Below is a training-grade analytical methods module (GC, HPLC, MS) designed specifically for Supercritical Fluid Extraction (SFE) programs and aligned with QbD, GMP, and scale-up validation. This is suitable for Oud Academia / SFE Technology Training Courses, including perfumery, nutraceutical, pharmaceutical, and environmental applications.
Analytical Methods for SFE Products
(GC, HPLC, MS – Principles, Selection, and Best Practices)
1. Role of Analytical Methods in SFE
Analytical chemistry in SFE answers three critical questions:
- What compounds were extracted? (identity)
- How much of each compound is present? (quantity)
- Is the extract consistent, safe, and compliant? (quality)
These methods support:
- Process optimization
- Fractionation decisions
- QbD implementation
- GMP batch release
- Commercial valuation
2. Method Selection Matrix
| Compound Type | Best Method | Why |
|---|---|---|
| Volatile / semi-volatile | GC-MS / GC-FID | High resolution, sensitivity |
| Non-volatile / thermolabile | HPLC / UPLC | No thermal degradation |
| Trace / unknown compounds | LC-MS / GC-MS | Structural identification |
| Fingerprinting / QC | GC-FID / HPLC-DAD | Reproducibility |
| Adulteration detection | GC-MS, FTIR, NMR | Pattern recognition |
3. Gas Chromatography (GC)
3.1 What GC Is Best For
- Essential oils
- Terpenes & sesquiterpenes
- Agarwood volatiles
- Fragrance ingredients
3.2 GC Detectors
| Detector | Use |
|---|---|
| FID | Quantitative profiling |
| MS | Compound identification |
| TCD | Limited use in SFE |
📌 GC-FID = Quantification
📌 GC-MS = Identification
3.3 Typical GC Conditions (SFE Extracts)
| Parameter | Range |
|---|---|
| Column | HP-5MS / DB-5MS |
| Carrier gas | Helium |
| Injection | Split / splitless |
| Oven program | 40–300 °C |
| Sample prep | Dilution in hexane / DCM |
3.4 GC Strengths & Limitations
✅ High resolution
✅ Excellent for volatile profiling
❌ Not suitable for non-volatile compounds
❌ Thermal degradation risk
4. High-Performance Liquid Chromatography (HPLC)
4.1 What HPLC Is Best For
- Polyphenols
- Chromones (agarwood)
- APIs
- Nutraceutical actives
- Heavy fragrance components
4.2 Common HPLC Detectors
| Detector | Application |
|---|---|
| UV-Vis / DAD | Quantitative, routine QC |
| FLD | High sensitivity |
| RID | Sugars, limited use |
| MS | Identification & trace analysis |
4.3 Typical HPLC Conditions
| Parameter | Range |
|---|---|
| Column | C18 reverse phase |
| Mobile phase | Water + acetonitrile / methanol |
| Gradient | Yes (most botanicals) |
| Flow rate | 0.5–1.0 mL/min |
| Detection | 210–280 nm (varies) |
4.4 HPLC Strengths & Limitations
✅ Handles non-volatile compounds
✅ GMP-friendly
❌ Lower resolution for volatiles
❌ Requires solvents
5. Mass Spectrometry (MS)
5.1 Role of MS
MS is not a standalone technique — it is a detector providing:
- Molecular weight
- Fragmentation pattern
- Structural clues
5.2 GC-MS vs LC-MS
| Feature | GC-MS | LC-MS |
|---|---|---|
| Sample volatility | Required | Not required |
| Ionization | EI (hard) | ESI/APCI (soft) |
| Fragmentation | High | Controlled |
| Libraries | Extensive | Limited |
| Use | Volatiles | Complex actives |
5.3 MS in SFE QbD
- Identify fraction-specific compounds
- Confirm target CQAs
- Detect degradation or co-extraction
- Support impurity profiling
6. Sample Preparation for Analysis
| Step | GC | HPLC |
|---|---|---|
| Dilution | Non-polar solvent | Polar solvent |
| Filtration | Optional | Required |
| Derivatization | Sometimes | Rare |
| Internal standard | Recommended | Required (GMP) |
📌 Poor sample prep = poor data
7. Quantification Strategies
Acceptable for GMP
✔ External calibration
✔ Internal standards
✔ Linearity (R² ≥ 0.995)
Not Acceptable
🚫 Area % only
🚫 Single-point calibration
🚫 Unvalidated methods
8. Method Validation Parameters
| Parameter | Requirement |
|---|---|
| Accuracy | 95–105% |
| Precision | RSD ≤ 5% |
| LOD / LOQ | Documented |
| Linearity | Verified |
| Robustness | Assessed |
9. Analytical Challenges in SFE Extracts
⚠ Co-extracted waxes
⚠ Matrix effects (LC-MS)
⚠ Fraction overlap
⚠ Column fouling
⚠ Detector saturation
➡ Solution: Fraction-resolved analysis
10. Application-Specific Notes
Perfumery & Agarwood
- GC-MS for sesquiterpenes
- HPLC-MS for chromones
- Ratio profiling > total yield
Nutraceuticals
- HPLC-DAD / LC-MS
- Marker-based standardization
Pharmaceuticals
- LC-MS/MS
- Full impurity profiling
11. Data Integrity & Documentation
✔ Raw data archived
✔ Calibration records
✔ SOP-based workflows
✔ Audit-ready reports
12. Key Takeaways
SFE without analytics is blind extraction.
- GC = volatile profiling
- HPLC = non-volatile quantification
- MS = identity confirmation
- Validation = credibility
- Profiling > yield
Next Modules I Can Prepare
- GC-MS method SOP for agarwood SFE
- HPLC fingerprinting template
- GMP analytical validation protocol
- Fraction-resolved analytical reporting format
- Investor-friendly analytics dashboard
Just tell me which one you want next.