B Pharmacy Sem 4: Pharmacognosy & Phytochemistry I

Subject 3. Pharmacognosy & Phytochemistry I

1. Introduction to Pharmacognosy (History, Scope & Sources of Drugs)
2. Biosynthesis & Classification of Secondary Metabolites
3. Carbohydrates & Glycosides (Structure, Occurrence & Pharmacological Importance)
4. Alkaloids (Classification, Isolation Techniques & Pharmacological Activities)
5. Terpenoids & Essential Oils (Extraction, Analysis & Therapeutic Uses)
6. Phytochemical Screening & Chromatographic Techniques (TLC, HPTLC, GC, HPLC)

Unit 1: Introduction to Pharmacognosy (History, Scope & Sources of Drugs)

A foundational exploration into the study of natural drugs—covering its definition, historical development, breadth of activities, and the diverse origins of pharmacologically active substances.

1.1 Definition & Core Concepts

1.1.1 Pharmacognosy

• Derived from Greek pharmakon (drug) and gnosis (knowledge)

• Pharmacognosy: Science of drugs of natural origin, encompassing their biological sources, chemical constituents, extraction, isolation, and standardization.

1.1.2 Key Terms

• Crude Drug: Unrefined plant, animal, or mineral material containing therapeutic constituents.

• Active Constituent: Specific chemical moiety responsible for pharmacological activity.

• Herbal Medicine: Preparations from plant materials for therapeutic use.

1.2 Historical Evolution

1.2.1 Ancient Civilizations

• Egypt & Mesopotamia (c. 3000 BCE): Ebers Papyrus listing >700 plant remedies (e.g., willow bark for pain).

• India & China:

  • Ayurveda (Charaka Samhita, ~1st millennium BCE)—herbal formulations;

  • Traditional Chinese Medicine (Shennong Ben Cao Jing, ~1st century CE)—365 materia medica entries.

1.2.2 Medieval to Renaissance

• Islamic Golden Age: Avicenna’s Canon of Medicine (1025 CE) systematized drug monographs.

• European Herbals: 16th–17th centuries—Dioscorides’s De Materia Medica resurgence; Paracelsus’s chemical remedies.

1.2.3 Modern Era

• 19th Century: Isolation of morphine (1804), quinine (1820)—birth of alkaloid chemistry.

• 20th Century:

  • Discovery of penicillin (1928) launching microbial pharmacognosy.

  • Standardization protocols (pharmacopoeias) and bioassay development.

• 21st Century: Integration with genomics, metabolomics, and sustainable sourcing.

1.3 Scope of Pharmacognosy

1.3.1 Drug Discovery & Lead Identification

• Bioprospecting—screening biodiversity for novel bioactive compounds.

• Ethnopharmacology—leveraging traditional knowledge to guide research.

1.3.2 Phytochemistry & Bioactive Profiling

• Isolation, structure elucidation (NMR, MS) of secondary metabolites.

• Quantitative analysis for standardization (marker compounds in plant extracts).

1.3.3 Quality Control & Standardization

• Macroscopic/macroscopic authentication of raw materials.

• Physicochemical parameters: moisture content, ash values, extractive values.

1.3.4 Formulation & Dosage Forms

• Development of herbal formulations: tinctures, extracts, capsules, topical gels.

• Ensuring stability, bioavailability, and reproducibility.

1.3.5 Regulatory & Conservation Aspects

• Compliance with WHO guidelines, national pharmacopeias, and Good Agricultural and Collection Practices (GACP).

• Conservation of endangered species and sustainable harvesting.

1.4 Sources of Natural Drugs

1.4.1 Plant Sources

• Leaves (e.g., digitalis leaves → cardiac glycosides)

• Bark (e.g., Cinchona → quinine)

• Roots & Rhizomes (e.g., ginseng → ginsenosides)

• Flowers & Fruits (e.g., saffron stigmas → crocin; citrus peels → flavonoids)

• Whole Plant (e.g., St. John’s wort for hypericin)

1.4.2 Animal Sources

• Venoms & Toxins (e.g., captopril development from Bothrops jararaca venom peptides)

• Glandular Products (e.g., insulin from porcine pancreas)

• Marine Invertebrates (e.g., ziconotide from cone snail toxin)

1.4.3 Microbial Sources

• Antibiotics: Penicillium spp. (penicillin), Streptomyces spp. (streptomycin, tetracyclines)

• Mycoproteins & Metabolites: Lovastatin from Aspergillus terreus

1.4.4 Mineral & Inorganic Sources

• Clays & Earths: Kaolin, bentonite in antidiarrheals

• Heavy Metals: Bismuth subsalicylate in gastrointestinal therapies

1.5 Integration in Pharmaceutical Sciences

1.5.1 Interdisciplinary Collaboration

• Coordination among botanists, chemists, pharmacologists, and process engineers.

