Molecular Absorption Spectroscopy:

Ultraviolet (UV) – Visible

Spectrophotometry

By:
Ari Hardianto, Ph.D.
Dr. Anni Anggraeni, M.Si
Electromagnetic Radiation
The interactions of electromagnetic radiation and matter are the subject of
the science called spectroscopy.
Spectroscopic analytical methods are based on measuring the amount of
radiation produced or absorbed by molecular or atomic species of interest
Electromagnetic radiation can be
treated as discrete packets of
energy or particles called photons
or quanta.

Frequency
Wave number
Wave length
Electromagnetic Spectrum
Interaction of Electromagnetic and Mater
Interaction of Electromagnetic and Mater

Type of spectroscopy Wavelength Range

UV 180 - 380 nm
Visible 380 - 780 nm
Spectroscopic Measurements
Absorption spectroscopy :

Emission spectroscopy Chemiluminescence spectroscopy

Photoluminescence Fluorescence Phosphorescence

spectroscopy spectroscopy spectroscopy
Absorption spectroscopy

Atomic Absorption
Analytes are gaseous atoms. Atomic absorption is measured at a single
wavelength using a very narrow, nearly monochromatic source.
Spectroscopic method: Atomic Absorption Spectroscopy.

Molecular Absorption
Analytes are molecules in a solution.
Spectroscopic method:
• Infrared spectroscopy
• Visible spectroscopy
• Ultraviolet spectroscopy

Type of spectroscopy Wavelength Range

UV 180 - 380 nm
Visible 380 - 780 nm
 Absorption Process
Interactions between the photons and
absorbing particles decreases the radiant
power of the beam from P0 to P.
The transmittance T of the solution is the
fraction of incident radiation transmitted by
the solution.
Transmittance is often expressed as a
percentage and called the percent
transmittance.

The absorbance, A, of a solution is related to

the transmittance in a logarithmic manner
Measuring Transmittance and Absorbance

To compensate reflection and

scattering effects:
• Compare analyte solutions and
solvent or a reagent blank
• Light beam is perpendicular to
cuvettes
Beer-Lambert law
When a monochromatic light is passed through a solution containing the
absorbing substance, the attenuation of light intensity is directly proportional
to the concentration of the absorbing species, c, and to the path length, b

a is a proportionality constant called the

absorptivity.

When we express the concentration in moles

per liter and b in cm, the proportionality
constant is called the molar absorptivity and is
given the symbol ε.
Limits to Beer’s Law
1. Real Limitations to Beer’s Law

Beer’s law describes the absorption behaviour only of dilute solutions and in
this sense is a limiting law.

At concentrations exceeding about 0.01 M, the average distances between

ions or molecules of the absorbing species are diminished to the point where
each particle affects the charge distribution and thus the extent of absorption
of its neighbours.
Because the extent of interaction depends on concentration, the occurrence
of this phenomenon causes deviations from the linear relationship between
absorbance and concentration. A similar effect sometimes occurs in dilute
solutions of absorbers that contain high concentrations of other species,
particularly electrolytes
Limits to Beer’s Law
2. Chemical Deviations

Deviations from Beer’s law appear when the absorbing species undergoes
association, dissociation, or reaction with the solvent to give products that
absorb differently from the analyte.
Limits to Beer’s Law
3. Instrumental Deviations: Polychromatic Radiation

Deviations from Beer’s law often occur when polychromatic radiation is used
to measure absorbance
Limits to Beer’s Law
4. Instrumental Deviations: Stray Light

Stray radiation, commonly called stray light, is defined as radiation from the
instrument that is outside the nominal wavelength band chosen for the
determination.
This stray radiation often is the result of scattering and reflection off
the surfaces of gratings, lenses or mirrors, filters, and windows.
Limits to Beer’s Law
5. Mismatched Cells

This error can be avoided either by using carefully matched cells or

by using a linear regression procedure to calculate both the slope and
intercept of the calibration curve.
UV-Visible Spectrophotometer
UV-Visible Spectrophotometer
Single-beam UV-Vis Spectrophotometer
UV-Visible Spectrophotometer
Single-beam UV-Vis Spectrophotometer

Source: https://www.youtube.com/watch?v=pxC6F7bK8CU
UV-Visible Spectrophotometer
Double-beam UV-Vis Spectrophotometer
UV-Visible Spectrophotometer
Multichannel Instruments
UV-Visible Spectrophotometer
Multichannel Instruments
Sample Containers (cuvettes)
Spectroscopic Sources

D2 Lamp H2 Lamp Tungsten Lamp Xenon Lamp

Quantitative applications: Why UV-Vis
Procedure details
λmax

Wavelength Selection
Wavelength of maximum
absorption (λmax)

Variables That Influence Absorption

Temperature, pH, electrolyte concentration, and the presence of interferences.

