CHAPTER ONE

SPECTROSCOPY
1.0 Introduction to Spectroscopy
Spectroscopy is the investigation and measurement of spectra produced by matter interacting
with or emitting electromagnetic radiation. Originally, spectroscopy was defined as the study of
the interaction between radiation and matter as a function of wavelength. Now, spectroscopy is
defined as any measurement of a quantity as a function of wavelength or frequency. During a
spectroscopy experiment, electromagnetic radiation of a specified wavelength range passes from
a source through a sample containing compounds of interest, resulting in absorption or emission.
During absorption, the sample absorbs energy from the light source. During emission, the sample
emits light of a different wavelength than the source’s wavelength.
In absorption spectroscopy, the sample’s compounds are excited by the electromagnetic radiation
provided by a light source. Their molecules absorb energy from the electromagnetic radiation,
become excited, and jump from a low energy ground state to a higher energy state of excitation.
A detector, usually a photodiode, on the opposite side of the sample records the sample’s
absorption of wavelengths, and determines the extent of their absorption. The spectrum of a
sample’s absorbed wavelengths is known as its absorption spectrum, and the quantity of light
absorbed by a sample is its absorbance.
Each molecule within a sample will only absorb wavelengths with energies corresponding to the
energy difference of the present transition. In simpler terms, this means that a molecule that
jumps from ground state 1 to excited state 2, with an energy difference of ΔE, will allow other
wavelengths to pass through until it can absorb radiation from a wavelength that corresponds to
ΔE. Light that passes through to the photodiode without any absorption is called Stray Radiant
Energy, or stray light. Absorption that occurs due to an energy difference between the two states
is called an absorption line, and a collection of absorption lines creates an absorption spectrum.
The frequency of each absorption line in an absorption spectrum tells us about the sample’s
molecular structure, and can be influenced by factors such as stray light, environmental
temperature, and electromagnetic fields.
Spectroscopy began with Isaac Newton splitting light with a prism; a key moment in the
development of modern optics. Therefore, it was originally the study of visible light that we call
color that later under the studies of James Clerk Maxwell came to include the entire
electromagnetic spectrum. Although color is involved in spectroscopy, it is not equated with the
color of elements or objects that involve the absorption and reflection of certain electromagnetic
waves to give objects a sense of color to our eyes. Rather spectroscopy involves the splitting of
light by a prism, diffraction grating, or similar instrument, to give off a particular discrete line
pattern called a "spectrum" unique to each different type of element. Most elements are first put
into a gaseous phase to allow the spectra to be examined although today other methods can be

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used on different phases. Each element that is diffracted by a prism-like instrument displays
either an absorption spectrum or an emission spectrum depending upon whether the element is
being cooled or heated,
Spectroscopy is used in physical and analytical chemistry because atoms and molecules have
unique spectra. As a result, these spectra can be used to detect, identify and quantify the
molecular and/or structural composition of a sample. Each type of molecule and atom will
reflect, absorb, or emit electromagnetic radiation in its own characteristic way. Spectroscopy
uses these characteristics to deduce and analyze the composition of a sample.

1.1 Types of Spectrometers and Spectroscopy

While there are many types of spectrometers, all spectrometers take in light, split it into its
spectral components, digitize the signal as a function of wavelength, and display it through a
computer. The design of a spectrometer changes depending on the scope and intentions of the
experiment, allowing researchers to measure molecular vibrations, absorbance, mass-to-charge
ratios, and much more.

 Monochromator

A monochromator is structurally similar to a spectrometer, but provides a much smaller window

of data. A monochromator captures one measurement in the UV-Visible spectrum at a particular,
predetermined, wavelength or bandwidth. Alternatively, a spectrometer captures the entire UV-
Visible spectrum in the same amount of time, and provides values for each wavelength.

 Radiometer

Radiometers, or light meters, are used to measure light in the UVA/VIS, UVA, and VIS spectra.
Radiometers consist of a meter body that measures current voltage from an internal or external
detector. A sensor or photodiode is used to measure a specific band of light, and filters are added
to the sensor to block unwanted wavelengths. Radiometer sensors are calibrated at the desired
peak intensity and measure all of the light under the curve to generate a single reading.

