MD100 Biochemistry Lab: TLC Introduction

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Questions and Answers

What is the primary function of the silica gel in thin layer chromatography (TLC)?

To serve as the stationary phase for separation (correct)

To provide qualitative data on the sample

To act as the mobile phase for dissolving the sample

To enhance the solubility of all compounds in the mixture

Which factor does NOT affect the separation of components during chromatography?

Size of the chromatography plate (correct)

Temperature of the environment

Nature of the compounds in the mixture

Equilibrium distribution between stationary and mobile phases

What kind of data can be obtained through careful TLC analysis?

Quantitative data only

Neither qualitative nor quantitative data

Qualitative data only

Both qualitative and quantitative data (correct)

What role does the mobile phase play in thin layer chromatography?

It facilitates the movement of compounds up the TLC plate. Signup and view all the answers

In TLC, how does polarity influence the separation of compounds?

Different polarities affect how compounds interact with the stationary and mobile phases. Signup and view all the answers

What happens when compounds are subjected to a mobile phase during chromatography?

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

Which type of chromatography uses a thin layer of silica for support?

Thin layer chromatography Signup and view all the answers

What characteristic of a solvent mixture is ideal for effective separation in TLC?

It should allow the compounds to be soluble to different degrees. Signup and view all the answers

How do polar compounds behave in thin layer chromatography (TLC) relative to the stationary phase?

They interact strongly with the stationary phase and move more slowly. Signup and view all the answers

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

To calculate the distance traveled by the solvent for RF value calculations. Signup and view all the answers

What role does Ninhydrin play in the TLC process?

It visualizes amino acids by producing a colored reaction. Signup and view all the answers

What is the likely effect of the solvent depth during the TLC process?

The solvent should not touch the spots to prevent sample dissolution or interference. Signup and view all the answers

What would be the outcome if a nonpolar compound is used on a polar stationary phase in TLC?

The compound would rapidly ascend the plate, eluting easily. Signup and view all the answers

Which step in the TLC procedure is crucial for ensuring accurate results?

Carefully marking the starting point of the samples before solvent application. Signup and view all the answers

In thin layer chromatography, which factor primarily determines the distance a compound travels up the plate?

The polarity of the compound relative to the stationary phase. Signup and view all the answers

Why is it important to dry the TLC plate after development and before visualization?

To prevent the solvent from affecting the results during detection. Signup and view all the answers

Study Notes

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

Objectives:
- Introduces general principles of chromatography.
- Explains use of Thin Layer Chromatography (TLC).
- Shows how to separate and identify amino acids using TLC.

Chromatography

A technique for separating closely related compounds in a mixture.
Different types exist: paper chromatography, thin layer, column, size-exclusion, ion exchange, affinity, and High-Performance Liquid Chromatography (HPLC).
Separation relies on differences in equilibrium distribution between stationary and mobile phases.
Equilibrium distribution depends on the nature and interaction of compounds with the phases.

Principle of Chromatography

Different molecules in a mixture separate as they move through a stationary phase with the aid of a mobile phase.
Some components stay longer in the stationary phase and move slowly.
Other components move rapidly into the mobile phase and leave quickly.
Separated components appear as spots at different levels on the plate.
Detection techniques help identify components.

Thin Layer Chromatography (TLC)

A technique for separating and identifying compounds.
Provides qualitative information; with attention to detail, quantitative data is achievable.
TLC plate is a thin layer of silica on a glass or aluminum support.

Stationary and Mobile Phases

Silica gel acts as the stationary phase.
Solvent mixture functions as the mobile phase.
Ideal solvents allow compounds to dissolve to varying degrees.
Separation occurs due to the partition equilibrium of the components in the mixture.

Separation of Compounds

Differential partitioning occurs as the solvent moves up the plate.
Compounds interact differently with stationary and mobile phases based on polarity.
Polar compounds have strong affinity for the polar stationary phase (e.g., silica gel) and move slowly.
Less polar compounds interact less with the stationary phase, moving further up the plate.

Experimental Procedure

Step 1: Draw horizontal lines on the TLC plate (1 cm from top, bottom, and edge).
Step 2: Spot samples along the baseline, with equal space between samples.
Step 3: Apply sample spots onto the plate using a capillary tube. Space samples equally from the bottom edge of the plate.
Step 4: Pour solvent into a glass chamber to a depth of less than 1 cm..
Step 5: Place the TLC plate in the chamber; ensure spots are not immersed in the solvent.
Step 6: Remove plate from chamber before the solvent front reaches the top of the stationary phase and air-dry.
Step 7: Mark the solvent front (furthest extent of solvent).

Ninhydrin Amino Acid Detection

To detect spots: Remove the TLC plate, dry it, and spray with Ninhydrin reagent.
Ninhydrin reacts with the α-amino group of primary amino acids; color is purple.

Rf Calculation

The Rf value reflects a compound's relative mobility.
Formula: Rf = Distance moved by substance from origin / Distance moved by solvent from origin

RF Values Table

Presents Rf values and detection limits for various amino acids. Color changes after heating are noted.

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Description

This quiz focuses on the principles and applications of Thin-Layer Chromatography (TLC) in medical biochemistry. Participants will learn how to separate and identify amino acids using TLC techniques, exploring the interaction of compounds with stationary and mobile phases.