Select LocaleEvery lab wants to run the most efficient methods for their analyses to increase sample throughput and increase revenue but it’s important when choosing a method to know your instruments’ capabilities to set yourself up for success. As the market evolves and becomes more advanced, traditional HPLC methods that may have long instrument run times or high solvent usage may be traded in for more advanced and economical methodologies.
UHPLC (ultra-high-performance liquid chromatography) is a term used in chromatography to describe analytical columns and instruments. When referring to analytical columns the term typically indicates shorter length and internal diameter columns. UHPLC instrumentation differs from HPLC instrumentation not only through increased maximum pressure capabilities, but also more importantly in that these systems have been optimized to reduce extra column volume, allowing columns to approach their maximum intrinsic efficiencies. If this is the first time hearing of extra column volume and the effects it can have on analytical separations, check out this technical article for some examples specific to potency analysis.
UHPLC methods can be instrumental in reducing analytical run times while achieving desired resolution of analytes, but to use these methods it takes some vetting of instrumentation. One of the biggest contributors to extra column volume that results in unforgiving band broadening (widening of analyte peak) is flow cell volume. Analytical flow cells come in many different volumes ranging from approximately 500 nL to 10 µL. If using a UHPLC method, especially with columns containing sub-2 micron particles or with an internal diameter of 2.1 mm, having a flow cell on the smaller side of the range (i.e. <1 µL) will reduce peak dispersion and allow the user to achieve superior resolution.
As the analyte list for required cannabinoids continues to expand, UHPLC methods are a valuable resource that can be used to keep the analysis time at a minimum while potentially also cutting back on solvent usage when compared to traditional HPLC methods. Some states, such as Colorado, are now requiring that total THC content include delta-8-THC, delta-9-THC, exo-THC, and delta-10-THC for reporting potency test results. To address these requirements, a UHPLC method to detect 18 cannabinoids that includes both delta-10-THC epimers and exo-THC has been developed.
| Peaks | tR (min) | |
|---|---|---|
| 1. | Cannabidivarinic acid (CBDVA) | 1.039 |
| 2. | Cannabidivarin (CBDV) | 1.160 |
| 3. | Cannabidiolic acid (CBDA) | 1.449 |
| 4. | Cannabigerolic acid (CBGA) | 1.550 |
| 5. | Cannabigerol (CBG) | 1.638 |
| 6. | Cannabidiol (CBD) | 1.719 |
| 7. | Tetrahydrocannabivarin (THCV) | 1.870 |
| 8. | Tetrahydrocannabivarinic acid (THCVA) | 2.361 |
| 9. | Cannabinol (CBN) | 2.557 |
| Peaks | tR (min) | |
|---|---|---|
| 10. | Cannabinolic acid (CBNA) | 2.923 |
| 11. | Exo-tetrahydrocannabinol (exo-THC) | 3.103 |
| 12. | d9-Tetrahydrocannabinol (9-THC) | 3.201 |
| 13. | d8-Tetrahydrocannabinol (8-THC ) | 3.310 |
| 14. | (6aR,9S)-delta-10-Tetrahydrocannabinol (9S-10-THC) | 3.719 |
| 15. | (6aR,9R)-delta-10-Tetrahydrocannabinol (9R-10-THC) | 3.916 |
| 16. | Cannabichromene (CBC) | 4.042 |
| 17. | δ-9-Tetrahydrocannabinolic acid-A (THCA) | 4.369 |
| 18. | Cannabichromenic acid (CBCA) | 4.838 |
| Column | Raptor ARC-18 (cat.# 931421E) | ||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Dimensions: | 100 mm x 3 mm ID | ||||||||||||||||||||||||
| Particle Size: | 1.8 µm | ||||||||||||||||||||||||
| Pore Size: | 90 Å | ||||||||||||||||||||||||
| Guard Column: | Raptor ARC-18 EXP guard column cartridge 5 mm, 3.0 mm ID, UHPLC µm (cat.# 9314U0253) | ||||||||||||||||||||||||
| Temp.: | 30 °C | ||||||||||||||||||||||||
| Standard/Sample | Cannabinoids acids 7 standard (cat.# 34144) | ||||||||||||||||||||||||
| Cannabidivarin (CBDV) (cat.# 34123) | |||||||||||||||||||||||||
| Cannabigerol (CBG) (cat.# 34091) | |||||||||||||||||||||||||
| Cannabidiol (CBD) (cat.# 34011) | |||||||||||||||||||||||||
| Tetrahydrocannabivarin (THCV) (cat.# 34100) | |||||||||||||||||||||||||
| Cannabinol (CBN) (cat.# 34010) | |||||||||||||||||||||||||
| d9-Tetrahydrocannabinol (d9-THC) (cat.# 34067) | |||||||||||||||||||||||||
| d8-Tetrahydrocannabinol (d8-THC) (cat.# 34090) | |||||||||||||||||||||||||
| Cannabichromene (CBC) (cat.# 34092) | |||||||||||||||||||||||||
| Compounds not present in these mixes were obtained separately. | |||||||||||||||||||||||||
| Diluent: | 25:75 Water:acetonitrile | ||||||||||||||||||||||||
| Conc.: | 50 µg/mL | ||||||||||||||||||||||||
| Inj. Vol.: | 1 µL | ||||||||||||||||||||||||
| Mobile Phase | |||||||||||||||||||||||||
| A: | 25:75 Water:acetonitrile, 2.5 mM ammonium formate, 0.1% formic acid | ||||||||||||||||||||||||
| B: | Methanol, 0.1% formic acid | ||||||||||||||||||||||||
|
| Detector | UV/VIS @ 228 nm |
|---|---|
| Instrument | UHPLC |
| Sample Preparation | Standards were prepared in a 2 mL screw-thread vial (cat.# 21143) and capped with a short-cap, screw-vial closure (cat.#24498). |
| Notes | Flow cell = 500 nL |
The total cycle time of this analysis is 6 minutes allowing for high throughput of samples. It is important to note that this chromatogram was obtained using a 500 nL flow cell and an LC otherwise optimized to reduce extra column volume.
When considering switching to a UHPLC method, it’s important to know the details of instrumentation in order to set yourself up for success. If the flow cell volume is too large for UHPLC analyses it’s good to note that vendors will oftentimes offer flow cells in a variety of different volumes in order to best to fit your needs. If you need help assessing your instrument’s capabilities please reach out to Restek Technical Support to get our help to understand your instrument capabilities and a good starting place of a UHPLC column dimension selection.
