What is UV-Visible Spectroscopy? (Definition of UV-Visible Spectroscopy)
UV-Visible spectroscopy is an analytical method in which a substance is exposed to ultraviolet (UV) or visible light, and the amount of light absorbed by the substance is measured.
The commonly used regions are:
- UV region: 200–400 nm
- Visible region: 400–700 nm
This absorption can be measured using a UV-Visible spectrophotometer.
The instrument measures this absorption and gives the result as absorbance or transmittance.
Principle of UV-Visible Spectroscopy
- The principle is based on the absorption of UV or visible radiation by molecules of sample.
- When a molecule absorbs UV or visible light, its electrons gain energy and move from a lower energy level to a higher energy level.
- This absorption can be measured using a UV-Visible spectrophotometer.
What is a UV-Visible Spectrophotometer?
A UV-Visible spectrophotometer is the instrument used to measure the absorption of UV or visible radiation by a sample.It is commonly used in pharmaceutical laboratories.
- Testing of drug purity and stability.
- Measurement of active pharmaceutical ingredients (APIs).
- Learn the drug releases study, etc.
A simple UV-Visible spectrophotometer consists of:
Let's understand them one by one.
- Light source
- Monochromator
- Sample holder
- Detector
- Display/readout system
Let's understand them one by one.
Light Source:
The light source produces the radiation required for analysis.Common sources:
Deuterium lamp: It is mainly used for the UV region.
Tungsten or tungsten-halogen lamp: It is commonly used for the visible region.
Monochromator:
- The light source produces a range of wavelengths.
- The monochromator selects the required wavelength from this range.
- A diffraction grating is commonly used for wavelength selection in modern instruments.
Sample Holder:
The sample is usually placed in a small container called a cuvette.For UV measurements, quartz or fused-silica cuvettes are commonly used because they allow UV radiation to pass through.
For visible measurements, glass or suitable plastic cuvettes can often be used.
Detector:
After passing through the sample, the remaining light reaches the detector.The detector converts the light signal into an electrical signal.
The instrument then processes this signal and displays the result.
Display System:
The final result can be displayed as:- Absorbance
- % Transmittance
- Spectrum
- Wavelength of maximum absorption
Working of UV-Visible Spectrophotometer
The working can be understood in a few simple steps.- Step 1: The light source produces UV or visible radiation.
- Step 2: The monochromator selects the required wavelength.
- Step 3: The selected light passes through the sample solution.
- Step 4: The molecules in the sample absorb some of the light.
- Step 5: The remaining light reaches the detector.
- Step 6: The detector converts the light into an electrical signal.
- Step 7: The instrument displays the absorbance or transmittance.
What is Absorbance?
Absorbance tells us how much light is absorbed by the sample.It is represented by A.
If a sample absorbs more light, its absorbance will be higher.
The formula is:
A = log (I₀/I)
Where:
I₀ = intensity of incident light
I = intensity of transmitted light
Higher absorption means higher absorbance.
It is represented by T.
I₀ = intensity of incident light
I = intensity of transmitted light
Higher absorption means higher absorbance.
What is Transmittance?
Transmittance tells us how much light passes through the sample.It is represented by T.
[T = \frac{I}{I_0}]
Percentage transmittance is:
[\%T = \frac{I}{I_0} \times 100]
There is an important relationship between absorbance and transmittance:
[A = -\log T]
Therefore: Absorbance increases → Transmittance decreases
Beer-Lambert's Law
Beer-Lambert's law is one of the most important topics in UV spectroscopy for B.Pharm students.
It states that:
The absorbance of a solution is directly proportional to the concentration of the absorbing substance and the path length of the solution, under suitable conditions.
[\%T = \frac{I}{I_0} \times 100]
There is an important relationship between absorbance and transmittance:
[A = -\log T]
Therefore: Absorbance increases → Transmittance decreases
Beer-Lambert's Law
Beer-Lambert's law is one of the most important topics in UV spectroscopy for B.Pharm students.
It states that:
The absorbance of a solution is directly proportional to the concentration of the absorbing substance and the path length of the solution, under suitable conditions.
The equation is:
A = \varepsilon bc
Where:
For exam purposes, remember:
A ∝ c
when other conditions remain constant.
A = \varepsilon bc
Where:
- A = Absorbance
- ε= Molar absorptivity
- b = Path length
- c = Concentration
For exam purposes, remember:
A ∝ c
when other conditions remain constant.
