KROMATOGRAFI

16ANK4034
Silabus:
1. Pendahuluan
2. Konsep dan Teori Kromatografi
3. Karakteristik Pemisahan dan Penggunaan
Krom.
4. Kromatografi Kolom dan Modifikasinya
5. Kromatografi Kertas dan Kromatografi Lapis
Tipis
6. Kromatografi Gas
7. Kromatografi Cair Kinerja Tinggi (HPLC)
Rujukan:
Sumar Hendayana, “Kimia Pemisahan”,
Penerbit Rosda, Bandung, 2006, 2010
Bonneli, “Pengantar Kromatografi”, terj: K.
Padmawinata dan I. Soediro, Penerbit ITB,
1990
Ibnu Gholib Gandjar dan Abdul Rohman,
“Kimia Farmasi Analisis”, cetakan ke-7,
Pustaka Pelajar, Yogyakarta, 2010
Rujukan Lebih Lanjut:
Chromatographic Methods 5th Edition - A. Braithwaite
and F.J. Smith, Kluwer, Dordrecht, 1999
Chromatography and Separation Science, Satinder
Ahuja, Academic Press, Amsterdam, 2003
Chromatography_Concepts and Contrasts 2nd ed,
James Miller, John Wiley & Sons, 2005
Introduction to Modern Liquid Chromatography,
Joseph J. Kirkland, and John W. Dolan, Wiley, 2010.
Einführung in die Laborpraxis Basiskompetenzen für
Laborneulinge, Springer 2011
Bab 1 : Pendahuluan
Definisi Umum:
Kromatografi merupakan suatu metoda
pemisahan campuran komponen
(senyawa) dalam komponen (senyawa)
penyusunnya dengan cara komponen
(senyawa) penyusunnya tersebut
terdistribusi di antara fasa diam atau fasa
gerak
Bab 1: Pendahuluan
Definisi IUPAC
A Method used primarily for the separation of
components of a sample, in which the
components are distributed between two
phases, one of which is stationary while other
moves. The Stationary phase may be solid,
or a liquid supported on a solid, or a gel. The
stationary phase may be packed in a column,
spread as a layer, or distributed as a film, etc.
Bab 1: Pendahuluan
Kromatografi Fasa diam Fasa gerak contoh
Kromatografi
padat gas GSC
gas-padat
Kromatografi
cair gas GLC (GC)
gas-cair

Kromatografi kolom
Kromatografi
padat cair Kromatografi lapis tipis
cair-padat
Kromatografi kertas

Kromatografi
cair cair HPLC
cair-cair
Sejarah Kromatografi
Mikhail Tswett, Russian, 1872-1919
Botanist
In 1903 Tswett used to chromatography to
separate plant pigments
He called the new technique chromatography
because the result of the analysis was 'written
in color' along the length of the adsorbent
column
Chroma means “color” and graphein means to “write”
 Original Chromatography Experiment
Start: A glass End: A series of
column is filled colored bands is
with powdered seen to form,
limestone corresponding to
(CaCO3). the different
An EtOH extract Later pigments in the
of leaf pigments original plant
is applied to the extract. These
top of the column. bands were later
determined to be
EtOH is used to
chlorophylls,
flush the pigments
xanthophylls and
down the column.
carotenoids.
Sejarah Kromatografi
The Russian botanist Mikhail
Tswett coined the term
chromatography in 1906 to
describe his experiments in
separating different colored
constituents of leaves by
passing an extract of the
leaves through a column
Penggolongan Metode Kromatografi

Different methods were attempted for classification of

chromatography:
A: Berdasar Fase Gerak
B: Berdasar mekanisme pemisahan
C: Berdasar teknik pemisahan
D: Berdasar tujuan penggunaan
A- ACCORDING TO MOBILE PHASE:
In this regard chromatography is classified into:

1- Liquid Chromatography (LC):

The mobile phase is liquid. In case of separation by
adsorption the stationary phase is solid so it is called:
Liquid-Solid Chromatography (LSC). If separation occurs
through partition the stationary phase is liquid so it is called:
Liquid -Liquid Chromatography (LLC).

