Instrumental Methods of Analysis UNIT 1: OVERVIEW AND SYLLABUS as Per PCI

Instrumental Methods of Analysis Unit I covers two major topics: UV–Visible Spectroscopy and Fluorimetry.

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Instrumental Methods of Analysis UNIT 1

I. UV–VISIBLE SPECTROSCOPY

Introduction and Fundamental Concepts

• Definition: UV–Visible spectroscopy deals with the recording of radiation absorption in the ultraviolet (UV) and visible regions of the electromagnetic spectrum.
• Regions:
• UV Region: 10–400 nm
  • Near UV (Quartz region): 200–400 nm
  • Far/Vacuum UV: 10–200 nm
• Visible Region: 400–800 nm
• Principle: Absorption of electromagnetic radiation induces electronic excitation from a lower to a higher molecular orbital — hence it is an electronic spectroscopy.
• Electromagnetic Radiation (EMR):
  • Nature: Dual character — behaves as waves and photons.
  • Types of EM Radiation (in order of increasing wavelength):
    γ-rays < X-rays < UV < Visible < IR < Microwaves < Radio waves

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Electronic Transitions

• Types of Electrons in Organic Molecules:
  • σ (sigma) electrons – present in single bonds
  • π (pi) electrons – present in double/triple bonds
  • n (non-bonding) electrons – lone pairs
  • σ* (antibonding sigma) electrons
  • π* (antibonding pi) electrons
• Types of Electronic Transitions:
  • σ → σ* – Highest energy transition (requires UV region, < 150 nm)
  • n → σ* – Lower energy transition (seen in compounds with lone pairs, e.g., amines)
  • π → π* – Lower energy than n → σ* (seen in unsaturated compounds)
  • n → π* – Lowest energy transition (seen in compounds with heteroatoms, e.g., aldehydes, ketones)

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Chromophores and Auxochromes

• Chromophore: Part of a molecule responsible for absorption of radiation (e.g., –C=C–, –C=O, –NO₂).
• Independent Chromophore: Shows absorption by itself.
• Dependent Chromophore: Shows absorption only when conjugated.
• Auxochrome: Functional group with one or more lone pairs of electrons attached to a chromophore.
• Function: Alters both wavelength and intensity of absorption.
• Types: Acidic (–OH, –COOH) and Basic (–NH₂).

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Spectral Shifts

• Bathochromic shift (Red shift): Absorption maximum (λmax) moves to a longer wavelength.
• Hypsochromic shift (Blue shift): λmax moves to a shorter wavelength.
• Hyperchromic effect: Increase in absorption intensity.
• Hypochromic effect: Decrease in absorption intensity.

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Absorption Bands

• K-bands: π → π* transitions in conjugated systems (intense).
• R-bands: n → π* transitions (less intense).
• B-bands & E-bands: π → π* transitions in aromatic compounds.

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Beer–Lambert Law

• Beer’s Law:
  Decrease in intensity ∝ concentration (c)
• Lambert’s Law:
  Decrease in intensity ∝ path length (l)
• Combined Beer–Lambert Law:
  [A = \varepsilon c l ]Where:
  • (A) = Absorbance
  • ( \varepsilon ) = Molar extinction coefficient
  • (c) = Concentration
  • (l) = Path length
• Transmittance (T):

  T = \frac{I}{I_0} \quad \text 

Meaning:

• (T) = Transmittance
• (%T) = Percentage transmittance
• (I) = Intensity of transmitted light
• (I_0) = Intensity of incident light
• Absorbance (A):
      A = log₁₀ (I₀ / I) 
• Deviations:
  Caused by chemical (association, dissociation, solvent reaction) and instrumental factors (stray light, polychromatic radiation).

 

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Instrumentation

Essential Components of a Spectrophotometer:

1. Radiation Source:
   • Hydrogen or Deuterium lamp (UV region)
   • Tungsten lamp (Visible region)
2. Wavelength Selector:
   • Filters: Absorption or interference filters
   • Monochromators: Prism or grating type
3. Sample Cells (Cuvettes):
   • Quartz/Fused silica (for UV), Glass (for Visible), Plastic (for routine work)
   • Standard path length = 1 cm
4. Detectors:
   • Phototube
   • Photomultiplier tube (most sensitive)
   • Photovoltaic cell
   • Silicon photodiode array
5. Recording System: Chart recorder or digital display
6. Power Supply: Converts AC to DC and stabilizes voltage

Types of Spectrophotometers:

• Single-beam: Measures sample absorbance directly.
• Double-beam: Splits beam into reference and sample for simultaneous measurement.

