Chromatography
Optimising two-step purifi cation: Key considerations for sample pump setups Ulrika Krop and Kate Monks on behalf of Knauer and Verulam Scientifi c
When purifying proteins and other actives in production or research environments the aim is to achieve the purest possible compound with the highest yield to ensure the preparation is cost effective. FPLC (Fast Protein Liquid Chromatography) uses the principles of chromatography to separate the target molecule from the remaining ingredients or compounds present in the reaction mixture. A good separation allows for a fraction of the chromatographed material to be collected increasing yields and removing contaminants. However, when you are trying to separate closely related compounds then this fraction may have to be separated using a different separation mechanism to ensure the best purity. This can mean using two FPLC systems with different chromatography set ups which can slow production and increase costs. This article shows that setting up an FPLC system and using Two Step Purifi cation can enable a single system to produce high yields and purities with a single run.
What is two step purifi cation?
Two step purifi cation is a special multicolumn chromatography solution. Two independent methods, each with their associated specifi c column, are used to realise the purifi cation of the target molecule without manual interference. The principle here is that the protein sample is applied on the fi rst column. During elution of the protein, the protein peak is detected triggering the collection of the eluted protein in a storage loop or storage vessel/container. The protein is then automatically applied on the second column to further enhance the quality and or purity of the purifi ed protein. Several systems set ups can be used to automate the purifi cation. In this Technote a two-step purifi cation with the sample pump set up is discussed.
Set up with sample pump: what do you need?
The FPLC system is set up as in Figure 1. A sample pump or simple injection valve is used to introduce the samples to the separation system. Gradient pumps are used with fl ow rates of up to 50 mL/min. Detection in this article is with UV detection and a conductivity monitor is used to monitor the salt gradient. Fraction collection can be undertaken with either a valve or a fraction collector for more smaller volumes. The essential module for two step separation is the column selection valve which enables the system to change between stationary phase / separation mechanism without the intervention of the scientist.
Figure 2: Flow scheme for the sample pump set up for two step purifi cation.
The column selection valve is placed between the multi-injection valve and the UV detector. The column outlet port (Col) of the multi-injection valve is connected via a PEEK capillary with the inlet port (IN) of the column selection valve. The outlet port (OUT) is connected to the UV detector fl ow cell, which in turn is connected to the conductivity monitor. The columns are installed according to Figure 3.
Figure 3: Connection of the column selection valve. Figure 1: Typical components of an FPLC system for automated 2 step separation.
The sample pump is used to apply the sample on the fi rst column. The peak eluting from the fi rst column is collected in the sample loop of the injection valve and redirected to the second column. This set ups allows the loading of large sample volumes and minimises the risk of cross contamination during fi rst peak collection because the sample loop is only used for the eluting peak. The injection of small sample volumes is not supported (Figure 2).
The outlet valve is placed between the conductivity monitor and the fraction collector. The conductivity monitor is connected via the PEEK capillary with the middle port of the outlet valve. Port 1 of the outlet valve goes by default to the fraction collector or waste container. Port 3 to 8 can be used for the collection of large fractions. Port 2 (reinjection) of the outlet valve is connected to the syringe port (Syr) of the multi-injection valve (Figure 4). The syringe port is no longer accessible for sample injection.
INTERNATIONAL LABMATE - APRIL 2025
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