PAPER CHROMATOGRAPHY

(PC)
Bentuk paling sederhana dari kromatografi

Dipakai secara luas untuk identifikasi kualitatif

Prinsip:
Sampel diteteskan pada kertas saring dengan mikropipet, selanjutnya
ujung kertas dicelupkan pada pelarut yang sesuai (jangan sampai
mengenai sampel).
Solven akan merambat ke atas dengan daya kapiler dan komponen sampel
akan bergerak naik dengan kecepatan yang berbeda sesuai dengan tingkat
retensinya pada kertas. Masing-masing komponen dapat dilihat dengan
mereaksikan kertas (menyemprot) dengan pereaksi yang dapat
membentuk warna.
Tingkat pemisahan
komponen dalam
sampel dinyatakan
dengan harga Rf

Each component is
characterized by its Rf
value, the ratio of a and b:
Rf = a/b: 0 ~ 1

Rf= Retention factor

Kertas saring selulosa yang dipakai biasanya sangat
hidrofilik (suka air) sehingga akan terlapisi oleh air yang
terserap oleh atmosfir. Jadi mekanisme pemisahannnya
partisi cair-cair, dimana komponen sampel terdistribusi
antara fasa tetap air dan pelarut yang dipakai (eluen).
Pelarut umumnya adalah campuran pelarut organik dan
air dengan pH yang dikontrol oleh suatu buffer.
Keuntungan:
Dapat dilakukan pemisahan yang lebih efektif dengan
metode pengembangan kromotogram 2 dimensi: dimensi
kertas kromatogram dibuat segi empat sama-sisi dan
sampel diteteskan pada salah satu pojok kertas. Setelah
diperoleh kromatogram dengan solven yang pertama.
Kertas diputar 90o dan selanjutnya dibuat kromatogram
lagi dengan solven yang lain. Jadi bila ada 2 komponen
atau lebih dalam sampel yang belum terpisahkan oleh
solven I, mungkin akan dapat dipisahkan oleh solven ke-
2.
 Deteksi sampel
-Jika sampel terdiri dari komponen yang
Dapat berfluoresensi . Noda dapat dide-
teksi dengan cara menyinari noda de-
ngan lampu UV dan bulatan yang ter-
bentuk ditandai dengan pensil
- Reagen pembentuk Warna
Asam amino & amina dideteksi dengan
menyemprotkan larutan ninhidrin pada
kertas sehingga terbentuk warna biru
atau purple
- Untuk analisis kuantitatif bulatan pensil
pada kertas dapat digunting dan dilarut-
Kan dalam pelarut yang sesuai, kemudi-
ditentukan kandungannya secara kuan-
titatif.
THIN LAYER CHROMATOGRAPHY
(TLC)
 THIN LAYER CHROMATOGRAPHY
•Thin layer chromatography (TLC) is an important
technique for identification and separation of mixtures
of organic compounds. It is useful in:

•Identification of components of a mixture (using appropriate

standards)
•following the course of a reaction,
•analyzing fractions collected during purification,
•analyzing the purity of a compound.

•In TLC, components of the mixture are partitioned

between an adsorbent (the stationary phase, usually
silica gel, SiO2) and a solvent ( the mobile phase) which
flows through the adsorbent.
 THIN LAYER CHROMATOGRAPHY

In TLC, a plastic, glass or aluminum sheet is coated

with a thin layer of silica gel.

A very small amount of a solution of the substance

to be analyzed is applied in a small spot with a A B U C D

capillary tube, ~1cm from the bottom of the

TLC plate
filter paper

The TLC is developed in a chamber

which contains the developing solvent
(the mobile phase). A truncated filter
paper placed in the chamber serves to
saturate the chamber with mobile phase.

A B U C D
 Stationary Phase: Alumina

O OH OH OH OH

Al Al Al Al Al
O O O O O O

Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-
Stationary Phase: Silica (SiO2)
OH
OH
OH
OH
OH Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si Si
O O
Si
Si O O O
Si O O
O
O O
O
Si
Si O
O
O O
O
 Reverse phase chromatography
Silica is alkylated with long chain hydrocarbon groups, using 18
carbons long (ODS). This is usually referred to as C-18 silica.
 THIN LAYER CHROMATOGRAPHY

