Medical Biochemistry I: TLC Techniques

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Questions and Answers

What is the primary purpose of Thin Layer Chromatography (TLC)?

To separate and identify compounds in a mixture. (correct)

To quantify the concentration of a compound.

To analyze the molecular weight of substances.

To determine the solubility of chemicals.

Which component acts as the stationary phase in a TLC system?

Glass support plate

Solvent mixture

Silica gel (correct)

Amino acid mixture

Which of the following describes how molecules separate in chromatography?

By undergoing a chemical reaction.

Only by their temperature stability.

Due to differences in their interaction with stationary and mobile phases. (correct)

Based solely on their molecular weight.

What happens to components of a mixture as the solvent moves up the TLC plate?

Some components remain in the stationary phase longer than others. Signup and view all the answers

What kind of information does TLC primarily provide?

Qualitative information about components Signup and view all the answers

Which solvent characteristic is essential for effective separation in TLC?

The solvent should have varying degrees of solubility for compounds. Signup and view all the answers

In the process of differential partitioning during TLC, which factor influences the rate at which compounds travel?

The polarity of the compounds. Signup and view all the answers

What role do the spots observed on the TLC plate serve after the separation has occurred?

They provide a means to identify the nature of the components. Signup and view all the answers

What is the effect of the polarity of a compound on its movement in thin layer chromatography (TLC)?

Less polar compounds travel farther up the plate. Signup and view all the answers

In the experimental procedure, where should the spots of the sample be placed on the TLC plate?

At an equal distance from the bottom edge. Signup and view all the answers

What happens after the TLC plate is placed in the solvent chamber?

The solvent moves up the plate by capillary action. Signup and view all the answers

What should be done with the TLC plate before the solvent front reaches the top?

It should be removed and dried immediately. Signup and view all the answers

Which of the following reagents is used for amino acid detection on the TLC plate?

Ninhydrin reagent. Signup and view all the answers

How is the RF value calculated in thin layer chromatography?

Distance traveled by the compound divided by the distance traveled by the solvent front. Signup and view all the answers

What is the purpose of using a pencil to mark the starting point on a TLC plate?

To provide a stable point for sample application. Signup and view all the answers

Why should samples on the TLC plate not touch the surface of the eluent in the chamber?

It can result in uneven sample migration. Signup and view all the answers

Study Notes

Introduction to Thin-Layer Chromatography (TLC)

Medical Biochemistry I, Lab Exercise 2
Fall Semester 2024
European University Cyprus

Objectives

Introduce the general principles of chromatography
Explain the use of Thin Layer Chromatography (TLC)
Separate and identify amino acids in a mixture using TLC

Chromatography

An analytical technique separating closely related compounds in a mixture.
Various types exist: paper, thin layer, column, size-exclusion, ion exchange, affinity, and HPLC.
Separation depends on equilibrium distribution differences between stationary and mobile phases.
These differences result from the nature and interaction degree of compounds with both phases.

Principle of Chromatography

Different molecules in a mixture separate on a solid or fluid stationary phase while being moved by a mobile phase.
Some components spend more time in the stationary phase, moving slower.
Other components move quickly into the mobile phase.
Separation is visualized as spots at different levels on a plate.
Spot characteristics are determined using detection techniques.

Thin Layer Chromatography (TLC)

A technique separating and identifying compounds of interest.
Provides qualitative and, with precision, quantitative data.
Silica gel adhered to a glass or aluminum plate forms the stationary phase.
Mobile phase is a solvent or solvent mixture.

Stationary and Mobile Phase

Silica gel is the stationary phase.
Solvent mixture forms the mobile phase.
Ideal solvent solubility differences between compounds.
Separation caused by the equilibrium distribution of components in the mixture.

Separation of Compounds

Differential partitioning: Compounds interact differently with the stationary and mobile phases based on their polarity.
Higher polarity compounds strongly adhere to the stationary phase (e.g., silica gel) and travel slower.
Lower polarity compounds interact less with the stationary phase and travel further.

Experimental Procedure

Draw horizontal lines using a pencil at 1 cm intervals on the TLC plate.
Create spots of equal distance at the spotting line. Each spot represents a sample.
Use a capillary tube, apply the sample solution to the plate, 1 cm from the bottom.
Pour an appropriate solvent into a glass chamber to a depth less than 1 cm.
Place the TLC plate in the chamber without eluent contact.
Close the chamber lid and allow the solvent to move up the plate via capillary action.
Remove the plate before the solvent reaches the stationary phase top edge and dry.
Mark the solvent front (farthest solvent reach).

Ninhydrin Amino Acid Detection

Remove the dried TLC plate.
Spray the plate with Ninhydrin reagent.
Ninhydrin reacts with the amino group of primary amino acids, creating purple spots.

R_f Calculation

R_f value = distance traveled by the substance / distance traveled by the solvent front from the origin.
Used to identify compounds based on their mobility.

R_f Values

Indicates different separated amino acid color reactions after exposure to Ninhydrin.
Detection limits and R_f values are provided for various amino acids.

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Description

This quiz explores the fundamentals of Thin Layer Chromatography (TLC) as part of Medical Biochemistry I, Lab Exercise 2. Students will learn about the principles of chromatography, its various types, and its application in separating and identifying amino acids. Perfect for those studying in the Fall Semester 2024 at European University Cyprus.