Instrumental Analysis

UV-vis and AAS

Marie Kristie B. Reyes,RCh

Spectroscopy
• Study of light
• Study of the interaction of light with particles and matter
Wave Properties of Radiation

Wavelength (λ)- linear distance between any two

equivalent points (ex. crest to crest)
- expressed in nm, µm, Å

Angstrom Å 10-10m

Nanometer nm 10-9m

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Particle Properties of Radiation
• Photon – a particle of electromagnetic radiation that has zero
mass and an energy of hυ

hc
E = hν ; E=
λ

Where h = Planck’s constant (6.63x10-34J•s)

c = speed of light, 3 x 108 m/sec
v = frequency of light, c/λ
λ = wavelength of light
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UV-Vis Spectroscopy
• Measurement of attenuation of a beam of light after it passes through a
sample
• Follows Beer-Lambert Law

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Electromagnetic Spectrum

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UV-Vis Spectroscopy
• Principle

• Absorption of UV or visible light by chemical compounds ; results in the production of

distinct spectra

• Samples are analyzed using light in the ultraviolet and visible regions of the
electromagnetic spectrum

• Chromophores are usually added to the samples

UV-Vis Spectroscopy

Main components of a UV-vis Spectrophotometer

https://www.technologynetworks.com/analysis/articles/uv-vis-spectroscopy-principle-strengths-and-limitations-and-applications-
349865#:~:text=Ultraviolet%2Dvisible%20(UV%2DVis,beverage%20industry%20and%20chemical%20research.
UV-Vis Spectroscopy
• Light Source

• Brightness across a wide wavelength range

• Stability
• Long service life
• Low cost
UV-Vis Spectroscopy
• Light Source

• Halogen Lamp

Shimadzu Tungsten halogen Lamp

UV-Vis Spectroscopy
• Light Source

• Deuterium Lamp

Shimadzu Deuterium Lamp

https://www.ssi.shimadzu.com/service-support/faq/uv-vis/instrument-design/3/index.html
UV-Vis Spectroscopy
• Monochromator

https://www.ssi.shimadzu.com/service-support/technical-support/analysis-basics/fundamentals-uv/single_double.html#1
UV-Vis Spectroscopy
• Sample Holder

https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fundamentals-uv/structure.html#4
 UV-Vis Spectroscopy
Single-beam
Spectrophotmeter

Double-beam
Spectrophotmeter

https://www.ssi.shimadzu.com/service-support/faq/uv-vis/instrument-design/20/index.html
 UV-Vis Spectroscopy
• Detector

• Phototubes
• Photomultiplier
• Photodiode
Photomultiplier tube

Energy Model of Silicon Photodiode

phototube

http://people.whitman.edu/~dunnivfm/FAASICPMS_Ebook/CH2/2_2_9.html
UV-Vis Spectroscopy
• Applications
• Bacteria culturing
• Drug identification
• Nucleic Acid purity check and quantitation
• Quantitation of chemical compounds
• Quality control in chemical research and experimentation
UV-Vis Spectroscopy
• Sample Problem Application
• Plot Absorbance (y-axis) vs Concentration (x-axis)

https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/basic/absorbance_detector.html
UV-Vis Spectroscopy
SAMPLE PROBLEM
A river water sample was tested for ammonia using the Phenate Colorimetric Method. In
this method, the formation of an intensely blue compound, indophenol, is formed by the
reaction of ammonia, hypochlorite, and phenol, catalyzed by a manganous salt.
Calculate the ppm ammonia-nitrogen content of the water sample given the following
data:
ABSORBANCE CONCENTRATION (µg N)
BLANK 0.0165
STANDARD 1 0.0930 0.5346
SATNDARD 2 0.1855 2.6730
STANDARD 3 0.3375 5.3459
SAMPLE (10 ml used) 0.2100 Unknown
Atomic Spectroscopy
• Absorption and Emission of radiation by atoms or atomic ions
• Flame Atomic Absorption Spectroscopy (FAAS)
• Flame Emission Spectroscopy (FES)
• Electrothermal

https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/basic/absorbance_detector.html
 Atomic Absorption
e- e-
external excitation

energy

Ground state
Excited state
Excited state

Energy, eV Energy absorbed

Ground State

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Quantitative Measure of Radiation of Absorption
• Transmittance- fraction of incident radiation (P) that passes through the
sample; often expressed as %

