Separation techniques- Chromatography, Slides of Analytical Chemistry

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Analytical

Chemistry

Unit III

Separation Techniques: Chromatography

Introduction to Separation Techniques ● (^) Separation techniques are used to separate mixtures into its constituent elements and/or compounds. Recall that a mixture contains elements and/or compounds which are not chemically combined together. ● (^) Depending on the physical and chemical properties of the substances in the mixture, we can choose the most appropriate separation technique to isolate them from the mixture.

Chromatography

Fundamentals of Chromatography: ● (^) Chromatography is a widely used method for the separation, identification, and determination of the chemical components in complex mixtures.. ● (^) Chromatography is a method used to separate the individual components of a mixture on the basis of their interactions with a stationary and a mobile phase. Each component interacts differently with the two phases which causes them to move at different speeds and separate. ● (^) The main principle of chromatography is that different substances interact differently with the stationary and mobile phases and this difference in interaction separates the compounds. As the mobile phase moves with the sample through the fixed stationary phase, compounds interact differently with the stationary phase based on their polarity and binding affinity and moves at a different speed based on its properties like size, charge, or affinity to the stationary phase resulting in separation. The separated components are represented as peaks on a chromatogram and identified by its retention time which is the time a compound takes to move through the system. ● (^) It is a powerful method in industry, where it is used on a large scale to separate and purify the intermediates and products in various syntheses.

Basic separation mechanisms/principles

● (^) All chromatographic methods require one static part (the stationary phase) and one moving part (the mobile phase).

● The techniques rely on one of the following phenomena/ mechanisms: adsorption; partition; ion

exchange; or molecular exclusion.

Adsorption : ● (^) It has a solid stationary phase and a liquid or gaseous mobile phase. ● (^) Each solute has its own equilibrium between adsorption onto the surface of the solid and solubility in the solvent. Upon passing over or through the adsorbent, molecules adsorb onto the surface of the solid stationary phase through weak, non-ionic attractive forces such as Van der Waals force, hydrogen bonding, and steric interactions. The least soluble or best adsorbed ones travel more slowly. ● (^) Liquid chromatography using a column containing silica gel or alumina and TLC are examples of adsorption chromatography ● (^) The type of interaction majorly depends upon the nature of the interacting molecules and differences in the compound flow rate eventually lead to the separation

Partition : ● (^) In partition chromatography the stationary phase is a non-volatile liquid which is held as a thin layer (or film) on the surface of an inert solid. ● (^) The mixture to be separated is carried by a gas or a liquid as the mobile phase. The solutes distribute themselves between the moving mobile phase and the stationary phases, with the more soluble component in the mobile phase reaching the end of the chromatography column first. ● (^) Paper chromatography is an example of partition chromatography.

Elution Chromatography on Columns Analogy- For understanding purpose

Chromatographic terms:

● Chromatograph - equipment that enables a sophisticated

separation EX. Gas chromatography or Liquid chromatography

● Stationary phase - Immobilized phase that is fixed in place

either in a column or on a planar surface. Examples : Silica layer - Thin Layer Chromatography

● Mobile phase - a phase that moves over or through the

stationary phase carrying with it the analyte mixture. The mobile phase may be a gas, a liquid, or a supercritical fluid.Moves in a definite direction. Liquid (LC), Gas (GC)

● Eluent - Fluid entering column/ solvent that carries the

analyte.

● Eluate - Mobile phase leaving the column.

● Retention time t

R -^ Time taken for a particular analyte to pass through the system (from the column

inlet to the detector) under set conditions. The retention time, tR , is the time between injection of a sample and the appearance of a solute peak at the detector of a chromatographic column

● Retardation factor (R/F Value) - a ratio representing the distance traveled by a compound divided by

the distance traveled by the solvent front, used to identify components, with values typically ranging from 0 to 1.

● Retention Factor - It is the amount of time a solute spends in

the stationary phase relative to the time it spends in mobile phase.

● capacity factor - The capacity factor also known as the retention factor, measures an analyte's

retention in chromatography, representing the ratio of time a compound spends in the stationary phase versus the mobile phase. It is calculated from the retention time divided by the time for an unretained peak (tM). ➔ (^) tR (Retention Time): Time from injection to peak detection. ➔ (^) tM (Void/Dead Time): Time for an unretained compound to pass through the column. ➔ (^) tR - tM : Adjusted retention time or reduced retention time. The capacity factor serves to normalize the retention time of an analyte to the dimensions of the analytical column. Performance and efficiency terminologies:

● Resolution - Resolution (R) is the term used in chromatography to define the quality of the

separation between two adjacent peaks. Both the distance between peaks and the width of each individual peak contribute to this separation. ● (^) Factors Affecting Resolution:

1. Efficiency - Column efficiency relates to peak width; narrower, tighter peaks (higher efficiency) improve resolution. 2. Selectivity - This represents the distance between the peaks; higher selectivity means further separation.

● Selectivity Factor (or Separation Factor)

The ratio of the capacity factors of two adjacent peaks. It Measures the ability of the system to distinguish between two analytes. It is considered the most effective tool for optimizing resolution. Where k2 is the retention factor of the second peak, and k2 is the retention factor of the first peak. The value must always be greater than 1.0 for a separation to occur.

● Column Efficiency

A measure of the sharpness of a peak, typically represented by the number of theoretical plates. A higher means narrower peaks. Higher efficiency (more plates) reduces band broadening.

● Peak Symmetry (Asymmetry Factor or Tailing Factor)

Compares the shape of the front half of the peak to the back half, usually measured as at 10% peak height, where is the trailing edge and is the leading edge. Ideal symmetry is 1.0. Values indicate tailing (peak drags), while values indicate leading (rare).

Different types of Chromatography

Applications of Chromatography Chromatographic methods have many applications including:

● Chromatography can be used in environmental testing to monitor air and water quality.

● It has applications in forensic science to detect toxic substances in the body and analyze

samples like blood, urine, or saliva.

● It can be used in bioprocessing or product purification like monoclonal antibodies and

vaccines.

● It has applications in the food industry to check the safety and quality of food products. It

helps to detect contaminants and additives in food products. It can also determine the

ingredient composition which is used for nutritional assessment and accurate labeling.

● It has applications in the chemical industry to check the purity and determine the chemical

properties of different compounds.

● It has applications in the pharmaceutical industry to develop drugs and check the quality of

the products.