2- Gas Chromatography (GC)

Where the mobile phase is inert gas nitrogen or helium.
Again if the stationary phase is solid it is called: Gas–Solid
Chromatography (GSC). When stationary phase is liquid it is
called: Gas-Liquid Chromatography (GLC).
B – According to mechanism of separation:
The mechanism of separation depends mainly
on the nature of the stationary phase. Based on
separation mechanisms chromatography can
be classified into:
1. Adsorpsi
2. Partisi
3. Pertukaran ion (ion-exchange)
4. Eksklusi/Permeasi (pengusiran/pendesakan)
5. Afinitas
6. Kiralitas
7. Elektroforesis
1- Adsorption Chromatography:

It is the oldest and most common type of

chromatography. The stationary phase is a solid
with adsorption power. Mixture components will
be adsorbed on the surface of the stationary
phase with different powers and that account for
separation. Silica gel is the most common
stationary phase in adsorption chromatography.
 Adsorption Chromatography
Very similar to partition
chromatography
Adsorption just on
surface, partition into thin
layer
Not used as widely as
partition used mainly in
TLC & very small
particles in LC
 2- Partition Chromatography:
The stationary phase is a liquid forming a thin film on an
inert solid acts as support. The stationary liquid is
usually more polar than the mobile liquid. The two
liquids must be immiscible with each other. Cellulose
powder and wet silica gel are examples of supports in
partition chromatography that carry film of water act as
stationary phase. Partition chromatography is preferable
over adsorption when dealing with polar compounds.
Partition Chromatography
Used in GC & LC
Molecules will partition into the
stationary phase based upon
affinity for stationary phase &
eventually partition into mobile
phase again
Thin layer is coated onto inside
of GC column or on small
particles on LC column
 3- Ion Exchange Chromatography:

It is used for separation of charged molecules. The stationary

phase is an ion exchange resin to which a cationic or anionic
groups are covalently bonded. Ions of opposite charges
(counter ions) in the mobile phase will be attracted to the resin
and compete with the components of the mixture for the
charged group on the resin. Both the mixture components and
the mobile phase must be changed. Mixture of Alkaloids
(compounds with positive charges) can be separated on
anionic exchanger, while mixture of organic acids (negative
charges) can be separated using cationic exchanger. Both
types are used for desalination of water.
Ion Exchange Chromatography
Separation of either
cations or anions
Separtion based on
relative strength of ionic
bond
Anion exchange has
cations on surface
Used in LC exclusively
 4- Molecular Exclusion
( Size Exclusion ) Chromatography:
 Molecular Exclusion
Chromatography
Separation based on
size
Small molecules get
trapped in pores &
take longer to get out
5- Affinity Chromatography:
It uses the affinity of proteins to specific ligands
such as enzymes. The ligand is attached to suitable
polysaccharide polymer such as cellulose -
agarose – dextran.
6- Chiral Chromatography:
In this type we can separate enantiomers – we used
chiral stationary phase that react with one
enantiomer more then the other so separation takes
place.
 Affinity Chromatography
Very selective
Specific binding site is
used to concentrate
analyte on column
Used a lot in
biological applications
7- Zone Electrophoresis:
C- ACCORDING TO THE TECHNIQUE (methods of
holding the stationary phase):

1- Planar or Plane Chromatography:

In this type of chromatography the stationary phase is used in the form of
layer. Plane chromatography is further classified into:
a- Thin Layer Chromatography (TLC):
The stationary phase in the form of fine powder is spread on glass or
plastic or aluminum sheets.
b- Paper Chromatography (PC):
A specific type of papers is used as stationary phase in the form of sheets.

2- Columnar or Column Chromatography (CC):

The stationary phase is held in to a tube made of glass or metal.
 D- ACCORDING TO PURPOSE OF USE:
Chromatography can be used for analytical work and also to obtain pure
materials from mixtures.

