Spectroscopy is a technique used to study the interaction between light and matter.
In UV-Visible
(Ultraviolet-Visible) spectroscopy, this technique focuses on the absorption of ultraviolet and
visible light by a substance, which can provide information about its electronic structure and
concentration.
1. UV-Visible Spectroscopy
1. Principle:
○ UV-Visible spectroscopy involves measuring the intensity of light absorbed by a
sample as a function of wavelength. The sample absorbs light at specific
wavelengths, and this absorption can be correlated with the electronic transitions
within molecules.
2. Wavelength Range:
○ UV-Visible spectroscopy covers the wavelength range from about 200 nm (in the
UV range) to 800 nm (in the visible range).
3. Electronic Transitions:
○ Absorption in UV-Visible spectroscopy is typically due to electronic transitions in
molecules. These can include:
■ π to π* transitions: Common in conjugated systems.
■ n to π* transitions: Occur in molecules with lone pair electrons.
■ σ to σ* transitions: Less common, occur in saturated compounds.
4. Beer-Lambert Law:
○ This law relates the absorbance (A) of a solution to the concentration (c) of the
absorbing species, path length (l), and molar absorptivity (ε):
A=log(I0I)=ε⋅c⋅lA=log(II0)=ε⋅c⋅l Where I0I0is the intensity of the incident light,
II is the intensity of the transmitted light.
5. Instrumentation:
○ Light Source: UV-Visible spectrometers use deuterium lamps (for UV) and
tungsten lamps (for visible light).
○ Monochromator: A device that isolates specific wavelengths of light.
○ Sample Holder: Usually a cuvette through which the light passes.
○ Detector: Measures the intensity of transmitted light.
6. Applications:
○ Quantitative Analysis: Determining the concentration of a solute in a solution.
○ Qualitative Analysis: Identifying substances based on their absorption spectra.
○ Kinetics: Studying reaction rates by monitoring changes in absorbance over time.
7. Spectrum:
○ The resulting UV-Visible spectrum displays absorbance versus wavelength. Peaks
in the spectrum correspond to wavelengths where the sample absorbs light,
providing insights into the electronic structure and concentration of the sample.
8. Sample Preparation:
○ Samples should be in a suitable solvent that does not absorb significantly in the
UV-Visible range. The concentration should be within a range where absorbance
is measurable but not too high to cause saturation.
UV-Visible spectroscopy works by analyzing how a sample absorbs ultraviolet (UV) and visible
light.
1. Light Source:
○ The spectrometer uses two types of lamps: a deuterium lamp for UV light
(200-400 nm) and a tungsten-halogon lamp for visible light (400-800 nm). These
lamps produce a broad spectrum of light.
2. Monochromator:
○ The light from the source passes through a monochromator, which disperses it
into its component wavelengths. The monochromator selects specific wavelengths
of light to pass through the sample.
3. Sample Holder:
○ The selected wavelength of light is directed through the sample, which is placed
in a cuvette or sample cell. The sample absorbs light at certain wavelengths
depending on its electronic structure.
4. Detector:
○ After passing through the sample, the remaining light (transmitted light) is
detected by a photodetector or photodiode. The detector measures the intensity of
light that passes through the sample.
5. Data Analysis:
○ The intensity of the transmitted light is compared to the intensity of the incident
light. This comparison is used to calculate absorbance using the Beer-Lambert
Law: A=log(I0I)A=log(II0) where I0I0is the intensity of the incident light and II
is the intensity of the transmitted light.
6. Spectrum Generation:
○ The process is repeated for a range of wavelengths, generating a spectrum that
plots absorbance (or transmittance) versus wavelength. Peaks in the spectrum
indicate wavelengths where the sample absorbs light strongly.
2. Infrared (IR) spectroscopy
It is an analytical technique used to identify and study chemicals through their interaction with
infrared light. It is widely used in chemistry, material science, and biology to determine the
structure, composition, and purity of substances.
1. IR Source
● Function: Produces a broad spectrum of infrared radiation.
● Common Types:
○ Globar Source: A silicon carbide rod heated electrically to produce IR radiation.
○ Nernst Glower: Made of a mixture of rare earth oxides that glows when heated.
○ Tungsten Filament: Used for near-infrared (NIR) spectroscopy.
2. Sample Handling System
● Function: Contains the sample in a manner that allows IR radiation to interact with it.
● Types:
○ Transmission Cells: For liquid or thin solid samples.
○ Attenuated Total Reflectance (ATR): A crystal is used to obtain spectra of solid or
liquid samples.
