B Pharmacy Sem 7: Instrumental Methods of Analysis

Subject 1. Instrumental Methods of Analysis

1. UV-Visible Spectroscopy
2. Infrared Spectroscopy
3. Flame Photometry & Atomic Absorption Spectroscopy
4. Fluorimetry & Nepheloturbidometry
5. High-Performance Liquid Chromatography (HPLC)
6. Gas Chromatography (GC)
7. Electrophoresis & Radiochemical Methods
8. Introduction to Mass Spectrometry

Unit 1 : UV–Visible Spectroscopy

1.1 UV–Visible Spectroscopy

Measures absorption of UV/visible light by chromophores.

• Principle: Electronic transitions (π→π*, n→π*) obeys Beer–Lambert Law

• Key Components: Deuterium/Tungsten lamp; monochromator; quartz cuvette; photodiode/PMT detector

• Applications: Drug assay, dissolution testing, impurity screening

• Strengths: High sensitivity (10⁻⁶ M), rapid, non‑destructive

• Limitations: Spectral overlap; requires transparent solvents

1.2 Infrared (IR) Spectroscopy

Detects molecular vibrations to identify functional groups.

• Principle: Molecule absorbs IR → vibrational excitation (stretching/bending)

• Key Components: IR source (Globar); interferometer or dispersive monochromator; sample compartment (neat, KBr disc); DTGS/TGS detector

• Applications: Structural elucidation, purity check, polymorph identification

• Strengths: Specific fingerprint region (600–1,500 cm⁻¹), minimal prep

• Limitations: Less quantitative; overlapping bands in mixtures

1.3 Flame Photometry & Atomic Absorption Spectroscopy (AAS)

Quantifies metal ions via light emission or absorption.

1.3.1 Flame Photometry

• Principle: Excited atoms in flame emit light at characteristic λ

• Applications: Na⁺, K⁺ determination in formulations

• Strengths: Simple, low cost

• Limitations: Limited to easily excited metals

1.3.2 Atomic Absorption Spectroscopy

• Principle: Ground‑state atoms absorb light from element‑specific lamp

• Applications: Trace metal analysis (Pb, Cd, Fe)

• Strengths: High specificity, detection down to ppb

• Limitations: Requires hollow‑cathode lamp for each element

1.4 Fluorimetry & Nepheloturbidometry

Exploits light emission or scattering for trace analysis.

• Fluorimetry

  • Principle: Molecule absorbs UV → emits lower‑energy photon

  • Use: Trace impurities, drug–protein binding studies

• Nepheloturbidometry

  • Principle: Suspended particles scatter incident light

  • Use: Particle size monitoring, protein aggregation assays

Strengths: Extremely sensitive (fluorescence), real‑time monitoring
Limitations: Fluorimeters need high‑purity solvents; scatter methods affected by particle shape

1.5 High‑Performance Liquid Chromatography (HPLC)

Separates components in a liquid mobile phase under high pressure.

• Principle: Differential partitioning between stationary (column) and mobile phase

• Key Components: Pump; injector; column (C18, etc.); detector (UV, PDA); data system

• Applications: Assay of multi‑component formulations, stability studies

• Strengths: High resolution, versatile detectors

• Limitations: Expensive columns; requires degassed solvents

1.6 Gas Chromatography (GC)

Analyzes volatile compounds by vapor‑phase separation.

• Principle: Partitioning between inert gas mobile phase and coated capillary column

• Key Components: Carrier gas supply; injector; capillary column; flame ionization (FID) or mass detector

• Applications: Residual solvents, volatile impurities

• Strengths: Excellent for volatile analytes, fast runs

• Limitations: Not suitable for non‑volatiles; requires derivatization

1.7 Electrophoresis & Radiochemical Methods

1.7.1 Electrophoresis

• Principle: Charged analytes migrate in electric field through gel

• Use: Protein isoform separation, peptide mapping

1.7.2 Radiochemical Methods

• Principle: Radioisotope‑labeled compounds detected by scintillation or GM counters

• Use: Tracer studies, pharmacokinetics

Strengths: High sensitivity; structural/kinetic insights
Limitations: Safety/regulatory concerns with radioisotopes; gel methods are low‑throughput

1.8 Introduction to Mass Spectrometry (MS)

Couples with GC/LC to provide molecular weight and structural information.

• Principle: Ionization (EI, ESI) → mass‑to‑charge separation → detector

• Applications: Drug identification, impurity profiling, metabolite detection

• Strengths: Ultra‑high sensitivity, precise mass data

• Limitations: Complex spectra; expensive instrumentation

Key Exam Tips

• UV–Vis vs. IR: UV–Vis for concentration; IR for functional groups.

• HPLC vs. GC: HPLC for non‑volatiles; GC for volatiles (with or without derivatization).

• Photometry vs. AAS: AAS more selective and sensitive than flame photometry.

• MS Coupling: Always note the ionization method—EI for GC, ESI for LC.

• Pre‑IND/Pre‑NDA meetings: Align with regulator.

• Rolling submissions: Submit sections as completed.

• Inspection & queries: Address regulator questions promptly.

2.5 Process Validation & Equipment Qualification

Ensures processes and equipment consistently produce quality products.

2.5.1 Equipment Qualification (IQ/OQ/PQ)

1. Installation Qualification (IQ): Verify correct installation.

2. Operational Qualification (OQ): Confirm performance across ranges.

3. Performance Qualification (PQ): Demonstrate consistent output under real‑world conditions.

2.5.2 Process Validation Lifecycle

• Stage 1 – Process Design: Define CQAs/CPPs (critical process parameters).

• Stage 2 – Process Performance Qualification (PPQ): Run consecutive batches.

• Stage 3 – Continued Process Verification: Ongoing monitoring (trend analysis, control charts).

2.5.3 Pitfalls & Tips

• Insufficient data points → weak statistical power.

• Ignoring scale effects → invalid conclusions.

• Tip: Use risk assessment (FMEA) to prioritize validation efforts.

2.6 Quality by Design (QbD) Principles

Proactive approach to build quality into products.

