Metoda Pemisahan

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.ht
ml

WhyChromatography?

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.
2.

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:Herethe
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:Herethe

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
* Partisi

*
*
*

: Kromatografi adsorpsi
: 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
Development) Pengusiran

(Displacement

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