Medical Biochemistry I - TLC Lab Exercise 2

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Questions and Answers

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

More polar compounds move faster due to lower interaction with the stationary phase.

Less polar compounds move slower because they interact more with the stationary phase.

Less polar compounds are not affected by the stationary phase.

More polar compounds move slower as they interact strongly with the stationary phase. (correct)

What is the purpose of marking the starting point on a TLC plate?

To indicate where the solvent would be applied.

To prevent the samples from moving off the plate.

To ensure samples are applied evenly along the plate. (correct)

To help in accurately measuring the solvent front.

What happens if the TLC plate touches the solvent in the chamber?

The results will not be affected and can be interpreted normally.

The solvent will not move up the plate due to obstruction.

The samples will dissolve completely, making results invalid. (correct)

The stationary phase will absorb the solvent, altering the analysis.

Which step should be taken immediately after the optimal development time for a TLC plate?

Remove the plate and allow it to dry. Signup and view all the answers

Why is Ninhydrin used in the detection process of a TLC plate?

It reacts with the α-amino group to produce a visual color change. Signup and view all the answers

What is the significance of the RF value in chromatography?

It compares the distance traveled by the compound to the solvent front. Signup and view all the answers

What is a crucial step before applying the sample to the TLC plate?

Dissolve the sample in an appropriate solvent. Signup and view all the answers

What does closing the lid of the chamber during TLC prevent?

Evaporation of the solvent, which may alter concentrations. Signup and view all the answers

What is the primary role of the stationary phase in chromatography?

To provide a surface for separation of compounds Signup and view all the answers

What is the main principle that drives separation in Thin Layer Chromatography?

Differential partitioning of compounds between phases Signup and view all the answers

Which of the following describes the ideal solvent system in Thin Layer Chromatography?

A solvent that allows for varying solubility among compounds Signup and view all the answers

What type of information does Thin Layer Chromatography primarily provide?

Qualitative data and potential quantitative insights Signup and view all the answers

In the context of TLC, what does moving faster in the mobile phase indicate about a compound?

It is less polar compared to other components Signup and view all the answers

What materials are commonly used to construct a TLC plate?

Glass or aluminum coated with silica gel Signup and view all the answers

Why is it necessary for the components of a mixture to interact differently with the stationary and mobile phases in chromatography?

To achieve effective separation of the compounds Signup and view all the answers

What effect does the polarity of compounds have on their movement in Thin Layer Chromatography?

More polar compounds tend to move slower in the mobile phase Signup and view all the answers

Study Notes

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

Objectives:
- Introduce general chromatography principles
- Understand Thin Layer Chromatography (TLC)
- Separate and identify amino acids in a mixture using TLC

Chromatography

Definition: An analytical technique used to separate closely related compounds in a mixture.
Types: Paper, thin layer, column, size-exclusion, ion exchange, affinity, and high-performance liquid chromatography (HPLC).
Separation Mechanism: Differences in equilibrium distribution between the stationary and mobile phases. This is due to the nature and interaction between the components and the two phases.

Principle of Chromatography

Separation Principle: Different molecules in a mixture separate as they move through the stationary phase with the help of the mobile phase.
Differential Movement: Some components stay longer in the stationary phase, moving slowly, while others move quickly through the mobile phase.
Visualization: After separation, components are visually seen as spots at different heights on the plate. Analysis of their colour and position are used for identification.

Thin-Layer Chromatography (TLC)

Technique: Used to separate and identify compounds of interest.
Qualitative and Quantitative Data: Provides qualitative data; with sufficient care, can also determine quantitative data.
TLC plate: A thin layer of silica adhered to a glass or aluminium support.

Stationary and Mobile Phase

Stationary Phase: Silica gel within TLC.
Mobile Phase: Solvent or solvent mixture.
Compound Solubility: Ideal solvents allow components to have varying degrees of solubility.
Partition Equilibrium: Separation arises from the partitioning equilibrium of components in the mixture.

Separation of Compounds

Differential Partitioning: Compounds interact differently with the stationary and mobile phases based on polarity.
Affinity for Stationary Phase: More polar compounds interact strongly with (polar) silica, moving slower
Affinity for Mobile Phase: Less polar compounds interact less with silica and more with the solvent, allowing them to move further up the plate.

Experimental Procedure

Spotting: Draw a horizontal line with a pencil at the bottom, top, and edge of the TLC plate (1cm). Draw spots at the line equally spaced apart. Each spot represents a sample..
Sample Application: Apply a small spot of solution using a capillary tube (1 cm from bottom edge), Place samples in a row, each moving separately .
Chamber Filling & Placement: Pour appropriate solvent into a chamber to a depth of less than 1cm; put the TLC plate in the chamber; lid on,.
Solvent Movement: The solvent moves by capillary action up the TLC plate; meeting and carrying samples; avoid solvent touching the spots.
Chamber Removal/Drying: Remove the plate from the chamber before solvent reaches the silica surface and dry.
Solvent Front Marking: Mark the solvent front (highest point of solvent travel)

Ninhydrin Amino Acid Detection

Detection: Remove and dry the TLC plate; use Ninhydrin reagent to spray; primary amino acids turn purple

R_f Calculation

Definition: R_f value is the ratio of the distance a substance travels to the distance the solvent travels.
Calculation: distance moved by sample / distance moved by solvent

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Description

This quiz introduces the fundamental principles of chromatography, with a focus on Thin Layer Chromatography (TLC). You will explore the separation and identification of amino acids in a mixture using TLC techniques. Test your understanding of chromatography and its mechanisms of separation.