2.6.1 Core Elements

1. Quality Target Product Profile (QTPP): Desired performance criteria.

2. Critical Quality Attributes (CQAs): Material properties impacting QTPP.

3. Critical Process Parameters (CPPs): Controls affecting CQAs.

4. Design Space: Multi‑dimensional region of acceptable operation.

2.6.2 Tools & Techniques

• Risk Assessment (e.g., Ishikawa, FMEA)

• Design of Experiments (DoE) for factor screening/optimization

• Control Strategy: Real‑time monitoring, PAT (Process Analytical Technology)

2.6.3 Benefits

• Robust processes with built‑in flexibility.

• Regulatory flexibility: Post‑approval changes within design space may not require new filings.

2.7 Pharmaceutical Packaging Technology

Protects product integrity, ensures patient safety.

2.7.1 Packaging Functions

• Barrier protection: Moisture, oxygen, light.

• Containment & identification: Child‑resistant closures, labeling.

• Convenience: Unit‑dose vs. bulk.

2.7.2 Materials & Systems

1. Primary packaging: Glass bottles, HDPE containers, blister packs.

2. Secondary packaging: Cartons, inserts with instructions.

3. Tertiary packaging: Shipping cartons, palletization.

2.7.3 Regulatory & Testing

• Stability studies: Packaging–product interaction (ICH Q1A).

• Closure integrity: Leak tests, microbial ingress.

• Child‑resistant/elderly‑friendly assessments.

Key Exam Tips

• Scale‑Up: Focus on similarity principles (geometric, kinetic, thermal).

• Tech Transfer vs. Validation: Transfer = knowledge hand‑off; Validation = proving consistency.

• QbD: Remember QTPP→CQA→CPP→Design Space flow.

• Packaging: Know primary vs. secondary vs. tertiary functions and tests.

Unit 3: Pharmacy Practice

This unit focuses on the roles and responsibilities of pharmacists in various practice settings—community, hospital, and clinical—emphasizing management, patient care, information services, and monitoring to ensure safe, effective medication use.

3.1 Community Pharmacy Organization & Management

Defines structure and daily operations of retail pharmacies.

• Purpose: Provide OTC and prescription medications, counsel patients, ensure regulatory compliance.

• Key Components:

  • Premises layout: Dispensing area, counseling space, OTC display

  • Staff roles: Pharmacist-in-charge, dispensers, assistants

  • Legal requirements: Licensing, record‑keeping (Controlled Substances Act)

• Workflow: Prescription receipt → verification → dispensing → patient counseling → documentation

• Common Pitfalls: Incomplete prescriptions, labeling errors, inadequate patient privacy

3.2 Hospital Pharmacy Organization & Drug Distribution

Covers formulary management, centralized vs. decentralized services, and sterile compounding.

• Purpose: Supply medications safely to inpatients/outpatients; support clinical teams.

• Key Components:

  • Pharmacy layout: Inpatient ward service, satellite dispensing units

  • Drug distribution systems: Floor stock, unit dose, IV admixture services

  • Formulary committee: Reviews and approves medications

• Workflow: Medication order → pharmacist review → preparation (unit dose/IV) → nursing delivery → documentation

• Common Pitfalls: Look‑alike/sound‑alike drugs, interruptions during order entry, miscommunication with nursing

3.3 Drug Store Management & Inventory Control

Ensures uninterrupted supply and cost‑effective stocking.

• Purpose: Optimize inventory levels, minimize wastage, control costs.

• Key Components:

  • ABC analysis: Classify items by consumption value

  • VED analysis: Categorize by criticality (Vital, Essential, Desirable)

  • Reorder policies: Economic order quantity (EOQ), safety stock

• Workflow: Demand forecasting → order placement → receiving/inspection → stock rotation (FEFO) → records update

• Common Pitfalls: Stock‑outs, expired inventory, overstocking high‑cost items

3.4 Drug Information Services & Sources

Provides evidence‑based information to healthcare professionals and patients.

• Purpose: Answer queries on drug therapy, interactions, dosing, adverse effects.

• Key Sources:

  • Primary: Original research articles

  • Secondary: Indexing services (PubMed, Embase)

  • Tertiary: Textbooks, compendia (Martindale, AHFS DI)

• Workflow: Receive query → literature search → evaluate evidence → provide written/oral response → document query

• Common Pitfalls: Using outdated references, misinterpretation of study data

3.5 Patient Medication History Interview & Counseling

Gathers accurate medication histories and provides tailored counseling.

• Purpose: Improve adherence, identify drug‑related problems, enhance therapeutic outcomes.

• Key Steps:

  1. Introduction & consent

  2. Gather history: Prescription, OTC, herbal, allergy, adherence

  3. Assess understanding: Indications, dosing, administration

  4. Counsel: Purpose, schedule, side effects, storage

  5. Follow‑up plan

• Common Pitfalls: Leading questions, assumptions about literacy, lack of cultural sensitivity

3.6 Health Screening Services

Offers preventive care through basic clinical tests and referrals.

• Purpose: Early detection of risk factors (e.g., hypertension, diabetes).

• Common Screenings: Blood pressure, blood glucose, lipid profile, BMI measurement

• Workflow: Patient interview → test performance → result interpretation → referral or counseling

• Common Pitfalls: Inaccurate technique (improper cuff size, calibration), inadequate follow‑up

3.7 Clinical Pharmacy & Therapeutic Drug Monitoring (TDM) Basics

Integrates pharmacists into clinical teams and optimizes drug therapy through TDM.

• Purpose: Ensure drug levels remain within therapeutic window to maximize efficacy and minimize toxicity.

• Key Components:

  • Pharmacokinetic sampling: Peak/trough timing

  • Assay methods: HPLC, immunoassay

  • Dose adjustment: Based on clearance, volume of distribution, patient factors

• Workflow: Order TDM → collect sample at correct time → lab analysis → interpret results → recommend dosing changes

• Common Pitfalls: Incorrect sampling time, failure to consider drug interactions or organ dysfunction

Key Exam Tips

• Community vs. Hospital: Community focuses on retail operations and patient counseling; hospital adds formulary and sterile services.

• Inventory Control: Remember ABC (value) and VED (criticality) analyses for prioritization.

• TDM: Always note whether sample is peak or trough and adjust dose accordingly.

• Counseling: Use “3C’s”—Check understanding, Communicate clearly, Confirm follow‑up.

Unit 4: Novel Drug Delivery Systems

This unit explores advanced carriers and strategies designed to optimize drug release, target specific sites, and improve therapeutic outcomes. Each section highlights core concepts, representative systems, mode of action, advantages, and evaluation methods.