I
%T= (100)
Io

I = transmitted light
Io = incident light
https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/basic/absorbance_detector.html
Quantitative Measure of Radiation of Absorption
• Absorbance- amount of radiation absorbed

1 Po
A = -log T = log = log = abc
T P

T= transmittance
P = light that passes through absorbing species
Po = light from the source
a = absorptivity
b = length of absorption path
c = concentration
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Basic Principles of AAS
• All atoms absorb light
• The wavelength at which light is absorbed is specific for a particular element
• The amount light absorbed is proportional to the concentration of absorbing
atoms

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 Basic Parts of AAS
• Light Source

Hallow Cathode Lamp

Kenkel, 2014

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Basic Parts of AAS

Background Correction

Shimadzu Deuterium Lamp

https://www.ssi.shimadzu.com/service-support/faq/uv-vis/instrument-design/3/index.html
 Basic Parts of AAS
• Optical System

Monochromator detector display

Light Burner
source
lens lens

single beam optical system

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 Basic Parts of AAS
• Optical System

Burner

Monochromator detector display

Light
source Reference

lens lens

double beam optical system

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 Basic Parts of AAS
• Atomization Chamber

• Generates a population of free analyte atom so that absorption can be measured

• Flame Atomization
• Electrothermal Atomization
• Cold Vapor Atomization
• Hydride Vapor Atomization

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 Basic Parts of AAS
• Monochromator

• Adjustable filter that selects a specific, narrow region of spectrum for the transmission
to the detector

• Rejects all wavelengths outside this region

• Examples= Reflection grating and Prism

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 Basic Parts of AAS
• Detector

• Measures the light intensity and amplifies the signal

• Examples:
• Photon Detectors (ex: photomultiplier tube)
• Heat Detectors

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 Flame Atomization Process

free atoms
Optical path Atomization

molecular species
Vaporization

liquid melt
solid particles
Desolvation

Aerosol-fuel-oxidant

Excess soln to waste

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 Interferences in AAS analysis
Ideally Light absorbed by
the
target element
Light absorbed
From Cathode Lamp
Light absorbed by the
target element
Reality Light absorbed from
coexisting elements

Light absorbed
From Cathode Lamp
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 Interferences in AAS analysis
• Physical Interference

• Due to fluid characteristics of the sample (viscosity, surface tension)

• Can be corrected by standard addition or sample dilution

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 Interferences in AAS analysis
• Chemical Interference

• Due to fluid characteristics of the sample (viscosity, surface tension)

generation of various compounds from chemical reaction between
components of the sample

• Can be corrected by target element extraction, or solvent extraction of the

obstructing element, or addition of suppressant or releasing agents

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 Interferences in AAS analysis
• Molecular Absorption
• Due to continuous absorption of other elements in the sample

• Spectral Interference
• Due to influence of other elements with absorption lines near the absorption lines of
the target element

• Can be corrected by sample treatment (extraction, digestion) and background

correction

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 Elements and Wavelengths
with Spectral Interference
Target Spectral line Interfering Spectral line
element (nm) elment (nm)
Al 308.215 V 308.211
Ca 422.673 Ge 422.657
Cd 228.802 As 228.812
Co 252.136 In 252.137
Cu 324.754 Eu 324.753
Fe 271.903 Pt 271.904
Ga 403.298 Mn 403.307
Hg 253.652 Co 253.649
Mn 403.307 Ga 403.298
Sb 217.023 Pb 216.999
Si 250.690 V 250.690
Zn 213.856 Fe 213.859

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 Background Correction Methods
• Deuterium (D2) Method
• Self Reversal Method
• Zeeman Correction Method

