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Introduction To Spectrophotometry

Spectrophotometry involves using a spectrophotometer to measure how much light is absorbed by a sample. Light interacts with matter through absorption, transmission, or emission. A spectrophotometer directs visible, ultraviolet, or infrared light through a sample and measures how much light is absorbed. Beer's Law states that absorbance is directly proportional to concentration, with absorbance increasing as concentration or path length increases. Creating a standardization graph of absorbance versus known concentrations produces a straight line used to determine unknown sample concentrations from their absorbance readings.

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Mikael Akif
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56 views25 pages

Introduction To Spectrophotometry

Spectrophotometry involves using a spectrophotometer to measure how much light is absorbed by a sample. Light interacts with matter through absorption, transmission, or emission. A spectrophotometer directs visible, ultraviolet, or infrared light through a sample and measures how much light is absorbed. Beer's Law states that absorbance is directly proportional to concentration, with absorbance increasing as concentration or path length increases. Creating a standardization graph of absorbance versus known concentrations produces a straight line used to determine unknown sample concentrations from their absorbance readings.

Uploaded by

Mikael Akif
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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Introduction to

Spectrophotometry
Spectroscopy
 Is the study of the
interaction of light & matter
 Spectrophotometer –
instrument that uses
electromagnetic radiation
from UV, visible or IR to
analyze the absorption or
transmission of a sample
 We will use visible in our
lab
Properties of Light

 Electromagnetic radiation moves in


waves
Electromagnetic Spectrum
Electromagnetic Spectrum
Colors & Wavelengths
COLOR WAVELENGTH (λ in nm)
Ultraviolet < 380

Violet 380 – 435


Visible Light

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


What is Colorimetry?

 The solutions of many compounds have


characteristic colors.

 The intensity of such a color is


proportional to the concentration of the
compound.
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
Instruments of Measurement

 Two most common:


1. Visible Spectrophotometer
 Spect 20, Spect 88
 Uses Xe or W lamps as light sources
 Glass cuvettes hold the sample

2. Atomic-Absorption Spectrophotometer
Instruments of Measurement
 What do visible spectrophotometers
measure?
– Amount of light absorbed by the dissolved
substance

– Qualitative – color gives info about the solution


composition
– Quantitative – provides numerical data for the
concentration
Absorption of Light

 White light
– All colors
– Polychromatic light
Absorption of Light

 Monochromatic light
– Light of one color
Monochromator-spreads out light into its
component wavelength

Red light is
absorbed
by the green
solution
The Spectrophotometer
Success of spectrophotometry…
 Requires sample to absorb light differently to the
other chemicals in the solution
 How is the correct wavelength selected?
– The amount of light absorbed depends on the
energy difference between 2 electron energy
levels
– Optimum wavelength for spectrophotometric
analysis is selected by measuring the visible
spectrum of the substance
– This is done by plotting absorbance (A) versus
wavelength (λ)
Food Dyes
 Only 7 dyes are
approved by the FDA
for use in foods,
drugs & cosmetics
 All artificial food
colors are mixtures of
these 7 dyes
 We will be using
FD&C Blue in this lab
 A solution containing this dye is
blue in white light FD&C Blue 1
 The colors absorbed by
solution are complementary to
the transmitted color
 Blue solution absorbs yellow,
orange, & red light
 So expect dye solution to peak
at 580 – 650 nm
 Optimum wavelength is
determined from wavelength of
max. absorption λmax = 630 nm
for Blue 1
 This is given for the blue
solution but you will have to
claculate this for the red
Wavelength of light absorbed:
 Is related to electronic structure of substance
 Intensity of light absorbed depends on the
concentration of solution
 More concentrated, the more intense color & the
greater intensity of light absorbed
 When light is absorbed, the radiant power (P) of light
beam decreases
Transmittance (T)
 This is the fraction of
incident light (P/Po)
that passes through
the sample
 T=P
Po
 Po = intensity of
“blank”
 Blank – is solution
identical to sample
but without solute
Definitions & Symbols

 Intensity (I)
 Transmittance (T)
– It’s also referred to as %T or T x 100
– T = P/Po
• Where Po is the intensity of the blank
• Can also use I = Intensity instead of Power
• T = I / Io
Graphical Relationship

 % transmission and % absorption are


not linearly related to concentration

 For a graph to be useful, a straight line


is needed

 ABSORBANCE = log(1/T) = -log(T)


The amount of light absorbed
depends upon:
 Concentration (c)
 Path length of
sample cell (b) thru
c
which light passes
 Defined by Beer’s
Law
P0 P

b
Beer’s Law
 The intensity of a ray of
monochromatic light
decreases exponentially
as the concentration of
the absorbing medium %T = Tx100 = P/P0x100%
increases
 A = - log T
 More dissolved
substance = more  A=εbc
absorption and less
transmittance
 ε = molar absorptivity
coefficient and is
constant for a substance
Spectral Transmission Curve

 Optimum wavelength
Standardization Graph

- Standards (solutions of known


concentration) of the compound of
interest are made, treated, and their
absorbances (ABS) and concentration
values are used to create a
Standardization Graph.
Standardization Graph

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