4.1 Sustained & Controlled Release Dosage Forms

Formulations engineered to release drug at a predetermined rate over an extended period.

4.1.1 Reservoir Systems

• Design: Drug core surrounded by rate‑controlling membrane

• Example: Theophylline osmotic tablets

• Release Mechanism: Diffusion through membrane pores or osmotic pumping

• Advantage: Zero‑order kinetics; constant plasma concentration

4.1.2 Matrix Systems

• Design: Drug uniformly dispersed in polymer matrix (hydrophilic or hydrophobic)

• Example: Ibuprofen in HPMC matrix

• Release Mechanism: Polymer swelling/erosion controls drug diffusion

• Advantage: Simple manufacturing; flexibility in release profile

4.1.3 Monolithic vs. Multiparticulate

• Monolithic: Single unit—risk of “dose dumping” if ruptured

• Multiparticulate (pellets, micropellets): Multiple small units—more uniform GI transit, lower variability

4.2 Targeted Drug Delivery Systems

Strategies to deliver therapeutics to specific tissues or cell types, thereby enhancing efficacy and reducing off‑target effects.

4.2.1 Passive Targeting

• Mechanism: Exploits enhanced permeability and retention (EPR) effect in tumors

• Systems: Long‑circulating liposomes (PEGylated doxorubicin)

• Benefit: Higher tumor uptake; reduced cardiotoxicity

4.2.2 Active Targeting

• Mechanism: Surface ligands (antibodies, peptides) bind to receptors on target cells

• Example: Folate‑conjugated nanoparticles for cancer cells overexpressing folate receptors

• Advantage: Selective cell uptake; minimal healthy tissue exposure

4.3 Vesicular Systems: Liposomes & Niosomes

Closed bilayer or multilayer vesicles capable of encapsulating hydrophilic and/or lipophilic drugs.

4.3.1 Liposomes

• Composition: Phospholipid bilayer enclosing aqueous core

• Example: Amphotericin B liposomal formulation

• Advantages: Biocompatible, can encapsulate both polar and nonpolar drugs, modifiable surface (PEGylation)

4.3.2 Niosomes

• Composition: Non‑ionic surfactant vesicles

• Example: Diclofenac niosomal gel for topical delivery

• Advantages: More stable, lower cost than liposomes, easy scale‑up

4.4 Micro‑ & Nanoparticulate Systems

Solid particles in the micron to nanometer range for controlled and targeted delivery.

4.4.1 Microspheres & Microcapsules

• Polymeric materials: PLA, PLGA

• Use: Depot injections (e.g., Lupron depot microspheres)

• Release: Degradation‑controlled or diffusion‑controlled

4.4.2 Nanoparticles

• Types: Polymeric nanoparticles, solid lipid nanoparticles, nanocrystals

• Applications: Brain delivery via BBB penetration, oral bioavailability enhancement

• Benefits: Improved solubility, surface modification for targeting, reduced clearance

4.5 Implants & Transdermal Drug Delivery

Implants and skin patches that bypass first‑pass metabolism and provide long‑term therapy.

4.5.1 Implantable Devices

• Types: Biodegradable (e.g., PLGA rods releasing leuprolide) and non‑biodegradable (e.g., silicone implants for contraception)

• Release Mechanism: Polymer erosion and diffusion

4.5.2 Transdermal Patches

• Design: Drug reservoir or matrix layer laminated to backing and adhesive

• Example: Nicotine, fentanyl patches

• Permeation Enhancers: Chemical (ethanol), physical (microneedles)

4.6 Evaluation of Novel Delivery Systems

Key tests to ensure safety, efficacy, and quality of NDDS.

Key Exam Tips

• Sustained vs. Controlled: Sustained = prolonged effect; Controlled = constant rate.

• Passive vs. Active Targeting: EPR effect vs. ligand‑receptor binding.

• Liposomes vs. Niosomes: Phospholipids vs. non‑ionic surfactants.

• Particle Size: Nano (<100 nm) for systemic delivery; micro (1–100 µm) for depot formulations.

• Evaluation Order: Start with in vitro release → stability → in vivo PK → biodistribution.

Unit 2: Infrared (IR) Spectroscopy

Infrared Spectroscopy exploits molecular vibrations to identify functional groups and assess compound purity by measuring absorption of IR radiation (400–4,000 cm⁻¹).

1. Principle

• Vibrational transitions

  • Molecules absorb IR photons matching vibrational mode energies (stretching, bending).

  • Each bond type has characteristic absorption frequencies.

• Beer–Lambert Law (in IR)

   

  $$A_{\tilde{\nu}} = \log_{10}\!\bigl(\tfrac{I_0}{I}\bigr)$$Aν~​=log10​(II0​​)

  where

  • $A_{\tilde{\nu}}$Aν~​ = absorbance at wavenumber

    $\tilde{\nu}$ν~ (cm⁻¹)

  • $I_0$I0​,

    $I$I = incident and transmitted intensities

2. Instrumentation

1. IR Source

   • Globar (silicon carbide rod) or Nernst glower (rare‐earth oxides): emits broad IR continuum.

2. Sample Compartment & Sampling Techniques

   • Neat (liquid films) on salt plates (NaCl, KBr).

   • KBr pellets: grind solid with KBr → transparent pellet.

   • ATR (Attenuated Total Reflectance): crystal (ZnSe, diamond) with drop of sample—simplest prep.

   • Mull: disperse solid in Nujol oil between salt plates.

3. Dispersive vs. FT‐IR

   • Dispersive: monochromator (prism/grating) scans wavenumbers sequentially.

   • FT‑IR: interferometer (Michelson) collects all wavenumbers simultaneously → faster scans, higher sensitivity, digital resolution.

4. Detector

   • DTGS (deuterated triglycine sulfate) or MCT (mercury cadmium telluride; cooled) converts IR to electrical signal.

5. Readout

   • Absorbance or %T vs. wavenumber plot (400–4,000 cm⁻¹), with a characteristic “fingerprint region” (600–1,500 cm⁻¹).

3. Sample Preparation Tips

• Dryness: KBr and salt plates must be moisture‑free—water absorbs strongly in IR.

• Thin films: Avoid overly thick samples—leads to total absorption (“flat” baseline).

• ATR alignment: Ensure good contact between sample and crystal; clean crystal between runs.