1- Analytical Chromatography:
a- Qualitative Chromatography
In this case Chromatography can be used to:
1- Confirm the absence or probable presence of certain constituent
in the sample under investigation
2- Give an idea about the complexity of the mixture and the least
number of compounds present.
3- Check purity and identity of any compound.
4- Establish a (finger print ) pattern for extracts , volatile oils or
pharmaceutical preparations. These finger prints can be then used to
check the identity and purity in the future.
5- Monitor both column chromatography and organic chemical
reactions.
 b- Quantities Chromatography:

The development of modern instruments enable the

use of chromatography to determine the amount of
any component in a mixture as absolute amount or
relative to another component HPLC/ GC/ HPTLC
can be used for there applications.
2- Preparative application:

This was the first and is the main application of

chromatography. The technique was developed
primarily for this purpose.
Chromatography is used to obtain reasonable
quantities of pure compounds from mixtures.
BAB 02

KONSEP DAN TEORI

KROMATOGRAFI
How Does Chromatography Work?
In all chromatographic separations, the sample is transported in
a mobile phase. The mobile phase can be a gas, a liquid, or a
supercritical fluid.

The mobile phase is then forced through a stationary phase

held in a column or on a solid surface. The stationary phase
needs to be something that does not react with the mobile
phase or the sample.

The sample then has the opportunity to interact with the

stationary phase as it moves past it. Samples that interact
greatly, then appear to move more slowly. Samples that interact
weakly, then appear to move more quickly. Because of this
difference in rates, the samples can then be separated into
their components.
Konsep dan teori kromatografi
Konsep dan teori kromatografi
Chromatography is based on a physical equilibrium
that results when a solute is transferred between the
mobile and a stationary phase.
K = distribution
A coefficient or
partition ratio
A A
A A
CS
A A K =
A CM
A Where CS is the molar
A concentration of the solute in
A
A the stationary phase and CM is
the molar concentration in the
mobile phase.
Cross Section of Equilibrium in a column.
“A” are adsorbed to the stationary phase.
“A” are traveling in the mobile phase.
Konsep dan teori kromatografi
Konsep dan teori kromatografi

Column Chrom.

Chromatogram
Konsep dan teori kromatografi
Konsep dan teori kromatografi
Skema linarut
Konsep dan teori kromatografi
Konsep dan Teori Kromatografi
Konsep dan Teori Kromatografi
Konsep dan Teori Kromatografi
Konsep dan Teori Kromatografi
Konsep dan Teori Kromatografi
Tugas Kelompok
A B C D
The analytes interacting most
strongly with the stationary
phase will take longer to pass
through the system than those
with weaker interactions.

These interactions are usually

chemical in nature, but in some
cases physical interactions can
also be used.
 Konsep dan teori kromatografi
Flow

Flow

Flow

Flow

In a mixture, each component has a different distribution coefficient, and thus spends a
different amount of time absorbed on the solid packing phase vs being carried along with
the mobile phase.
If a detector is used to determine when the components
elute from the column, a series of Gaussian peaks are
obtained, one for each component in the mixture that was
separated by the column.

Note: The first two components were not completely separated.

Peaks in general tend to become shorter and wider with time.
Konsep dan teori kromatografi
Kromatogram
Kromatogram merupakan hasil rekaman
yang menggambarkan urutan keluarnya
komponen campuran dari kolom.
Dari kiri ke kanan dalam kromatogram
menyatakan waktu, biasanya dalam menit.
Sumbu vertical menyatakan intensitas
komponen.
Jumlah peak yang muncul menyatakan
jumlah komponen yang terdapat dalam
campuran.
Konsep dan teori kromatografi
Contoh Kromatogram
Konsep dan teori kromatografi
The Theoretical Plate (Lempeng Teori)
Theoretical plate is a term coined by Martin & Synge.
It is based on a study in which they imagined that
chromatographic columns were analogous to distillation
columns and made up or numerous discrete but connected
narrow layers or plates. Movement of the solute down the
column then could be treated as a stepwise transfer.

