Pendahuluan

Fithriani Armin, M.Si., Apt.

What on Earth did scientist do before
Chromatography?
- Extraction
is based on the difference in solubility material
is grounded, placed with a solvent which
dissolves soluble compounds. A second
extract solvent . The mixture is placed in a
separatory funnel
- Crystallization
also based on the difference of solubility. The
solubility is solved in a fixed volume of solvent.
The purified compound crystallizes as solution
cools, evaporates or diffuses
- Distillilation
separates components based on their volatility
typically via vaporization-condensation
method
Filtration
separate components of a mixture based on
their particle size. Used most often to
separate a liquid from a solid
www.chemguide.co.uk/.../idealfract.html
Brief History of Chromatography
1903 Tswett, a Russian botanist coined
the term chromatography. He passed plant
tissue extracts through a chalk column to
separate pigments by differential
adsorption chromatogrpahy
1915 R.M Willstatter, German Chemist win
Nobel Prize for similar experiement
1922 L.S Palmer, American scientist used
Tswetts techniques on various natural
products
1931 Richard Kuhn used chromatography to
separate isomers oh polyene pigments; this
is the first known acceptance of
chromatographic methods
http://www.chemgeo.uni-hd.de/texte/kuhn.html
Why Chromatography?

Chromatography is a very important technique in

chemistry. The reason is that it can separate one type
of molecules from others.
 Chromatography is an analytical technique used to
determine the purity of a substance or to separate a
mixture into its components.
 Chromatography is a non-destructive procedure for
resolving a complex mixture into its individual
fractions or compounds.
This is a separation procedure and the separated
entities are identified by other analytical techniques
like UV-visible, Infrared, NMR (nuclear magnetic
resonance), Mass spectrometry etc. In applications for
quantitative analysis the measurement of the area
under the curve in chromatogram is done.
Definition & Principle in chromatography

Definition:

Chromatography is defined as the process of

separation of the individual components of a mixture
based on their relative affinities towards stationary and
mobile phases.
 The stationary phase refers to the solid or liquid to
which components in a mixture bind or adsorb.

The mobile phase refers to the liquid or gas that moves

the components in a mixture over the stationary
phase.
 Menurut Farmakope Indonesia IV
Kromatografi adalah suatu teknik atau prosedur
pemisahan zat terlarut oleh suatu proses migrasi
diferensial dinamis dalam sistem yang terdiri
dari dua fase atau lebih yang salah satu
diantaranya bergerak secara berkesinambungan
dalam arah tertentu dan didalamnya zat-zat itu
menunjukkan perbedaan mobilitas disebabkan
adanya perbedaan dalam adsorpsi, partisi,
kelarutan, tekanan uap, ukuran molekul atau
kerapatan muatan ion.
 Menurut IUPAC
Kromatografi adalah metode yang
digunakan untuk pemisahan
komponen dalam sampel dimana
komponen itu terdistribusi dalam
dua fase yang salah satunya diam
dan yang lainnya bergerak.
Principle:
The samples are subjected to flow by mobile liquid
phase onto or through the stable stationary phase. As
in the definition the principle involved is separation of
fractions of mixture based on their relative affinity
towards the two phases during their travel.

The fraction with greater affinity to stationary phase

travels slower and shorter while that with less affinity
travels faster and longer.
 Components in a mixture are separated based on their
different abilities to bind or adsorb to the stationary
phase, and on their different abilities to desorb or
dissolve in the mobile phase.
Types of Chromatography.

Based on the mode or method employed in separation

chromatography is broadly classified as

1. Adsorption mode: Here the stationary phase is a

solid while the mobile phase is liquid. The compounds
travel on the stationary phase under the influence of
mobile phase based on their relative adsorption to the
solid stationary phase.
2. Partition mode: In this mode both the stationary
and mobile phase are liquids. So the compounds have
affinity based on their partition into the individual
liquid phases. The one with greater partition to
stationary phase has higher affinity to stationary phase
and vice versa.
Based on the nature of stationary phase it is of
two types:

a) Normal phase chromatography: Here the

stationary phase is polar in nature and hence the
compounds with higher polarity elute out last while
non polars come out first.
b) Reverse phase chromatography: Here the
stationary phase is non-polar in nature and hence the
compounds with lower polarity elute out last and vice-
versa.