• Pellet clarity: Grind uniformly; apply even pressure to avoid scattering artifacts.

4. Spectral Interpretation

• Functional Group Region (1,600–4,000 cm⁻¹):

  • O–H stretch (broad, 3,200–3,600 cm⁻¹)

  • N–H stretch (sharper, 3,300–3,500 cm⁻¹)

  • C–H stretches (alkanes ~2,850–2,970 cm⁻¹; aromatics ~3,000–3,100 cm⁻¹)

  • C=O carbonyl (sharp, 1,680–1,750 cm⁻¹)

• Fingerprint Region (600–1,500 cm⁻¹):

  • Complex pattern unique to each molecule; compare with reference spectra for identification.

5. Applications

• Structural elucidation: Confirm presence/absence of functional groups.

• Polymorph detection: Different IR patterns for crystalline forms.

• Purity assessment: Identify organic contaminants by extra peaks.

• Reaction monitoring: Follow disappearance of reactant peaks or appearance of product peaks.

• Quantitative IR: Use ATR‑IR or transmission IR with calibration curves (limited linear range).

6. Advantages & Limitations

• Advantages

  • Rapid, non‑destructive analysis.

  • Minimal sample required (especially ATR).

  • Rich structural information in fingerprint region.

• Limitations

  • Overlapping bands in complex mixtures.

  • Water and CO₂ interference—need dry environment.

  • Less sensitive for trace-level quantitation (µg‑mg range).

7. Key Exam Tips

• Draw and label a FT‑IR spectrometer schematic (source, interferometer, detector).

• Memorize characteristic wavenumber ranges for major functional groups.

• Compare dispersive vs. FT‑IR: speed, sensitivity, resolution differences.

• Practice interpreting simple spectra—identify at least three functional groups and discuss purity.

Unit 3: Flame Photometry & Atomic Absorption Spectroscopy

This unit covers two complementary atomic spectroscopic techniques for quantifying metallic elements in pharmaceutical and biological samples. Both rely on atomization of analyte atoms and measurement of light—either emitted or absorbed—at element‑specific wavelengths.

3.1 Flame Photometry

3.1.1 Principle

• Emission process: Atoms of alkali and alkaline earth metals (e.g., Na⁺, K⁺, Ca²⁺) are excited in a flame; as they return to ground state they emit light at characteristic wavelengths.

• Intensity–Concentration relationship: Emitted light intensity ∝ concentration of the element in the sample (within linear range).

3.1.2 Instrumentation

1. Nebulizer & Burner Head

   • Sample solution aspirated into fine aerosol by nebulizer; directed into a fuel–oxidant flame (commonly air–acetylene or nitrous‑oxide–acetylene).

2. Monochromator

   • Prism or diffraction grating selects the specific emission line (e.g., 589 nm for Na, 766 nm for K).

3. Detector

   • Photomultiplier tube (PMT) measures emitted photon intensity.

4. Readout & Calibration

   • Digital display or recorder plots intensity vs. concentration using standard calibration curve.

3.1.3 Sample Preparation & Calibration

• Matrix matching: Prepare calibration standards in a matrix similar to the sample to minimize chemical interferences.

• Dilution: Ensure concentrations fall within the linear dynamic range (typically 0.1–10 ppm for Na/K).

• Blank measurement: Use solvent blank to zero the instrument before analysis.

3.1.4 Applications

• Electrolyte analysis: Na⁺ and K⁺ in injection fluids or biological fluids.

• Excipient testing: Quantification of metal‑based salts (e.g., CaCO₃) in formulations.

• Quality control: Rapid screening for metal‑ion contamination.

3.1.5 Strengths & Limitations

• Strengths:

  • Simple operation and low cost.

  • Fast analysis (seconds per sample).

• Limitations:

  • Limited to easily excited elements (primarily Group 1 and 2).

  • Susceptible to chemical interferences (ionization, refractory compounds).

  • Lower sensitivity compared to AAS (detection limits ~ppm).

3.2 Atomic Absorption Spectroscopy (AAS)

3.2.1 Principle

• Absorption process: Ground‑state atoms in an air–acetylene or nitrous‑oxide flame (or graphite furnace) absorb light at element‑specific resonance wavelengths generated by a hollow‑cathode lamp.

• Beer–Lambert Law: Absorbance of the atomized sample ∝ concentration of the element.

3.2.2 Instrumentation

1. Hollow‑Cathode Lamp

   • Contains cathode made of the element of interest; emits sharp resonance lines when current passes.

2. Atomizer

   • Flame: sample aspirated into flame; good for routine analyses.

   • Graphite Furnace (GFAAS): offers higher sensitivity (ppt–ppb level) by electrically heating a small sample pellet.

3. Monochromator

   • High-resolution grating isolates narrow absorption lines (0.1–0.2 nm bandwidth).

4. Detector

   • Photomultiplier tube measures decrease in light intensity transmitted through the atomized cloud.

5. Readout & Data System

   • Digital display of absorbance; software constructs calibration curves and computes concentrations.

3.2.3 Sample Preparation & Calibration

• Acid digestion: Convert samples (e.g., tablets, biological fluids) to clear solutions; remove organic matrix.

• Standard additions / matrix standards: Compensate for matrix effects.

• Dilution & blanking: Bring analyte levels within working range (often ppb to low ppm) and zero with reagent blank.

3.2.4 Applications

• Trace metal analysis: Pb, Cd, Fe, Cu in raw materials, formulations, and biological samples.

• Environmental testing: Detection of heavy‑metal contaminants in water or packaging materials.

• Pharmacokinetics: Monitoring metal‑based drugs (e.g., lithium).

3.2.5 Strengths & Limitations

• Strengths:

  • High specificity (element‑only absorption lines).

  • Excellent sensitivity: ppb–ppt levels (especially with graphite furnace).

• Limitations:

  • Requires a separate lamp for each element.

  • Graphite furnace mode has slower throughput and requires careful method development.

  • Potential spectral interferences from overlapping lines—necessitates background correction.

3.3 Comparison & Practical Tips

• Tip 1: Always match the sample matrix to standards to reduce chemical interferences.

• Tip 2: Use background correction (deuterium lamp or Zeeman) in AAS to account for non‑specific absorption or scattering.

• Tip 3: For ultra-trace work, prefer graphite furnace AAS but validate furnace temperature program carefully to avoid analyte loss.

