Instrumentation of Affinity Chromatography

A typical setup for affinity chromatography consists of the following components:

Here's a colored diagram illustrating the instrumentation of Affinity Chromatography:

1. Pump:

• Ensures a consistent and controlled flow of the mobile phase (sample solution and elution buffer) through the affinity column. Modern systems utilize high-pressure pumps for faster runs.

2. Sample Injector:

• This allows for the precise introduction of the sample into the chromatography system.

• Auto-samplers can load multiple samples sequentially for high-throughput.

3. Affinity Column:

• The core of the system, it contains the immobilized ligand specific for the molecule of interest.

• The column material typically consists of agarose, Sepharose, or other polymeric beads covalently linked with the ligand.
  

4. Detectors:

• These monitor the eluted compounds from the column. Common detectors include:

  1. Ultraviolet (UV) Detector: Detects compounds based on their absorbance at specific wavelengths, useful for most organic molecules.

  2. Fluorescence Detector: For molecules that fluoresce or can be labeled with fluorescent tags.

  3. Conductivity Detector: Monitors changes in solution conductivity, useful when elution involves changes in ionic strength.

5. Fraction Collector:

• Collects separated compounds in distinct fractions for further analysis or use.

6. Temperature Control Unit:

• Some procedures require the temperature to be maintained at specific levels, either for stability or to optimize binding/elution conditions.

7. Recorder/Data System:

• Modern systems interface with computers that allow for real-time data acquisition, analysis, and visualization. This is essential for peak identification, quantification, and data storage.