KROMATOGRAFI

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KROMATOGRAFI
Kromatografi adalah suatu cara pemisahan senyawa dari campurannya melalui elusi
sepanjang fasa lain yang tak bergerak.

Eluen dapat berupa fasa gas atau cair yang selanjutnya disebut fasa gerak (mobile
phase), sedangkan fasa tak bergerak atau fasa stasioner (fasionery phase) dapat berupa
padatan atau cairan yang diselaputkan pada permukaan padatan pendukung
(supporting solid) yang innert.

Chromatography involves a sample (or sample extract) being dissolved in a mobile
phase (which may be a gas, a liquid or a supercritical fluid).

The mobile phase is then forced through an immobile, immiscible stationary phase. The
phases are chosen such that components of the sample have differing solubilities in
each phase.

A component which is quite soluble in the stationary phase will take longer to travel
through it than a component which is not very soluble in the stationary phase but very
soluble in the mobile phase. As a result of these differences in mobilities, sample
components will become separated from each other as they travel through the
stationary phase.
SEJARAH KROMATOGRAFI
1834 Runge F F :pemisahan campuran zat warna dan ekstrak tanaman
1906 Tswett M. : pemisahan pigmen kloropil pada CaCO
3
dengan eluen
petroleum eter kromatografi
1931 Kuhn, R et al. : kromatografi cair-padat, pemisahan xanthophyl kuning telur
1941 Tiselius A.: kromatografi cair, frontal analisis
1941 Martin & Synge : model efisiensi kolom
1944 Consden, Gordon, Martin : kromatografi kertas
1947 Boyd et al.: kromatografi penukar ion
1951 Kirchner : Kromatografi lapis tipis (TLC)
1952 James & Martin : Kromatografi gas
1956 van Dempter : fungsi distribusi gausian
1964 J C. Moore : kromatografi gel permeation
Klasifikasi kromatografi
Fasa gerak Fasa diam Mekanisme Istilah/nama
Gas padat adsorbsi GSC
Cair partisi GLC
Cair cair partisi LLC
padat adsorbsi LSC
Penukar ion Penukar ion IEC
Gel Exclusion EC
Cairan
superkritik
Spesi organik
pada padatan
partisi SFC
Kromatografi
Kromatografi
gas
Kromatografi
cair
Gas - Cair Gas - padat Liqiud-liquid Lquid-solid Ion-exchange Exclusion
Klasifikasi : peralatan
Kromatografi kolom
Kromatografi lapis tipis (TLC = Thin Layer Chromatography)
Kromatografi kertas (paper chromatography)
Kromatografi gas (GC= gas chromatography)
Kromatografi cair kinerja tinggi (HPLC = high performance liquid
chromatography)
KROMATOGRAFI KOLOM
Kromatografi Kolom
Kesetimbangan distribusi
Fasa gerak
Fasa diam
K = -----------
Cd
Cg
K = koefisien distribusi
Cg = Konsentrasi dalam fasa gerak
Cd = konsentrasi zat dalam fasa diam
Fraksi waktu tinggal dalam fasa gerak =-------------------------------------------------
Jumlah molekul dalam fasa gerak
Total molekul yang ada
= ------------------------
Cg Vg
Cg Vg + Cd Vd
= ------------------------
1
1 + K Vd/Vg
= -----------
1
1 + k
k = faktor kapasitas
Vg =volume fasa gerak
Laju pergerakan
Fraksi = -----------
1
1 + k
Laju linier =
Laju pergerakan =
-----------
1
1 + k
Laju pergerakan rata-rata ,molekul ditentukan oleh:

1. Laju fasa gerak
2. Rasio volume fasa diam terhadap volume fasa gerak
3. Koefisien distribusi: khas untuk setiap komponen
Waktu retensi, t
R
Waktu yang diperlukan oleh komponen menempuh perjalanan sepanjang kolom
t
R
= ------------------ = -------- (1+k)
panjang lintasan
Laju pergerakan
L

= t
M
(1+k)
t
M
= waktu yang diperlukan oleh molekul fasa gerak sepanjang
lintasan/kolom
Volume retensi
Volume fasa gerak yang diperlukan untuk mengelusi komponen sampel
Vol = waktu x laju alir
V
R
= t
R
x F
V
R
= V
M
(1+k) = V
M
+ KVd
Retensi relatif:

