Chromatography Overview and Principles

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Questions and Answers

What characteristics can be utilized for the separation of compounds in chromatography?

Electrical conductivity and toxicity

Boiling point and color

Molecular size and shape (correct)

Viscosity and density

In Thin Layer Chromatography (TLC), what is the role of the stationary phase?

To provide a liquid medium for the separation

To impede the flow of ions in the mixture

To coat the separating compounds on the plate (correct)

To reduce the speed of the mobile phase

What is the elution process in chromatography?

The separation of compounds based on pH

The movement of the mobile phase through the column (correct)

The process of compressing the stationary phase

The cooling of the chromatographic system

Which of the following best defines the term 'eluent' in chromatography?

Fluid that enters the chromatography column Signup and view all the answers

How does Gel Exclusion chromatography separate molecules?

By size through a sieving effect Signup and view all the answers

What role does the mobile phase play in chromatography?

It facilitates the movement of compounds through the stationary phase Signup and view all the answers

What is the function of the stationary phase in Thin Layer Chromatography?

To provide a fixed surface for sample application Signup and view all the answers

What is the formula for calculating the retention factor (Rf) in chromatography?

Rf = dist.travelled by sample / dist.travelled by solvent Signup and view all the answers

In paper chromatography, what acts as the stationary phase?

Cellulose filter paper Signup and view all the answers

Which condition is NOT measured using Thin Layer Chromatography (TLC)?

Diabetes mellitus Signup and view all the answers

What is the purpose of Ninhydrin in paper chromatography?

To form a colored complex with amino acids Signup and view all the answers

How far should the solvent front migrate in paper chromatography before removing the chromatogram?

About 1 cm below the top edge Signup and view all the answers

What type of chromatography is characterized by solid-liquid partitioning?

Paper Chromatography Signup and view all the answers

Which amino acids are not processed properly in Maple syrup disease?

Leucine, isoleucine, and valine Signup and view all the answers

What is the main application of Thin Layer Chromatography in biochemical analysis?

Separation or isolation of metabolites Signup and view all the answers

Study Notes

Chromatography Overview

Chromatography is an analytical technique used to separate closely related compounds in a mixture.
It's used to separate proteins, peptides, amino acids, lipids, carbohydrates, vitamins, and drugs.

Physical Basis of Separation

Separation occurs based on differences in physical characteristics of the compounds, including:
- Molecular size
- Shape
- Charge
- Volatility
- Solubility
- Adsorptivity

Essential Components of Chromatographic Systems

Stationary Phase: A porous solid matrix where the sample, dissolved in the mobile phase, is absorbed.
Mobile Phase: The mixture of substances to be separated, dissolved in a liquid or gas.

General Chromatography Principle

All forms of chromatography rely on the same principle.
- A stationary phase
- A mobile phase
The mobile phase moves through the stationary phase, carrying the mixture's components.
Components separate due to differing interactions with the stationary and mobile phases (redistribution).

Elution Process

Eluent: The fluid entering the column.
Eluate: The fluid exiting the column.
Elution: The process of moving the mobile phase through the column.

Gel Exclusion Chromatography

Separates molecules based on size.
Molecules are separated by sieving through a porous matrix.
Larger molecules are eluted faster (bypass the matrix).
Smaller molecules are eluted slower (trapped in the matrix).

Ion Exchange Chromatography

Separates ions based on their charges.
Anions or cations are attracted to oppositely charged groups on the stationary phase.
Separates ionic compounds.

Thin Layer Chromatography (TLC)

Stationary Phase: A thin layer of silica gel, cellulose, or alumina coated onto a plate (e.g., glass or plastic).
Mobile Phase: A liquid solvent.
Components separate based on their relative affinities for the stationary and mobile phases.
Compounds with higher affinity for the stationary phase move slower; those with higher affinity for the mobile phase move faster.
The resulting spots on the plate are identified and quantified.

Measuring R_f Values (TLC)

Rf value (Retention Factor): A measure of how far a compound travels relative to the solvent front on the TLC plate.
R_f = Distance travelled by the compound/ Distance travelled by the solvent.
R_f values are used to identify compounds.

Paper Chromatography

Stationary Phase: Cellulose paper.
Mobile Phase: A liquid solvent.
Components separate based on their polarity towards the mobile and stationary phases.
Separation is based on the relative affinities of compounds for the solvents and the paper fibers.
R_f values can be used to identify substances separated.

Paper Chromatography Process

Sample spotted onto the paper.
Paper placed in a developing chamber with the mobile phase.
Mobile phase moves up the paper via capillary action.
Components separate based on their interactions with the paper and solvent.

Applications of TLC in Specific Conditions

TLC is used to detect and measure amino acid concentrations in various conditions like phenylketonuria, alkaptonuria, and maple syrup disease.
- These conditions involve specific amino acid abnormalities detectable via TLC.

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Description

This quiz covers the fundamental concepts of chromatography, an analytical technique vital for separating substances in various mixtures. It explores the physical basis of separation, essential components of chromatographic systems, and the general principles involved in the process.