Virtual HPLC
Run a reversed-phase separation and watch the bands pull apart inside the column. Change the mobile phase, pH, flow, temperature and column, break something on purpose, and get the system suitability verdict at the end.
- Live chromatogram
- 8 fault scenarios
- System suitability
- Printable report
HPLC simulation report
The coloured bands inside the column are the four components separating as they travel. Watch them pull apart, then arrive at the detector as peaks.
Chromatogram
Integration
System suitability
This is a teaching simulation. Retention, efficiency and pressure follow standard chromatographic relationships, but the sample and the column are invented and no result here applies to a real product or method. Confirm all system suitability requirements in your own method and in USP <621> under your own licence.
Eight ways an HPLC run goes wrong
Set everything to a passing run first, then introduce one fault at a time. The point is to learn the signature of each failure, because the instrument never tells you which one you have.
- Mobile phase too strong. Everything elutes early and the critical pair closes up. The most common cause of a failed resolution.
- Worn column. Plates fall, tailing climbs, resolution follows. Push the column history slider up and watch it happen.
- Air bubble. Retention wanders and the baseline carries spikes.
- Leak after the pump. Retention runs late while pressure reads low — that combination is the diagnosis.
- Not equilibrated. Each chromatogram looks fine but the replicate %RSD fails.
- Carryover. A ghost peak at the main retention time, and impurities that read high.
- Tired lamp. Big peaks look normal while the small ones vanish into the noise and S/N fails.
- Wrong wavelength. Every area falls together and nothing else looks wrong.
Print each one and keep the set. Eight annotated chromatograms teach a new analyst more than any written procedure.
The chromatography behind the picture
Retention
Each component follows the linear solvent strength relationship, the model that underlies reversed-phase method development:
φ is the organic fraction, kw the retention in pure water and S how strongly that component responds to organic. Because the four components have different S values, changing the organic percentage changes their order, not just their speed — drag the organic slider and watch the main peak and Impurity B swap places. That is selectivity, and it is why a 5% error in mobile phase preparation destroys a separation.
The main peak is a weak acid with a pKa near 4.2. Raise the buffer pH past it and the molecule ionises, loses most of its retention and runs early. That is why methods specify the buffer pH, and why "about pH 4" is not a specification.
Efficiency and pressure
Plate height follows van Deemter, so there is an optimum flow: too slow and diffusion broadens the band, too fast and mass transfer cannot keep up. Move the flow slider and watch plate count peak and then fall. Backpressure comes from mobile phase viscosity, which is why methanol runs at a markedly higher pressure than acetonitrile at the same flow, and why a hot column runs at a lower one.
System suitability
At the end of the run the usual tests are applied: resolution of the critical pair, tailing factor, plate count, the %RSD of five replicate injections, signal-to-noise on the smallest peak and the capacity factor. The limits shown are common defaults — your method and USP <621> decide the real ones, so read them under your own licence.
Use Copy peak table and paste straight into the System Suitability calculator to work the numbers properly.
Training analysts on HPLC?
We run this as a live seminar, with your methods, your columns and your real failures.