Set Up the Method :

Open Unicorn or the software interface used to control the AKTA Purifier.

Define the Gradient or Elution Program :

Set the flow rate and gradient based on your purification protocol (this depends on the sample and buffer conditions). For example, a linear gradient might be used, but the exact settings depend on your specific method.

Configure the UV Detection :

Go to the UV detector settings to set the parameters for absorbance measurement :

Wavelength : Set the wavelength to 280 nm (this is typical for detecting proteins or nucleic acids).

Range : Set the range of the UV detector, often 0–2.000 AU (adjust based on expected peak sizes).

Sensitivity : Adjust the sensitivity of the UV detector. You want the sensitivity to be high enough to detect small absorbances but not too high to cause noise.

Configure the Fraction Collection Trigger :

In the Method Editor, navigate to the Fraction Collection settings.

Enable Fraction Collection : Make sure fraction collection is turned on.

Trigger Based on UV1 : Set the fractionation trigger based on UV1.

Set Trigger Level : Set the trigger level to 10 mAU (this will trigger fractionation once the UV absorbance exceeds 10 mAU).

Fractionation Parameters :

Set up your fractionation parameters under the Fraction Collector section :

Fraction Size : Define the fraction size (e.g., 1 mL, 2 mL, etc.) depending on the volume of your elution.

Trigger Condition : Set the condition to trigger fractionation when UV1 exceeds 10 mAU.

Check the Rest of the Method :

Ensure that the method includes proper settings for the pump, gradient (if applicable), pressure limits, and temperature settings (if applicable).

Also, verify that your column and sample conditions are optimal for the UV detection at 280 nm.

Run the Method :

Once everything is set up, run the method and monitor the process to ensure that fraction collection is triggered when the UV absorbance exceeds 10 mAU at 280 nm.

During the run, you should see fractions collected automatically when the threshold is reached, and you can monitor the UV trace for confirmation.