○ Diffuse Reflectance: For powdered or rough-surfaced solid samples.
○ Gas Cells: For gases, typically with longer path lengths to enhance absorbance.
3. Monochromator
● Function: Disperses the IR radiation into its component wavelengths and allows only a
specific wavelength to pass through.
● Types:
○ Gratings: Rotating gratings that separate light into different wavelengths.
○ Prisms: Less common in modern instruments, used to disperse light by refraction.
4. Detector
● Function: Detects the IR radiation that has interacted with the sample.
● Common Types:
○ Thermocouple: Measures temperature changes due to absorbed IR radiation.
○ Bolometer: Measures the temperature rise in a material as it absorbs IR radiation.
○ Pyroelectric Detector: Uses materials that generate an electric charge when
heated.
○ Photoconductive Detector: Utilizes semiconductors that change their conductivity
based on IR absorption.
5. Interferometer (in FTIR)
● Function: Used in Fourier Transform Infrared (FTIR) spectroscopy to generate an
interference pattern from the IR source.
● Michelson Interferometer: The most common type, consisting of a beam splitter, a
moving mirror, and a fixed mirror.
6. Computer and Software
● Function: Controls the instrument, collects data, and processes the spectrum.
● Software Capabilities:
○ Spectral Analysis: Identifies functional groups or materials.
○ Quantitative Analysis: Measures the concentration of compounds in a mixture.
7. Beam Splitter (in FTIR)
● Function: Splits the incoming IR beam into two paths in an interferometer.
● Common Materials: KBr (potassium bromide) or CaF2 (calcium fluoride) for different
wavelength ranges.
Types of IR Spectroscopy
● Dispersive IR Spectroscopy: Uses a monochromator to filter the IR light.
● Fourier Transform Infrared (FTIR) Spectroscopy: More modern and widely used, it
collects all wavelengths simultaneously and uses mathematical transformation (Fourier
transform) to obtain the spectrum.
How IR Spectroscopy Works:
● Infrared Light Absorption: Molecules absorb infrared light at specific wavelengths,
which corresponds to the vibrations of the chemical bonds within the molecule. Different
bonds absorb different frequencies of IR radiation.
● Vibrational Modes: The absorbed energy causes the bonds to vibrate in various ways,
such as stretching, bending, or twisting. These vibrations are characteristic of specific
functional groups (like -OH, -CH, -CO, etc.).
● Spectrum Generation: The instrument records the intensity of light absorbed at each
wavelength, producing an IR spectrum. The x-axis represents the wavelength (often in
wavenumbers, cm⁻¹), and the y-axis shows the absorbance or transmittance.
● Interpretation: The resulting spectrum has peaks corresponding to the specific vibrations
of the bonds in the sample. By analyzing these peaks, one can infer the presence of
certain functional groups and gain insights into the molecular structure.
Key Regions in an IR Spectrum:
● Fingerprint Region (600-1500 cm⁻¹): Contains complex patterns of many peaks, unique to
individual molecules. It’s often used for substance identification.
● Functional Group Region (1500-4000 cm⁻¹): More straightforward, with peaks
corresponding to specific functional groups.
Applications:
● Identification: Determining the chemical structure of unknown compounds.
● Quality Control: Ensuring the purity of a substance by checking for impurities.
● Studying Interactions: Understanding how molecules interact with each other, such as in
drug binding studies.
● Monitoring Reactions: Tracking chemical reactions by observing changes in the spectrum
over time.
3. Fluorescence spectroscopy
Fluorescence spectroscopy is a highly sensitive analytical technique that measures the
fluorescence emitted by a substance upon excitation by a specific wavelength of light. This
technique is widely used in various fields, including chemistry, biology, medicine, and
environmental science, for its ability to detect and quantify minute amounts of substances and to
study molecular interactions and dynamics.
Principles of Fluorescence Spectroscopy
Fluorescence occurs when a molecule absorbs light at one wavelength (excitation) and
subsequently emits light at a longer wavelength (emission). The process involves several key
steps:
1. Excitation: When a molecule absorbs a photon of light, it is excited from its ground state
to a higher energy electronic state.
2. Relaxation: The excited molecule undergoes internal conversion and vibrational
relaxation, where it loses some energy non-radiatively, typically through heat.
3. Emission: The molecule returns to the ground state by emitting a photon of light. The
emitted light has a longer wavelength than the absorbed light due to the loss of energy
during the relaxation process. This difference in wavelength is known as the Stokes shift.