 Spectrograph

A spectrograph is an instrument that separates light by its wavelength or frequency and records
the resulting spectral range in a multichannel detector, such as a photographic plate. Light
entering a spectrograph through a small opening in the spectrograph hits a collimating mirror that
lines up the entering rays of light parallel to each other. Then, the rays hit a diffraction grating,
passing through or bouncing off into their constituent wavelengths, each with their own speed

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and direction that are dependent on their spectral color. The grating bends each wavelength in a
different direction, separating red from orange, orange from yellow, and so on. The diffraction
grating controls can be rotated to change which wavelengths of light reach a second mirror,
which then focuses them onto a photodetector that converts photons into electrical signals for
computer analysis.

 Spectroradiometer

Spectroradiometers are ideal for measuring the spectral energy distribution of small, precise light
sources. Light is dispersed using prisms or diffraction gratings. Spectroradiometers record the
radiation spectrum of a light source and calculate parameters such as luminance and
chromaticity. Factors such as sensitivity, linearity, stray light, and polarization error are less
influential on spectroradiometry than spectrometry, making spectroradiometry a more efficient
method for measuring narrow-band emitters.

 Spectroscope

A spectroscope is a hand-held device used to identify the spectral composition of light. Light
passes through a slit at one end, enters a prism, and is observed as a spectrum by the user’s eye.
Early astronomers used spectroscopes to study the composition of planets and stars. The
spectrums observed by these astronomers played a key role in dozens of hypotheses about the
gaseous nature of planets within our solar system.

 Spectrophotometry

Spectrophotometry measures how much light is absorbed by, reflected off, or transmitted
through a chemical substance by measuring the intensity of light as the beam passes through a
sample. Electromagnetic energy from the sample, enters the device through the aperture and is
separated into its component wavelengths by holographic grating. The separated light rays are
focused onto a CCD array detector which determines the intensity of each wavelength using a
pixel of the array. Spectrophotometry has broad applications within science and is used within
biochemistry, physics, material and chemical engineering, clinical application, and chemistry.
Spectrophotometers can be divided into two categories that are dependent on the wavelength of
the light source.

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 CHAPTER TWO
SPECTROPHOTOMETER

2.0 Introduction to Spectrophotometer

A spectrophotometer is an instrument that measures the amount of light that passes through a
solution. It is used to determine the concentration of a substance in a solution by measuring the
amount of light absorbed by the substance.
A spectrophotometer works by shining a beam of light through a solution and measuring the
amount of light that passes through. The amount of light that is absorbed by the solution is
proportional to the concentration of the substance in the solution.

The spectrophotometer consists of a light source, a monochromator, a sample holder, and a

detector. The light source emits a beam of light that is passed through the monochromator. The
monochromator selects a specific wavelength of light to pass through the sample. The sample
holder holds the solution in place. The detector measures the amount of light that passes through
the sample.

2.1 Types of spectrophotometers

1. UV-Visible spectrophotometer

UV-Visible (UV-Vis) spectrophotometers are widely used analytical instruments that measure
the absorbance and transmittance of light in the ultraviolet range (185-400nm) and visible range
(400–800 nm) of the electromagnetic spectrum.

Principle: The principle behind UV-Visible is spectrophotometry is based on the interaction

between light and matter. When UV or visible light passes through a sample, molecules in the
sample can absorb certain wavelengths of light, causing electronic transitions within the
molecules. The amount of light absorbed is proportional to the concentration of the absorbing
substance in the sample, which is described by Beer-Lambert Law:

A = εcl

Where;

 A is Absorbance
 ε is the molar absorptivity coefficient (L·mol⁻¹·cm⁻¹)
 c is the concentration of the analyte (mol/L)
 l is path length of the sample cuvette (cm) (Skoog et al., 2018).

