UNIT 5.3
Chromatographic Techniques
Separating and identifying plant compounds
🎯 After this unit, you will be able to:
- Explain the basic principle of chromatography
- Perform paper and thin-layer chromatography (TLC)
- Calculate Rf values and identify compounds
- Understand the principles of HPLC and GC
🧪 What is Chromatography?
Chromatography is a technique for separating mixtures into their individual components. All chromatographic methods rely on two phases:
- Stationary phase: A solid or liquid fixed in place (e.g., paper, silica gel)
- Mobile phase: A liquid or gas that moves through the stationary phase, carrying the sample
Key insight: Compounds separate because they interact differently with the stationary and mobile phases. Those that interact more strongly with the stationary phase move more slowly .
🧪 [Diagram: Basic principle of chromatography showing separation of components — to be inserted]
📊 Types of Chromatography
📄
Paper Chromatography
Stationary phase: paper
Simple, inexpensive, good for plant pigments, amino acids
🪟
Thin-Layer Chromatography (TLC)
Stationary phase: silica on glass/plastic
Faster, better resolution than paper; widely used
💧
Column Chromatography
Stationary phase in glass column
For preparative separations; can collect fractions
⚡
High-Performance Liquid Chromatography (HPLC)
High pressure, automated
High resolution, quantitative, expensive
💨
Gas Chromatography (GC)
Mobile phase: gas; stationary phase: liquid
For volatile compounds; often coupled with MS
📄 Part 1: Paper Chromatography
Paper chromatography is the simplest and least expensive form of chromatography. It's excellent for teaching and for quick separations of plant pigments, amino acids, and sugars .
Principle
- Stationary phase: Water molecules trapped in cellulose paper
- Mobile phase: Organic solvent (e.g., petroleum ether, acetone, water mixtures)
- Separation: Based on partition coefficient between water and solvent
Paper Chromatography of Plant Pigments
🌿 Leaf Pigment Separation Protocol
- Cut a strip of chromatography paper
- Draw a pencil line 2 cm from the bottom (never use pen—ink runs!)
- Grind a leaf with acetone or ethanol to extract pigments
- Spot the extract on the pencil line using a capillary tube; let dry
- Place paper in a chromatography chamber with solvent (e.g., petroleum ether:acetone 9:1)
- Cover and let solvent rise until near the top
- Remove, mark solvent front, and let dry
- Observe separated pigments (carotenes yellow-orange, xanthophylls yellow, chlorophyll a blue-green, chlorophyll b yellow-green)
🌿 [Diagram: Paper chromatography setup and pigment separation — to be inserted]
Rf Values
The Rf (retention factor) is the ratio of distance moved by the compound to distance moved by the solvent front:
Rf = distance traveled by compound / distance traveled by solvent front
Rf values are characteristic for each compound under specific conditions (solvent, temperature, paper type). They can be used for tentative identification .
🧪 Identifying Amino Acids by Paper Chromatography
A mixture of amino acids is spotted on paper and developed in butanol:acetic acid:water (4:1:1). After drying, the paper is sprayed with ninhydrin, which turns amino acids purple. Rf values are calculated and compared to standards:
- Alanine: Rf ≈ 0.38
- Glycine: Rf ≈ 0.26
- Leucine: Rf ≈ 0.73
- Proline: Rf ≈ 0.43 (yellow with ninhydrin)
🪟 Part 2: Thin-Layer Chromatography (TLC)
TLC is similar to paper chromatography but uses a thin layer of adsorbent (usually silica gel) coated on a glass, plastic, or aluminum plate. It offers better resolution and faster development .
Advantages of TLC over Paper Chromatography
| Feature |
Paper Chromatography |
TLC |
| Stationary phase |
Cellulose (paper) |
Silica gel, alumina, cellulose |
| Resolution |
Lower |
Higher |
| Development time |
Hours |
Minutes |
| Sensitivity |
Lower |
Higher |
| Visualization |
Limited |
Many options (UV, iodine, sprays) |
Normal-Phase vs. Reversed-Phase TLC
- Normal-phase: Polar stationary phase (silica), nonpolar mobile phase. Polar compounds move slower .
- Reversed-phase: Nonpolar stationary phase (C18), polar mobile phase. Nonpolar compounds move slower .
TLC Protocol
🔬 General TLC Procedure
- Cut or use a pre-made TLC plate (handle by edges only!)
- Draw a pencil line 1 cm from bottom
- Apply samples as small spots using capillary tube; keep spots small and concentrated
- Let spots dry completely
- Place plate in developing chamber with solvent (solvent level below spots!)
- Cover and let solvent rise until near top
- Remove, mark solvent front, let dry
- Visualize: UV light, iodine vapor, or spray reagent
- Calculate Rf values
🔍 Did you know? TLC plates can be visualized under UV light if compounds absorb UV or if the plate contains a fluorescent indicator. Compounds appear as dark spots against a fluorescent background .
🧴 Part 3: Column Chromatography
Column chromatography is used for preparative separations—you can collect fractions containing pure compounds for further analysis .
Principle
- Stationary phase packed in a glass column (silica gel, alumina, etc.)
- Sample applied to top
- Mobile phase (solvent) flows through column, eluting compounds
- Fractions collected and analyzed
🧴 [Diagram: Column chromatography setup — to be inserted]
Applications in Plant Biochemistry
| Application |
Stationary phase |
Mobile phase |
| Chlorophyll separation |
Sugar or cellulose |
Petroleum ether with increasing acetone |
| Flavonoid purification |
Sephadex LH-20 |
Methanol or ethanol |
| Protein purification |
Ion exchange, gel filtration |
Buffers with increasing salt |
⚡ Part 4: High-Performance Liquid Chromatography (HPLC)
HPLC is an advanced form of column chromatography that uses high pressure to push solvent through very small particle sizes, achieving high resolution and speed .