Components of a Fluorescence Spectroscopy System
1. Light Source
● Function: Provides the excitation light needed to excite the fluorophores in the sample.
● Common Types:
○ Xenon Arc Lamp: Provides a continuous spectrum of light, commonly used for
broad-range fluorescence measurements.
○ Mercury Vapor Lamp: Emits specific wavelengths suitable for exciting particular
fluorophores.
○ LEDs: Provide specific excitation wavelengths with low power consumption and
long life.
○ Lasers: Provide intense, monochromatic light for high sensitivity and selectivity
in fluorescence measurements.
2. Monochromator or Filter (Excitation)
● Function: Selects the specific wavelength of light used to excite the sample.
● Types:
○ Grating Monochromator: Disperses light into its component wavelengths and
selects a specific wavelength.
○ Interference Filters: Allow only a narrow band of wavelengths to pass through,
blocking others.
○ Dichroic Mirrors: Reflects specific wavelengths while transmitting others,
commonly used in confocal microscopy.
3. Sample Holder
● Function: Contains the sample in the path of the excitation light.
● Types:
○ Cuvettes: Typically made of quartz or glass, used for liquid samples.
○ Microscope Slides: Used in fluorescence microscopy for solid samples or thin
films.
○ Microplates: Used in high-throughput screening applications for small-volume
samples.
4. Monochromator or Filter (Emission)
● Function: Selects the specific wavelength of light emitted by the sample after excitation.
● Types:
○ Emission Monochromator: Scans through wavelengths to detect the emission
spectrum.
○ Emission Filters: Select specific emission wavelengths for detection, often used in
filter-based fluorometers.
5. Detector
● Function: Detects the fluorescence emitted from the sample and converts it into an
electrical signal.
● Common Types:
○ Photomultiplier Tube (PMT): Highly sensitive to low levels of light, making it
ideal for detecting weak fluorescence.
○ Photodiode: Less sensitive than PMTs but more rugged and compact, suitable for
higher light levels.
○ CCD (Charge-Coupled Device): Used in imaging applications to detect and
record fluorescence across a large area.
6. Optics
● Function: Directs and focuses light from the source to the sample and from the sample to
the detector.
● Types:
○ Lenses: Focus the excitation light onto the sample and collect emitted light.
○ Beam Splitters: Direct part of the light to the sample and part to the detector or
reference.
○ Optical Fibers: Used to transmit light to and from the sample, allowing flexible
setup configurations.
7. Computer and Software
● Function: Controls the instrument, acquires data, and processes the fluorescence spectra.
● Software Capabilities:
○ Spectral Analysis: Identifies specific fluorophores and quantifies their
concentrations.
○ Kinetic Studies: Monitors changes in fluorescence over time.
○ Imaging: In fluorescence microscopy, software is used to capture and analyze
fluorescence images.
8. Reference Detector (Optional)
● Function: Monitors the intensity of the excitation light to correct for fluctuations in the
light source.
● Placement: Often positioned to monitor the excitation beam before it interacts with the
sample.
9. Polarizers (Optional)
● Function: Used to study fluorescence polarization, which can give information about
molecular orientation and dynamics.
● Types:
○ Excitation Polarizer: Polarizes the light before it reaches the sample.
○ Emission Polarizer: Analyzes the polarization state of the emitted light.
Applications of Fluorescence Spectroscopy
1. Biochemical and Medical Research
Fluorescence spectroscopy is indispensable in studying biological molecules, such as proteins,
nucleic acids, and enzymes. Fluorescent tags and dyes are commonly used to label these
molecules, enabling researchers to track their behavior in cells, tissues, or whole organisms. This
technique is also crucial in DNA sequencing, where fluorescently labeled nucleotides are used.
2. Environmental Monitoring
In environmental science, fluorescence spectroscopy helps in detecting and quantifying
pollutants, such as polycyclic aromatic hydrocarbons (PAHs) and heavy metals, in water, soil,
and air. It is particularly useful for monitoring oil spills and detecting trace amounts of
contaminants.
3. Material Science
Fluorescence spectroscopy is employed to study the properties of new materials, such as
quantum dots, polymers, and nanomaterials. The technique provides insights into the electronic
and optical properties of these materials, guiding the development of new technologies.
4. Clinical Diagnostics
In medicine, fluorescence spectroscopy is used in diagnostic tests, such as immunoassays, where
it helps in the detection of specific proteins, pathogens, or biomarkers in patient samples. The
high sensitivity and specificity of fluorescence-based methods make them suitable for early
disease detection.