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Applications: UV spectrophotometry has a wide range of applications across various industries;
i. Pharmaceutical Analysis
 It is used to determine drug concentration, purity, and dissolution rates.
 It helps in the quantitative analysis of antibiotics, vitamins, and hormones
ii. Biochemical and Molecular Biology Studies
 DNA and RNA quantification is performed by measuring absorbance at 260 nm.
 Proteins are analyzed at 280 nm due to the presence of aromatic amino acids
iii. Environmental Monitoring
 Detection of pollutants such as nitrates, phosphates, and organic contaminants in water.
 It is used to monitor air pollution by measuring the absorption spectra of atmospheric
pollutants
iv. Food and Beverage Industry
i. Analysis of food colorants, preservatives, and contaminants.
ii. It is used for quality control of beverages, dairy products, and edible oils (Holler et al.,
2020).
Types of UV Spectrophotometers
UV spectrophotometers are classified based on their operational design:
1. Single-Beam Spectrophotometer
i. Measures the intensity of light before and after passing through the sample.
ii. The reference and sample measurements are taken separately, making the process slower.
iii. More affordable and commonly used in basic laboratory applications (Holler et al.,
2020).
2. Double-Beam Spectrophotometer
i. Simultaneously measures both the sample and reference light intensities using a beam
splitter.
ii. Provides more accurate and stable readings, reducing errors caused by fluctuations in the
light source.
iii. Preferred for high-precision analysis in research and industrial applications (Skoog et al.,
2018).

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 2. Infrared (IR) Spectrophotometer
An infrared spectrophotometer is an analytical instrument used to identify materials including
organic polymers. Infrared spectrophotometers record the relative amount of energy as a function
of the wavelength/frequency of the infrared radiation when it passes through a sample.
Principle: Measures absorption in the infrared region (700-15000nm). Absorbed infrared
radiation causes molecular vibrations. Different bonds absorb at characteristic frequencies,
creating a distinct absorption spectrum.

Applications: Here are some of the applications:

i. Chemical and Pharmaceutical Industries

 Identification of compounds: IR spectra act like fingerprints, allowing for the

identification of unknown substances and verifying the identity of known ones.
 Structure elucidation: IR spectroscopy provides information about the functional
groups present in a molecule, aiding in determining its structure.

ii. Environmental Analysis

 Monitoring air and water pollution: IR spectroscopy can detect and measure the
concentration of various pollutants in air and water samples.
 Greenhouse gas monitoring: IR is used to measure the levels of greenhouse gases in the
atmosphere.

iii. Food Science: IR spectroscopy is used to assess the quality of food products by
analyzing their composition and detecting contaminants.

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 iv. Material Science: IR spectroscopy is used to study the structure and properties of
polymers.
v. Medical Diagnostics

 Disease detection: IR spectroscopy is being explored as a tool for the early detection of
diseases by analyzing biological fluids.
 Personalized medicine: IR may be used to analyze patient-specific responses to
treatments, leading to more personalized medicine approaches.

vi. Agricultural Science: IR spectroscopy can be used to analyze soil composition, health
and ensure the quality and safety of agricultural products.

3. Atomic Absorption Spectrophotometer (AAS)

Atomic Absorption Spectrophotometers (AAS) are widely used analytical tools that measure the
concentration of elements in a sample by analyzing the absorption of light by free atoms.

Principle: It operates on the principle that atoms absorb radiation at specific wavelengths,
corresponding to the energy differences between their atomic energy levels. When a sample is
exposed to radiation, the atoms absorb energy and transition from a ground state to an excited
state. The absorbed radiation is directly proportional to the concentration of the absorbing atoms.

Applications: Some common applications include:

i. Environmental Monitoring: AAS is used to measure the levels of heavy metals (e.g.,
lead, mercury, cadmium) in water, soil, and air samples. This helps in assessing pollution
levels and developing remediation strategies.
ii. Food Safety: AAS is employed to determine the concentration of metals in food
products, ensuring compliance with safety regulations and protecting consumer health.
iii. Clinical Diagnostics: AAS can measure the levels of metals in blood, urine, and other
bodily fluids, aiding in the diagnosis and monitoring of various medical conditions.
iv. Industrial Applications: AAS is used in various industries to analyze the composition of
materials, ensuring product quality and optimizing manufacturing processes. For
example, it can be used to measure the concentration of metals in alloys, ceramics, and
polymers.
v. Pharmaceutical Analysis: AAS plays a role in quality control and research in the
pharmaceutical industry, where it is used to determine the concentration of elements in
drug formulations and monitor impurities.