The Relationship between Absorbance and Concentration.

The calibration standards for a photometric or a spectrophotometric method
should approximate as closely as possible the overall composition of the actual
samples and should encompass a reasonable range of analyte concentrations.

Standard Addition Method

The standard addition method can be helpful in counteracting matrix effects.
 External Standard
yi = absorbance
xi = Konsentrasi

sampel Larutan standar Persamaan regresi linier (kurva kalibrasi):

y = m.x + b m = slope
y - b = m.x b = intersep
residu
yi - m.x + b 𝑦−𝑏 Cx = Konsentrasi
𝐶𝑥 = 𝑥 = sampel
1,2
𝑚
1,0
absorbance

0,8
yi

0,6
0,4
Sample Sample
absorbance 0,2 concentration
0,0
10 20 30 40 50 60
Concentration (ppm)
xi
Capability of Detection
Capability of Detection
Limit of Detection (LoD) The smallest amount or concentration of analyte in the
test sample that can be reliably distinguished from
blank.

Based on visual detection by the analysis of samples with known concentrations

of analyte and by establishing the minimum level at
which the analyte can be reliably detected.
𝑆
Based on signal-to-noise =3
𝑁

Based on the Standard Deviation 3.3 × σ𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡

𝐿𝑜𝐷 =
of the Response and the Slope 𝑠𝑙𝑜𝑝𝑒

Based on the Standard 𝐿𝑜𝐷 = 3σ𝑏𝑙𝑎𝑛𝑘 from the absorbance

Deviation of the Blank of ten blanks

Based on the Calibration Curve

Capability of Detection
Limit of Quantification The lowest analyte concentration in the sample of
(LoQ) analyte that can be determined with an
acceptable repeatability and trueness

𝑆
Based on signal-to-noise = 10
𝑁

Based on the Standard Deviation 10 × σ𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡

𝐿𝑜𝐷 =
of the Response and the Slope 𝑠𝑙𝑜𝑝𝑒

Based on the Standard 𝐿𝑜𝐷 = 10σ𝑏𝑙𝑎𝑛𝑘 from the absorbance

Deviation of the Blank of ten blanks

Based on the Calibration Curve

 UV lab practical
Determination of NO3- in water
UV lab practical
Determination of NO3- in water

Concentration of KNO3 Absorbance

(ppm)

1 Weight quatitatively
0.025 gram KNO3
1000 2.073

2 Add water to
the mark,
up to 25 mL
500
250
2
1.7
125 1.4
62.5 1.201
31.25 0.774
15.625 0.536
7.8125 0.413

1000 ppm KNO3 3.90625 0.357

1.953125 0.338
Blank 0.273
Sample ?
 UV lab practical
2.5
Concentration of KNO3 Absorbance
(ppm)
2 1000 2.073
500 2
1.5
250 1.7
Absorbace

125 1.4
62.5 1.201
1
31.25 0.774
15.625 0.536
0.5
7.8125 0.413
3.90625 0.357
0
0 200 400 600 800 1000 1.953125 0.338
ppm
blank 0.273
UV lab practical
1.8

Concentration of KNO3 Absorbance

1.6 (ppm)
1.4 1000 2.073

1.2
500 2
250 1.7
Absorbace

1
125 1.4
0.8
62.5 1.201
0.6 31.25 0.774

0.4
15.625 0.536
7.8125 0.413
0.2
3.90625 0.357
0
0 25 50 75 100 125 150
1.953125 0.338
ppm
Blank 0.273
UV lab practical

Concentration of KNO3 Absorbance

(ppm)

The random
errors in the
y-direction 62.5 1.201
31.25 0.774
15.625 0.536
σ𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡
7.8125 0.413
3.90625 0.357
1.953125 0.338
3.3 × σ𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 0 0.273
𝐿𝑜𝐷 =
𝑠𝑙𝑜𝑝𝑒
UV lab practical