As the mobile phase rises up the TLC plate by

capillary action, the components dissolve in the
solvent and move up the TLC plate.
Individual components move up at different rates,
depending on intermolecular forces between the
component and the silica gel stationary phase and
the component and the mobile phase.
http://www.instructables.com/id/EW1YDCYF4REC0IU/
The stationary phase is SiO2 and is very “polar”.
It is capable of strong dipole-dipole and H-bond donating and accepting
interactions with the “analytes” (the components being analyzed).
More polar analytes interact more strongly with the stationary phase in move
very slowly up the TLC plate.
By comparison, the mobile phase is relatively nonpolar and is capable
of interacting with analytes by stronger London forces, as well as by dipole-
dipole and H-bonding.
More nonpolar analytes interact less strongly with the polar silica gel and more
strongly with the less polar mobile phase and move higher up the TLC plate.
 THIN LAYER CHROMATOGRAPHY

Once the solvent is within ~1-2 cm of the top of

the TLC sheet, the TLC is removed from the
developing chamber and the farthest extent of
the solvent (the solvent front) is marked with a
pencil.
The solvent is allowed to evaporate from the
TLC sheet in the hood.
The spots are visualized using a UV lamp.

A fluorescent compound, usually Manganese-

activated Zinc Silicate, is added to the adsorbent
that allows the visualization of spots under a
blacklight (UV254). The adsorbent layer will
fluoresce light green by itself, but spots of analyte
quench this fluorescence and appear as a dark spot.
http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html
 THIN LAYER CHROMATOGRAPHY - Visualization
Chromatogram of 10 essential oils,
Stained with vanillin reagent.
As the chemicals being separated may be
colorless, several methods exist to visualize
the spots:
• Visualization of spots under a UV254 lamp. The
adsorbent layer will thus fluoresce light green by
itself, but spots of analyte quench this
fluorescence.
• Iodine vapors are a general unspecific color.
• Specific color reagents exist into which the TLC
plate is dipped or which are sprayed onto the
plate.
• Once visible, the Rf value of each spot can be
determined
Paper/TLC
Chromatography
Animation
 THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
2.0 cm
Rf (A) = = 0.40
5.0 cm
Solvent Front

Rf (B) = 3.0 cm = 0.60

5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Distance A
migrated = 3.0 cm Rf (C) = 0.8 cm = 0.16
5.0 cm

Distance B
migrated = 2.0 cm 3.0 cm Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
0.8 cm Rf (U1) = 3.0 cm = 0.60
Origen
x x x x x 5.0 cm
A B U C D
0.8 cm
Rf (U2) = = 0.16
5.0 cm

The Rf is defined as the distance the center of the spot moved divided
by the distance the solvent front moved (both measured from the
origin)
 THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
2.0 cm
Rf (A) = = 0.40
5.0 cm
Solvent Front

Rf (B) = 3.0 cm = 0.60

5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Distance A
migrated = 3.0 cm Rf (C) = 0.8 cm = 0.16
5.0 cm

Distance B
migrated = 2.0 cm 3.0 cm Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
0.8 cm Rf (U1) = 3.0 cm = 0.60
Origen
x x x x x 5.0 cm
A B U C D
0.8 cm
Rf (U2) = = 0.16
5.0 cm
The Rf is defined as the distance the center of the spot moved divided
by the distance the solvent front moved (both measured from the
origin)
 THIN LAYER CHROMATOGRAPHY – Rf’s

Rf values can be used to aid in the identification of a

substance by comparison to standards.

The Rf value is not a physical constant, and

comparison should be made only between spots on
the same sheet, run at the same time.

Two substances that have the same Rf value may be

identical; those with different Rf values are not
identical.
 THIN LAYER CHROMATOGRAPHY – Rf’s

Absorption of Solutes
The adsorption strength of compounds increases with increasing polarity of
functional groups, as shown below:

-CH=CH2, -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONR2, -CO2H.
(weakly adsorbed) (strongly adsorbed)
(nonpolar) (more polar)
Elution Strength of Mobile Phase (
Elution strength is generally considered to be equivalent to polarity. A solvents
elution strength depends on Intermolecular Forces between the solvent and the
analytes and between the solvent and the stationary phase.
A more polar (or more strongly eluting solvent) will move all of the analytes to a
greater extent, than a less polar, weakly elution solvent.
For example, the elution strength of hexane is very low;  = 0.01.
the elution strength of ethyl acetate is higher;  = 0.45
the elution strength of ethanol is even higher;  = 0.68
Solvent Properties and Elution Strengths
 Resolution

The separation between two analytes on a

chromatogram can be expressed as the
resolution, Rs and can be determined using
the following equation:

Rs = (distance between center of spots)

(average diameter of spots)

In TLC, if the Rs value is greater than 1.0, the

analytes are considered to be resolved.
x x
254 nm 366 nm