Independent guide to the UNICORN™ method editor

Method structure

A UNICORN™ method is built from phases, shown in the Method Outline and taken from the Phase Library. Each phase contains instructions whose length is set in the method base (for example volume, column volumes or time), or ended by a Watch condition. A typical bind–elute outline:

1. Equilibration — run start buffer until UV, conductivity and pH are stable; the volume needed depends on the column and buffers
2. Sample application — load sample at a defined flow rate and volume
3. Column wash — remove unbound material
4. Elution — gradient or step elution to release bound protein
5. Strip or cleaning — remove tightly bound material, using conditions allowed for the resin
6. Re-equilibration — return the column to starting conditions

Gradients

A Gradient instruction reads as “form a gradient to reach Target after Length”, starting from the current eluent composition. With a non-zero length the %B changes linearly; a length of zero gives a step. A program such as the one below is therefore a series of linear segments. Illustrative IEX program, not a recommended method:

Fractionation

Fractionation is set in the phase properties of the Sample Application, Column Wash and Elution phases, to the fraction collector or an outlet valve position. Collection can be at fixed volumes or signal-based, and a Watch instruction can start collection when a peak elutes. Level and slope criteria depend on detector path length, wavelength, sample concentration and baseline noise, so set them from a scouting run rather than a fixed mAU value, and account for the delay volume between the UV monitor and the collector. Available settings vary with the fraction collector.

Watch instructions and alarms

Watch instructions let the progress of a run depend on monitor signals. A Watch tests a signal (for example UV, conductivity, pH, pressure or flow) against a condition such as greater than, less than, slope, valley or peak maximum, and performs an action when the condition is met; Hold until waits for a condition before continuing, and Watch off cancels a watch. AStable signal test, for example, can end equilibration once a signal has settled.

Pressure protection is set with alarm limits in the system settings or the method. When an alarm limit is exceeded, UNICORN™ pauses the run and stops the pumps until the alarm is acknowledged; alarms act only when enabled. Take pressure limits from the column instructions, taking into account where the system measures pressure.

Saving and reusing methods

Save methods with descriptive names such as SEC_column_PBS_analytical_v1. Version your methods — when you optimise conditions, save as v2 rather than overwriting. For records, print the method’s text instructions and variables; choose which items to include in the Print dialog. Menus and options vary with UNICORN™ version and system configuration.

Sources and scope

Cytiva™, UNICORN™ 7.5 Method Manual (official manual 29452484 AA, accessed 2026-09-25) supports the phase, Gradient, Watch, alarm and print descriptions. Other UNICORN™ releases and instrument configurations may differ; check the manual for your version. If the direct manual link is unavailable, use Cytiva™’s official documentation finder and select the matching software version and instrument.