Using the EZLC Modeler for Cannabinoid Separations–Part 1: Optimization of an Existing Method
24 Mar 2025Developing analytical methods for the analysis of cannabinoids is no easy task. Along with keeping up with changing regulations, there is also a constant emergence of new cannabinoids, with current highlights focusing on THC isomers. It can be challenging to keep methods up to date when needing to add these new compounds to testing scopes. Traditional method development for cannabinoids includes determining the appropriate column chemistry and dimensions, organic modifiers, buffers, and flow rates. Often this means purchasing primary and secondary standards, as well as performing method development and validating a new method. Production-based labs do not always have the bandwidth to perform the necessary method development to add in these new compounds.
In early 2024, Restek released a new library in our EZLC chromatogram modeler to assist with the separation and optimization of methods for cannabinoids, with the goal to help those looking to develop or optimize their cannabinoid separations by allowing users to virtually model separations without needing to enter the lab.
In this blog series, we will discuss how to use EZLC modeling software to develop a method for the analysis of cannabinoids by LC-UV.
Accessing the EZLC Modeler:
To access EZLC software, go to www.restek.com/resource-hub. Click the icon for EZ Suites & Calculators and select the Pro EZLC Chromatogram Modeler. A username and password will need to be created. Note: This is the same username and log in as the Restek website, if you do not have one, a new one will need to be created. Once created, a new screen will appear showing a drop-down menu for compound class/library selection (Figure 1).
Figure 1: EZLC screen
Part one of this series will discuss the optimization of an existing method (Figure 2). This step-by-step guide will walk you through how to use EZLC software for this task.
Figure 2: 16 Cannabinoids on Raptor ARC-18 2.7 μm by LC-UV
| Peaks | tR (min) | |
|---|---|---|
| 1. | Cannabidivarinic acid (CBDVA) | 1.877 |
| 2. | Cannabidivarin (CBDV) | 2.086 |
| 3. | Cannabidiolic acid (CBDA) | 2.592 |
| 4. | Cannabigerolic acid (CBGA) | 2.750 |
| 5. | Cannabigerol (CBG) | 2.912 |
| 6. | Cannabidiol (CBD) | 3.084 |
| 7. | Tetrahydrocannabivarin (THCV) | 3.391 |
| 8. | Tetrahydrocannabivarinic acid (THCVA) | 4.279 |
| Peaks | tR (min) | |
|---|---|---|
| 9. | Cannabinol (CBN) | 4.609 |
| 10. | Cannabinolic acid (CBNA) | 5.437 |
| 11. | Δ9-Tetrahydrocannabinol (Δ9-THC) | 5.815 |
| 12. | Δ8-Tetrahydrocannabinol (Δ8-THC) | 6.002 |
| 13. | Cannabicyclol (CBL) | 6.916 |
| 14. | Cannabichromene (CBC) | 7.263 |
| 15. | Tetrahydrocannabinolic acid A (THCA-A) | 7.612 |
| 16. | Cannabichromenic acid (CBCA) | 8.510 |
| Column | Raptor ARC-18 (cat.# 9314A65) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Dimensions: | 150 mm x 4.6 mm ID | ||||||||||||
| Particle Size: | 2.7 µm | ||||||||||||
| Pore Size: | 90 Å | ||||||||||||
| Guard Column: | Raptor ARC-18 EXP guard column cartridge 2.7 µm (cat.# 9314A0250) | ||||||||||||
| Temp.: | 30 °C | ||||||||||||
| Standard/Sample | |||||||||||||
| Tetrahydrocannabivarin (cat.# 34100) | |||||||||||||
| Cannabidiolic acid (CBDA) (cat.# 34094) | |||||||||||||
| Cannabichromene (CBC) (cat.# 34092) | |||||||||||||
| Cannabigerol (CBG) (cat.# 34091) | |||||||||||||
| delta-9-Tetrahydrocannabinolic acid A (THCA-A) | |||||||||||||
| delta-8-Tetrahydrocannabinol ( | |||||||||||||
| delta-9-Tetrahydrocannabinol ( | |||||||||||||
| Cannabinol (CBN) (cat.# 34010) | |||||||||||||
| Cannabidiol (CBD) (cat.# 34011) | |||||||||||||
| Compounds not present in these mixes were obtained separately. | |||||||||||||
| Diluent: | 25:75 Water:methanol | ||||||||||||
| Conc.: | 50 µg/mL | ||||||||||||
| Inj. Vol.: | 5 µL | ||||||||||||
| Mobile Phase | |||||||||||||
| A: | Water, 5 mM ammonium formate, 0.1% formic acid | ||||||||||||
| B: | Acetonitrile, 0.1% formic acid | ||||||||||||
|
| Detector | UV/Vis @ 228 nm |
|---|---|
| Instrument | HPLC |
| Notes | Standard cat.# 34093 was used to produce this chromatogram, but has since been discontinued. For assistance choosing a replacement for this application, contact Restek Technical Service or your local Restek representative. |
Once logged into the EZLC modeler, use the drop-down arrow for Compound Class to select Cannabinoids. Choose compounds by either scrolling down the list or by typing the name or CAS number into the search bar. After the compounds are selected, Target All must be checked in order to ensure the software optimizes the separation. Prior to clicking Generate Model, a Phase must be selected.