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Types of Atomic Absorption Spectrophotometer

Atomic Absorption Spectrophotometers (AAS) are classified based on the atomization technique
used to convert a sample into free atoms. The main types include:

1. Flame Atomic Absorption Spectroscopy (FAAS)

 Uses a flame (typically air-acetylene or nitrous oxide-acetylene) to atomize the sample.

 Suitable for detecting metals in solutions with moderate sensitivity.
 Commonly used for environmental, clinical, and food analysis.

2. Graphite Furnace Atomic Absorption Spectroscopy (Furnace AAS)

 Uses an electrically heated graphite tube for atomization instead of a flame.

 Provides higher sensitivity and lower detection limits than FAAS.
 Ideal for trace metal analysis in biological, environmental, and pharmaceutical samples.

3. Hydride Generation Atomic Absorption Spectrophotometer (HGAAS)

 Specialized for detecting elements like arsenic (As), selenium (Se), and antimony (Sb) by
converting them into volatile hydrides.
 Increases sensitivity for certain elements that form stable hydrides.

4. Cold Vapor Atomic Absorption Spectrophotometer (CVAAS)

 Specifically designed for mercury (Hg) detection.

 Uses chemical reduction to convert mercury into a vapor, which is then analyzed.

5. High-Resolution Continuum Source AAS (HR-CS-AAS)

 Uses a high-intensity continuum light source instead of a traditional hollow cathode

lamp.

 Allows for multi-element analysis and improved spectral resolution.

4. Near-Infrared (NIR) Spectrophotometer

A near-infrared (NIR) spectrophotometer is a specialized instrument that measures the
absorption and reflection of near-infrared light by a sample. This region of the electromagnetic

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spectrum falls between visible light and mid-infrared light, roughly from 700 to 2500 nanometers
(nm).
Principle: Measures absorption in the near-infrared region (700–2500 nm). It detects overtones
and combinations of vibrational modes of molecules, particularly organic compounds like water
and proteins.

Applications: Used in agriculture for grain quality control and in pharmaceuticals for drug
formulation.

i. Food and Agriculture: NIR spectroscopy is used extensively to analyze the composition
of food products, including moisture, protein, fat, carbohydrates, and fiber content. This
helps ensure consistent quality and nutritional value.
ii. Pharmaceuticals: NIR can quickly verify the identity of incoming raw materials,
analyze composition and preventing errors in drug manufacturing.
iii. Chemicals and Polymers:
 Chemical Identification: NIR can be used to identify, quantify and monitor
various chemical compounds and their reactions.
 Polymer Analysis: NIR is used to study the structure and properties of polymers,
including composition, molecular weight, and crystallinity.

iv. Environmental Science

 Soil Analysis: NIR can be used to analyze soil composition, including organic matter
content and nutrient levels.
 Water Quality: NIR can be used to monitor water quality by detecting various
contaminants.

2.2 Components of spectrophotometer

Light Source:

 Function: Provides the initial beam of light. The specific type of light source depends on
the wavelength range of the spectrophotometer.
 Types:

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 o Tungsten Lamp: Common for visible light and near-infrared (NIR)
spectrophotometers. Produces a broad spectrum of light.
o Deuterium Lamp: Used in ultraviolet (UV) spectrophotometers. Emits UV light.
o Xenon Arc Lamp: Can be used for both UV and visible light. Offers a high-
intensity, broad spectrum output.
o LEDs (Light Emitting Diodes): Increasingly used due to their stability, long life,
and ability to produce specific wavelengths. Common in some newer instruments.

2. Sample Holder/Compartment:

 Function: Holds the sample in the light path. Must be transparent to the wavelengths
being used.
 Types:
o Cuvettes: Small, rectangular containers made of glass, quartz (for UV), or plastic.
Used for liquid samples.
o Solid Sample Holders: Various holders designed for films, powders, or other
solid materials.
o Flow Cells: Used for continuous monitoring of flowing liquids, often in industrial
settings.