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Applications

• Spectrophotometric Titrations: Acid–base, redox, precipitation, complexometric titrations.
• Single-Component Analysis: Direct or indirect method.
• Multi-Component Analysis: Simultaneous estimation using mathematical treatment of spectra.

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Instrumental Method of Analysis UNIT 1

II. FLUORIMETRY

Theory and Principle

• Fluorescence: Emission of light when a molecule absorbs radiation and returns from excited singlet state to ground state.
• Fluorimetry: Measurement of fluorescence intensity using a filter fluorimeter or spectrofluorometer.

Electronic States:

• Singlet State: All electrons paired.
• Triplet State: Two electrons with parallel spins.
• Excited State: Electrons promoted from HOMO → LUMO.

Relaxation Processes:

• Fluorescence: S₁ → S₀ (singlet to singlet, light emitted)
• Phosphorescence: T₁ → S₀ (triplet to singlet, delayed emission)
• Internal Conversion: Non-radiative transition between singlet states
• Intersystem Crossing: S₁ → T₁ (spin flip)
• Collisional & External Conversion: Energy lost via molecular collisions or solvent interactions.

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Factors Affecting Fluorescence

• Concentration: Linear relation with intensity only if absorbance < 0.02
• Quantum Yield (Φ): Ratio of photons emitted/absorbed
• Intensity of Incident Light
• Oxygen: Quenches fluorescence
• pH: Affects ionization (e.g., aniline)
• Temperature & Viscosity: High temperature increases collisional quenching
• Photodecomposition: Decreases fluorescence
• Quenching: Reduction in fluorescence by other substances
  • Types: Self-quenching, Collisional, Static, Inner filter effect

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Instrumentation

• Light Source: Mercury vapor lamp, Xenon arc lamp (intense), Tungsten lamp
• Filters/Monochromators: For excitation and emission wavelength selection
• Sample Cells: Quartz (cylindrical/rectangular)
• Detectors: Photomultiplier tube (PMT), Barrier-layer cell
• Types of Instruments: Single-beam filter fluorimeter, Double-beam fluorimeter, Spectrofluorometer

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Applications

• Quantitative Analysis: Trace detection up to nanogram level
• Determination of:
  • Inorganic substances
  • Thiamine HCl, Phenytoin
  • Indoles, Phenols, Phenothiazines
  • Proteins, Plant pigments, Steroids
  • Boron, Manganese, Aluminum in alloys
  • Cadmium (with 2-(2-hydroxyphenyl) benzoxazole)

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🇮🇳 Key Pharma / B.Pharm Related Stats in India

1. Industry Growth & Market Size

   • India’s pharmaceutical industry is projected to reach USD 120-130 billion by 2030, up from approx USD 55 billion in recent years.

   • CAGR (Compound Annual Growth Rate) of the sector is estimated to be around 8-10% for FY25-FY30.

   • In 2024, the pharma market in India was valued at about USD 66.66 billion and is expected to reach nearly USD 88.86 billion by 2030.o

   • Domestic growth (internal demand) plus exports are both major contributors to this expansion.

2. Exports & Global Share

   • India’s pharma exports grew by ~9% in 2024.

   • India supplies around 20% of global demand for generic medicines.

   • By 2030, India’s share in global pharma market is expected to rise to approximately 5%.

3. Starting Salary for B.Pharm Graduates

   • Fresh B.Pharm graduates in India can expect ₹2.5 to ₹4.5 lakhs per annum as starting salary (≈₹20,000-₹38,000 per month depending on city, role, company).

   • Factors that make a difference: location (metros higher), company (MNC vs local), job role (quality control, medical rep, clinical research etc.)

4. Therapy & Market Trends

   • Anti-infectives continue to hold a significant share in the Indian pharma market; Oncology is expected to grow at a faster rate through 2030.

   • The formulation segment (tablets & capsules) dominates, but injectables and specialty drugs are projected to grow rapidly.