BP504T Unit V: Basics of Phytochemistry – Extraction, Spectroscopy, Chromatography & Electrophoresis

B.PHARM 5TH SEMESTER • BP504T • UNIT-V

Basics of Phytochemistry

Modern Extraction Techniques • Spectroscopy • Chromatography • Electrophoresis

Unit-V: Basics of Phytochemistry deals with modern approaches used for the extraction, isolation, purification, identification and analysis of phytoconstituents. The unit connects traditional pharmacognosy with modern analytical phytochemistry by introducing advanced extraction techniques and instrumental methods such as spectroscopy, chromatography and electrophoresis.

1. Unit-V at a Glance

Modern Extraction

Modern approaches for obtaining phytoconstituents efficiently from crude drugs and plant materials.

Spectroscopy

Application of UV-Visible, IR, NMR and mass spectrometric techniques in characterization and identification.

Chromatography

Separation, purification, identification and quantitative analysis of phytoconstituents.

Electrophoresis

Separation of charged molecules based on their electrophoretic mobility.

2. General Workflow of Phytochemical Investigation

Crude Drug → Authentication → Drying → Size Reduction → Extraction → Fractionation → Separation → Purification → Identification → Structural Characterization → Phytoconstituent

Key concept: Extraction separates constituents from plant material, whereas chromatographic and other separation techniques help separate individual components. Spectroscopic techniques are then used primarily for identification and structural characterization.

3. Modern Methods of Extraction

Extraction: Extraction is the process of removing soluble and desired chemical constituents from a crude drug or plant material using a suitable solvent or extraction medium.

Why Modern Extraction Techniques?

• Improved extraction efficiency.
• Reduced extraction time.
• Lower solvent consumption in many processes.
• Better recovery of thermolabile constituents in suitable conditions.
• Improved selectivity.
• Potential reduction in environmental burden.

Major Modern Extraction Approaches

Ultrasound-Assisted Extraction

Uses ultrasonic energy to improve solvent penetration and mass transfer between plant material and extraction solvent.

Microwave-Assisted Extraction

Uses microwave energy to accelerate heating and improve release of extractable constituents.

Supercritical Fluid Extraction

Uses fluids above their critical temperature and pressure, particularly supercritical CO₂, as an extraction medium.

Pressurized Liquid Extraction

Uses elevated temperature and pressure to enhance solvent penetration and extraction efficiency.

Enzyme-Assisted Extraction

Uses enzymes to disrupt plant cell-wall components and facilitate release of intracellular constituents.

Accelerated Solvent Extraction

Uses elevated temperature and pressure to improve extraction with organic or aqueous solvents.

4. Ultrasound-Assisted Extraction (UAE)

Principle

Ultrasound produces acoustic cavitation in the extraction medium. Formation and collapse of microscopic bubbles can increase cell disruption, solvent penetration and mass transfer.

Plant Material → Solvent Addition → Ultrasonic Energy → Cavitation → Cell Disruption → Enhanced Mass Transfer → Extract

Advantages

• Reduced extraction time.
• Enhanced mass transfer.
• Can reduce solvent requirement.
• Useful for many classes of phytoconstituents.

5. Microwave-Assisted Extraction (MAE)

Principle

Microwave energy interacts with polar molecules and produces rapid volumetric heating. The resulting heating can increase pressure within plant cells and facilitate release of intracellular constituents.

Plant Material + Solvent → Microwave Energy → Rapid Heating → Cell Disruption → Constituent Release → Extract

Precaution: Microwave conditions must be optimized because excessive temperature or prolonged exposure can degrade thermolabile phytoconstituents.

6. Supercritical Fluid Extraction (SFE)

Principle

A substance above its critical temperature and critical pressure can exist in the supercritical state. Supercritical fluids possess properties intermediate between gases and liquids and can penetrate plant matrices while dissolving suitable compounds.

Plant Material → Supercritical Fluid → Pressurization → Extraction → Pressure Reduction → Separated Extract

Supercritical CO₂

Supercritical carbon dioxide is particularly useful for extraction of relatively non-polar compounds such as volatile oils and some lipophilic constituents.

