MD100 Medical Biochemistry I - TLC Lab Exercise

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Questions and Answers

What is the primary role of the stationary phase in Thin Layer Chromatography?

To separate compounds based on their solubility

To provide a mobile medium for the compounds

To interact with compounds differently based on polarity (correct)

To act as a detector for the compounds present

Which of the following is NOT a type of chromatography mentioned?

Column chromatography

Electrophoresis (correct)

Thin layer chromatography

Paper chromatography

What effect does the polarity of compounds have in TLC?

It causes compounds to move at the same rate.

It determines the interaction level with stationary and mobile phases. (correct)

It ensures all compounds separate evenly.

It prevents any component from entering the mobile phase.

What is typically used as the stationary phase in a TLC plate?

Silica gel Signup and view all the answers

In the context of TLC, what does differential partitioning refer to?

The unique rates at which each compound adheres to the stationary phase. Signup and view all the answers

What is the purpose of using a suitable detection technique after separation in TLC?

To accurately identify the nature of separated compounds. Signup and view all the answers

How does the mobile phase contribute to the separation of compounds in TLC?

It facilitates the movement of compounds across the stationary phase at various rates. Signup and view all the answers

What kind of information can Thin Layer Chromatography primarily provide?

Qualitative and, with care, quantitative information Signup and view all the answers

How does the polarity of a compound affect its movement on a TLC plate?

More polar compounds interact more strongly with the stationary phase, resulting in slower movement. Signup and view all the answers

Which step is crucial for ensuring that the sample spots do not touch the solvent in the TLC chamber?

The TLC plate should be placed in the chamber with the spots above the solvent level. Signup and view all the answers

What role does Ninhydrin play in the detection of amino acids on the TLC plate?

It produces a color change upon reacting with amino acids. Signup and view all the answers

What is the purpose of marking the solvent front on a TLC plate?

To calculate the Rf value of the samples accurately. Signup and view all the answers

What happens to the solvent on the TLC plate once it reaches the top of the stationary phase?

The TLC plate should be removed and dried immediately. Signup and view all the answers

What is the significance of using equal distances between sample spots on the TLC plate?

It prevents cross-contamination between samples. Signup and view all the answers

Which of the following statements about RF values is incorrect?

RF values are always greater than 1.0 for all compounds. Signup and view all the answers

What is the role of the stationary phase in TLC?

It interacts with the compounds, affecting their movement. Signup and view all the answers

Study Notes

MD100 Medical Biochemistry I - Lab Exercise 2: Introduction to Thin-Layer Chromatography

This lab exercise introduces the general principles of chromatography, focusing on Thin Layer Chromatography (TLC).
Students will learn to separate and identify amino acids in a mixture using TLC.

Objectives

Understand general principles of chromatography.
Learn how to use Thin Layer Chromatography (TLC).
Separate and identify amino acids in a mixture using TLC.

Chromatography

Chromatography is an analytical technique used to separate closely-related compounds within a mixture.
Various types of chromatography exist, including paper, thin layer, column, size-exclusion, ion exchange, affinity, and High-Performance Liquid Chromatography (HPLC).
Separation is due to differences in the equilibrium distribution of components between the stationary and mobile phases.
These differences are driven by the nature and degree of interaction between the compounds and the two phases.

Principle of Chromatography

Chromatography works on the principle that different molecules in a mixture will interact differently with the stationary and mobile phases.
Components interacting more strongly with the stationary phase move more slowly.
Components interacting more strongly with the mobile phase move more quickly.
Once separated, components are visualized as spots on a plate (different levels of travel) and identified via suitable detection techniques.

Thin Layer Chromatography (TLC)

TLC is a technique for separating and identifying compounds of interest.
It is both qualitative (identifying components) and, with care, quantitative (measuring amounts).
The TLC plate consists of a thin layer of silica gel adhered to a glass or aluminum support.

Stationary and Mobile Phase

Silica gel acts as the stationary phase.
The solvent or solvent mixture acts as the mobile phase.
In an ideal solvent system, compounds of interest have different solubilities in the mobile and stationary phase.
Separation results from the partition equilibrium of the compounds in the mixture.

Separation of Compounds

Differential partitioning occurs as the solvent travels up the plate in TLC.
Different affinity for stationary phase: more polar compounds interact strongly with the polar stationary phase (like silica) and move more slowly.
Different affinity for mobile phase: less polar compounds interact less with the stationary phase and thus move more quickly up the plate.

Experimental Procedure

Draw horizontal pencil lines on the plate, at the top, bottom, and edge (1 cm). These function as markers.
Place small, equally-spaced spots on the pencil line marking the starting point.
Each spot will represent a different sample.
Use a capillary tube to apply the spots (1 cm from the bottom edge), with each sample having its own lane to avoid interference.
Using a solvent, fill a chamber with a small depth (less than 1 cm).
Place the TLC plate into the chamber, ensuring spots are not submerged. Close the lid.
The solvent rises via capillary action; once it's approaching the top edge of the plate, remove the plate to prevent solvent from reaching the top of the silica.
Mark the solvent front and allow the plate to dry.
(Note: a suitable solvent is necessary; the exact solvent will be mentioned in lab instructions.)

Ninhydrin Amino Acid Detection

After allowing the separated compounds optimal time, and drying, the spots are detected using a Ninhydrin reagent.
Ninhydrin reacts with the alpha-amino group of amino acids to produce a purple colour.

Rf values

The Rf value is used to identify amino acids in mixtures.
Calculated by dividing the distance the component moved by the distance the solvent moved.

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Description

This lab exercise introduces the principles of Thin Layer Chromatography (TLC) within the context of medical biochemistry. Students will learn how to separate and identify amino acids in a mixture using TLC techniques, understanding the foundational concepts of chromatography in the process.