Key Exam Tips

• Mechanisms: Distinguish emission (flame photometry) vs. absorption (AAS).

• Instrumentation: Know the role of the hollow‑cathode lamp and why you need element‑specific lamps in AAS.

• Sensitivity range: Be able to rank methods by detection limit (Flame FP < Flame AAS < GFAAS).

• Interferences: Understand ionization in flame photometry and spectral/background in AAS, and list correction strategies.

Unit 4: Fluorimetry & Nepheloturbidometry

This unit presents two light‑based techniques—Fluorimetry for measuring emitted fluorescence and Nepheloturbidometry for quantifying light scattering—to detect and quantify trace analytes with high sensitivity.

4.1 Fluorimetry

4.1.1 Definition & Principle

Fluorimetry (or fluorescence spectroscopy) measures the emission of light by a substance that has absorbed photons. After excitation at a specific wavelength, fluorophores relax to ground state by emitting lower‑energy photons—the intensity of this emission is proportional to analyte concentration.

4.1.2 Key Terms

• Excitation wavelength (λₑₓ): Wavelength used to excite electrons.

• Emission wavelength (λₑₘ): Wavelength at which emitted light is measured.

• Stokes shift: Difference between λₑₓ and λₑₘ; indicates energy loss.

• Quantum yield (Φ): Ratio of emitted photons to absorbed photons; a measure of fluorescence efficiency.

4.1.3 Instrumentation

1. Light Source: Xenon arc lamp (broad UV–Vis emission) or LED.

2. Excitation Monochromator: Selects λₑₓ.

3. Sample Holder: Fluorescence cuvette (quartz, low fluorescence background).

4. Emission Monochromator: Isolates λₑₘ.

5. Detector: Photomultiplier tube (PMT) with high gain for low‑level light detection.

6. Readout: Fluorescence intensity vs. λₑₘ spectrum; can also monitor intensity at fixed λₑₘ for kinetics.

4.1.4 Sample Preparation

• Avoid quenching agents (oxygen, heavy metals, high ionic strength) that reduce fluorescence.

• Use dilute solutions (typically 10⁻⁹–10⁻⁶ M) to prevent inner‑filter effects.

• Maintain consistent temperature (fluorescence is temperature‑sensitive).

4.1.5 Applications

• Trace impurity detection: Down to parts‑per‑billion (ppb).

• Protein–ligand binding studies: Intrinsic fluorescence of tryptophan residues.

• Enzyme assays: Coupled reactions yielding fluorescent products (e.g., AMC).

• Clinical diagnostics: Fluorescent immunoassays (ELISA formats).

4.1.6 Advantages & Limitations

• Advantages:

  • Exceptional sensitivity (10⁻¹²–10⁻⁹ M).

  • High selectivity via choice of λₑₓ/λₑₘ.

  • Capable of real‑time kinetic measurements.

• Limitations:

  • Many compounds are non‑fluorescent—require derivatization.

  • Susceptible to photobleaching (loss of signal over time).

  • Quenching effects from sample matrix can complicate quantitation.

4.2 Nepheloturbidometry

4.2.1 Definition & Principle

Nepheloturbidometry measures light scattering by suspended particles in a sample. As particles scatter incident light, detectors placed at an angle (commonly 90°) quantify scattered intensity, which correlates with particle concentration or turbidity.

4.2.2 Key Terms

• Nephelometry: Measures scattered light at an angle (sensitive to small particles).

• Turbidimetry: Measures decrease in transmitted light (sensitive to larger particles).

• Scattering intensity (Iₛ): Proportional to particle size and concentration (Rayleigh scattering for very small particles, Mie scattering for larger).

4.2.3 Instrumentation

1. Light Source: Tungsten lamp or laser diode (visible region).

2. Sample Cell: Standard cuvette or disposable vial.

3. Detector Arrangement:

   • Nephelometer: Detector at 90° to incident beam.

   • Turbidimeter: Detector inline at 0° for transmitted light decrease.

4. Readout: Scattered light intensity vs. time or converted to turbidity units (NTU).

4.2.4 Sample Preparation

• Uniform dispersion: Vortex or sonication to prevent aggregation.

• Appropriate dilutions: Ensure scattering intensity falls within linear range.

• Avoid air bubbles: They cause spurious scattering signals.

4.2.5 Applications

• Protein aggregation assays: Monitor formation of colloidal aggregates.

• Suspension stability testing: Evaluate shelf‑life of suspensions/emulsions.

• Immunonephelometry: Quantify antigen–antibody complexes in serology.

• Bioprocess monitoring: Measure cell culture density via optical density.

4.2.6 Advantages & Limitations

• Advantages:

  • Simple, rapid measurements.

  • Non‑destructive; minimal sample preparation.

  • Suitable for turbid or opaque samples.

• Limitations:

  • Non‑specific: all particles scatter light, so requires selective precipitation or labeling for specificity.

  • Sensitive to particle size distribution; calibration must match sample characteristics.

  • Air bubbles and dust can introduce large errors.

4.3 Key Exam Tips

• Fluorimetry vs. UV–Vis: Fluorimetry measures emitted light; UV–Vis measures absorbed light.

• Stokes shift: Always explain its significance in avoiding stray excitation light in emission measurement.

• Quenching mechanisms: Be able to list dynamic vs. static quenching and their effects on fluorescence intensity.

• Nephelometry vs. Turbidimetry: Know detector placement (90° vs. 0°) and typical applications for each.

Unit 5: High‑Performance Liquid Chromatography (HPLC)

High‑Performance Liquid Chromatography (HPLC) is a versatile separation technique used to identify, quantify, and purify components in complex mixtures. By pumping a liquid mobile phase through a tightly packed column under high pressure, HPLC achieves excellent resolution and reproducibility.

5.1 Principle

• Partitioning Mechanism: Components distribute between a stationary phase (solid packing) and a mobile phase (liquid solvent).

• Retention Factor (k’):

   

  $$k’ = \frac{t_R – t_0}{t_0}$$k′=t0​tR​−t0​​

  where

  • $t_R$tR​ = retention time of analyte

  • $t_0$t0​ = column dead time (unretained solvent front)

• Selectivity (α): Ratio of retention factors of two analytes; determines separation quality.