In most HPLC analysis, the mode used is reverse phase

chromatography as many of the biological,
phytochemical compounds and even drugs are polar in
nature.
Berdasarkan sifat fisika fase diam dan fase gerak
Berdasarkan bentuk kemasan atau geometrik fase
diam

Kromatografi planar, dimana fase diam tersebar

dalam bentuk lapis tipis pada lempeng kaca,
plastic atau aluminium (KLT) atau dalam bentuk
lembaran bahan selulosa (KK).
Kromatografi kolom, dimana fase diam dikemas
dalam suatu kolom gelas atau logam (KG, KC,
dan KCSK).
Berdasarkan mode pemisahan

* Adsorpsi : Kromatografi adsorpsi

* Partisi : Kromatografi Partisi
- Kromatografi fase terikat
- Kromatografi pasangan ion
- Kromatografi penekanan ion
* Pertukaran ion : Kromatografi pertukaran ion
* Eksklusi ukuran : Kromatografi eksklusi ukuran
* Afinitas : Kromatografi afinitas
Berdasarkan cara pengembangan / elusi

Pengembangan elusi (Elution Development)

Analisis frontal (Frontal Analysis)
Pengembangan pemindahan (Displacement
Development) Pengusiran
technique basis for separation apply this technique to:
adsorption / phase transfer to a solid surface liquid or gaseous mixtures that contain at
desorption least one component that adsorbs
chromatography phase transfer from a mobile mixture to liquid or gaseous solutions that contain
a stationary phase several components with differing affinities
for the stationary phase
condensation phase separation by condensing gases gaseous mixtures containing at least one
in the mixture to liquids gas with a much higher boiling point than
the others
dialysis phase transfer through a porous solutions containing small molecules
membrane that allows some molecules mixed with very large molecules
to pass through, but not others
effusion gases with faster molecules flow through tiny gaseous mixtures containing
pinholes faster than gases with slow molecules gases with different molecular
weights
dissolution (washing, soluble components can be washed away, mixtures of solids with different
solvent extraction) leaving behind insoluble components (phase solubilities
transfer to a washing solvent)
electrorefining separate a metal from impurities by dissolving solid mixtures with a metal as
it and then plating it onto an electrode one component
filtration collect solid particles on a filter heterogeneous mixture
containing a solid phase
floatation dense components sink, and lighter ones float heterogeneous mixture with
phases with different densities
ion exchange ions in the mixture bind to surfaces with
oppositely charged sites (phase transfer to an
ion exchange resin)
precipitation convert solutes to an easily separated solid
form
scrubbing bubble mixture through a solution that
selectively absorbs a component (phase
transfer from gas to solution)
stripping a gas bubbled through the mixture carries off
the most volatile components
(phase transfer from solution to gas)
volatilization components with widely differing volatility can
be driven out of the mixture by heating (phase
(drying, change from solid or liquid to gas)
distillation,
sublimation)
solutions containing ions
solutions containing a solute that can
be precipitated
gaseous mixtures containing a
solute that can be selectively
absorbed by a scrubbing solution
a liquid mixture containing at least
one volatile component
a mixture containing components
with differing volatility
Advantages of chromatography

can separate very complex mixtures

drugs, plastics, flavorings, foods, pesticides,
tissue extracts, fuels, air samples, water
samples, ...
very small sample sizes
separated components can be collected
individually
analyses can be highly accurate and precise
Sheet chromatography

paper chromatography (PC)

stationary phase is liquid soaked into a sheet or
strip of paper
mobile phase is a liquid solvent
some components spend more time in the
stationary phase than others
components appear as separate spots spread out
on the paper after drying or "developing"
 thin layer chromatography (TLC)
stationary phase is a thin layer of adsorbent
(Al2O3 or SiO2, usually) coating a sheet of plastic
or glass
some components bond to the adsorbent
strongly; others, more weakly
as with paper chromatography, components
appear as spots on the sheet
Column chromatography
gas chromatography (GC)
sample mixture is injected into a long tube (the
column)
mobile phase is an inert gas that sweeps the sample
down the tube
stationary phase lining the tube selectively adsorbs or
dissolves components
the stationary phase is a solid or very syrupy liquid

silicone polymers (like Silly Putty!) are often used as

stationary phases in gas chromatography
a detector responds to separated components as
they leave the tube
What is chromatography used for?

1. finding concentrations
gas chromatogram of gasoline
ion chromatogram of orange juice
 each peak corresponds to a separate component in the
mixture
area of each peak is proportional to concentration
2. chemical fingerprinting
species identification
"killer" bees can be distinguished from native
bees by comparing gas chromatograms of
cuticle extracts
tracing contraband sources
detecting drugs in urine