Theoretical plates (N) measure how efficiently a

column can separate a mixture into its components.
This efficiency is based on the retention time of the
components and the width of the peaks.
Konsep dan teori kromatografi
The Theoretical Plate (Lempeng Teori)
Hubungan Efisiensi Kolom dan Lempeng Teori

L = length of column packing

s standard deviation s2/L variance per unit length.

Konsep dan teori kromatografi

Chromatogram
Konsep dan teori kromatografi
The Theoretical Plate (Lempeng Teori)
Hubungan Efisiensi Kolom dan Lempeng Teori

W1/2
BAB 03

KARAKTERISTIK PEMISAHAN
DAN DINAMIKA KROMATOGRAFI
Karakteristik Pemisahan dan
Dinamika Kromatografi
 Parameter kromatografi
1. Waktu Retensi (tR)
Ukuran waktu mulai injeksi cuplikan
hingga suatu komponen campuran keluar
kolom.
 2. Faktor Kapasitas (k’)
Factor kapasitas merupakan suatu ukuran kekuatan inetarksi suatu
komponen dengan fasa diam yang diformulasi sebagai berikut:

k’= (tR-t0)/t0 = ns/nm = K(Vs/Vm)

k’= factor kapasitas

tR = waktu retensi
t0 = waktu yang diperlukan oleh suatu komponen yang tidak
berinteraksi dengan fasa diam untuk meninggalkan kolom
ns = jumlah mol suatu senyawa di dalam fasa diam
nm = jumlah mol suatu senyawa di dalam fasa gerak
K = Koefisien partisi
Vs = volume fasa diam
Vm= volume fasa gerak
 3. SELEKTIVITAS

Secara umum, slektivitas dapat diartikan

sebagai ukuran keterpilihan dua
komponen campuran yang dipisahkan,
diformulasikan sebagai berikut:
α = k’2/k’1
k’1 dan k’2 masing-masing adalah factor
kapasitas komponen pertama dan
komponen kedua
 4. EFESIENSI
Tingkat efesiensi pemisahan dengan
kromatografi tercermin pada peak-peak
kromatogram yang dihasilkan. Semakin
lebar suatu peak kromatogram maka
dapat dikatakan pemisahan semakin
kurang efisien. Secara kuantitatif,
efesiensi ini dapat dijelaskan dengan teori
plat (N).
Harga N berbentuk kurva gaussan

N= tR2/ δ2
Secara praktis standar deviasi (δ) dapat
diganti dengan lebar peak (w) sehingga
dapat ditulis N = 16tR2 / w2 atau N= 5,55tR2
/ w21/2.
 4. EFESIENSI
Efesiensi pemisahan dapat juga
dinyatakan dalam bentuk parameter lain
yaitu HETP (Height Equivalent to a
theoretical Plat) yang diformulasikan
sebagai berikut:
HETP = L/N
L menyatakan panjang kolom dalam
centimeter. Kebalikan dari harga N,
semakin kecil harga HETP semakin efisien
 5. RESOLUSI
Resolusi adalah derajat pemisahan dua
komponen campuran dalam proses
kromatografi dinyatakan dengan istilah
resolusi (Rs) yang diformulasikan sebagai
berikut:
R = (N1/2/4) ((α-1)/ α)((k’2/(1+k’2))

N = Efesiensi rata-rata
α = selektivitas
k’ = retensi
 5. RESOLUSI
Resolution (Rs)
Resolution (R) defines the degree of separation of 2 adjacent bands.
Base line resolution is achieved when R>1.5 and it is a fundamental goal
for quantitative analysis.

Pemisahan sempurna jika Rs= 1,5

70
5. RESOLUSI

W1/2
5. RESOLUSI
5. RESOLUSI
5. RESOLUSI
2. Diketahui suatu kromatogram dari analisis
campuran yang mengandung isomer
nitroanilin sbb.:
Parameter o-nitroanilin m-nitoanilin p-nitroanilin
Waktu retensi (tR) 5 menit 30 detik 5 menit 40 detik 6 menit 25 detik
Luas alas (Wb) 11 detik 13 detik 16 detik
Luas alas ½ tinggi 6,3 detik 7,2 detik 8,7 detik
(Wh/2)

a. Hitunglah bilangan/jumlah lempeng dari setiap puncaknya!