1.5.2 Modern Technologies

• High‑throughput screening of natural extracts.

• Metabolomics & Cheminformatics for rapid dereplication and lead optimization.

1.5.3 Ethical & Sustainability Considerations

• Fair benefit sharing with indigenous communities.

• Cultivation and tissue culture to reduce wild‐harvest pressure.

1.6 Key Points for Exams

1. Define pharmacognosy and list its principal activities.

2. Trace History: Match key milestones (e.g., morphine isolation, Canon of Medicine).

3. Classify Sources: Provide two examples each of plant, animal, microbial, and mineral drugs.

4. Quality Tests: Describe three physicochemical parameters used to standardize crude plant drugs.

5. Scope Essay: Outline how ethnopharmacology informs modern drug discovery and the role of sustainability.

Unit 2: Biosynthesis & Classification of Secondary Metabolites

A comprehensive analysis of how plants and other organisms synthesize bioactive “secondary” metabolites—detailing their biosynthetic pathways, structural classes, and relevance to drug discovery.

2.1 Definition & Biological Role

2.1.1 Primary vs. Secondary Metabolites

• Primary Metabolites: Universal compounds essential for growth (e.g., sugars, amino acids, lipids).

• Secondary Metabolites: Species‑ or tissue‑specific compounds not directly required for basic metabolism but conferring ecological advantages—defense, signaling, attraction.

2.1.2 Physiological Functions

• Defense: Phytoalexins (e.g., camalexin), tannins deter herbivores and pathogens.

• Attraction: Pigments (anthocyanins), volatile terpenes attract pollinators.

• Allelopathy: Release of compounds (e.g., juglone) to inhibit neighboring plants.

2.2 Major Biosynthetic Pathways

2.2.1 Shikimate Pathway

• Precursor: Phosphoenolpyruvate + Erythrose‑4‑phosphate → chorismate.

• Produces: Aromatic amino acids (phenylalanine, tyrosine, tryptophan) → phenolics, flavonoids, alkaloids.

• Key Enzymes: DAHP synthase, chorismate mutase, phenylalanine ammonia‑lyase (PAL).

2.2.2 Acetate (Polyketide) Pathway

• Precursor: Acetyl‑CoA units → polyketide chains via successive Claisen condensations.

• Produces: Tetracyclines, anthraquinones (e.g., emodin), macrolides (via polyketide synthases in microbes).

• Key Features: Modular enzyme complexes (PKS I, II, III) dictate chain length and cyclization pattern.

2.2.3 Mevalonate & Methylerythritol Phosphate (MEP) Pathways

• Mevalonate Pathway (cytosolic in plants, most animals): Acetyl‑CoA → mevalonate → isopentenyl pyrophosphate (IPP).

• MEP Pathway (plastidic in plants, bacteria): Glyceraldehyde‑3‑P + pyruvate → 1‑deoxyxylulose phosphate → IPP.

• Produces: Terpenoids (monoterpenes, diterpenes, triterpenes, steroids), carotenoids.

• Key Enzymes: HMG‑CoA reductase (mevalonate), DXS (DXP synthase in MEP).

2.3 Classification of Secondary Metabolites

2.3.1 Alkaloids

• Definition: Nitrogen‑containing, basic compounds derived mainly from amino acids.

• Subclasses:

  • Tropane (e.g., atropine, scopolamine)

  • Isoquinoline (e.g., morphine, berberine)

  • Indole (e.g., reserpine, vinblastine)

• Biosynthetic Origin: Tyrosine (isoquinolines), tryptophan (indoles), ornithine (tropanes).

2.3.2 Phenolics & Polyphenols

• Definition: Aromatic ring compounds with one or more hydroxyl groups, derived from phenylalanine via the shikimate pathway.

