Thin_layer_chromatography zoology pg student

Slide 1: Title Slide
Title: Thin Layer Chromatography (TLC) in Zoology
Subtitle: An Overview for Postgraduate Students
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Date
Slide 2: Introduction to Thin Layer Chromatography (TLC)
Definition: A technique for separating compounds based on their different interactions with a stationary phase and a mobile phase.
Purpose: Used for analyzing and purifying compounds in various fields, including zoology.
Slide 3: Historical Background
Origins: Developed by German chemist Fritz Mikhailov in the 1950s.
Evolution: Advances in TLC techniques and its applications in various scientific fields.
Slide 4: Principles of TLC
Basic Concept: Separation based on differential partitioning between a stationary phase (solid or liquid on a plate) and a mobile phase (solvent).
Mechanism: Adsorption or partition chromatography.
Slide 5: TLC Components
Stationary Phase: Typically a thin layer of adsorbent (e.g., silica gel, alumina) coated on a plate.
Mobile Phase: Solvent or solvent mixture that moves through the stationary phase.
Sample: Analytes that are to be separated.
Slide 6: Types of TLC Plates
Silica Gel Plates: Most common; used for a wide range of compounds.
Alumina Plates: Useful for different types of compounds.
Cellulose Plates: Used for specific applications.
Slide 7: TLC Procedure - Preparation
Sample Application: Spotting the sample onto the plate.
Development: Placing the plate in a developing chamber with the mobile phase.
Slide 8: TLC Procedure - Development
Solvent Front: The front line of the solvent moving up the plate.
Migration of Spots: Movement of sample spots as the solvent moves.
Slide 9: Detection Methods
UV Light: For compounds that absorb UV light.
Chemical Reagents: Specific reagents that react with the compounds to produce visible spots.
Visual Observation: Color changes or spot appearance.
Slide 10: Rf Value
Definition: The ratio of the distance traveled by the compound to the distance traveled by the solvent front.
Formula: Rf = (Distance traveled by compound) / (Distance traveled by solvent front)
Importance: Used to identify compounds based on their Rf values.
Slide 11: Applications in Zoology
Metabolite Analysis: Identifying metabolic products in animal tissues.
Drug Analysis: Testing animal drugs or compounds.
Toxin Detection: Detecting toxins in animal samples.
Slide 12: Case Study 1: Metabolite Analysis
Objective: To analyze and separate metabolites from animal tissue samples.
Method: Procedure followed, types of samples, results, and interpretations.
Slide 13: Case Study 2: Drug Analysis
Objective: To determine the presence and concentration of drugs in animal tissues.
Method: Procedure, types of drugs tested, results, and their significance.
Slide 14: Limitations of TLC
Resolution: Limited by the thickness of the stationary phase and the type of mobile phase.
Sensitivity: May not detect very low concentrations of analytes.
Reproducibility: Variability in results due to different experimental condi

1. Dr. S.D. SARASWATHY
Assistant Professor
Department of Biomedical Science
BharathidasanUniversity
Tiruchirappalli
Thin Layer
Chromatography (TLC)
Biotechniques

2. KEY CONCEPTS
•Introduction
•General Principle
•TLC Technique
•Applications

3. INTRODUCTION
•Thin Layer Chromatography (TLC) is an important
technique used for identification and separation of
mixture of chemical compounds into its individual
components.
•TLC is a form of liquid chromatographyconsisting
of two phases: A mobile phase (liquid) and A
stationary phase (solid).
•Differences in the interactions between the solutes
and stationary and mobile phases enable separation.

4. •TLC technique involves the distribution of
components of a mixture to be separated between
two phases.
•The components of the mixture are partitioned
between an adsorbent(stationary phase), and a
solvent (mobile phase).
•Different compounds will have different solubility
and adsorption to the two phases between which
they are to be partitioned.
•In TLC separation of the individual substances is
based on their relative affinities towards stationary
and mobile phases.
PRINCIPLE

5. •The stationary phase: is a thin layer of adsorbent
(usually silica gel or alumina) coated on a plate.
•The mobile phase: is a developing liquid which
flows through the stationary phase, carrying the
samples with it.
•Components with more affinity towards stationary
phase travels slower.
•Components with less affinity towards stationary
phase travels faster.
PRINCIPLE

