Purify biomolecules easily with IEC

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Purifying proteins easily using ion exchange chromatography (IEC)

Dear Reader,

Ion exchange chromatography (IEC) is one of the most important chromatographic separation methods in the biochemical laboratory for the purification of proteins. Learn more about the principle and application of the method below.

Get to know our compact AZURA® Bio Lab system, which is not only suitable for ion exchange chromatography, but also for the automation of most FPLC methods such as Affinity (AC), Hydrophobic Interaction (HIC), Ion Exchange (IEC), and Size Exclusion Chromatography (SEC).

Enjoy reading!

Basics

The IEC method

Since proteins have a specific charge density distribution due to the zwitterionic nature of their building blocks and their individual structure, they can interact in a differentiated manner with a charged stationary phase.

Ion exchange chromatography separates molecules based on their net charge. The stationary phase, an ion exchanger, is selected according to the charge characteristics of the target proteins. For molecules with a positive total charge, a cation exchanger (see Fig. 1) is used. The stationary phase itself is of the opposite charge (negative in this case).

Figure 1:
Cation exchanger (stationary phase, grey)
with symbolized protein molecules (light blue)

Procedure:

In the initial phase, charged proteins bind to the oppositely charged stationary phase at low ionic strength of the binding buffer. After a washing step, the target protein is eluted from the column by a high salt concentration. The ions of the elution buffer compete with the bound proteins for the charged binding sites of the column matrix. Weakly charged proteins that bind less strongly to the stationary phase elute at low salt concentrations, while stronger charged proteins leave the column only at higher salt concentrations.

Ion exchange chromatography has a high binding capacity and is therefore often used for protein purification in the early "capture" step of multi-step purification methods.

For a reproducible IEC, an FPLC system is needed that can precisely generate buffer gradients.

Application

Separation of model proteins by IEC

The three substances α-chymotrypsinogen A, cytochrome C and lysozyme are eluted from the cation exchange phase by means of a salt gradient. The blue trace shows the elution profile (chromatogram) measured on the basis of the UV signal at a wavelength of 280 nm. The red trace shows the conductivity signal. The linear increase in conductivity due to the programmed salt gradient can be clearly seen.

This application was made, using an AZURA® Bio Lab Purification system with a cation exchanger column.

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FPLC all-rounder

The AZURA® Bio Lab Purification system for FPLC methods such as IEC features a high-precision binary gradient pump for flow rates up to 50 ml/min with two selectable buffers per channel.
The system with its metal-free flow path meets demanding FPLC requirements and is suitable for Affinity (AC), Hydrophobic Interaction (HIC), Ion Exchange (IEC), and Size Exclusion Chromatography (SEC).

Compatible with all popular columns
Variable wavelength UV detector
Conductivity monitor
Precise / robust fraction collector
Intuitive PurityChrom® software

The AZURA® Bio Lab system is available at a special price for a limited time. Contact us.

Inquiries to sales@knauer.net or

Learn more about this FPLC system ...

AZURA^® Bio Lab system

AZURA Bio Lab purification system for FPLC

*Technical data are subject to change without notice.
Prices may vary by country and do not include taxes,
customs duties or delivery.

Training / Workshop

FPLC training

Nov. 14, 2018 | 09:00 - 17:00

In theoretical as well as practical parts of this course users will learn the basics of FPLC and will also get useful tips for a successful purification of biomolecules.

Target audience: Users who want to purify proteins using FPLC. The course is suitable for beginners and for users with basic knowledge.

This course date is planned for users who speak German, but we can also offer it in English on request.

For questions and registration, please contact us via academy@knauer.net

More about the FPLC training ...

Oliver Gültzow
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On behalf of the KNAUER team I wish you a nice day!