• Subclasses:

  • Simple Phenols (e.g., eugenol)

  • Flavonoids (e.g., quercetin, rutin)

  • Tannins (hydrolyzable: gallotannins; condensed: procyanidins)

• Functions: Antioxidant, UV protection, enzyme inhibition.

2.3.3 Terpenoids (Isoprenoids)

• Definition: Structures built from C₅ isoprene units (IPP and DMAPP).

• Subclasses:

  • Monoterpenes (C₁₀; e.g., limonene, menthol)

  • Sesquiterpenes (C₁₅; e.g., artemisinin)

  • Diterpenes (C₂₀; e.g., taxol)

  • Triterpenes/Steroids (C₃₀; e.g., saponins, cholesterol)

• Roles: Membrane components, hormones, fragrance, defense.

2.3.4 Glycosides

• Definition: Aglycone (non‑sugar moiety) linked to one or more sugar residues.

• Subclasses:

  • Cardiac Glycosides (e.g., digoxin—steroid aglycone + digitoxose)

  • Anthraquinone Glycosides (e.g., senna)

  • Cyanogenic Glycosides (e.g., amygdalin)

2.4 Integration & Pharmaceutical Relevance

2.4.1 Lead Discovery & Semi‑Synthesis

• Understanding biosynthetic route enables pathway engineering (e.g., overexpressing taxadiene synthase for taxol).

• Precursor‑Directed Biosynthesis: Feeding analogs to cultures to generate novel derivatives.

2.4.2 Metabolic Engineering

• Transfer of entire biosynthetic clusters into microbial hosts (e.g., artemisinic acid in yeast).

2.4.3 Chemodiversity & SAR

• Structural diversification via tailoring enzymes (hydroxylases, methyltransferases, glycosyltransferases) yields analog libraries.

2.5 Key Points for Exams

1. Pathway Mapping: Draw schematic of shikimate vs. mevalonate pathways, showing key intermediates.

2. Classify Examples: Given structures, identify as alkaloid, terpenoid, or phenolic and state precursor.

3. Biosynthetic Enzymes: Describe role of HMG‑CoA reductase and phenylalanine ammonia‑lyase in secondary metabolism.

4. Pharma Applications: Explain how artemisinin biosynthesis understanding led to improved production in yeast.

5. Integrative Question: Propose a semi‑synthetic modification on a polyketide scaffold to enhance water solubility.

Unit 3: Carbohydrates & Glycosides (Structure, Occurrence & Pharmacological Importance)

An in‑depth treatment of plant‑derived carbohydrates and their glycosidic derivatives—covering structural features, natural distribution, types of glycosides, biological roles, and therapeutic applications.

3.1 Carbohydrate Fundamentals

3.1.1 Definition & General Formula

• Carbohydrates: Polyhydroxy aldehydes or ketones (monosaccharides) and their oligomeric/polysaccharide forms.

• General empirical formula: Cₙ(H₂O)ₘ, where typically m = n or closely related.

3.1.2 Monosaccharide Classification

• By Carbon Number: Trioses (n=3), tetroses, pentoses (e.g., D‑ribose), hexoses (e.g., D‑glucose, D‑fructose).

• By Functional Group: Aldoses (aldehyde at C‑1) vs. ketoses (ketone at C‑2).

3.1.3 Ring Structures & Stereochemistry

• Haworth Projections: Five‑membered furanoses vs. six‑membered pyranoses.

• Anomeric Carbon: C‑1 in aldopyranoses; gives rise to α/β anomers via hemiacetal formation.

• Epimers & Enantiomers: Difference at one stereocenter (e.g., glucose vs. galactose at C‑4); mirror‑image D/L forms.

3.2 Occurrence of Carbohydrates in Plants

3.2.1 Storage Polysaccharides

• Starch: Amylose (linear α(1→4)) and amylopectin (branched α(1→4), α(1→6)); energy reserve in seeds/tubers.

• Inulin: Fructan polymer in chicory and artichoke—prebiotic and diuretic uses.

3.2.2 Structural Polysaccharides

• Cellulose: Linear β(1→4)-linked glucose in cell walls—industrial source of microcrystalline cellulose.

• Hemicelluloses & Pectins: Heteropolysaccharides (galacturonans, arabinoxylans) with gelling properties (e.g., jam setting).

