Understanding Chromatography: Principles, Phases, and Methodology

Understanding Chromatography

Chromatography is used in column chromatography. The y-axis represents the intensity of the signal provided by the detector, and the x-axis represents time. Peaks can give us sample information, both qualitative and quantitative. Detectors are usually connected to computers that provide the different peak parameters (height, amplitude, etc.), relating them to the concentration. The position of the peak (time) provides qualitative information, and the areas or heights of the peak provide quantitative information.

The retention time, tR, is the time it takes for a component to come out from the injection of the sample into the column up to the middle of its peak. The areas of the peaks are proportional to the concentration of the analytes. To make a quantitative determination, chromatographic analysis of analyte standards are performed to determine the relationship between the peak area and the standard concentration. The areas of the peaks will give the concentration of the chromatographed analytes.

Mobile and Stationary Phases

The mobile phase is a moving liquid and is characterized by its composition, solubility, UV transparency, viscosity, and miscibility with other solvents. The stationary phase is typically in the form of a column packed with very small porous particles, and the liquid mobile phase is moved through the column by a pump.

Mobile Phase Treatment

Solvent treatment is necessary to remove gases and particles. They must be pre-treated to remove dissolved gases. Degassing can be done by bubbling an inert gas (argon, helium, etc.), by vacuum degassing, or by ultrasound. The particles are removed by filtration, with suitable filters according to the hydrophilic solvent (acetate, cellulose nitrate) or hydrophobic solvent (Teflon).

Sample Injection Systems

These systems introduce a small volume (microliters) of liquid sample between the pumping system and the column. They can be manual or automatic.

Columns

Columns come in different diameters and lengths. The filling is usually silica, although there are also synthetic polymer fillers.

Detectors

Various detectors are used, including refractive index, UV-Vis detector, fluorescence detector, electrical conductivity detector, and electrochemical detector.

Methodology

1. Sample treatment: previously diluted liquid or solid samples. The sample must be filtered before introducing it into the system, with appropriate filters.
2. Choice of the mobile phase.
3. Mobile phase treatment.
4. Purge system.
5. Choice and placement of the column.
6. Development of the method. Calculations and results.
7. Cleaning and maintenance of equipment.

Carbon dioxide IR spectrum

Water IR spectrum

Pentane spectrum