3. Monochromator (or Wavelength Selector):

 Function: Isolates a specific wavelength of light from the source's broad spectrum. This
is crucial for measuring absorbance or transmittance at a particular wavelength.
 Types:
o Prism: Disperses light into its component wavelengths using refraction. Older
technology, less common now.
o Grating: A reflective surface with closely spaced grooves that diffract light,
separating it into wavelengths. More common due to better resolution and
efficiency.
o Filters: Allow only a specific range of wavelengths to pass through. Simpler and
less precise than gratings or prisms, often used in less expensive instruments.

4. Detector:

 Function: Measures the intensity of the light that passes through the sample.
 Types:
o Photomultiplier Tube (PMT): Extremely sensitive, used for UV and visible
light. Converts light photons into an electrical signal.
o Photodiode: A semiconductor device that generates a current proportional to the
light intensity. Common for visible and NIR.

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 o Charge-Coupled Device (CCD): An array of light-sensitive pixels, often used in
spectrophotometers that can acquire a full spectrum at once.

5. Amplifier and Readout System:

 Function: Processes the signal from the detector and displays the results.
 Components:
o Amplifier: Increases the weak electrical signal from the detector.
o Analog-to-Digital Converter (ADC): Converts the analog signal to a digital
signal for computer processing.
o Computer/Display: Displays the results, usually as absorbance, transmittance, or
concentration. Software often provides tools for data analysis and manipulation.

6. Optics (Mirrors, Lenses):

 Function: Direct and focus the light beam through the instrument.
 Mirrors: Reflect the light.
 Lenses: Focus the light onto the sample, monochromator, and detector.

7. Control System:

 Function: Manages the operation of the spectrophotometer, including wavelength

selection, data acquisition, and instrument settings. Often integrated with the computer.

2.3 Advantages of spectrophotometer

1. High Sensitivity and Accuracy:
Spectrophotometers provide precise and reliable quantitative measurements of absorbance and
transmittance in a sample.
2. Wide Range of Applications: They are used in biochemistry, pharmaceutical analysis,
environmental monitoring, and food science to analyze compounds like proteins, nucleic acids,
and metal ions.
3. Non-Destructive Analysis: Samples can be analyzed without being consumed or altered,
making it useful for repeated measurements.
4. Rapid and Simple Operation: Modern spectrophotometers offer quick analysis with minimal
sample preparation.
5. Capability for Qualitative and Quantitative Analysis: It allows the identification of
unknown compounds and the determination of their concentrations.
Limitations

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1. Interference from Impurities: Contaminants or unwanted substances in the sample can lead
to inaccurate readings.
2. Limited Sensitivity for Low-Concentration Samples: Some spectrophotometers struggle
with detecting extremely low analyte concentrations without enhancement techniques.
3. Requirement for Proper Calibration: Regular calibration is necessary to maintain accuracy,
which can be time-consuming.
4. Dependence on Sample Transparency: Turbid or highly concentrated samples may cause
scattering or absorption errors.
5. Cost and Maintenance: High-performance spectrophotometers, especially UV-Vis and FTIR
models, can be expensive and require routine maintenance.

2.4 Benefits of Spectrophotometers

The benefit of a spectrophotometer is:
1. High Sensitivity and Accuracy: Spectrophotometers provide precise and reliable
measurements of absorbance and transmittance, allowing for the accurate quantification of
substances, even at very low concentrations. This makes them essential in biochemical,
pharmaceutical, and clinical research.
2. Wide Range of Applications: They are used in various fields, including medical diagnostics
(e.g., blood tests, enzyme activity assays), environmental monitoring (e.g., detecting pollutants in
water and air), and the pharmaceutical industry (e.g., assessing drug purity and concentration).
3. Fast and Non-Destructive Analysis: Spectrophotometric methods require minimal sample
preparation and provide rapid results. Additionally, they do not alter or destroy the sample,
allowing for further testing if needed.
4. Qualitative and Quantitative Analysis: Spectrophotometers help identify unknown
compounds based on their absorbance spectra (qualitative analysis) and determine the precise
concentration of a known substance using Beer-Lambert’s Law (quantitative analysis).
5. Versatility in Wavelength Selection: Modern spectrophotometers can analyze samples
across a broad range of wavelengths, from ultraviolet (UV) to visible (Vis) and infrared (IR)
light, making them suitable for various chemical and biological analyses.
6. Cost-Effective and Efficient: Compared to other analytical techniques like chromatography
or mass spectrometry, spectrophotometry is relatively affordable, requires less sample
preparation, and provides quick results, making it a cost-effective method for routine laboratory
analysis.