Kromatografi lapis tipis
TLC
Thin-layer chromatography
(TLC) is a chromatographic
technique that is useful for
separating organic compounds.
Because of the simplicity and
rapidity of TLC, it is often used
to monitor the progress of
organic reactions and to check
the purity of products.
Teknik Elusi KLT/KK
Elusi Ascending : dari bawah ke atas
Elusi Discending : dari atas ke bawah
Elusi dua dimensi
Gambar teknik elusi

Elusi dua dimensi
Two-dimensional TLC uses the TLC method twice to separate spots
that are unresolved by only one solvent. After running a sample in
one solvent, the TLC plate is removed, dried, rotated 90
o
, and run in
another solvent. Any of the spots from the first run that contain
mixtures can now be separated. The finished chromatogram is a two-
dimensional array of spots.
Identifikasi spot hasil elusi
Spot berwarna
Spot tak berwarna
Spot tak berwarna:

- Diuapi dengan amoniak
- Diuapi dengan iodium (I
2
)
- Disemprot dengan larutan pengembang (pewarna)
- Disemprot dengan asam sulfat pekat
Nilai Rf
Aplikasi pemisahan TLC
No Senyawa yang
dipisahkan
Solven (Fasa gerak) Sorben (fasa
diam)
Reagent deteksi
1. Kloroplast
pigmens
Isooktan-aceton-eter (3:1:1) -Silika gel
-Polyamida
-
Petroleum eter-aseton (4:6) Alumina
2. Alkaloid Bensen-etanol (9:1) Silika gel Bromocresol green
Dragendorff
Iodine
CHCl
3
-aseton-dietilamin (5:4:1) Silika gel
3. Amina Etanol 90% - NH
3
25% (4:1) Silika gel Asetoasetilfenol
Alizarin
Aseton-H
2
O (99:1) Kielsguhr G
4. Gula Benzena-as.asetat-metanol
(1:1:3)
Silika gel dg bufer
as. borat
Butanol-piridin-H
2
O (6:4:3) Selulosa
5. Food Dyes Metil etil keton-as asetat-metanol
(40:5:5)
Silika gel G
Butanol-etanol-H
2
O (9:1:1) Alumina
No Senyawa yang dipisahkan Solven (Fasa gerak) Sorben (fasa diam)
6. Tserpenoid Heksana-etil asetat (85:15) -pati-asam asalisilat
Benzena petroleum eter Alumina
7. Vitamin Metanol, CCl
4
, atau petroleum eter Alumina
Metanol, propanol atau CHCl
3
Silika gel G
8. Fenol Benzena, atau dietil eter alumina
Heksana-etilasetat (4:1 atau 3:2) Silkika gel + as oksalat
9. Asam amino Butanol-asamasetat-H2O (4:1:1) Silika gel G
N-butanol-aseton-NH3-H2O (10:10:5:2) Selulosa
Iso propanol-asam format-H2O (20:1:5) selulosa
10. Flavonoid dan
coumarin
Metanol-H
2
O (8:2 atau 6:4) Polyamida
Petroleum eter etil asetat (2:1) Silika gel G
11. Steroid dan sterol Benzene-etil asetat (9:1) Silika gel G
CHCl
3
etanol (96:4) alumina
Zweig G & Joseph S, 1972, Handbook of Chromatography, Vol II. CRC Press USA
Pereaksi pengembang (detection Reagent)
Aplikasi pemisahan

Kromatografi Kertas

Aplikasi Pemisahan

Kromatografi Gas
Gas
Chomatography
GC
PRINSIP GAS-KROMATOGRAFI
Gas chromatography - specifically gas-liquid
chromatography - involves a sample being
vapourised and injected onto the head of the
chromatographic column. The sample is
transported through the column by the flow
of inert, gaseous mobile phase. The column
itself contains a liquid stationary phase which
is adsorbed onto the surface of an inert solid.
Ekstraksi
Skema peralatan GC
Plotter kromatogram
1
2
3
4
5
Carrier gas

- The carrier gas must be chemically inert.
- Commonly used gases include nitrogen,
helium, argon, and carbon dioxide.
- The choice of carrier gas is often dependant
upon the type of detector which is used.
- The carrier gas system also contains a
molecular sieve to remove water and other
impurities.
Sample injection port