3.2.3 Oligosaccharides

• Raffinose Family: Raffinose, stachyose in legumes—implicated in flatulence but potential prebiotic effects.

3.3 Glycoside Definition & Formation

3.3.1 Glycosides

• Definition: Compounds in which a sugar moiety (glycone) is O‑, N‑, or C‑linked to a non‑sugar aglycone (genin).

• Biosynthesis: Catalyzed by glycosyltransferases using activated sugar donors (e.g., UDP‑glucose).

3.3.2 Glycosidic Bond

• O‑Glycosides: Sugar–OH bonds to aglycone (most common).

• N‑Glycosides: Sugar attached via nitrogen (e.g., cyanogenic glycosides).

• C‑Glycosides: Direct C–C linkage (stable to acid hydrolysis, e.g., puerarin).

3.4 Major Glycoside Classes & Examples

3.5 Pharmacological Roles & Mechanisms

3.5.1 Cardiac Glycosides

• Inhibit Na⁺/K⁺‑ATPase in myocytes → increased intracellular Ca²⁺ → enhanced cardiac contractility.

• Narrow therapeutic index; monitoring of plasma levels essential.

3.5.2 Anthraquinone Glycosides

• Hydrolyzed by colonic flora → aglycone induces peristalsis and inhibits water absorption.

• Used as stimulant laxatives; dosing adjusted to avoid cramping.

3.5.3 Flavonoid Glycosides

• Scavenge free radicals; stabilize capillary walls and reduce permeability.

• Employed in venotonic and anti‑inflammatory herbal preparations.

3.5.4 Cyanogenic Glycosides

• Upon tissue damage, β‑glucosidase cleaves to release HCN → toxic at high doses.

• Low‑dose dietary exposure underlie traditional uses and safety concerns.

3.5.5 Saponin Glycosides

• Amphipathic; form soap‑like foams.

• Exhibit membrane‑permeabilizing, cholesterol‑binding activities → adjuvant or expectorant effects.

3.6 Isolation & Analysis

3.6.1 Extraction

• Aqueous/Alcoholic Decoction & Infusion for polar glycosides.

• Percolation under controlled pH to preserve labile bonds.

3.6.2 Hydrolysis & Derivatization

• Acid/Base Hydrolysis: Releases aglycone for identification; sugar analysis by TLC or GC (as alditol acetates).

3.6.3 Chromatographic Techniques

• TLC/HPTLC: Identification via Rf values and color reagents (e.g., Ehrlich’s reagent for cardiac glycosides).

• HPLC–DAD/MS: Quantitative marker assays (e.g., digoxin in Digitalis extract).

• GC–MS: Volatile sugar derivatives or aglycones after silylation.

3.7 Key Points for Exams

1. Draw & Explain: Structure of an O‑glycosidic bond and mechanism of enzymatic hydrolysis by β‑glucosidase.

2. Classify: Given an aglycone structure, predict appropriate glycoside class and likely sugar donor.

3. Describe: Pharmacodynamics of digoxin and monitoring parameters.

4. Outline Extraction: Step‑by‑step procedure to isolate anthraquinone glycosides from senna leaves.

5. Analytical Plan: Design an HPLC method to quantify rutin in a herbal preparation, including choice of column, mobile phase, and detection wavelength.

Unit 4: Alkaloids (Classification, Isolation Techniques & Pharmacological Activities)

An exhaustive exploration of nitrogen‑containing plant secondary metabolites—covering their structural definition, biosynthetic classes, isolation and purification methods, diverse pharmacological actions, analytical techniques, and exam‑relevant summaries.

4.1 Definition & General Properties

4.1.1 Definition

• Alkaloids: Naturally occurring organic compounds containing one or more basic nitrogen atoms—usually heterocyclic—and biosynthesized from amino acids.

4.1.2 Physicochemical Characteristics

• Basicity: pKₐ typically 7–10; exist as salts under slightly acidic to neutral pH.

• Solubility: Free bases are lipophilic (soluble in organic solvents); salts are water‑soluble.

• Optical Activity: Many are chiral with significant stereochemical complexity.