b. Hitung pula tinggi setara lempeng teoritis dari puncak2 tsb.
(panjang kolom: 1,5 meter)!
c. Hitung daya pisah di antara dua solut yang berdekatan!
d. Apa yang dapat disimpulkan dari kromatogram tersebut
dengan melihat nilai daya pisah yang diperoleh?
Profll Pelebaran Puncak
Peak (Band Broadening)
 Konsep dan teori kromatografi
Flow

Flow

Flow

Flow

In a mixture, each component has a different distribution coefficient, and thus spends a
different amount of time absorbed on the solid packing phase vs being carried along with
the mobile phase.
Profll Pelebaran Puncak
Peak (Band Broadening)
1) Why broadening?
a) diffusion
b) slow equilibration of solute between the
m.p and s.p.
c) irregular flow paths.
Profll Pelebaran Puncak
Peak (Band Broadening)
2) Longitudinal
diffusion :
the faster the flow
 the less a band
spends in column.
the less time for
diffusion.
 broadening
Profll Pelebaran Puncak
Peak (Band Broadening)
3) solute requires time to equilibrate
between phases.
m.p.

s.p.

(s.p.m.p.) with temp.

broadening  u
Can’t equilibrate rapidly enough.
Profll Pelebaran Puncak
Peak (Band Broadening)
Profll Pelebaran Puncak
Peak (Band Broadening)
4) An optimum rate : flow rate for the best
separation.
Profll Pelebaran Puncak
Peak (Band Broadening)
5) Multiple paths
 Profll Pelebaran Puncak
Peak (Band Broadening)
Plate height equation
Profll Pelebaran Puncak
Peak (Band Broadening)
Kinetic Processes

Van - Deemter Equation

Profll Pelebaran Puncak
Peak (Band Broadening)
Kinetic Processes
Van - Deemter
Equation
λ and γ are constants that
depend on quality of the
packing.

B is coefficient of
longitudinal diffusion.

Cs and Cm are coefficients

of mass transfer in
stationary and mobile
phase, respectively.
Profll Pelebaran Puncak
Peak (Band Broadening)
6) Plate height equation
BAB IV
KROMATOGRAFI KOLOM DAN
MODIFIKASINYA
IV. Kromatografi Kolom dan Modifikasinya

A. Krom. Kolom Klasik (Kolom Terbuka)

B. Krom. Kolom dengan modifikasi fase diam
1) Kr. Fase terbalik (RP-reverse phase)
2) Kr. Pertukaran ion
3) Kr. Permeasi gel (eksklusi ukuran/molekul)
4) Kr. Afinitas
C. Krom. Kolom dengan modifikasi operasi
1) VLC (KCV-krom cair vakum)
2) Flash chrom
3) Krom tekanan (rendah, sedang, tinggi)
A. Krom. Kolom Klasik (Kolom
Terbuka)
A.1 Kolom
- terbuka, bekerja karena gaya tarik bumi
- Terbuat dari kaca, polimer, stainless steel
- Perbandingan diameter dan panjang
(antara 10 s.d 100 kali)

A.2 Fase Diam

A.3 Fase Gerak
A. Krom. Kolom Klasik (Kolom
Terbuka)
 Types of columns:
1- Gravity Columns:
The mobile phase move through the stationary phase by gravity
force.
2- Flash Columns (Air or nitrogen pressure):
The mobile phase is pushed by stream of air or nitrogen using
special using special values (Adaptors).
3-Low and Medium Pressure Columns (pumped):
The movement of mobile phase is accelerated by using pumps
that generate low or medium pressure. The increase in the flow
rate shorten the time of separation.
4-Vacuum Columns [Vacuum liquid chromatography (VLC)]:
The adsorbent is applied dry into a sintered glass funnel. The
sample is applied by dry method or as solution. Then the mobile
phase is added portion by portion and vacuum is applied after each
portion to collect each fraction.
5- High pressure Columns (HPLC):
In this columns we use very fine silica gel so great
increaser in separation power. However, the flow
rate of the mobile phase is severely decreased.
High pressure pumps are used to push the solvent
through the column which in this case must be made
of stainless steel.
 Backing of Columns:
The adsorbent is applied to the Column in two ways:

Slurry packing (Wet method):

The adsorbent is suspended in the mobile phase
and stirred very well to drive off all air bubbles.
The resulted slurry is then poured into the column.
At the tap end of the column a piece of glass wool
or cotton must be added before the slurry
application. Sand may be added after the slurry.
After slurry application the column must be allowed
to settle overnight.