For optimum column efficiency, the sample should not be too large, and
should be introduced onto the column as a "plug" of vapour - slow
injection of large samples causes band broadening and loss of
resolution.
Microsyringe is used to inject sample through a rubber septum into a
flash vapouriser port at the head of the column.
The temperature of the sample port is usually about 50C higher than
the boiling point of the least volatile component of the sample.
For packed columns, sample size ranges from tenths of a microliter up
to 20 microliters. Capillary columns, on the other hand, need much less
sample, typically around 10
-3
mL. For capillary GC, split/splitless
injection is used. Have a look at this diagram of a split/splitless injector;
A small amount of liquid (microliters) is injected through a silicon
rubber septum (using a special microliter syringe) into the hot
(usually 200+ degrees C) GC injector that is lined with an inert glass
tube. The injector is kept hot by a relatively large, metal heater block
that is thermostatically controlled.
The injector can be used in one of two modes; split or splitless.
The injector contains a heated chamber containing a glass liner
into which the sample is injected through the septum. The carrier
gas enters the chamber and can leave by three routes (when the
injector is in split mode). The sample vapourises to form a mixture
of carrier gas, vapourised solvent and vapourised solutes. A
proportion of this mixture passes onto the column, but most exits
through the split outlet. The septum purge outlet prevents septum
bleed components from entering the column.
Injector
Diagram skematik
Columns

There are two general types of column, packed and capillary (also known as open tubular).
Packed columns contain a finely divided, inert, solid support material (commonly based on
diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m
in length and have an internal diameter of 2 - 4mm.

Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one
of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-
coated columns consist of a capillary tube whose walls are coated with liquid stationary
phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of
support material such as diatomaceous earth, onto which the stationary phase has been
adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of
capillary column are more efficient than packed columns.

Kolom Kapiler dalam oven
Kolom packing Kolom kapiler
Column temperature

For precise work, column temperature must be controlled
to within tenths of a degree.

The optimum column temperature is dependant upon the
boiling point of the sample. As a rule of thumb, a
temperature slightly above the average boiling point of the
sample results in an elution time of 2 - 30 minutes. Minimal
temperatures give good resolution, but increase elution
times. If a sample has a wide boiling range, then
temperature programming can be useful. The column
temperature is increased (either continuously or in steps)
as separation proceeds.
capillary gas chromatography : high resolution.
Capillary columns :
1) more theoretical plates per meter as compared to packed columns and
2) since they have less resistance to flow they can be longer than packed
columns. This means that the average capillary column (30 meters long)
has approximately 100,000 theoretical plates while the average packed
column (3 meters) has only 2500 plates
But with this separation power comes some limitations: 1) Capillary
columns, because they have smaller diameters (0.05 to 0.53 mm) than
packed columns (2 to 4 mm), require relatively specialized injectors and
ancillary flow and pressure controllers and 2) capillary column require a
smaller amount of sample than packed columns. While the average
sample mass of each component in a mixture that is separable by packed
column GC can be in the microgram range (10
-6
grams) per injection,
capillary columns routinely only handle 50 nanograms (10
-9
grams) of a
particular component or less.
DETEKTOR
FID = Flame ionisation detector
ECD = Electron chapture
FPD = Flame photometry detector
AED = Atomic Emission Detector
PID = Photo ionisation detector


There are many detectors which can be used in gas chromatography. Different
detectors will give different types of selectivity. A non-selective detector
responds to all compounds except the carrier gas, a selective detector
responds to a range of compounds with a common physical or chemical
property and a specific detector responds to a single chemical compound.

