5.2: Chromatography simulation
- Page ID
- 539653
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Get familiar with the simulator
- Open the Multi-Dimensional Separations Website (do not use Safari)
Figure \(\PageIndex{1}\): Access the Multi-Dimensional Separations Simulator. (CC-NC-BY-SA; Jack Malek 2025) - Click “login” on the top left (red arrow) .
- Username: jcm166
- password: Duke401L
- Select tools, then MultiSim LC [BETA]
- Click on Compounds to View options. Click “add compound.” In the Select Compounds popup window, deselect the default compounds and select caffeine, acetophenone, phenol, and pyridine
Figure \(\PageIndex{1}\): Copy and Paste Caption here. (Copyright; author via source) - In the Mobile Phase options, you can adjust the type of method (isocratic and gradient); and you can adjust the mobile phase conditions.
- Select ACN as Solvent B.
- "Mobile Phi" is the percent of Solvent B in the mobile phase mixture.
- In the stationary phase tab, you can change the column parameters. For our applications, we will be using the Agilent Zorbax SB C18 column, with 100mm x 4.6mm dimensions with 5μm particle size and 0.12 internal porosity. The other parameters can be ignored for now.
- In the Chromatographic Properties tab, set the flow rate to 1mL/min and keep the rest of the parameters as they are.
Guidance for Exploring the HPLC Simulator
- Start with a Simple Isocratic method: Understand %B and Retention
- Begin with an isocratic method in the Mobile Phase tab (constant %B).
- Set %B to a low value (e.g., 10%) and observe: Are the peaks well separated? What happens to retention time as %B decreases?
- Now increase %B (e.g., 50%, then 80%). How do the retention times change? Do peaks spread apart or crowd together?
- Introduce Gradients
- Try a gradient that increases %B slowly over time (e.g., 10% → 40% over 5 min). How do retention times shift and how to peak shapes change compared to isocratic runs? Does it change whether peaks are co-eluting?
- Compare a steep gradient (e.g., 10% → 90% over 5 min) with a shallow gradient (slow increase). Which gives better separation? Which is faster overall?