• Resolution (Rₛ):

   

  $$R_s = \frac{2(t_{R2} – t_{R1})}{W_1 + W_2}$$Rs​=W1​+W2​2(tR2​−tR1​)​

  where

  $W_1, W_2$W1​,W2​ are peak widths at baseline.

5.2 Instrumentation

1. Solvent Reservoirs:

   • Mobile phase bottles (aqueous and organic).

   • Degassing (ultrasonic or in‑line degasser) to remove dissolved gases.

2. Pump System:

   • Delivers constant flow (isocratic) or programmed gradient of solvents under high pressure (up to 400 bar).

   • Types: Piston pumps (most common).

3. Injector:

   • Manual loop injector or autosampler for reproducible, small-volume (1–100 µL) injections.

4. Column:

   • Stationary phases:

     • Reversed‑phase: C18, C8 (nonpolar) – most widely used.

     • Normal‑phase: Silica (polar) – for lipophilic analytes.

     • Ion‑exchange: Charged resins – for ionic species.

     • Size‑exclusion: Porous beads – for biomolecules.

   • Dimensions: 4.6 mm i.d. × 150 mm × 3–5 µm particle size is common.

5. Detector:

   • UV/Vis absorbance (wavelength-specific) – universal for chromophores.

   • Photodiode array (PDA) – full-spectrum acquisition.

   • Refractive index (RI) – for compounds lacking UV absorbance.

   • Fluorescence – high sensitivity for fluorescent analytes.

   • Mass spectrometry (LC‑MS) – structural identification and trace analysis.

6. Data System:

   • Software for chromatogram visualization, peak integration, and quantitation via calibration curves.

5.3 Modes of Operation

• Isocratic Elution: Constant mobile phase composition—simple, reproducible, but limited separation power for complex mixtures.

• Gradient Elution: Gradual change in solvent strength (e.g., increasing organic percentage)—improves resolution and reduces run time for mixtures with wide polarity range.

5.4 Sample Preparation

• Filtration: Remove particulates (>0.45 µm filters) to protect column integrity.

• Dilution & pH Adjustment: Match sample matrix to mobile phase to prevent peak distortion.

• Solid‑Phase Extraction (SPE): Concentrate and clean up samples from complex matrices (plasma, environmental samples).

• Derivatization: Enhance detectability or retention of non‑UV chromophores (e.g., pre‑column o‑phthalaldehyde for amines).

5.5 Applications

• Pharmaceutical Assay: Quantitate active ingredients and impurities in formulations.

• Stability Studies: Monitor degradation products over time under stress conditions.

• Bioanalysis: Measure drug and metabolite levels in biological fluids (plasma, urine) using LC‑MS.

• Purification: Collect fractions of purified compounds for further use.

• Method Validation: Demonstrate linearity, precision, accuracy, specificity, LOD/LOQ per ICH guidelines.

5.6 Advantages & Limitations

• Advantages:

  • High resolution and reproducibility.

  • Broad applicability to polar and nonpolar compounds.

  • Capability for quantitative and preparative separations.

• Limitations:

  • Relatively high cost (columns, pumps, detectors).

  • Requires careful solvent selection and degassing.

  • Column lifetime limited by sample cleanliness and operating conditions.

5.7 Key Exam Tips

• Column Selection: Know when to choose reversed‑phase vs. normal‑phase vs. ion‑exchange.

• Calculate k’ and Rₛ: Practice using retention times and peak widths.

• Gradient vs. Isocratic: Understand pros/cons for complex vs. simple mixtures.

• Validation Parameters: Be ready to list and define accuracy, precision, linearity, LOD, LOQ.

• Troubleshooting: Common issues—ghost peaks (carryover), peak tailing (column overload or silanol interaction), baseline drift (mobile phase or detector problems).

Unit 6: Gas Chromatography (GC)

Gas Chromatography (GC) separates and analyzes volatile and semi‑volatile compounds by partitioning them between a gas mobile phase and a liquid or solid stationary phase coated inside a capillary column. It’s prized for its high resolution, speed, and sensitivity in analyzing small organic molecules.

6.1 Definition & Principle

• Definition:
  GC is a chromatographic technique in which a sample is vaporized and carried by an inert gas through a column; components separate based on their volatility and affinity for the stationary phase.

• Partitioning Mechanism:
  Each analyte continually equilibrates between the mobile gas phase and the stationary liquid (or solid) phase lining the column walls.

   

  $$K = \frac{C_{\text{stationary}}}{C_{\text{mobile}}}$$K=Cmobile​Cstationary​​

  where

  $K$K is the distribution constant. Compounds with lower volatility or higher affinity for the stationary phase elute later.

6.2 Instrumentation

1. Carrier Gas Supply

   • Typically helium, nitrogen, or hydrogen—chosen for inertness and suitable flow characteristics.

2. Injector

   • Split: Diverts a portion of the sample to waste to prevent column overload.

   • Splitless: Entire sample directed onto column for trace-level analysis.

   • On‑column: Direct deposition of liquid sample onto column (for thermally sensitive analytes).

3. Column

   • Capillary (open tubular): Fused-silica with internal coating (0.1–0.25 µm film thickness), 0.1–0.32 mm i.d., 10–60 m length.

   • Packed: Less common; larger diameter, packed with solid support coated with liquid stationary phase.

4. Oven & Temperature Control

   • Isothermal: Maintain constant temperature.

   • Temperature programming: Ramp temperature during run to separate compounds of varying boiling points efficiently.

5. Detector

   • Flame Ionization Detector (FID): Universal for organic compounds; excellent linear range.

   • Thermal Conductivity Detector (TCD): Universal but less sensitive; detects changes in thermal conductivity.

   • Electron Capture Detector (ECD): Highly sensitive for electronegative compounds (e.g., halogenated pesticides).

   • Mass Spectrometer (GC–MS): Provides structural identification via mass spectra.

6. Data System

   • Software integrates peaks, calculates retention times, and quantifies concentrations against calibration standards.

6.3 Sample Preparation

• Volatilization: Sample must be in a volatile form—dissolve solids/liquids in a volatile solvent (e.g., hexane, dichloromethane).

• Derivatization: Convert non‑volatile or polar analytes into volatile derivatives (e.g., silylation of alcohols, acylation of amines).

• Filtration: Remove particulates that could block injector or column.