7. Pressurized Liquid Extraction

Pressurized liquid extraction uses elevated temperature and pressure to maintain the extraction solvent in a liquid state and increase its ability to penetrate the plant matrix.

Plant Matrix → Solvent → Elevated Pressure → Elevated Temperature → Enhanced Solubility & Diffusion → Extract

8. Enzyme-Assisted Extraction

Plant cell walls contain structural polymers such as cellulose, hemicellulose and pectin. Suitable enzymes can partially degrade these barriers and improve release of intracellular constituents.

Plant Tissue → Cell-wall Enzymes → Cell-wall Disruption → Enhanced Release → Improved Extraction

9. Comparison of Modern Extraction Techniques

Technique	Main Principle	Major Advantage	Important Consideration
UAE	Acoustic cavitation	Rapid mass transfer	Ultrasound intensity and temperature
MAE	Microwave heating	Rapid heating	Thermal degradation
SFE	Supercritical fluid	Low solvent residue and tunable selectivity	High-pressure equipment
PLE	Pressure + temperature	Efficient solvent extraction	Optimization of temperature and pressure
EAE	Enzymatic cell-wall disruption	Improved release	Enzyme specificity and stability

10. Spectroscopy in Phytochemistry

Spectroscopy involves the study of interaction between electromagnetic radiation and matter. In phytochemistry, spectroscopic methods are important for identification and structural characterization of isolated compounds.

UV-Visible

Useful for chromophoric compounds, conjugated systems and many quantitative assays.

IR / FTIR

Useful for identifying characteristic functional groups and comparing samples.

NMR

Provides detailed information about the molecular framework and chemical environment of nuclei.

Mass Spectrometry

Provides molecular mass information and characteristic fragmentation patterns useful for structural characterization.

11. UV-Visible Spectroscopy

Principle

Molecules absorb ultraviolet or visible radiation when electrons undergo transitions between different energy levels.

Sample → UV/Visible Radiation → Electronic Transition → Absorbance → Spectrum / Quantification

Applications in Phytochemistry

• Detection of chromophoric constituents.
• Quantitative estimation using Beer–Lambert relationship.
• Monitoring chromatographic fractions.
• Characterization of conjugated systems.

12. Infrared Spectroscopy

Principle

Molecular bonds absorb infrared radiation at characteristic frequencies corresponding to vibrational transitions.

Sample → IR Radiation → Molecular Vibrations → Characteristic Absorption → IR Spectrum

Important Functional Groups

Functional Group	Typical IR Region*
O–H	Broad absorption in the ~3200–3600 cm⁻¹ region
C=O	Approximately ~1650–1750 cm⁻¹ depending on functionality
C–H	Approximately ~2850–3000 cm⁻¹ for many aliphatic C–H bonds
C=C	Approximately ~1600–1680 cm⁻¹ depending on structure

*Approximate ranges; exact absorption depends on molecular environment.

13. Nuclear Magnetic Resonance Spectroscopy

NMR spectroscopy provides information about the chemical environment of nuclei such as ¹H and ¹³C. It is one of the most powerful techniques for structural elucidation of isolated phytoconstituents.

Compound → Magnetic Field → Radiofrequency Irradiation → Nuclear Resonance → Chemical Shift → Spin–Spin Coupling → Structural Information

Major NMR Information

Parameter	Information Obtained
Chemical shift (δ)	Chemical environment of nuclei
Integration	Relative number of contributing nuclei in ¹H NMR
Multiplicity	Coupling relationship between neighboring nuclei
Coupling constant (J)	Information about spin-spin coupling
¹³C NMR	Carbon framework and carbon environments

14. Mass Spectrometry

Principle

Mass spectrometry converts molecules into ions and separates the ions according to their mass-to-charge ratio (m/z).

Sample → Ionization → Ion Formation → Mass Separation → Detection → Mass Spectrum

Applications

• Determination of molecular mass.
• Detection of molecular ions or related ion species.
• Structural information through fragmentation.
• Useful in combination with chromatography, e.g. LC-MS.