Higher concentration → Higher absorbance
What is λmax?
λmax (lambda max) is the wavelength at which a substance shows its maximum absorption.For example, if a drug shows maximum absorbance at 254 nm:
λmax = 254 nm
λmax is important because it is often used for quantitative analysis of a drug.
Simple way to remember:
λmax = wavelength of maximum absorption.
Some common groups associated with UV absorption include:
C=C
C=O
N=N
λmax = 254 nm
λmax is important because it is often used for quantitative analysis of a drug.
Simple way to remember:
λmax = wavelength of maximum absorption.
What is a Chromophore?
A chromophore is the part of a molecule responsible for its absorption of UV or visible radiation.Some common groups associated with UV absorption include:
C=C
C=O
N=N
What is an Auxochrome?
An auxochrome is a group that can modify the absorption characteristics of a chromophore.Common examples include:
–OH
–NH₂
–OR
Electronic Transitions
When a molecule absorbs UV radiation, an electron can move from a lower energy orbital to a higher energy orbital.The major electronic transitions are:
σ → σ
n → σ
π → π
n → π
Applications of UV-Visible Spectroscopy in Pharmacy
UV-Visible spectroscopy has many uses in pharmaceutical laboratories.1. Assay of Drugs: It can be used to determine the amount of drug present in a pharmaceutical preparation.
For example, a suitable UV method may be used for the analysis of a drug in a tablet solution.
2. Quantitative Analysis: UV spectroscopy can be used to determine the concentration of a substance in a solution.
This is commonly done using Beer-Lambert's law or a calibration curve.
3. Identification of Drugs: The absorption spectrum of a substance can provide useful information for identification.
However, UV spectroscopy alone may not always be sufficient for complete identification.
4. Dissolution Testing
UV spectroscopy can be used in some dissolution studies to determine how much drug has been released from a dosage form.
UV spectroscopy can be used in some dissolution studies to determine how much drug has been released from a dosage form.
For example: Tablet → Dissolution medium → Sample withdrawn → UV analysis
5. Determination of λmax
UV spectroscopy is used to find the wavelength at which a drug shows maximum absorption.
This is useful during analytical method development.
This is useful during analytical method development.
6. Research Work
UV-Visible spectroscopy is also used in pharmaceutical research for studying compounds, reactions and certain degradation processes.
UV-Visible spectroscopy is also used in pharmaceutical research for studying compounds, reactions and certain degradation processes.
Advantages of UV-Visible Spectroscopy
Some important advantages are:- Simple technique
- Rapid analysis
- Relatively inexpensive
- Requires a small amount of sample
- Useful for quantitative analysis
- Easy to perform
- Commonly available in pharmaceutical laboratories
Limitations of UV-Visible Spectroscopy
UV spectroscopy also has some limitations.1. Not all substances absorb UV or visible light strongly
A suitable absorbing group may be required.
2. Interference can occur
Other substances present in the sample may also absorb at the selected wavelength.
3. Low selectivity
Two different substances can sometimes have similar absorption characteristics.
4. Sample preparation is important
Dirty cuvettes, bubbles, turbidity or incorrect dilution can affect the result.
UV-Visible Spectroscopy: Important Viva Questions
What is UV-Visible spectroscopy?It is an analytical technique used to measure the absorption of UV or visible radiation by a substance.
What is the principle of UV spectroscopy?
It is based on the absorption of UV or visible radiation by molecules, resulting in electronic excitation.
What is λmax?
λmax is the wavelength at which maximum absorption occurs.
What is Beer-Lambert's law?
It states that absorbance is directly proportional to concentration and path length under suitable conditions.
Which cuvette is commonly used for UV analysis?
Quartz or fused-silica cuvette.
Which lamp is used for the UV region?
Deuterium lamp.
Which lamp is commonly used for the visible region?
Tungsten or tungsten-halogen lamp.
What is a chromophore?
The part of a molecule responsible for UV or visible absorption.
What is absorbance?
Absorbance indicates the amount of radiation absorbed by a sample.
What happens to absorbance when concentration increases?
Under suitable Beer-Lambert conditions, absorbance increases with concentration.
Absorbance indicates the amount of radiation absorbed by a sample.
What happens to absorbance when concentration increases?
Under suitable Beer-Lambert conditions, absorbance increases with concentration.
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