Concentration of KNO3 Absorbance

(ppm)

Determine concentration of sample by

plugin the absorbance to linear
regression equation 62.5 1.201
31.25 0.774
Y = bx + a
15.625 0.536
7.8125 0.413
3.90625 0.357
1.953125 0.338
0 0.273
UV lab practical
Spiking

𝐴𝑠𝑎𝑚𝑝 𝐴𝑠𝑝𝑖𝑘𝑒
=
𝑉𝑜 𝑉 𝑉𝑠𝑡𝑑
𝐶𝐴 𝑉 𝐶𝐴 𝑉𝑜 + 𝐶𝑠𝑡𝑑 𝑉
𝑓 𝑓 𝑓
UV lab practical
Spiking

𝐴𝑠𝑎𝑚𝑝 𝐴𝑠𝑝𝑖𝑘𝑒
=
𝑉𝑜 𝑉 𝑉𝑠𝑡𝑑
𝐶𝐴 𝑉 𝐶𝐴 𝑉𝑜 + 𝐶𝑠𝑡𝑑 𝑉
𝑓 𝑓 𝑓
UV lab practical
Spiking

𝐴𝑠𝑎𝑚𝑝 𝐴𝑠𝑝𝑖𝑘𝑒
=
𝑉𝑜 𝑉 𝑉𝑠𝑡𝑑
𝐶𝐴 𝑉 𝐶𝐴 𝑉𝑜 + 𝐶𝑠𝑡𝑑 𝑉
𝑓 𝑓 𝑓
 Vis lab practical
Determination of Fe3+ in water
 Vis lab practical
Determination of Fe3+ in water 1 Weight
quatitatively
86.4 gram
Fe3+(aq) + CNS-(aq) [FeCNS]2+(aq)
𝐹𝑒𝑁𝐻4 (𝑆𝑂4 )2 2 Add 10 mL
concentrated
HCl
Colourless Red

𝐹𝑒 3+
𝑜𝑓 100 𝑝𝑝𝑚 =
𝐴𝑟 𝐹𝑒
𝑀𝑟 𝐹𝑒𝑁𝐻4 (𝑆𝑂4 )2 .12𝐻2 𝑂
×
𝑚𝑔 𝐹𝑒𝑁𝐻4 (𝑆𝑂4 )2
1𝐿 3 Add water to
the mark,
up to 100 mL
𝑀𝑟 𝐹𝑒𝑁𝐻4 (𝑆𝑂4 )2 .12𝐻2 𝑂 100 𝑚𝑔/𝐿
𝑚𝑔 𝐹𝑒𝑁𝐻4 (𝑆𝑂4 )2 = × 1𝐿
𝐴𝑟 𝐹𝑒
482.25 100 𝑚𝑔/𝐿
𝑚𝑔 𝐹𝑒𝑁𝐻4 (𝑆𝑂4 )2 = × 1𝐿
55.85
𝑚𝑔 86.347 𝑚𝑔
𝑚𝑔 𝐹𝑒𝑁𝐻4 (𝑆𝑂4 )2 = 863.47 =
𝐿 100 𝑚𝐿 100 ppm Fe3+

4 M HCl = 1 portion of concentrated HCl (12 M) added to 2 portion of distilled water

2 M KCNS = 20 g KCNS per L of distilled water
 Vis lab practical
Determination Fe3+ in water

Fe3+(aq) + CNS-(aq) [FeCNS]2+(aq) Concentration of Fe in Absorbance

[FeCNS]2+ form (ppm)
Colourless Red
50

1 Transfer x mL
Fe3+ of
100 ppm 2 Add 2 mL
HCl 4 M
40
25
20
10
Bring your own

4 Add water to
the mark,
up to 100 mL
3 Add 5 mL
KCNS 2M
5
2
sample, water from
your home which has
reddish brown colour.
1
Blank
sample

25 mL

Note: To minimize pipetting error, the dilution factor is maximum 10.

Vis lab practical
Determination Fe3+ in water

Concentration of Fe in Absorbance
[FeCNS]2+ form (ppm)
50
Calculate the LoD and LoQ
40
Determine the linearity range
Perform spiking procedure 25
Calculate the % R of external standard 20
10
5
2
1
Blank
Sample