15. Chromatography in Phytochemistry

Chromatography is a separation technique in which components distribute differently between a stationary phase and a mobile phase.

Complex Plant Extract → Stationary Phase + Mobile Phase → Differential Migration → Separated Components

16. Major Chromatographic Techniques

Paper Chromatography

Useful for simple analytical separation and identification of certain polar compounds.

TLC

Rapid analytical technique for monitoring extracts, fractions and purity.

HPTLC

Instrumentally enhanced TLC providing improved documentation, densitometry and quantitative applications.

Column Chromatography

Useful for preparative separation and purification of natural products.

HPLC

High-resolution separation and quantitative estimation of phytoconstituents.

GC

Important for volatile and thermally suitable compounds, including many components of essential oils.

17. Thin Layer Chromatography (TLC)

Basic Principle

Components move across a stationary phase under the influence of a mobile phase. Different components travel at different rates.

Sample Application → Developing Chamber → Solvent Migration → Component Separation → Visualization → Rf Value

Rf Value

Rf = Distance travelled by solute ÷ Distance travelled by solvent front

Remember: Rf is characteristic only under defined experimental conditions. Changing stationary phase, solvent system, temperature or other conditions can change the observed value.

18. HPTLC

High Performance Thin Layer Chromatography is an advanced form of TLC involving controlled sample application, standardized plate development and instrumental detection/documentation.

Extract → Automated Application → Chromatographic Development → Detection → Densitometric Evaluation → Fingerprint / Quantification

Applications

• Herbal drug fingerprinting.
• Detection of adulteration.
• Identification of phytoconstituents.
• Quality control of herbal preparations.
• Quantitative estimation of marker compounds.

19. Column Chromatography

Column chromatography is primarily a preparative separation technique in which a stationary phase is packed into a column and the sample is eluted using an appropriate mobile phase.

Crude Extract → Column Packing → Sample Loading → Elution → Fraction Collection → TLC Monitoring → Pooled Fractions

Practical principle: Fractions with similar chromatographic profiles can be combined after TLC/HPTLC monitoring, followed by further purification when necessary.

20. High Performance Liquid Chromatography (HPLC)

Principle

HPLC separates components based on their differential interactions with the stationary phase and the mobile phase under high-pressure liquid flow.

Solvent Reservoir → Pump → Injector → Column → Detector → Chromatogram

Major Components of HPLC

Component	Function
Solvent reservoir	Contains mobile phase
Pump	Delivers mobile phase at controlled high pressure
Injector / autosampler	Introduces sample
Column	Contains stationary phase and performs separation
Detector	Detects separated compounds
Data system	Records and processes chromatographic signals

21. Gas Chromatography (GC)

Gas chromatography separates compounds that can be vaporized without decomposition. It is especially useful for volatile constituents and components of essential oils.

Sample → Injector → Carrier Gas → Column → Detector → Chromatogram

Applications in Pharmacognosy

• Analysis of essential oils.
• Identification of volatile constituents.
• Quality control of volatile oil-containing crude drugs.
• Fingerprinting of complex volatile mixtures.

22. Chromatographic Techniques – Quick Comparison

Technique	Typical Role	Major Application
TLC	Analytical	Identification, purity and monitoring
HPTLC	Analytical / quantitative	Fingerprinting and marker estimation
Column Chromatography	Preparative	Isolation and purification
HPLC	Analytical / preparative	Separation and quantitative estimation
GC	Analytical	Volatile constituents and essential oils

23. Electrophoresis

Electrophoresis is a separation technique in which charged molecules migrate through a medium under the influence of an electric field.

Charged Molecules → Electric Field → Differential Migration → Separation → Bands / Zones

Factors Affecting Electrophoretic Mobility

• Net electrical charge.
• Molecular size.
• Molecular shape.
• pH of the medium.
• Electric field strength.
• Nature and concentration of supporting medium.