Advantages and Limitations
Fluorescence spectroscopy offers several advantages, including high sensitivity, specificity, and
the ability to study dynamic processes in real time. It is non-destructive, allowing for the analysis
of small sample volumes without altering the sample's properties. However, the technique has
limitations, such as photobleaching, where the fluorescent signal decreases over time due to
prolonged exposure to light. Additionally, fluorescence quenching, where the fluorescence
intensity is reduced due to various factors (e.g., solvent effects, temperature, or the presence of
quenchers), can complicate data interpretation.
4. Raman spectroscopy
It is a powerful analytical technique used to study vibrational, rotational, and other
low-frequency modes in a system. It relies on the inelastic scattering of monochromatic light
(usually from a laser) to provide information about molecular vibrations that can be used to
identify molecules and study molecular interactions.
Principles of Raman Spectroscopy:
Raman spectroscopy is based on the Raman effect, where incident photons interact with a
molecule and are scattered with a shift in energy corresponding to the vibrational modes of the
molecule. This shift in energy is what gives rise to the Raman spectrum, which is characteristic
of the molecular structure of the sample.
1. Components of Raman Spectroscopy
a. Laser Source:
● Purpose: Provides the monochromatic light required for the Raman effect.
● Common Laser Types:
○ Argon ion (488 nm, 514.5 nm)
○ Nd
(1064 nm)
○ Helium-Neon (632.8 nm)
○ Diode lasers (various wavelengths)
● Considerations: The wavelength of the laser is crucial; shorter wavelengths generally
produce stronger Raman signals but can also cause fluorescence, while longer
wavelengths minimize fluorescence but may reduce signal intensity.
b. Sample Illumination and Collection System:
● Microscope: Used to focus the laser onto the sample and collect the scattered light.
Micro-Raman setups are common for small sample areas.
● Optical Filters:
○ Notch Filters: Remove the Rayleigh scattered light and allow only the Raman
scattered light to pass.
○ Edge Filters: Used to filter out unwanted wavelengths and isolate the Raman
signal.
c. Spectrometer:
● Purpose: Disperses the Raman scattered light into its constituent wavelengths (Raman
spectrum).
● Types of Spectrometers:
○ Grating Spectrometers: Use diffraction gratings to disperse light. They offer
high resolution but can be complex.
○ Interferometers: Used in Fourier-transform Raman spectroscopy (FT-Raman) for
high sensitivity.
d. Detector:
● Types of Detectors:
○ Charge-Coupled Devices (CCDs): Highly sensitive detectors that convert the
Raman signal into a digital format for analysis.
○ Photomultiplier Tubes (PMTs): Older technology, still used for certain
applications requiring high-speed detection.
● Considerations: The detector's sensitivity and noise characteristics directly affect the
quality of the Raman spectrum.
2. Additional Considerations:
a. Spectral Resolution:
● Determined by the spectrometer and the detector, it defines how well the instrument can
distinguish between closely spaced Raman peaks.
b. Laser Power and Spot Size:
● The power of the laser and the size of the focused spot on the sample influence the
intensity of the Raman signal. Higher power can improve signal strength but also risks
damaging sensitive samples.
c. Sample Preparation:
● Raman spectroscopy requires minimal sample preparation, but the sample’s physical state
(solid, liquid, gas) and the matrix in which it’s embedded can affect the quality of the
Raman signal.
d. Environmental Conditions:
● Ambient light, temperature, and humidity can influence Raman measurements,
necessitating controlled conditions or background correction techniques.
Applications of Raman Spectroscopy:
● Chemical Analysis: Identification of molecular compounds and analysis of their
structure.
● Material Science: Characterization of materials like polymers, semiconductors, and
nanomaterials.
● Biology and Medicine: Studying biomolecules, tissues, and cells for diagnostic
purposes.
● Environmental Science: Monitoring pollutants and contaminants in water, air, and soil.
Advantages of Raman Spectroscopy:
● Non-Destructive: Requires no sample preparation, preserving the sample for further
analysis.
● Versatility: Applicable to solids, liquids, and gases.
● Specificity: Provides detailed information about molecular vibrations, allowing for
precise identification of substances.
Challenges and Limitations:
● Fluorescence Interference: Fluorescence can overshadow the Raman signal, particularly
with shorter laser wavelengths.
● Weak Signal: Raman scattering is inherently weak, which can limit sensitivity.
● Expensive Equipment: The advanced optics and detectors required make Raman
instruments costly
