Instrumental Method of Analysis

Final Y. B. Pharm, Sem VII

“Thin Layer Chromatography”

Mrs. P. C. Kothale
Department of Pharmaceutical Chemistry
Assistant Professor

APPASAHEB BIRNALE COLLEGE OF PHARMACY, SANGLI

 Introduction:
✔ It is also known as Planer Chromatography.
✔ Thin layer chromatography is first introduced by Izmailov &
Shraiber in 1938.
✔ They used to separate plant extract on 2 mm thick firm
adhesive layer of alumina on glass plate.
✔ Consden, Gordon and Martin used filter papers for separating
amino acids.
✔ In 1950 Kirchner identified terpenes on filter paper & later
on fiber paper coated with alumina.
✔ In 1958 – stahl developed standard equipment for TLC.
Definition:
Thin layer chromatography is defined as method of
separation or identification of a mixture of components into
individual components by using finely divided adsorbent
coated or spread over a chromatographic plate.
Principle:
✔ Here, sample mixture is separated by using two phases

✔ Stationary phase – silica/alumina

✔ Mobile phase – Polar/non polar

✔ When thin layer plate is kept into development chember then solvent

flows through out the thin layer plate due to capillary action against

gravitational force. So, components are separated on the basis of

Adsorption mechanism.
Principle:
✔ The different component from sample having different affinity towards
stationary phase. The adsorption coefficient (often denoted as K) describes
how strongly a compound adsorbs (sticks) to the stationary phase relative to
its tendency to remain in the mobile phase.
✔ A higher adsorption coefficient means that the compound has a stronger
affinity for the stationary phase .Lower Rf Value
✔ A lower adsorption coefficient indicates that the compound is more
soluble in the mobile phase and has a weaker interaction with the
stationary phase. Higher Rf value.
Principle:
Silica (SiO2) is a solid with an extended structure of tetrahedral silica
atoms bridged together by bent oxygen atoms. The surface of silica
particles, silica contain very polar silanol (Si-O-H) groups.
Principle:
Normal Phase :- Stationary Phase – polar
Mobile phase – non polar
The non polar compounds elute first and the most polar
compounds elute last. The mobile phase consists of a nonpolar
solvent such as hexane or heptane mixed with a slightly more polar
solvent such as isopropanol, ethyl acetate or chloroform.
Principle:
Reverse Phase :- Stationary Phase – non polar
Mobile phase – polar
The polar compounds elute first and the non polar compounds elute
last. Methanol–water, ethanol–water, isopropanol–water,
acetonitrile–water.
 Experimental Techniques:
1. Coating Material
2. Preparation of thin layers in plates
3. Activation of adsorbent
4. Purification of Slilica Gel G layers
5. Sample application
6. Development tank
7. Solvent system
8. Plate development
9. Detection of component
10. Evaluation of chromatogram
1. Coating Material:
✔ Adsorbent do not adhere on plate so binder are added –
gypsum(CaSO4), starch, hydrated silicon dioxide.
✔ Silica gel G, Alumina G- Binder gypsum is present
✔ UV absorbent substance can be detected by addition of fluoroscent
additive (dark spot – green fluorescent indictor)
Components to be
Separation Mechanism
Adsorbent Acidic/Basic Activity separated

Silica gel Acidic Active Adsorption Acidic & neutral

Alumina Basic Active Adsorption Basic & neutral

Kiesleguhr Neutral Inactive Partition Strongly

hydrophilic

Cellulose Neutral more Partition Water soluble

Powder
1. Coating Material:
2. Preparation of thin layers in plates:

a. Pouring

b. Dipping

c. Spraying

d. Spreading
2. Preparation of thin layers in plates:
a. Pouring
Slurry is put on to slide, plate is tipped back & forth to form uniform
layer.
2. Preparation of thin layers in plates:
b. Dipping:
Peifer – 1962 plates are prepared by dipping them at a time back
to back in chloroform/chloroform-methanol slurry.
2. Preparation of thin layers in plates:
c. Spraying:
Reitsema, small point of sprayer was used to form uniform layer
on plate.
2. Preparation of thin layers in plates:
d. Spreading:
Slurry is placed in applicator, moved over the stationary plate.
2. Preparation of thin layers in plates:
e. Precoated plates
Precoated adsorbent layer on glass or plastic sheets, expensive, 0.1
mm – 0.2 mm thickness.
3. Activation of adsorbent:
Air dry, oven drying 110°c for 30 min. Drying made adsorbent layer
active. Alumina plates – 150 ° c for 40 min.
4. Purification of Silica gel G layer:

Silica gel G contain iron as impurity, distortion in chromatographs.

Iron free layers can be obtained by running drying plate into

methanol-conc. HCl (9:1 v/v), iron get migrated from upper edges

& plates are again dried in oven.