In gel chromatography the adsorbent must be

soaked in the mobile phase overnight to absorb
the mobile phase and swell.
2- Dry Packing:
In this method the dry adsorbent is poured to the
column directly. Vibration is the applied to get rid
of air bubbles then the mobile phase as passed
through the adsorbent. This method can not be
applied gel Chromatography.
Mobile phase:
It is a mixture of organic solvents (unusually one
solvent only) the choice of the column mobile
phase is achieved after TLC study in different
solvent systems. Good solvent system must
produce Rf value less than 0.6 for all materials to
be separated by the column. If the system
moves them more and produces higher Rf no
separation will occur. Systems that do not move
spots at all on TLC are not good for column
separation.
Isocratic system:
Means using the same mobile phase from the
beginning to the end of the separation.

Gradient:
The polarity of the system increased gradually
during separation by increasing the proportion of the
more polar solvent. A typical gradient may be start
with CHCl3, followed by CHCl3/MeOH mixtures with
gradual increase in % of MeOH till all spots are
eluted from the system.
Monitoring the column:
Usually fractions of certain volume are
collected evaporated to small volume and
spotted on TLC. Similar fractions are
collected together for more purification or
crystallization.
In bioassay guided fraction the fractions are
monitored by the bioassay then by TLC.
Monitoring the column:

Usually fractions of certain volume are collected

evaporated to small volume and spotted on TLC.
Similar fractions are collected together for more
purification or crystallization.

In bioassay guided fraction the fractions are

monitored by the bioassay then by TLC.
 Sample Application
1- Wet application: Dissolve the sample in
the initial mobile phase and apply by pipette
to the top of the column. This is very good
method but in most of cases the samples are
not soluble in the initial mobile phase.

2- Dry loading: Dissolve sample in any

volatile solvent. The sample solution is then
adsorbed on small weight of adsorbent and
the solvent is allowed to evaporate. The dry
adsorbent loaded with the sample is then
applied to the column.
Factors affecting separation:
 Factors due to Stationary Phase:
1- Particle size of the stationary phase: Reducing the
particle size increases the surface area and
improve separation. However, reduction of the
particle size will decrease the flow rate of the
mobile phase.

In HPLC we use very fine particles to get very good

separation. The flow rate problem is solved by the
use high pressure pumps to push the mobile phase
through the stationary phase. Columns are made of
stainless steel to withstand the high pressure.
2- Adsorbent activity: The choice of the suitable
adsorbent is very important.

3- Uniformity if packing of the column: If the

stationary phase is not packed uniformly then
the bands will be irregular and less uniform
resulting in poor separation.

4- Concentration of the mixture: the proper ratio

between sample to be separated and the
amount of stationary phase is very important
too much samples resulted in bad separation.
 Factors due to Mobile Phase:
1- Selection of the proper mobile phase: Very polar mobile
phase will wash out all components without any separation.
On the other hand very non polar mobile phase will result in
broad band and poor separation.

2- Rate of flow: Slower flow rate usually resulted in a better

separation and narrower bands.

3- Consistency of flow: The continuous flow of the mobile phase

during the whole experiment gives better separation than
interrupting the flow then continue it later.
 Factors due to Columns:

Column dimensions: Increasing the length of the

column improve separation. However, that
usually leads to slower flow rate. Also increasing
the column length some times is impractical.

Column temperature: Increasing the temperature

usually reduces the adsorption power of the
stationary phase and increase elution speed. This
may leads to decrease in the efficiency
separation.