4.2 Classification of Alkaloids

4.2.1 Based on Biosynthetic Precursors

4.2.2 Based on Ring Structure

• Monocyclic: Coniine

• Bicyclic: Tropane alkaloids

• Polycyclic: Indole‑diterpenoid alkaloids (e.g., vinblastine)

4.3 Occurrence & Examples

4.3.1 Plant Families & Sources

• Solanaceae: Atropa, Datura (tropanes)

• Papaveraceae: Papaver somniferum (isoquinolines)

• Rubiaceae: Rauwolfia serpentina (indoles)

• Leguminosae: Conium maculatum (piperidines)

• Theaceae & Malvaceae: Camellia sinensis, Theobroma cacao (purines)

4.3.2 Ecological Functions

• Defense against herbivores and pathogens

• Allelopathic interactions

4.4 Isolation & Purification Techniques

4.4.1 Extraction

1. Defatting: Remove lipids with nonpolar solvents (hexane).

2. Acidic Extraction: Grind plant material in dilute HCl → convert alkaloids to water‑soluble salts.

3. Basification & Back‑Extraction: Raise pH (pH 9–10) to free base → extract into organic solvent (chloroform, dichloromethane).

4.4.2 Purification

• Liquid–Liquid Partitioning: Stepwise pH adjustments to separate alkaloid classes by pKₐ differences.

• Vacuum Distillation: For volatile alkaloids (e.g., nicotine).

• Chromatography:

  • Column Chromatography on silica gel or alumina with gradient elution (ethyl acetate–methanol).

  • Preparative HPLC for high‑purity isolation.

4.5 Pharmacological Activities & Mechanisms

4.5.1 Dose & Therapeutic Index

• Many alkaloids have narrow therapeutic indices—dose titration and monitoring essential (e.g., morphine).

4.6 Analytical & Standardization Methods

4.6.1 Qualitative Tests

• Dragendorff’s Reagent: Orange‑red precipitate with alkaloid salts.

• Mayer’s & Wagner’s Reagents: White or brown precipitates.

4.6.2 Quantitative Analysis

• HPLC–UV/DAD: Gradient elution and detection at characteristic λ_max (e.g., morphine at 285 nm).

• GC–MS: For volatile alkaloids after derivatization (e.g., N‑acetylation).

• LC–MS/MS: Sensitive quantitation in complex matrices (biological fluids).

4.7 Key Points for Exams

1. Draw & Classify: Structures of atropine and morphine; assign to correct biosynthetic class.

2. Isolation Flowchart: Outline extraction and purification steps for a generic plant alkaloid.

3. Mechanism Description: Explain how reserpine lowers blood pressure via VMAT2 inhibition.

4. Analytical Protocol: Design an HPLC method to quantify codeine in a cough syrup, including sample prep and detection wavelength.

5. Safety Considerations: Discuss the narrow therapeutic window of tropane alkaloids and strategies to mitigate overdose risk.

Unit 5: Terpenoids & Essential Oils (Extraction, Analysis & Therapeutic Uses)

A comprehensive examination of terpenoid natural products and their volatile essential oils—covering structural principles, biosynthetic origins, extraction methods, analytical techniques, pharmacological applications, and exam‑worthy summaries.

5.1 Terpenoid Fundamentals & Classification

5.1.1 Isoprene Rule (C₅ Building Blocks)

• Isoprene Unit: C₅H₈ monomer; terpenoids arise from head‑to‑tail and head‑to‑head linkages of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).

5.1.2 Major Terpenoid Classes

5.1.3 Structural Diversity

• Cyclization Patterns: Linear chains vs. mono‑, bi‑, polycyclic frameworks.

• Functionalization: Hydroxylation, oxidation, glycosylation yield alcohols, ketones, acids, glycosides.

5.2 Biosynthesis & Occurrence

5.2.1 Biosynthetic Pathways

• Mevalonate Pathway (cytosol): Acetyl‑CoA → HMG‑CoA → mevalonate → IPP → DMAPP.

• MEP Pathway (plastidic in plants, bacteria): Pyruvate + G3P → DXP → MEP → IPP/DMAPP.

5.2.2 Natural Sources

• Plants: Essential oils in glandular trichomes (mint, lemon), resins (pine), latex (taxus).

• Microbes: Actinomycetes produce sesquiterpene antibiotics (e.g., albaflavenone).

• Marine Organisms: Diterpene macrolides with antitumor activity.

5.3 Essential Oils

5.3.1 Definition & Composition

• Essential Oil: Complex mixture of volatile terpenoids (monoterpenes, sesquiterpenes) and small phenolics.