5. Sample application:

Agla microsyringe is used to load the sample on TLC for quantitative

work. Applicator made from capillary are used for qualitative work.
6. Development tank:
Ascending chromatography is common technique.
6. Development tank: a. Process of Ascending chromatography
❖ Preparation: A small amount of sample is applied as a spot near the bottom edge of
a TLC plate (typically about 2 cm above the edge).
❖ Positioning the Plate: The TLC plate is then placed upright in a development tank
containing a shallow layer of solvent (mobile phase).
❖ Solvent Migration: The solvent is drawn up the plate by capillary action and based
on adsorption coefficient.
❖ Separation of Compounds: As the solvent ascends, it carries the compounds
depending on their affinities for the stationary phase and their solubility in the
mobile phase. Compounds that are more soluble in the mobile phase travel further,
while those that interact more strongly with the stationary phase travel less.
❖ Solvent Front: The solvent front is allowed to move up the plate until it reaches a
point near the top (usually about two-thirds or three-quarters of the way up the
plate), after which the plate is removed from the tank.
❖ Drying and Visualization: The TLC plate is then dried, and the separated spots can
be visualized under UV light or by using staining reagents.
6. Development tank:
b. Mechanism of Separation:
The compounds in the sample interact differently with the stationary phase,
leading to differential adsorption. Compounds with higher affinity for the
stationary phase adsorb more and travel slowly, while those with less affinity
move further with the solvent.
c. Why Use Ascending Chromatography?:
❖ Simple Setup: It requires minimal equipment—a TLC plate, development
tank, and a small amount of solvent.
❖ Effective for Small-Scale Analysis: It is effective for qualitative analysis of
mixtures, helping to determine the number of components and their relative
polarities.
❖ Visual Comparison: It allows for easy visual comparison of different
samples or to monitor the progress of a chemical reaction, since the
separated spots can be seen directly on the plate.
7. Solvent System:
❖ Trangle contain adsorbent activity (Stationary phase), nature mixture

(sample), nature of Eluent solvent (mobile phase)

❖ When compound is unknown then rotate M will result into inactive

adsorbent activity and polarity of mobile phase.

8. Plate development:
❖ Spot is given at the bottom with the help of applicator which is just
above the 2 cm from edges of plate. In such way that spot should not
be dipped into mobile phase.
❖ Prepared plate kept into development tank at 45° and run the mobile
phase two third part of plate.
9. Detection of component:

a. Iodine chamber: Shake the flask containing iodine and keep the

developed plate result into yellow colored stain will observed.

b. UV detection: For colorless compound.

10. Evaluation of chromatogram:
i. Qualitative: Rf value = Distance travelled by solute
Distance travelled by solvent
ii. Quantitative:
a. Direct Method:
Visual assessment of chromatogram
Measuring spot areas
Incorporating TLC in Densitometry
b. Indirect Method:
Gravimetry, Colorimetry, Polarography, Radiometry, Fluorimetry,
Flame photometry, Coulometry
Advantages of TLC over other chromatographic techniques:
1. Simple equipment:
The TLC requires simple equipment.
2. Short development time:
In TLC, development time, is only about 1 hour or so for good
separation on inorganic adsorbent layers. This is an advantage of
TLC over paper and column chromatography which require several
hours or days.
3. Wide choice of stationary phase:
The method may be employed for adsorption, partition (including
reversed phase) or ion exchange chromatography.
4. Early recovery of separated components.
It is possible to remove the powdery coating of the plates by
scraping with a knife. Means that a spot or zone may be removed
quantitatively & required compound dissolved in a suitable solvent is
determined by calorimetric or spectrophotometric analysis.
5. Separation effects.
The separation effects achieved by thin layer chromatography are
usually super available which are to those obtained by paper
chromatography.
6. Easy visualization of separated compounds:
Detection of fluorescence compounds under UV d is easier than on
paper because the inorganic background does not fluoresce.
7. Sensitivity:
Extremely sharp delineated spots are obtained & visualized by spray
reagent will be able to detect much smaller quantities in TLC than
paper chromatography. This increase in sensitivity is of the order of
10 to 100 folds as compared as of paper chromatography.
8. Variable thickness of thin layers.
preparative separation with the aid of thicker layers as well as to most
separations by column (chromatography.
9. Chemically inert stationary phase:
Inert, affording vigorous means of detection including the application
of strong heat or of corrosive reagent such as conc. H2SO4.
Application:
1) To check process
2) In organic chemistry:
I. Checking purity of sample
II. Purification process
III. Examination reaction
IV. Identifying organic compounds
• Acids
• Alcohols
• Glycols
• Alkaloids
• Amines
• Antibiotics
3. Identification of inorganic ions