24. Gel Electrophoresis

In gel electrophoresis, molecules migrate through a gel matrix under an electric field. Separation depends on factors such as molecular size and charge, depending on the specific electrophoretic system.

Sample Loading → Electric Field → Molecular Migration → Differential Mobility → Band Formation → Detection

Importance in Natural Products

Electrophoretic techniques are particularly relevant to analysis of proteins, enzymes and other charged biomolecules associated with plant materials.

25. Interactive: Which Technique Should You Choose?

Need to obtain constituents from plant material?

Use an appropriate extraction method such as conventional solvent extraction, UAE, MAE, SFE, PLE or enzyme-assisted extraction, depending on the nature of the target compound and matrix.

Need rapid monitoring of fractions?

TLC/HPTLC is useful for monitoring extracts, fractions and purification steps.

Need high-resolution separation or quantitative assay?

HPLC is commonly used for separation and quantitative estimation of phytoconstituents.

Need analysis of volatile constituents?

GC is particularly suitable for volatile and thermally stable compounds, including many essential-oil constituents.

Need information about chromophoric compounds or quantification?

UV-Visible spectroscopy can provide absorption information and can be used for quantitative assays under validated conditions.

Need functional-group information?

IR spectroscopy is useful for identifying characteristic functional groups.

Need detailed structural information?

NMR provides information about chemical environments, connectivity, coupling and molecular framework.

Need molecular-mass information?

Mass spectrometry provides m/z information and fragmentation data useful for molecular characterization.

Need separation of charged biomolecules?

Electrophoresis separates charged molecules according to their electrophoretic mobility under an applied electric field.

26. Complete Strategy for Isolation & Identification

Plant Material → Extraction → Preliminary Screening → Solvent Partition → TLC / HPTLC → Column Chromatography → HPLC → Purified Compound → MS → IR → NMR → Structure Identification

Important distinction: Chromatography primarily answers: “How can the mixture be separated?” Spectroscopy primarily answers: “What is the identity and structure of the compound?” In modern phytochemistry, these techniques are frequently combined rather than used in isolation.

27. Technique → Information Obtained

Technique	Main Information	Typical Phytochemical Application
UAE	Enhanced extraction	Recovery of phytoconstituents
MAE	Rapid extraction	Extraction of suitable phytochemicals
SFE	Selective extraction	Volatile/lipophilic compounds
TLC	Separation and Rf	Screening and fraction monitoring
HPTLC	Fingerprint and quantification	Herbal drug quality control
Column chromatography	Preparative separation	Isolation
HPLC	High-resolution separation	Isolation and assay
GC	Volatile compound separation	Essential oils
UV-Visible	Electronic absorption	Chromophoric compounds and assays
IR	Functional groups	Structural characterization
NMR	Molecular framework	Structure elucidation
MS	m/z and fragmentation	Molecular mass and structural information
Electrophoresis	Electrophoretic mobility	Proteins and charged biomolecules

28. Modern Phytochemical Analysis is Multidisciplinary

Step 1 – Extract

Use an appropriate extraction procedure based on polarity, stability and target constituent.

Step 2 – Separate

Use solvent partitioning and chromatographic techniques to reduce the complexity of the mixture.

Step 3 – Purify

Use preparative chromatography and repeated separation when necessary.

Step 4 – Characterize

Combine MS, IR, NMR and other appropriate spectroscopic information to establish molecular identity.

29. Role in Herbal Drug Quality Control

• Authentication of plant material.
• Detection of adulteration.
• Identification of marker compounds.
• Chemical fingerprinting.
• Quantitative estimation of active or marker constituents.
• Batch-to-batch consistency.
• Detection of degradation or instability.

Raw Herbal Drug → Extraction → Chromatographic Fingerprint → Marker Identification → Quantification → Quality-Controlled Herbal Product

30. One-Minute Revision

UAE

Ultrasound → cavitation → enhanced mass transfer.

MAE

Microwave → rapid heating → enhanced extraction.

SFE

Supercritical fluid → selective extraction.