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 Background Correction Methods
• Deuterium (D2) Method

from target
element

from background
component
Absorbance Absorbance
Absorbance from HCL from D2 lamp from target element

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 D2 Background Correction
(Double Beam Configuration)

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Background Correction
• Self Reversal Method
• Lamp is a self reversal lamp generating high and low current alternately

• Low current is absorbed by both target element and coexistent element

• High current is absorbed only as background

Background Correction
• Self Reversal Zeeman Correction
• Based on the splitting and polarization of the spectral line in a magnetic
field

• One component is absorbed by both element and coexistent background,

the other component is absorbed only by the background
 Comparison of Background Correction Methods
Method D2 Self-Reversal Zeeman

Wavelength 190-430 nm 190-900 nm 190-900 nm

Correction for Good Good Good

Molecular
Absorption
Correction for Not Good Good Good
Spectral
Interference
Light Intensity Strong Strong Not Strong
Sensitivity Good Not Good Not Good

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Standard Preparation
2.0 ppm

1.5 ppm

Stock Std. Intermediate 1.0 ppm

Soln. Soln.
Working
(1000 ppm) (100 ppm) Standards
normally > 4 std
Eqn. of a Line
Y = mX + b
where:
Y = absorbance
X = concentration
m = slope
b = y intercept
r  0.999
Standard Addition Method

Equal volumes of
unknown solution

Standard
Solution

CX CX+1 CX+2 CX+3

Standard Addition Curve

A
b
s

Cx 0 1.0 2.0 3.0

Conc. (ppm)
Sample Problem #1
A river sample was tested for copper concentration by atomic absorption
spectrometry using standard addition method. A volume of 100.0 µL of a 1000
µg/mL Cu standard was added to a 100.0 mL river sample. Calculate the copper
concentration in the sample using the following data:

Absorbance of reagent blank = 0.020

Absorbance of sample = 0.520
Absorbance of sample plus addition – blank = 1.020
Solution Add Vspike of Cspike

V1 V1
Substitute equation (3) into equation (2)

Ratio equation (4) to equation (1) to eliminate k

Computing for the concentration of sample:
Sample Problem #2
A 5.00 mL of a wastewater sample was tested for iron, diluted to 25.00 mL and
gave an absorbance of 0.132 nm (Flask A). Another 5.00 mL of the sample was
added with 3.00 mL of 100.3 ppm Iron standard, then diluted to 25.00 mL.(Flask
B) The resulting solution gave an absorbance of 0.855 nm. Calculate the Fe
concentration.
Solution
Let:
Add V1 of C1
Cx = concentration of Fe in the original sample
Flask A= C1= 5Cx/25
Flask B= C2 = (5Cx + 3(100.3))/25

Dilute to Vtotal

Flask A Flask B
Sample Problem #3
A wastewater sample was tested for Lead using AAS and multiple standard
addition. Concentration of stock Lead standard used is 100.0 ppm. Volume final
for the solutions is 50.00 mL Calculate the Pb concentration given the following
data:
Solution Volume sample Volume standard Absorbance
(mL) (mL) (nm)
1 5.00 0 0.0505
2 5.00 0.20 0.0577
3 5.00 0.40 0.0639
4 5.00 0.60 0.0712
5 5.00 0.80 0.0783
Plot concentration (x-axis) vs Absorbance (y-axis)
y=mx + b
Set y=0

Slope = 0.01728 (B)

y-intercept = 0.05050 (A)
r= 0.999700

X= concentration= 2.92 ppm Pb

Sample Problem #4
A wastewater sample was tested for Lead using AAS and multiple standard
addition. Concentration of stock Lead standard used is 100.0 ppm. Volume final
for the solutions is 50.00 mL Calculate the Pb concentration given the following
data:
Solution Volume sample Volume standard Absorbance
(mL) (mL) (nm)
1 25.00 0 0.0306
2 25.00 0.20 0.0378
3 25.00 0.40 0.0440
4 25.00 0.60 0.0513
5 25.00 0.80 0.0584