6. •Adsorbents used as Stationary Phase:
-Inorganic: Silica Gel, Kieselguhr, Aluminium Silicate,
Bentonite.
-Organic: Cellulose & its acetylates, Charcoal & activated
Charcoal, DextranGel, Polyamides.
•Solvents used as Mobile Phase:
-Petroleum ether, Benzene, Carbon tetrachloride.
•Selection of Adsorbents and Solvents:
Adsorbent should not adhere to glass plate.
Solvents should be of high purity.
Selected based on the nature of the compound to be separated
(polar or non polar.)
METHOD

7. •R
fvalue indicates the position of migrated spots on
chromatogram.
•In TLC the results are represented by R
fvalue which
represents the migration of solute relative to the
solvent front.
•The R
fvalue is calculated as:-
Distance travelled by the solute
R
fValue =
Distance travelled by the solvent front
R
fVALUE

8. •A plastic, glass or aluminum sheet is coated with a thin layer of
silica gel (adsorbent).
•Plates must be dried, activated and stored in desiccatoruntil used.
TLC -TECHNIQUE
STEP 1: Preparation of Slurry
STEP 2: Preparation of Tank
•Solvent mixtures should be freshly prepared for analysis.
•Solvent is poured down side of the tank (1.5cm depth).
•Tankis covered with the glass lid and kept for saturation.
•A very small amount of sample (solution) to be analyzed is
applied in a small spot with a capillary tube, ~1cm from the
bottom of the TLC plate.
STEP 3: Application of Sample (Spot)

9. TLC -TECHNIQUE
•The TLC is developed in
a chamber which
contains the mobile
phase (solvent).
•When the mobile phase
rises up the plate up by
capillary action, the
components dissolve in
the solvent and move.
http://nptel.ac.in/courses/102103047/module4/lec21/4.html
A TLC experimental set-up

10. TLC -TECHNIQUE
•Individual components in the sample
move up at different rates.
•Morepolar analytesinteract more
stronglywith the stationary phase
move very slowly up.
•Morenonpolaranalytesinteract less
strongly with the polar silica gel and
more strongly with the less polar
mobile phase move higher up.
•Once the solvent reaches the top (below
~1-2 cm) of the TLC sheet the plate is
removed from the developing chamber
and position of solvent front is marked.
http://classes.kvcc.edu/chm220/TLC%20
Lab/lab/procedures1.htm

11. TLC -TECHNIQUE
•The solvent is allowed to evaporate
from the TLC sheet.
•As the compound is colorless, it can
be visualized by suitable methods.
•Lipids -Iodine vapors
•Amino acids -Ninhydrinreagent.
•Also, manganese-activated zinc
silicate (fluorescent compound),is
added to the adsorbent that allows
the visualization of spots under a
black light (UV
254
lamp).
•Once visible, the R
f
value of each spot can be determined.
http://orgchemboulder.com/Technique/Procedures
/TLC/TLC.shtml

12. AB CU
x xxx
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
R
f (A) =
R
f (B) =
R
f (C) =
R
f (U
1) =
R
f (U
2) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
R
f (D) = = 0.80
4.0 cm
5.0 cm
4.0 cm TLC –CALCULATIONOFR
fVALUE
https://slideplayer.com/slide/6184680/18/images/21/THIN+LAYER+CHROMATOGRAPHY.jpg

13. •Qualitative results of TLC
–expressed as fractions of 1.0
–can be expressed from R
fvalues (Ex: R
fx 100)
–no more than two decimal places
•R
fvalues can be used to aid in the identification of a
substance by comparison to standards.
•Comparison should be made onlybetween spots on the
same sheet, run at the same time.
•Identical substances will have the same R
fvalue, whereas
non-identical compounds will differ in their R
fvalues.
RESULTSOFTLC -R
fVALUE

14. •TLC is used in qualitative and quantitative analysis to separate
organic compounds and to test the purity of compounds.
•This technique is useful for separation of lipids, amino acids
and sugars etc.
•It is useful in:
•Identification of components of a mixture.
•Following the course of a reaction,
•Analyzing fractions collected during purification,
•Analyzing the purity of a compound.
APPLICATIONS