• Principal Components: Major 70–90% (e.g., limonene in citrus); minor constituents impart aroma nuances.

5.3.2 Physicochemical Properties

• Volatility: Boiling points between 150–300 °C; vapor pressure suited for diffusive aroma.

• Solubility: Insoluble in water; soluble in ethanol, fixed oils.

5.4 Extraction Methods

5.4.1 Steam Distillation

• Principle: Co‑distillation of volatiles with water vapor at reduced effective boiling points; widely used for mint, lavender.

5.4.2 Hydro‑ or Dry‑Distillation

• Hydrodistillation: Plant material immersed in water; simple apparatus (Clevenger).

• Dry Distillation: Pyrolysis of resins (e.g., olibanum) to yield oleoresins.

5.4.3 Solvent Extraction & Enfleurage

• Cold Solvent: Hexane or supercritical CO₂ for temperature‑sensitive constituents.

• Enfleurage: Fat‐based absorption of aroma compounds; historically used for jasmine.

5.4.4 Expression (Cold Pressing)

• Mechanical rupture of oil glands (e.g., citrus peel) without heat.

5.5 Analytical Techniques

5.5.1 Gas Chromatography (GC & GC‑MS)

• GC‑FID: Quantitative profiling of major terpenoids.

• GC‑MS: Structural identification via fragmentation patterns; essential for fingerprinting.

5.5.2 High‑Performance Thin‑Layer Chromatography (HPTLC)

• Qualitative: Rapid screening of essential‑oil profiles; visible/derivatized spots.

5.5.3 HPLC & LC‑MS

• Non‑Volatile Terpenoids: Analysis of diterpene glycosides and triterpenoid saponins.

5.5.4 Spectroscopic Methods

• IR: Functional‑group analysis (C=C, OH) in terpenoids.

• NMR: Detailed structural elucidation (1D/2D) for novel diterpenes.

5.6 Therapeutic Uses & Mechanisms

5.7 Quality Control & Standardization

5.7.1 Organoleptic Evaluation

• Aroma & Color: Sensory profiling against reference oils.

5.7.2 Physicochemical Parameters

• Refractive Index, Optical Rotation, Density: Indicators of purity and adulteration.

• Specific Gravity: Measured at 20 °C for standardization.

5.7.3 Chromatographic Fingerprinting

• GC Profile: Match retention time and relative abundance of marker terpenoids.

• Chemometric Analysis: Multivariate statistics for geographical or cultivar differentiation.

5.8 Key Points for Exams

1. Define & Classify: Explain the isoprene rule and classify terpenoids by carbon number.

2. Extraction Rationale: Justify choice of steam distillation vs. supercritical CO₂ for temperature‑sensitive oils.

3. Analytical Design: Outline a GC‑MS method to fingerprint lavender oil, including column type and detector settings.

4. Mechanism Discussion: Describe how artemisinin’s endoperoxide moiety mediates antimalarial activity.

5. Quality Criteria: List three physicochemical tests to confirm purity of a commercial essential oil and interpret typical values.

Unit 6: Phytochemical Screening & Chromatographic Techniques (TLC, HPTLC, GC, HPLC)

A detailed overview of qualitative and quantitative methods for identifying and standardizing plant constituents—covering preliminary phytochemical tests and advanced chromatographic analyses.

6.1 Phytochemical Screening: Purpose & Preliminary Tests

6.1.1 Objectives of Screening

• Rapid identification of major secondary metabolite classes in crude extracts.

• Guidance for targeted isolation and bioactivity assays.

6.1.2 Common Phytochemical Tests

6.2 Chromatography: Fundamental Principles

6.2.1 Definition & Modes

• Chromatography: Separation of components between a mobile phase and a stationary phase based on differential affinity.

• Modes:

  • Adsorption: Silica/alumina stationary phase (TLC, HPTLC).

  • Partition: Liquid–liquid distribution (HPLC with reversed phase).

  • Gas–Liquid: Volatile analytes partition into stationary liquid film (GC).

6.2.2 Key Parameters

• Retention Factor (R_f) for TLC:

  $$R_f = \frac{\text{Distance traveled by spot}}{\text{Distance traveled by solvent front}}$$

• Retention Time (t_R) for GC/HPLC

• Selectivity (α), Resolution (R_s), Efficiency (N)

6.3 Thin‑Layer Chromatography (TLC & HPTLC)

6.3.1 Plate Composition & Preparation

• TLC: Silica gel or alumina on glass/plastic sheets.

