High Performance Liquid Chromatography (HPLC Simulator) of proteins

General Aim of HPLC Simulator

To separate and detect amino acids in a tissue using HPLC simulator.

 

Method

HPLC - Reversed phase column chromatography.

Theoretical Background / Context

HPLC in analytical chemistry is a chromatographic method used to separate the components of a mixture. 

An HPLC simulator is a valuable educational tool that replicates these processes virtually, allowing learners and researchers to understand instrument design, functionality, and chromatographic principles without requiring a physical lab setup.

The typical design of an HPLC usually involves these elements. 

1. A filtered solvent reservoir for mobile phase 
2. Degassing system to prevent bubbles in the mobile phase
3. A pump to maintain a constant flow of mobile phase in HPLC despite the backpressure applied by the resistance of flow through the packed column.
4. An injector to inject samples. This can be manual or automatic.
5. A column (solid phase). Length, internal diameter, and stationary phases in HPLC are to be chosen.
6. A detector/data system to plot the chromatogram. includes UV, diode, and fluorescent detectors.

 

• Samples are forced to flow under high pressure through the column (solid phase). Solvents (mobile phase) allow the flow of the sample into the column, where it is separated into its components according to their interaction with the solid phase. 
• Components are detected and recorded on a chromatogram. The resolution of various components is determined by the extent of interaction between the solute components and the stationary phase. 
• In normal phase chromatography, the mobile phase is nonpolar while the stationary phase is polar. In reversed-phase chromatography, the mobile phase is polar while the stationary phase is nonpolar.

Principle of Work | HPLC Simulator

Method development for an HPLC online training experiment usually follows these steps:

1. Choosing the mode. In a practical HPLC simulator online, we choose the reversed phase chromatography.

2. Choosing the column and column packing dimensions. For example in online HPLC, it is suitable to use a reversed-phase column Agilent ZORBAX Eclipse Plus C18 columns and recommended guard cartridges. 

3. Choosing the stationary phase chemistry.

4. Choosing the mobile phase solvents and their pH.

5. Running initial isocratic or gradient experiments to define boundary conditions and optimize the experimental conditions.

In the High Performance Liquid Chromatography experiment, analysis of amino acids in a tissue sample is done. ----

In chromatography simulation,The general steps for Amino Acid analysis include:

1. Preparation of HPLC mobile phases: filtration with vacuum pump.

2. Preparation of amino acid standards.

3. Preparation of Internal Standard (IS) stock solution.

4. Tissue sample preparation: sample is minced and prepared using perchloric acid for protein separation, then lyophilized. The dried protein then undergoes acid hydrolysis using 6 M HCL.  

5. Online ortho-phthalaldehyde/9-fluorenylmethyl chloroformate (OPA/FMOC) precolumn derivatization is performed.

6. Setting the parameters of detection in an online chromatography lab.