5
Figure 4: Connection of the outlet valve. How to set up a method
Setting up a method for two step purifi cation requires a logical progression of the target compound through the system adjusting the system in a systematic way to allow the most effective separation.
In this example the fi rst step a 1 ml ion exchange column and in the second step a 5 ml Desalting column was used. The sample pump with an air sensor is used for automatic sample application. A 2 ml sample loop was used for intermediate peak parking. Two separate methods were written for the two columns.
Figure 7: System diagram during collection of intermediate peak.
Figure 8: System Diagram for Desalting method during system wash.
After the ion exchange method, the desalting method starts. First, the system and the tubing are primed with buffer used for the desalting step. The conditioning is used to wash the system at a fl owrate of 2.5 ml/min with buffer A2 (Figure 8, Sections 1,2) for 1 minute. The column selection valve is in the bypass position (Figure 8, Section 3).
Figure 5: System diagram for the Ion Exchange method during the automatic sample injection.
First, the changes in the fl ow path are depicted in the fl ow scheme (Figure 5) and important aspects of the ion exchange method with automatic sample application are highlighted.
The sample is automatically applied via the sample pump. Therefore, the fl ow of the sample pump is set to 1 ml/min (Figure 5 Section 1) and the multi-injection valve switches to direct injection (Figure 5, Section 2). Make sure to choose the correct column in the beginning of the method (Figure 5, Section 3) and that the column is equilibrated with buffer A. During automatic sample injection the wait function is used to detect the end of the sample application via the air sensor (Figure 5, Section 4).
Figure 9: System Diagram of Desalting method.
After conditioning the system, the desalting step starts. Therefore, the column changes (Figure 9, Section 1). To inject the intermediate peak onto the desalting column the multi-injection valve is set to the inject position (Figure 9, Section 2). The eluting peak is precisely fractionated with the help of the threshold function (Figure 9, Section 3) using the fraction collector. At the end of the two-step purifi cation run the protein is purifi ed and collected in small fractions
To run both methods one after the other a sequence table is used. To enable this an additional washing and re-equilibration step must be run between samples for the Ion exchange column, but this is not detailed in this article.
Figure 6: System Diagram after sample application.
When the sample application is fi nished, the sample pump is turned off (Figure. 6, Section 1) and the system pump starts running (Figure 6, Section 2). The multi-injection valve is switching to the Manual Load position (Figure 6, Section 3). After an isocratic washing step, the elution starts after 5 ml with the beginning of the gradient.
To recognise the eluting peak two threshold functions are used. The thresholds are active during the gradient elution. Once a peak above 100 mAU is detected (threshold ‘Peak Storage Start’), the peak is rerouted to the sample loop. For this the outlet valve switches to the reinjection position (Figure 7, Section 1). The multi-injection valve was already in the manual load/reinjection position. If the peak is below 100 mAU (threshold ‘Peak Storage Stop’) the outlet valve switches back to collector. The annotation in the threshold function is used to mark the start and stop of the peak storage in the chromatogram. Please keep in mind to program an execution delay for the delay volume between the UV detector and the outlet valve for ‘Peak Storage Start’ and an execution delay for the delay volume between the UV detector and the multi-injection valve for “Peak Storage Stop”. At the end of the run purifi ed protein is stored in the injection loop and can be further purifi ed via the second column in the next step without manual interference.
This article explains the basic set up of a two-step separation using a basic FPLC system enabling higher yields and sample purity. The automated nature of this type of purifi cation allows the scientist to walk away from the system during the purifi cation process once initial method development is completed. All parameters of the separation model can be adjusted to maximise the yield using standard chromatographic method development and adjusting timings for the second separation.
This article is based on work carried out by Ulrika Krop and Kate Monks of Knauer Knauer Wissenschaftliche Geräte GmbH,
www.knauer.net.
The purifi cation was run and developed on a Knauer Multimethod FPLC system which was set up for bio-chromatography methods with an additional sample pump.
Verulam Scientifi c are the UK offi cial distributor and service provider for KNAUER, please direct any enquiries to enquiries@verulamscientifi
c.com or call 01234 381000.
WWW.LABMATE-ONLINE.COM
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