• Concentration: Evaporate solvent if analyte concentration is low; re‑dissolve in smaller volume.

• Matrix Cleanup: Solid‑phase extraction (SPE) or liquid–liquid extraction to remove interfering substances.

6.4 Applications

• Residual Solvent Analysis: Quantify trace organic solvents in drug substances and excipients.

• Volatile Impurity Profiling: Detect and measure volatile degradation products.

• Environmental Testing: Analyze pollutants (pesticides, hydrocarbons) in water or air samples.

• Flavor and Fragrance Characterization: Profile essential oils, aroma compounds.

• GC–MS in Drug Metabolism: Identify and quantify drug metabolites in biological matrices.

6.5 Advantages & Limitations

• Advantages:

  • High separation efficiency and resolution.

  • Fast analysis times with temperature programming.

  • Sensitive detectors down to pg levels (with ECD or MS).

  • Excellent reproducibility of retention times.

• Limitations:

  • Restricted to volatile or derivatizable compounds.

  • Thermal degradation risk for heat‑labile analytes.

  • Requires high‑purity carrier gas and rigorous leak‑free plumbing.

  • Derivatization adds extra sample‑prep steps and potential artifacts.

6.6 Key Exam Tips

• Split vs. Splitless: Know when to use splitless injection (trace analysis) versus split (routine, higher concentration).

• Temperature Programming: Explain how a ramp rate affects peak resolution and run time.

• Detector Selection:

  • FID for hydrocarbons and general organics.

  • ECD for halogenated compounds.

  • GC–MS for unequivocal identification.

• Derivatization Reactions: Be ready to outline a common silylation or acylation procedure for polar analytes.

• Retention Mechanisms: Relate boiling point and polarity to retention order under isothermal conditions.

Unit 7: Electrophoresis & Radiochemical Methods

This unit introduces two distinct analytical approaches—Electrophoresis for separation of charged biomolecules and Radiochemical Methods for tracing and quantifying radiolabeled compounds. You’ll learn each technique’s definition, underlying principle, instrumentation, sample considerations, applications, and pros/cons.

7.1 Electrophoresis

7.1.1 Definition & Principle

Electrophoresis is a separation technique in which charged particles migrate through a support medium under an electric field. Molecules separate based on their charge-to-mass ratio and the characteristics of the medium.

7.1.2 Key Terms

• Mobility (μ): Velocity per unit field strength; depends on charge (q) and frictional drag (f).

   

  $$\mu = \frac{q}{f}$$μ=fq​

• Support Media:

  • Agarose gel: Large pore size, ideal for DNA/RNA.

  • Polyacrylamide gel (PAGE): Small, tunable pores for proteins/peptides.

• Buffer System: Maintains pH and ionic strength to control molecule charge.

7.1.3 Instrumentation & Setup

1. Power Supply: Provides constant voltage or current (typically 50–300 V).

2. Gel Casting System: Plates or trays with spacers to form uniform gel.

3. Sample Wells: Created by combs; hold aliquots of sample mixed with loading dye.

4. Running Buffer: Covers gel; conducts current and maintains pH.

5. Detection:

   • Staining: Ethidium bromide or SYBR Green for DNA; Coomassie Blue or silver stain for proteins.

   • Imaging: UV transilluminator or gel documentation system.

7.1.4 Types of Electrophoresis

• Native PAGE: Proteins retain native conformation; separation by charge and size.

• SDS–PAGE: Sodium dodecyl sulfate denatures proteins and imparts uniform negative charge; separation primarily by molecular weight.

• Agarose Gel Electrophoresis: DNA/RNA sizing; compact gel matrix.

• Capillary Electrophoresis (CE): High-voltage separation in narrow capillaries; fast, high-resolution.

7.1.5 Sample Preparation

• Buffer exchange/dialysis: Remove interfering salts or impurities.

• Denaturation (for SDS–PAGE): Heat sample with SDS and reducing agent (β‑mercaptoethanol or DTT).

• Loading Dye: Contains glycerol (to sink into well) and tracking dye (bromophenol blue).

7.1.6 Applications

• Protein analysis: Molecular weight determination, purity checks, isoform separation.

• Nucleic acid analysis: DNA fingerprinting, restriction fragment sizing, RT‑PCR product verification.

• Capillary Electrophoresis: Chiral separations, pharmaceutical impurity profiling, biopharmaceutical quality control.

7.1.7 Advantages & Limitations

• Advantages:

  • High resolution for biomolecules.

  • Versatile: multiple formats (agarose, PAGE, capillary).

  • Quantitative when combined with densitometry or fluorescent detection.

• Limitations:

  • Time‑consuming gel preparation and staining.

  • Limited throughput for slab gels.

  • Artifacts like smiling bands or diffusion.

7.2 Radiochemical Methods

7.2.1 Definition & Principle

Radiochemical methods use radioisotope–labeled compounds as tracers. Detection relies on measuring emitted radiation (β, γ) to track the distribution, metabolism, or binding of labeled molecules.

7.2.2 Key Terms

• Tracer Principle: Introduce a small amount of radioisotope-labeled analog to follow its fate without altering system behavior.

• Isotopes Commonly Used:

  • ³H (Tritium): β emitter, low energy, used in ligand binding and metabolic studies.

  • ¹⁴C: β emitter, moderate half‑life, ideal for metabolic pathway tracing.

  • ³²P: β emitter, high energy, used in nucleic acid labeling.

  • ⁹⁹ᵐTc: γ emitter, used in imaging and diagnostic scans.

7.2.3 Instrumentation & Detection

1. Scintillation Counting (β emitters):

   • Sample mixed with scintillation cocktail; β particles excite scintillant → flash of light → photomultiplier tube detection.

2. Gamma Counters (γ emitters):

   • Shielded scintillation or solid‑state detectors record γ photons without the need for cocktail.

3. Autoradiography:

   • Radiolabeled sample on a gel or TLC plate is placed against X‑ray film; radiation darkens film to localize bands.

7.2.4 Sample Preparation

• Radiolabeling: Incorporate radioisotope into molecule via enzymatic or chemical synthesis.

• Purification: Remove unincorporated label by chromatography or dialysis.

• Safety Measures: Use shielding (lead bricks), monitor contamination, follow waste disposal regulations.

7.2.5 Applications

• Pharmacokinetics: Measure absorption, distribution, metabolism, and excretion (ADME) of drugs.

