FACULTY OF HEALTH & LIFE SCIENCES

KCH20501P
ANALYTICAL CHEMISTRY

1) NAME : SHARMILA D/O SURESH

MATRIC NUM : 012022021658

2) NAME : NAVITHA A/P MURUGAN

MATRIC NUM : 012021020495

3) NAME : NURUL HERLIN BINTI MUKHLIS

MATRIC NUM : 012020092323
 INTRODUCTION

Chromatography is essentially a physical method of separation in which the

components of a mixture are separated by their distribution between two phases;
one of these phases in the form of a porous bed, bulk liquid, layer or film is
generally immobile (stationary phase), while the other is a fluid (mobile phase)
that percolates through or over the stationary phase. A separation results from
repeated sorption/desorption events during the movement of the sample
components along the stationary phase in the general direction of mobile-phase
migration. Useful separations require an adequate difference in the strength of the
physical interactions for the sample components in the two phases, combined
with a favourable contribution from system transport properties that control
sample movement within and between phases. Several key factors are
responsible, therefore, or act together, to produce an acceptable separation.
PRICNIPLES OF CHROMATOGRAPGHY
 Physical method of separation that distributes components to separate
between two phases moves in a definite direction.
 Substances are separated based on their differential distribution between
two phases

CLASSIFICATION OF CHROMATOGRAPHY
TECHNIQUES BY CHROMATOGRAPHIC BED SHAPE
 Column chromatography
 Planar chromatography
 Paper chromatography
 Thin layer chromatography
TECHNIQUES BY PHYSICAL STATE OF MOBILE PHASE
 Gas chromatography
 Liquid chromatography
AFFINITY CHROMATOGRAPHY
 Supercritical fluid chromatography
 COLUMN CHROMATOGRAPHY
PRINCIPLES
 Solid materials (Adsorbants) – Ability to hold the molecules at their surface
 Attractive forces (Vanderwalls & Hydrogen )
 Functional groups (Hydroxyl/ Aromatic)
 Silica

B. PLANAR CHROMATOGRAPHY
Separation technique - Stationary phase is present as or on a plane. Paper – Paper

Chromatography

Layer of solid particles spread on a support such as a glass plate

 Thin layer Chromatography.
Different compounds in the sample mixture travel different distances according to how strongly they interact with
the stationary phase as compared to the mobile phase.

C. PAPER CHROMATOGRAPHY
Paper chromatography is a technique that involves placing a small dot or line of
sample solution onto a strip of chromatography paper. The paper is placed in a container with a shallow layer of
solvent and sealed. As the solvent rises through the paper, it meets the sample mixture, which starts to travel up
the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the
mixture travel further if they are less polar. More polar substances bond with the cellulose paper more quickly, and
therefore do not travel as far.
 D. THIN-LAYER CHROMATOGRAPHY (TLC)
 Thin-layer chromatography (TLC) is a widely employed laboratory technique used to
separate different biochemicals on the basis of their relative attractions to the stationary
and mobile phases. It is similar to paper chromatography.
 Used to identify the unknown compounds and to determine the purity of mixture.

Advantages :
 Simple, Rapid and Cheap
 Faster runs
 Better separations
 Choice between different adsorbents.
 Better resolution
 Allow for quantification
TLC is very versatile; multiple samples can be separated simultaneously on the same layer, making it very
useful for screening applications such as testing drug levels and water purity.

GAS CHROMATOGRAPHY
Gas-Liquid chromatography, (GLC)
Mobile phase – Gas (Helium) Carrier Gas Pressure = 4 kg/cm2 Stationary phase -
Column, which is typically "packed" or "capillary".
The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid
matrix inside a larger metal tube (a packed column).
Partition Coefficient of Volatile analyte between a solid stationary phase (Silicone) and a mobile gas (Helium).
AFFINITY CHROMATOGRAPHY

Based on specific & non-covalent binding of the proteins to other molecules – Ligands (His-tags, biotin or antigens)
Physical properties of the analyte.
Biochemistry in the purification of proteins (Enzymes) bound to tags.
After purification, some of these tags are usually removed and the pure protein is obtained.

SUPERCRITICAL FLUID CHROMATOGRAPHY

Used for the analysis and purification of low to moderate molecular weight, thermally labile molecules.
Principles are similar to those of (HPLC)
Mobile phase - High pressure liquid or Super critical Carbon Dioxide. Modifiers – Methanol, Ehanol, isopropyl alcohol, acetonitrile and Chloroform.

APPLICATION
 Use in industry primarily for separation of Chiral (Asymmetric Carbon atoms) molecules.
• Soman
• Glyceraldehyde
• Phosphours (Phosphine)
• Sulfar metal
• Cobalt
• Enkephalins

DETECTOR
 Gas Chromatography or liquid Chromatography
 To visualize components of the mixture being eluted off the chromatography column.

CONCLUSION
Chromatography is a process for separating components of a mixture. To get the process started, the mixture is dissolved in a
substance called the mobile phase, which carries it through a second substance called the stationary phase.
Keller, Roy A. and Giddings, J. Calvin. "chromatography". Encyclopedia
Britannica, 10 Nov. 2020,
https://www.britannica.com/science/chromatography. Accessed 1 June
2022.

C.F. Poole,
CHROMATOGRAPHY,
Editor(s): Ian D. Wilson,
Encyclopedia of Separation Science,
Academic Press,
2000,
Pages 40-64,
ISBN 9780122267703,
https://doi.org/10.1016/B0-12-226770-2/00021-1.
(https://www.sciencedirect.com/science/article/pii/B012226770200021
1)
INSTRUCTIONS FOR CARD GAME

1) A total of 15 questions on the topic will be provided to the players.

2) Cards which stated “ TRUE “ or “ FALSE “ will be distributed to the players as
well.
3) Show the card according to the questions asked.
4) Winners will be based on who had answer more to the right answers.
 Questions For The Card Game

1) Chromatography is technique of separating the components or solute of each

mixture.

- True / False

2) Stationary phase stands for the mobilized phase.

- True / False

3) Retention Factor ( Rƒ ) = Distance travelled by a solute

Distance travelled by a solvent

- True / False

4 ) Chromatogram is the graph that indicates the outcome of chromatography.

- True / False

5) Chromatography consists of Stationary Phase and Mobile Phase.

- True / False

6) Ink can be separated by a method of chromatography

- True / False
7) Chromatography separate the mixture of dyes on the basis of their gravity

- True/False

8) Mixtures can be separated using chromatography

- True/False

9) In paper chromatography , the LEAST soluble solute stays closest to the start point

- True/ False

10 ) Chromatography useful it can be used as medicine.

- True/False

11) TLC is used to identify the known compounds and to determine the purity of mixture.

- True/False

12) Affinity chromatography is consist of Supercritical fluid chromatography.

- True/False

13) The Stationary phase of Gas Chromatography is typically “packed” or “capillary”

- True/False

14) The advantage of TLC is Simple, Rapid and Cheap.

- True/False