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CHM256
CHM256
BASIC CHROMATOGRAPHY
Learning Outcomes
At the end of the chapter, student should be able to explain:
INTRODUCTION OF
CHROMATOGRAPHY
CHROMATOGRAPHY is Technique employed for separation
of the components of mixture by continuous distribution of the
components between 2 phases.
CLASSICAL SEPARATION METHODS
• Masking
• Precipitation
• Distillation
• Solvent extraction
• Ion exchange
• Chromatography
• Electrophoresis
CHROMATOGRAPHY
FUNCTION
The stationery phase can be paper, silica,
coated silica and derivatized silica.
Signal
grey
Stationary
phase: solid
Mobile
phase: liquid
Pink = surface of stationery.
Analyte attracted to the surface
Black dot
solute
2. Column chromatography
- Stationary phase: silica gel (solid)
- Mobile phase: solvent (liquid)
Macam oil & water campur lah camna pun akan terpisah
PARTITION CHROMATOGRAPHY
Mobile phase:
liquid
Stationary solute
phase: liquid
supported on
solid
PLANAR CHROMATOGRAPHY
COLUMN CHROMATOGRAPHY (CC)
TYPES OF CHROMATOGRAPHY
COLUMN AND PLANAR CHROMATOGRAPHY
2 TYPES OF CHROMATOGRAPHY
Column or elution
chromatography
Liquid solvent ;
benzene (liquid)
narrow tube
PC – an organic solvent
( in liquid )
Planar chromatography PC = water adsorbed
i. Paper on paper (liquid)
chromatography PC
TLC – a solid TLC – a solvent
ii. Thin Later
Chromatography TLC adsorbent ; alumina , ( in liquid )
Silica gel (solid)
Mobile phase sama cuma bezanya pada stationary phase
Separation column
Chromatography
Column Plana
chromatography
If SP polar
MP less polar
i) Paper
Chromatography
Principles and techniques of PC
Spotted
sample
Origin line
The more soluble a component,
The further it moves
solvent
Solvent front
(b) (a)
Origin line
Yg rendah
-less polar and strongly ad
a -More affinity so interact more strongl
Contains -So compound retarded and le
both
compound
b a&b
Rf a = 0.85
Rf b = 0.42
A = Polar compound. The compound is soluble.
B = nonpolar. The compound is less soluble
Same Rf value = Same
component present in the sample
Paper chromatography
Measuring
Retention
factor (Rf) value
Solvent:
spots move
mobile
upward by
phase
Filter capillary
paper: action
stationary
phase
• Retention factor (Rf) value
The distance travelled by the compound divided by the
distance travelled by the solvent.
Distance Solvent
by spot distance
Solvent front
blue
purple
10 cm
8 cm
6 cm
yellow
2 cm
Origin line
ii) Thin Layer Chromatography
(TLC)
Yg drawn guna
pencil td
TLC Developing Chambers Developing the TLC plates
Solvent must
below the line
(e) Visualizing the substance spots
eg; dia moving but we cant see so these are the methods
( 1-2 sec )
Sulfuric Acid
UV lamp
TLC plate under TLC plate under TLC plate inside the Spraying TLC plate
UV light (365 UV light (254 iodine chamber with ninhydrin
nm) nm)
Variety of TLC
Advantages of TLC
over PC
PC separation x smoothly
seperate
Tahan lasak
2. COLUMN CHROMATOGRAPHY
Mcm burette
but no marks
2. COLUMN CHROMATOGRAPHY
Eluent = mobile phase. Term shj berbeza.
Eluent is to carried the component of mixture through stationary phase
Adsorbtion = ada
interaction dgn SP
Macam MP
Depends on
Stationery Phase.
SP = powder
SP = slurry,
kepekatan tinggi
Analyze untuk tau component exist
in the sample?
eg; tryglicerine/ prot/ fat ke,
Principles of chromatographic separations
Sample Mobile phase
1) Sample injected in column.
Dlm coumn already ada SP.
2) masukkan MP as eluent
SAME macam TLC
& PC but this one
put in column Component A
Stationary
Component B
phase
Component C
If MP = polar, so SP = non-polar.
If sample polar so move faster. If non-polar slower sbb attracted to SP so retarded
Separation of compounds based on polarity
2. Perform a column chromatography using a polar
stationary phase and a non-polar mobile phase.
Stationary phase Stationary phase is Water trapped in the A solid adsorbent such as
placed in a narrow paper or paper strip alumina (Al2O3) or silica gel
tube or column. which made up of (SiO2).
cellulose fibers (made-
up of hydroxyl groups).
Hospital
- To detect types of blood or alcohol levels
Law Enforcement
- To compare a sample found at a crime scene with samples from
suspects
Environmental Agency
- To determine the level of pollutants in the water supply
Manufacturing Plant
- to purify a chemical needed to make a product
Applications of TLC
• To Analyze ceramides and fatty acids.