. Construct the absorbance spectrum by

plotting A vs λ. Determine λmax by locating the

wavelength at which the absorbance is greatest.

absorbtion spectrum

The spectrum itself is a plot of absorbance vs wavelength and is characterized by the wavelength (λmax) at which the absorbance is the greatest.

The value of λmax is important for several reasons. This wavelength is characteristic of each compound and provides information on the electronic structure of the analyte. In order to obtain the highest sensitivity and to minimize deviations from Beer's Law (see subsequent pages on this topic), analytical measurements are made using light with a wavelength of λmax.

Double Beam Spectrometer