5.4 Laboratory Practical: Agarwood Sampling, Extraction, and Chromatographic Profiling

Here’s a detailed Laboratory Practical Module for Agarwood (Oud) research or training programs, focusing on sampling, extraction, and chromatographic profiling:

Duration: 1–2 days
Target Participants: Researchers, lab technicians, students, plantation managers
Objectives:

  1. Learn proper sampling techniques for resin and wood.
  2. Conduct extraction of volatile compounds for analysis.
  3. Perform chromatographic profiling using GC-MS and HPLC.
  4. Interpret data for quality assessment and marker identification.

1. Sample Collection

Materials: Agarwood resin, chips, or induced heartwood sections; gloves; sterilized tools; labeled containers.

Procedure:

  1. Select representative trees or resin deposits (note tree ID, age, induction method).
  2. Collect resin chips or small heartwood sections (avoid excessive damage).
  3. Record metadata: tree age, resin grade, location, date of collection.
  4. Store samples in airtight containers, in cool, dark conditions to prevent degradation.

Tips:

  • Use clean, sterilized tools to avoid contamination.
  • Label all samples clearly for traceability.

2. Sample Preparation and Extraction

Materials: Grinder, solvent (ethanol, methanol, or hexane), Soxhlet/sonicator, rotary evaporator, glassware.

Procedure:

A. Powdering

  1. Air-dry or oven-dry agarwood chips at 40–50°C.
  2. Grind to a fine powder for maximum surface area.

B. Solvent Extraction

  1. Weigh ~2–5 g of powdered sample.
  2. Add appropriate solvent (1:10 w/v ratio).
  3. Sonicate for 30–60 min or macerate for 24–48 h.
  4. Filter the extract through Whatman No.1 paper.
  5. Concentrate extract using a rotary evaporator at low temperature.

C. Essential Oil Extraction (optional)

  • Use steam distillation for volatile oil collection if required for GC-MS analysis.

3. Chromatographic Profiling

A. Gas Chromatography-Mass Spectrometry (GC-MS)

Purpose: Identify volatile sesquiterpenes.

Procedure:

  1. Dissolve extract in suitable solvent (hexane or ethanol).
  2. Inject ~1–2 µL into GC-MS system.
  3. Use capillary column for separation (e.g., DB-5).
  4. Record retention times and mass spectra.
  5. Compare spectra with reference libraries (NIST, Wiley) for compound identification.

Output: Sesquiterpene profile and relative abundance of key aromatic compounds.

B. High-Performance Liquid Chromatography (HPLC)

Purpose: Quantify chromones and semi-volatile compounds.

Procedure:

  1. Dissolve extract in HPLC-grade methanol.
  2. Filter through 0.45 µm syringe filter.
  3. Inject 10–20 µL into HPLC system with C18 reverse-phase column.
  4. Use gradient elution (water + acetonitrile or methanol).
  5. Detect at 254–280 nm (UV) for chromones.
  6. Compare retention times with standard compounds for identification and quantification.

Output: Chromone content, chemical fingerprint for quality assessment.

4. Data Analysis

  1. Identify key sesquiterpenes and chromones for high-grade resin markers.
  2. Calculate relative abundance or peak area to compare samples.
  3. Record findings in standardized lab sheets or digital logs.
  4. Optional: Use chemical profiles to assign quality grade (A–D) for resin or oil.

5. Safety and Best Practices

  • Wear gloves, lab coat, and eye protection.
  • Work in fume hood when handling volatile solvents.
  • Dispose of solvents and waste according to safety regulations.
  • Label all chemicals and samples carefully.

6. Learning Outcomes

Participants will be able to:

  • Collect representative agarwood samples safely and systematically.
  • Extract volatile and semi-volatile compounds efficiently.
  • Perform GC-MS and HPLC analysis for chemical profiling.
  • Interpret chromatograms for quality assessment, genotype selection, and marker identification.