Components of an HPLC System
- Solvent reservoirs and pump: Deliver mobile phase at high pressure
- Injector: Introduces sample
- Column: Contains stationary phase (C18 most common for reversed-phase)
- Detector: Usually UV-Vis, fluorescence, or refractive index
- Computer/data system: Records chromatogram and controls system
⚡ [Diagram: HPLC system components — to be inserted]
Types of HPLC
| Type |
Stationary phase |
Applications |
| Reversed-phase (RP-HPLC) |
Nonpolar (C18) |
Most common; separates nonpolar to moderately polar compounds |
| Normal-phase (NP-HPLC) |
Polar (silica) |
Separates polar compounds, isomers |
| Ion-exchange (IEX) |
Charged resins |
Separates ions, proteins, nucleic acids |
| Size-exclusion (SEC) |
Porous beads |
Separates by molecular size |
Chromatogram Interpretation
An HPLC chromatogram plots detector response vs. time. Each peak represents a compound. Information obtained:
- Retention time (tR): Time from injection to peak maximum—identifies compound (compared to standards)
- Peak area/height: Proportional to concentration—used for quantification
- Peak shape: Indicates separation quality (sharp peaks are good)
💨 Part 5: Gas Chromatography (GC)
GC is used for volatile compounds that can be vaporized without decomposing. The mobile phase is an inert gas (helium, nitrogen), and the stationary phase is a liquid coated inside a capillary column .
Applications in Plant Biochemistry
- Essential oils: Terpenes, aroma compounds
- Fatty acid methyl esters (FAMEs): Oil composition analysis
- Volatile organic compounds (VOCs): Plant stress responses, aromas
- Pesticide residues: Detection and quantification
GC-MS (Gas Chromatography-Mass Spectrometry)
GC-MS combines separation with mass spectrometry for compound identification. The mass spectrometer provides structural information, allowing positive identification of unknown compounds .
🌿 Analyzing Essential Oils by GC-MS
Ethiopian frankincense (Boswellia species) essential oil can be analyzed by GC-MS to identify its components. The chromatogram shows peaks for α-pinene, limonene, and other terpenes. The mass spectrum of each peak provides a "fingerprint" that can be matched to databases for identification .
🤔 Choosing the Right Chromatographic Technique
| Sample type |
Recommended technique |
Why? |
| Plant pigments |
Paper or TLC |
Simple, visual, inexpensive |
| Amino acids |
Paper, TLC, or HPLC |
Ninhydrin visualization; HPLC for quantification |
| Sugars |
HPLC or TLC |
HPLC with RI detection for quantification |
| Fatty acids |
GC (as FAMEs) |
Volatile derivatives needed |
| Phenolics/flavonoids |
HPLC-UV or HPLC-MS |
High resolution, UV spectra aid identification |
| Volatile aromas |
GC-MS |
Best for volatiles; MS provides identification |
| Proteins |
Column chromatography (IEX, SEC) |
Gentle conditions preserve activity |
🇪🇹 Chromatography in Ethiopian Research
Coffee Analysis
Ethiopian coffee research uses HPLC to analyze chlorogenic acids, caffeine, and other compounds that affect flavor and quality. This helps characterize different coffee varieties and growing regions .
Essential Oils
Ethiopia has many aromatic plants (e.g., Ocimum, Thymus, Rosmarinus species). GC-MS is used to analyze essential oil composition, supporting the development of natural products for export .
Traditional Fermented Foods
HPLC and GC are used to study the biochemical changes during fermentation of traditional foods like injera (teff) and kocho (enset), helping to understand nutritional quality and safety .
☕ Characterizing Ethiopian Coffee by HPLC
Researchers at Jimma University use HPLC to analyze chlorogenic acid content in coffee from different regions. They find that Harrar coffee has higher chlorogenic acid than Yirgacheffe, which may contribute to its distinctive flavor. This type of analysis supports the development of coffee provenance and quality standards .
📌 Unit Summary
| Technique |
Principle |
Typical applications |
| Paper chromatography |
Partition between water in paper and solvent |
Pigments, amino acids |
| TLC |
Adsorption on silica gel |
Lipids, flavonoids, many organics |
| Column chromatography |
Adsorption, ion exchange, size exclusion |
Preparative separations, protein purification |
| HPLC |
High-pressure liquid on small particles |
Quantitative analysis of many compounds |
| GC |
Partition between gas and liquid |
Volatile compounds, fatty acids |
Rf value = distance moved by compound / distance moved by solvent front
Reflection question: You are studying the essential oil composition of a traditional Ethiopian medicinal plant. You want to identify both the major volatile components and quantify a specific non-volatile flavonoid. Which chromatographic techniques would you use for each part, and why?
📌 Key terms introduced
Stationary phase
Mobile phase
Rf value
Paper chromatography
Thin-layer chromatography (TLC)
Column chromatography
High-performance liquid chromatography (HPLC)
Gas chromatography (GC)
GC-MS
Retention time
Normal-phase
Reversed-phase
C18 column
✅ Check your understanding
- Define stationary phase and mobile phase. Give examples for paper chromatography.
- How is Rf calculated, and what does it tell you?
- What are the advantages of TLC over paper chromatography?
- Why is HPLC called "high-performance"?
- For analyzing fatty acid composition of niger seed oil, which technique would you choose and why?
Discuss your answers in the course forum.
Plant Biochemistry for Horticulture · HORT 202 · Dilla University · Last updated March 2026