• HPTLC: Higher‑performance plates with finer particles and uniform layers.

6.3.2 Sample Application & Development

1. Spotting: Microliter bands vs. discrete spots using capillary or automatic applicator.

2. Mobile Phase Selection: Binary/ternary solvent systems optimized for polarity (e.g., toluene–ethyl acetate–formic acid).

3. Development: Vertical chamber saturated with mobile phase vapor; allow capillary ascent.

6.3.3 Detection & Visualization

• UV Light (254/366 nm) for native fluorescence or after derivatization (e.g., NP/PEG for flavonoids).

• Chemical Reagents:

  • Dragendorff’s for alkaloids

  • Anisaldehyde–sulfuric acid for terpenoids (color development on heating)

• Densitometry (HPTLC) for semi‑quantitative measurement of band intensity.

6.4 Gas Chromatography (GC)**

6.4.1 Instrument Components

• Injector: Split/splitless for sample introduction.

• Column: Capillary fused‑silica coated with stationary phase (e.g., 5% phenyl–95% methyl polysiloxane).

• Detector:

  • FID (Flame Ionization) for hydrocarbons and terpenoids.

  • MS (Mass Spectrometry) for structural identification.

6.4.2 Sample Preparation

• Volatile Oils: Direct injection or dilution in solvent (e.g., hexane).

• Non‑Volatile Metabolites: Derivatization (silylation, methylation) to increase volatility and thermal stability.

6.4.3 Data Interpretation

• Chromatogram: Peaks identified by t_R and mass spectra; quantification via peak area relative to internal standard.

6.5 High‑Performance Liquid Chromatography (HPLC)**

6.5.1 Modes & Columns

• Reversed‑Phase (RP‑HPLC): C₁₈/C₈ columns; water–acetonitrile or water–methanol gradients for polar to non‑polar analytes.

• Normal‑Phase (NP‑HPLC): Silica columns for non‑polar compounds.

6.5.2 Detectors

• UV–Vis/DAD: Monitoring at characteristic λ_max (e.g., polyphenols at 280 nm).

• Fluorescence: High sensitivity for native or derivatized analytes.

• MS: LC–MS for structural confirmation and trace-level quantitation.

6.5.3 Method Development

1. Mobile Phase Composition & pH: Buffer choice (phosphate, acetate) to control analyte ionization.

2. Gradient vs. Isocratic: Gradient elution for complex mixtures; isocratic for routine assays.

3. Flow Rate & Temperature: Optimize for resolution vs. analysis time.

6.6 Method Validation & Quality Assurance

6.6.1 Validation Parameters (ICH Q2(R1))

• Specificity: Distinct separation of analyte from matrix.

• Linearity & Range: Correlation coefficient (r² > 0.999).

• Accuracy & Precision: Recovery (90–110%), %RSD <2%.

• LOD & LOQ: Based on signal‑to‑noise ratios (3:1 and 10:1).

• Robustness: Small deliberate changes in method parameters.

6.6.2 Documentation & Reporting

• Standard Operating Procedures (SOPs) for each technique.

• Batch records, chromatographic logs, calibration curves.

6.7 Integration into Standardization

• TLC Fingerprinting: Rapid batch-to-batch consistency check for herbal extracts.

• HPTLC Quantification: Semi‑automated assay of marker compounds (e.g., curcumin in turmeric).

• GC Profiling: Authentication of essential‑oil composition; detection of adulterants.

• HPLC Assays: Official monograph methods for quantifying active constituents (e.g., glycyrrhizin in licorice).

6.8 Key Points for Exams

1. Describe & Compare: Advantages and limitations of TLC vs. HPTLC.

2. Calculate R_f: Given solvent front and spot distances, compute retention factors for three phytoconstituents.

3. Design Chromatogram: Outline an HPLC method (column, mobile phase, detection λ) to quantify quercetin in an extract.

4. Method Validation: List five validation parameters and describe acceptance criteria.

5. Case Scenario: Propose a workflow—screening, isolation, and quantification—for alkaloids in an unknown plant sample using TLC and HPLC.