Detectors can also be grouped into concentration dependant detectors and
mass flow dependant detectors. The signal from a concentration dependant
detector is related to the concentration of solute in the detector, and does not
usually destroy the sample Dilution of with make-up gas will lower the
detectors response.
Mass flow dependant detectors usually destroy the sample, and the signal is
related to the rate at which solute molecules enter the detector. The response
of a mass flow dependant detector is unaffected by make-up gas. Have a look
at this tabular summary of common GC detectors:
Detectors
Detector
Type Support
gases
Selectivity Detectability Dynamic
range
FID Mass flow Hydrogen
and air
Most organic cpds. 100 pg 107
TCD Concentration Reference Universal 1 ng 107
ECP Concentration Make-up Halides, nitrates, nitriles,
peroxides, anhydrides,
organometallics
50 fg 105
FPD Mass flow Hydrogen
and air
possibly
oxygen
Sulphur, phosphorus, tin,
boron, arsenic,
germanium, selenium,
chromium
100 pg 103
PID Concentration Make-up Aliphatics, aromatics,
ketones, esters,
aldehydes, amines,
heterocyclics,
organosulphurs, some
organometallics
2 pg 107
NPD Mass flow

Hydrogen
and air

Nitrogen, phosphor
10 pg

FID
ECD
FPD
Photodiode dan photovoltaic diode
PID
AED
Kondisi Operasional analisis
Jenis kolom
Bahan kolom (fasa diam)
Laju alir gas pembawa (carrier gas)
Suhu injector
Suhu kolom
Mode pemanasan kolom (isotermal atau temperatur
terprogram)

Jenis detektor
Suhu detektor
Hasil Kromatografi Gas
Berupa kromatogram
Aspek kualitatif (nilai Rt)
Aspek kuantitatif


Metoda Standar:

Metoda standar eksternal
Internal
addisi
Standar eksternal
Sampel dibuat kromatogram diinjeksikan
sendiri)
Standar dibuat kromatogram
Dibandingkan antara nilai Rt standar dan Rt
sampel. Peak atau puncak sampel yang
memiliki nilai Rt yang sama berarti senyawa
sampel sama dengan senyawa standar
Sandar internal
Sampel dibuat kromatogram diinjeksikan
sendiri)
Standar + sampel dicampur dan dibuat
kromatogram
Puncak/peak sampel dan peak campuran
diperbandingkan. Peak yang semakin tinggi
menunjukkan senyawa sampel + sandar.
Liquid Chromatography (LC)
Liquid chromatography (LC) is an analytical
chromatographic technique that is useful for
separating ions or molecules that are dissolved in a
solvent. If the sample solution is in contact with a
second solid or liquid phase, the different solutes will
interact with the other phase to differing degrees
due to differences in adsorption, ion-exchange,
partitioning, or size. These differences allow the
mixture components to be separated from each
other by using these differences to determine the
transit time of the solutes through a column.
HPLC
Kolom HPLC
Waktu Retensi
Retention factor, k', is often used to describe the migration rate of an
analyte on a column. You may also find it called the capacity factor. The
retention factor for analyte A is defined as;

k'
A
= t
R
- t
M
/ t
M

t
R
and t
M
are easily obtained from a chromatogram.
The time taken for the
mobile phase to pass
through the column is
called t
M
.
TEORI PLAT


HETP = L / N
Kromatogram
Resolusi dan Selektivitas
selectivity factor, a , which describes the
separation of two species (A and B) on the
column;

a = k '
B
/ k '
A
Jumlah plat
TEORI LAJU ALIR
HETP = A + B / u + C u
where u is the average velocity of the mobile phase. A, B, and C are factors which
contribute to band broadening.
A - Eddy diffusion
The mobile phase moves through the column which is packed with
stationary phase. Solute molecules will take different paths through the
stationary phase at random. This will cause broadening of the solute
band, because different paths are of different lengths.

B - Longitudinal diffusion
The concentration of analyte is less at the edges of the band than at the
center. Analyte diffuses out from the center to the edges. This causes
band broadening. If the velocity of the mobile phase is high then the
analyte spends less time on the column, which decreases the effects of
longitudinal diffusion.

C - Resistance to mass transfer
The analyte takes a certain amount of time to equilibrate between the
stationary and mobile phase. If the velocity of the mobile phase is high,
and the analyte has a strong affinity for the stationary phase, then the
analyte in the mobile phase will move ahead of the analyte in the
stationary phase. The band of analyte is broadened. The higher the
velocity of mobile phase, the worse the broadening becomes.
Metoda Analisis
Kondisi operasional alat
Aspek kuantitaif: standar
- Standar ekxternal
- Standar internal
- Standar adisi
Hasil Analisis Kromatografi
Aspek Kualitatif
Aspek kuantitatif

Aplikasi Analisis kromatografi