Figure 3: Selected compounds for EZLC modeling
The software will generate a set of conditions that are catered to the selected compound list, including column dimension, ammonium formate concentration, %B, temperature, and flow rate. While the program can optimize the method automatically using the Optimize Gradient Slope function, the following is an example of manual optimization.
It is important to note that inputting your instruments dwell and extra column volume is necessary for retention times comparison from modeled data to translate to your specific instrument. If unsure how to calculate, check out our help file: Pro EZLC Chromatogram Modeler Help.
Figure 4: EZLC conditions: default solve conditions (left) and manually optimized conditions (right)
By manually changing the temperature, %B, and flow rate, the software models a shorter cycle time and provides a solution that reduces solvent consumption.
Figure 5: Comparison of 16 cannabinoids existing method versus modeler optimized conditions
| Peaks | Modeled (min) | Experimental (min) | Difference (sec) | |
|---|---|---|---|---|
| 1. | Cannabidivarinic acid (CBDVA) | 1.52 | 1.49 | 1.80 |
| 2. | Cannabidivarin (CBDV) | 1.68 | 1.64 | 2.40 |
| 3. | Cannabidiolic acid (CBDA) | 2.04 | 2.00 | 2.40 |
| 4. | Cannabigerolic acid (CBGA) | 2.12 | 2.12 | 0.00 |
| 5. | Cannabigerol (CBG) | 2.26 | 2.21 | 3.00 |
| 6. | Cannabidiol (CBD) | 2.41 | 2.32 | 5.40 |
| 7. | Tetrahydrocannabivarin (THCV) | 2.62 | 2.53 | 5.40 |
| 8. | Tetrahydrocannabivarinic acid (THCVA) | 3.23 | 3.14 | 5.40 |
| Peaks | Modeled (min) | Experimental (min) | Difference (sec) | |
|---|---|---|---|---|
| 9. | Cannabinol (CBN) | 3.45 | 3.33 | 7.20 |
| 10. | Cannabinolic acid (CBNA) | 4.01 | 3.91 | 6.00 |
| 11. | Δ9-Tetrahydrocannabinol (Δ9-THC) | 4.30 | 4.11 | 11.40 |
| 12. | Δ8-Tetrahydrocannabinol (Δ8-THC) | 4.45 | 4.24 | 12.60 |
| 13. | Cannabicyclol (CBL) | 5.07 | 4.83 | 14.40 |
| 14. | Cannabichromene (CBC) | 5.28 | 5.02 | 15.60 |
| 15. | Tetrahydrocannabinolic acid A (THCA-A) | 5.49 | 5.30 | 11.40 |
| 16. | Cannabichromenic acid (CBCA) | 6.07 | 5.87 | 12.00 |
| Column | Raptor ARC-18 (cat.# 9314A65) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Dimensions: | 150 mm x 4.6 mm ID | ||||||||||||
| Particle Size: | 2.7 µm | ||||||||||||
| Pore Size: | 90 Å | ||||||||||||
| Temp.: | 35 °C | ||||||||||||
| Standard/Sample | |||||||||||||
| Cannabinoids acids 7 standard, 1000 µg/mL, acetonitrile with 1% DIPEA and 0.05% ascorbic acid, 1 mL/ampul (cat.# 34144) | |||||||||||||
| Cannabinoids neutrals 9 standard, 1000 µg/mL, P&T methanol, 1 mL/ampul (cat.# 34132) | |||||||||||||
| Diluent: | Acetonitrile | ||||||||||||
| Conc.: | 50 ppm | ||||||||||||
| Inj. Vol.: | 5 µL | ||||||||||||
| Mobile Phase | |||||||||||||
| A: | Water, 4 mM ammonium formate, 0.1 % formic acid | ||||||||||||
| B: | Acetonitrile, 0.1 % formic acid | ||||||||||||
|
| Detector | UV/Vis @ 228 nm |
|---|---|
| Flow Cell Size: | 500 nL |
| Instrument | Waters ACQUITY UPLC H-Class |
| Sample Preparation | Working standard was prepared in a 2 mL, 9 mm amber vial (cat. 21142) by diluting 50 µL of each standard into 900 µL acetonitrile and capped with a 9 mm short screw cap (cat. 24497). |
By using EZLC modeling software for cannabinoids, a set of conditions was generated in minutes to optimize an existing method. This not only saved development resources, but it also reduced instrument run time and solvent consumption (Table 1).
Table 1: Comparison of an existing method versus an optimized method
| Conditions | Existing | Optimized |
| Analytical Column: | Raptor ARC-18 150 x 4.6 mm , 2.7 µm | Raptor ARC-18 150 x 4.6 mm , 2.7 µm |
| Flow (mL/min): | 1.5 | 1.7 |
| Temperature (°C): | 30 | 35 |
| % Mobile Phase A: | 25 (Water, 5 mM ammonium formate, 0.1% formic acid) | 23 (Water, 4 mM ammonium formate, 0.1% formic acid) |
| % Mobile Phase B: | 75 (Acetonitrile, 0.1% formic acid) | 77 (Acetonitrile, 0.1% formic acid) |
| Solvent Usage (mL): | 15 | 12 |
| Cycle Time (min): | 10 | 7 |
Tune in for part two of this series discussing the effects of ammonium formate concentrations on cannabinoid separations.
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