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2.5 Challenges associated with spectrophotometer
There are several challenges associated with using a spectrophotometer, including instrumental,
sample-related, and operational limitations. Some key challenges are:
1. Instrumental Challenges
 Baseline Drift & Noise: Over time, spectrophotometers may exhibit baseline drift due to
temperature fluctuations, electronic noise, or aging of optical components.
 Wavelength Accuracy: Calibration errors can lead to slight deviations in wavelength
accuracy, affecting measurements.
 Stray Light: Unwanted light can interfere with measurements, leading to inaccurate
absorbance values, especially at higher concentrations.
2. Sample-Related Challenges
 Interference from Other Components: Contaminants or overlapping absorption peaks
can affect accuracy.
 Path Length Variability: Improper cuvette placement or differences in cuvette
dimensions can lead to inconsistent readings.
 Low Sensitivity for Trace Analysis: Some spectrophotometers may struggle to detect
very low concentrations without specialized techniques.
3. Operational Challenges
 User Errors: Incorrect calibration, improper cuvette handling, or not using blank
solutions properly can result in inaccurate readings.
 Degradation of Light Source: The performance of lamps (e.g., tungsten, deuterium)
decreases over time, affecting measurement stability.
 Software & Data Processing Issues: Older models may lack advanced software
capabilities for data correction and processing.

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 2.6 Application of Spectrophotometer

1. It is useful in qualitative analysis, especially when identifying classes of compounds in

both biological and pure state.
2. The spectrophotometer is essential in quantitative analysis of biochemistry practical such
as in determining the unknown concentration of a given species through absorption
spectrometry. A perfect example is the nucleic acid in a protein.
3. Enzyme assay is the primary use of spectrophotometry.
4. Identifying the molecular weight of a particular sample such as amine picrates, ketone
compounds, aldehyde, and sugar, to name a few.

2.7 Maintenance of a spectrophotometer

Maintaining a spectrophotometer is essential to ensure accurate and reliable measurements.

Some key maintenance practices:

Daily Maintenance:

1. Warm-up Time: Allow the instrument to warm up before use (typically 10-30 minutes).
2. Clean the Sample Holder: Wipe the sample compartment and cuvette holder with a soft,
lint-free cloth.
3. Use Clean Cuvettes: Always use clean and scratch-free cuvettes. Rinse them with
distilled water after use.
4. Check for Light Path Obstructions: Ensure there is no dust or debris blocking the light
path.
5. Baseline Calibration: Perform baseline correction or blank measurement before running
samples.

Weekly Maintenance:

1. Inspect the Optical Components: Check mirrors, lenses, and detectors for dirt or
contamination.
2. Check the Lamp Intensity: Monitor lamp performance and replace if intensity decreases
significantly.
3. Wipe the Exterior: Clean the spectrophotometer’s surface using a soft cloth with mild
detergent.

Monthly Maintenance:

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 1. Check and Replace Lamps: If the instrument uses a halogen or deuterium lamp, replace it
according to manufacturer guidelines.
2. Calibrate the Wavelength Accuracy: Use a calibration standard or built-in calibration
function.
3. Verify Performance: Use a known standard sample to check accuracy and precision.

Annual Maintenance:

1. Professional Servicing: Schedule an annual professional calibration and servicing.

2. Software Updates: Update firmware and software if applicable.
3. Check Internal Components: Inspect electronic components, fans, and connectors.

Best Practices:

 Avoid Fingerprints on Optical Surfaces: Handle cuvettes with gloves or by the frosted
edges.
 Store in a Clean Environment: Keep the spectrophotometer covered when not in use to
protect from dust.
 Use High-Quality Reagents: Impure solvents or solutions can affect readings.
 Follow Manufacturer Guidelines: Always refer to the user manual for specific
maintenance procedures.

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