• Receptor Binding Assays: Determine affinity (Kₙ) and number of binding sites (Bₘₐₓ).

• Metabolic Pathway Elucidation: Trace metabolic intermediates in vitro and in vivo.

• Diagnostic Imaging: γ‑emitters in radiopharmaceuticals (e.g., ⁹⁹ᵐTc‑labelled compounds).

7.2.6 Advantages & Limitations

• Advantages:

  • Unmatched sensitivity (picomolar to femtomolar).

  • Direct quantitation without optical interferences.

  • Enables in vivo tracing and imaging.

• Limitations:

  • Regulatory and safety burdens handling radioactivity.

  • Short half‑lives require timely experiments.

  • Disposal of radioactive waste is costly and strictly controlled.

7.3 Key Exam Tips

• Electrophoresis: Be prepared to compare native PAGE vs. SDS–PAGE and outline the role of SDS and reducing agents.

• Capillary vs. Slab Gels: Highlight faster runs and higher resolution of CE.

• Radioisotope Selection: Match isotope properties (half‑life, emission type, energy) to application (e.g., ³H for binding assays, ⁹⁹ᵐTc for imaging).

• Safety Protocols: Always mention shielding, contamination monitoring, and waste disposal in radiochemical methods.

Unit 8: Introduction to Mass Spectrometry (MS)

Mass spectrometry is a powerful analytic technique that provides precise molecular-weight and structural information by ionizing chemical species and measuring their mass‑to‑charge ratios (m/z).

8.1 Definition & Principle

• Definition:
  MS is the measurement of the masses of atoms or molecules in a sample by converting them to ions and detecting those ions according to their m/z values.

• Basic Steps:

  1. Ionization: Converts neutral analyte molecules into gas‑phase ions.

  2. Mass Analysis: Separates ions by their m/z under electric or magnetic fields.

  3. Detection: Records the abundance of each ion, producing a mass spectrum (intensity vs. m/z).

8.2 Ionization Techniques

8.2.1 Electron Ionization (EI)

• Mechanism: High‑energy electrons (70 eV) bombard gaseous analyte → removes electron → radical cation (M⁺·).

• Use: GC–MS coupling; volatile, thermally stable compounds.

• Spectrum: Characteristic fragmentation pattern → structural clues.

8.2.2 Electrospray Ionization (ESI)

• Mechanism: Liquid sample pumped through a capillary under high voltage → charged droplets form → solvent evaporates → multiply charged ions.

• Use: LC–MS coupling; polar, high‑molecular‑weight biomolecules (peptides, proteins).

• Spectrum: Multiple charge states allow detection of large species within low m/z range.

8.2.3 Matrix‑Assisted Laser Desorption/Ionization (MALDI)

• Mechanism: Sample co‑crystallized with UV‑absorbing matrix; laser pulse desorbs and ionizes analyte with minimal fragmentation.

• Use: Analysis of large biomolecules (proteins, polymers).

• Spectrum: Predominantly singly charged ions; clean molecular‑ion peak.

8.3 Mass Analyzers

8.3.1 Quadrupole

• Principle: Four parallel rods generate oscillating electric fields; only ions of specific m/z pass through to detector at a given setting.

• Use: Routine quantitative analysis; tandem MS (MS/MS).

8.3.2 Time‑of‑Flight (TOF)

• Principle: Ions accelerated to same kinetic energy; lighter ions travel faster and reach the detector sooner.

• Use: High‑resolution, wide mass‑range applications (MALDI‑TOF).

8.3.3 Ion Trap

• Principle: Ions confined in a 3D or linear trap by RF fields; sequentially ejected by m/z for detection.

• Use: MSn experiments (multiple stages of fragmentation).

8.3.4 Orbitrap & FT‑ICR

• Orbitrap: Ions orbit a central spindle electrode; frequency of oscillation correlates with m/z.

• FT‑ICR: Ions trapped in a magnetic field; cyclotron frequency measured via Fourier transform.

• Use: Ultra‑high resolution and mass accuracy for complex mixtures.

8.4 Tandem Mass Spectrometry (MS/MS)

• Process:

  1. MS1: Select precursor ion of interest.

  2. Collision‑Induced Dissociation (CID): Precursor collides with inert gas → fragments.

  3. MS2: Analyze fragment ions to elucidate structure or confirm identity.

• Application: Structural elucidation, quantitation of trace analytes with high specificity, proteomics peptide sequencing.

8.5 Sample Preparation

• Desalting & Cleanup: Remove salts and buffers (e.g., via SPE) to prevent ion suppression in ESI.

• Concentration: Enrich low‑abundance analytes.

• Derivatization: Improve volatility (for EI) or ionization efficiency.

• Matrix Selection (MALDI): Choose appropriate UV‑absorbing matrix (e.g., α‑cyano‑4‑hydroxycinnamic acid).

8.6 Applications

• Pharmaceutical Analysis:

  • Drug identification and quantitation (LC–MS/MS assays).

  • Impurity profiling and degradation product characterization.

• Proteomics & Metabolomics:

  • Peptide sequencing, post‑translational modification mapping.

  • Small‑molecule metabolite identification in biological samples.

• Structure Elucidation:

  • Fragmentation patterns to determine functional groups and substructures.

• High‑Throughput Screening:

  • Rapid compound library analysis in drug discovery.

8.7 Advantages & Limitations

• Advantages:

  • Exceptional sensitivity (ppt–ppb levels).

  • High specificity via tandem MS.

  • Broad mass range (small molecules to large biopolymers).

  • Structural information from fragmentation.

• Limitations:

  • Instrument complexity and high cost.

  • Requires skilled operation and method development.

  • Matrix effects in ESI can cause ion suppression/enhancement.

  • Data analysis can be computationally intensive.

8.8 Key Exam Tips

• Ionization–Analyzer Pairing: Match EI with GC–MS, ESI with LC–MS, MALDI with TOF.

• Interpreting Spectra: Practice reading simple EI fragmentation patterns (alpha cleavage, McLafferty rearrangement).

• MS/MS Workflow: Be able to outline precursor selection, CID, and product‑ion scanning.

• Mass Accuracy vs. Resolution: Define each term and explain why high resolution is critical for isobar differentiation.

• Quantitative MS: Understand use of internal standards (stable‑isotope labeled) for precise quantitation.