TLC

Rapid separation → Rf → identification/monitoring.

HPTLC

Fingerprinting → densitometry → quantitative analysis.

HPLC

High-resolution separation → assay and purification.

GC

Volatile compounds → essential oils.

UV-Visible

Electronic transitions → absorption → quantification.

IR

Molecular vibrations → functional groups.

NMR

Chemical environment → molecular structure.

MS

m/z → molecular mass and fragmentation.

Electrophoresis

Electric field → differential mobility → separation.

31. Frequently Asked Differences

TLC vs HPLC

TLC: simple, rapid and inexpensive analytical separation on a planar stationary phase.

HPLC: high-resolution liquid chromatographic separation using a packed column and high-pressure mobile-phase delivery.

IR vs NMR

IR: mainly provides information about functional groups through molecular vibrational absorption.

NMR: provides detailed information about nuclear chemical environments and molecular connectivity.

HPLC vs GC

HPLC: suitable for a broad range of compounds, including many non-volatile and thermolabile constituents.

GC: particularly useful for volatile compounds that can be vaporized without decomposition.

Extraction vs Isolation

Extraction: transfers soluble constituents from plant material into an extraction medium.

Isolation: separates an individual constituent from a complex extract and obtains it in purified form.

32. Interactive Quick Quiz

Q1. Which technique uses acoustic cavitation?

Q2. Which technique is particularly useful for volatile compounds?

Q3. Rf value is associated with:

Q4. Which technique provides m/z information?

Q5. Which technique is especially important for detailed structural elucidation?

Q6. Which technique is primarily based on differential migration of charged molecules?

33. Important Examination Questions

Long Answer Questions

1. Explain the modern methods of extraction of phytoconstituents. Discuss their principles and advantages.
2. Describe the principle, instrumentation and applications of HPLC in phytochemical analysis.
3. Explain chromatography and discuss the applications of TLC, HPTLC, column chromatography, HPLC and GC in pharmacognosy.
4. Discuss the application of spectroscopy in the isolation and identification of phytoconstituents.
5. Explain the principle and applications of UV-Visible, IR, NMR and mass spectroscopy in phytochemistry.
6. Describe electrophoresis and its applications in phytochemical investigation.
7. Explain the complete process of isolation and identification of a phytoconstituent using modern analytical techniques.

Short Answer Questions

1. Define extraction.
2. What is ultrasound-assisted extraction?
3. What is microwave-assisted extraction?
4. What is supercritical fluid extraction?
5. Define chromatography.
6. What is Rf value?
7. Write the principle of HPLC.
8. Write two applications of HPTLC.
9. What is the use of GC in pharmacognosy?
10. What information is obtained from IR spectroscopy?
11. What is chemical shift in NMR?
12. What is m/z in mass spectrometry?
13. Define electrophoresis.
14. Write two applications of spectroscopy in phytochemistry.
15. Differentiate extraction and isolation.

34. Master Flowchart – Unit-V

Crude Drug → Modern Extraction → Preliminary Analysis → Chromatographic Separation → Purification → UV / IR → MS → NMR → Identification & Structure Elucidation

35. Final Revision Table

Topic	Key Point
Modern extraction	Improves efficiency, selectivity and/or reduces extraction time and solvent use depending on method.
UAE	Acoustic cavitation.
MAE	Microwave-assisted heating.
SFE	Supercritical fluid, commonly CO₂.
TLC	Rapid analytical separation and Rf determination.
HPTLC	Fingerprinting and quantitative densitometric analysis.
Column chromatography	Preparative separation and purification.
HPLC	High-resolution separation and quantitative analysis.
GC	Volatile and thermally suitable compounds.
UV-Visible	Electronic absorption and quantitative analysis.
IR	Functional-group identification.
NMR	Detailed structural characterization.
Mass spectrometry	Molecular mass and fragmentation information.
Electrophoresis	Separation based on electrophoretic mobility.

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B.Pharm 5th Semester • BP504T Pharmacognosy & Phytochemistry-II
Unit-V • Basics of Phytochemistry