Kuliah Metode Pemisahan (Semester Ganjil 2020/2021)

Rabu, 21-10-2020
Ni Made Widi Astuti
INTRODUCTION
— Chromatography is meant those processes which allow
the resolution of mixtures by effecting separation of
some or all their components in concentrated zones on
or in phases different from those in which they are
originally present, irrespective of the nature of the
force or forces causing the substances to remove from
one phase to another" (Williams, 1952)
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PRINSIP PEMISAHAN
— The resolution of mixtures of solutes on filter paper
may depend on surface adsorption, on ion exchange,
or on partition between solvents (Block, et al.,1955)

Paper chromatography makes the use of “Cellulose filter

paper” contains water in its pore as the stationary
phase whereas thin layer chromatography makes the
use of “Glass plate coated with silica gel” as the
stationary phase
PROSES PEMISAHAN SOLUT
The movement of a solute zone was explained conveniently as follows:
— The cellulose fibers have a strong affinity for the water present in the
solvent phase but very little for the organic liquid.
— The paper itself is thought of as an inert support holding a stationary
aqueous phase. As solvent flows through a section of the paper
containing the solute, a partition of this compound occurs between the
mobile organic phase and the stationary water phase. Thus, some of
the solute leaves the paper and enters the organic phase.
— When the mobile liquid reaches a section of the paper containing no
solute, partition again occurs. This time, solute is transferred from the
organic phase to the paper phase. With continuous flow of solvent, the
effect of this partition between the two phases is the transfer of a solute
from the point of its application to the paper to a point some distance
along the paper in the direction of solvent flow(Block, et al.,1955)
Properties Paper chromatography Thin layer chromatography
Principle Partition chromatography Adsorption chromatography
Preparation time Less Comparatively more
Stationary phase Water present in the pores of cellulose Glass coated with silica gel

Mobile phase Hydrophilic mobile phase: Pyridine, carbon-tetrachloride,

Isopropanol: Ammonia: Water acetone, glycerol etc.
Methanol: Water
Hydrophobic mobile phase:
Dimethyl ether: Cyclohexane
Kerosene: Isopropanol

Sample requirement Less amount of sample is required Comparatively requires little more
sample
Heat treatment Paper cannot be heated in an oven for TLC plate can be heated in an oven for
long time long time
Use of silica gel It does not use silica gel It makes the use of silica gel
Separation efficiency More efficient for polar water soluble Efficient for less polar compounds
compounds.
Physical separation Physical separation is found and It lacks the physical separation and
ascending technique is preferred descending technique is preferred

Use of corrosive reagents Can’t use It can be coated with corrosive

reagents
Evaluation Usuallnya Cannot evaluate under the Spots can be evaluate under the UV-
UV-light light
Time It takes less time for the particle It takes more time for the particle
separation separation
outlines the procedure generally
followed in paper
chromatography :
— 1. Choice of filter paper (chromatography system)
— 2. Preparation of sample.
— 3. Application of sample to paper.
— 4. Choice of solvents.
— 5. Development of chromatogram.
— 6. Drying of chromatogram after development and
detection of spots.
— 7. Quantitative estimation.
TYPES OF PAPER
— Pure cellulose
— Silica gel loaded
— Ion exchange cellulose (45% resin)à inorganic anions
and cations
— Resin loaded
Substance have been employed as supports for
stationary phase (SP) : rubber latex, olive oil, silicon oils
à reverse phase
PREPARATION OF SPECIMENS
— Sample à must be solution (~ 0,5% à 10 µL apllied)
à depend on complexity of sample and sensitivity of
detection sample
— Solid sample à extraction à Soxhlet
— Urine à extraction
— Removal of matrix interferants à extraction,
ultrafiltration, desalting
SOLVENTS
— two-phase systems or mixtures of miscible organic
solvents.
— The choice of a suitable developing solvent may be
governed partly by the observation that slower moving
solvents produce rounder and less diffuse spots
(Kowkabany, 1952).
— The rate of movement of the solvent is governed, among
other properties, by its viscosity, surface tension, and
density.
— It is also observed that the Rf increases by raising the water
content of a miscible pair of solvents, e.g., collidinel:utidine.
This Rf increase is also true for the component, which is
most soluble in the mobile phase.
The solvent used should ideally
have the following properties
— 1. The individual components of the solvent should be obtainable easily and at fairly low
cost, but should be of sufficient purity for direct use.
— 2. The solvent should be stable in air and when mixed with small quantities of acid and
alkaline vapours.
— 3. The solvent should be capable of being prepared as required by simple mixing, or of being
prepared in bulk and stored till required. Thus, it is valuable to be able to prepare solvents
composed of liquids as required, but where one of the components is solid it is more
valuable to be able to prepare and store large amounts.
— 4. The components should be relatively non-volatile, or their volatilities should be similar in
the closed apparatus so that they evaporate off the sheet at about the same rate.
— 5. The solvent should be capable of rapid, complete and easy removal from the sheet after
the chromatogram has been run. Any traces of unremoved or unremovable solvent should
be inert to the location reagents.
— 6. The solvent should remain homogeneous throughout the range of temperature
experienced in the particular laboratory.
— 7. The solvent should not react with any of the substances to be separated.
— 8. The substances to be separated should be spread through the whole length of the sheet
from just above the origin practically to the solvent front: i.e. the Rf values should vary
from 5-95. If the front has run off the sheet, the fastest component should be near the front
edge of the sheet. (Smith, 1976)
DRYING
— to remove excess solvent prior to any further
manipulations.
— In a hood at room temperature
— with the aid of a fan-type electric heater.
— an electric, forced-air oven
DETECTION OF SPOTS
CHEMICAL DERIVATISATION
— a suitable color reaction
— Corrosive agents (sulfuric acid cannot be used on paper)
PHYSICAL METHODS
— UV
— fluorescence or absorption
— Densitometer techniques
— Radioactive/radiochemical Or "tagged" compounds by
autoradiography
ISOLATED à further spectroanalytical studies
QUANTITATIVE METHOD??????
COLOUR REAGENTS
— for the amino acids utilizes three to four successive
color reagents: ninhydrin or isatin, Ehrlich reagent
(dimethylaminobenzaldehyde), Sakaguchi or diazo
reagents (Jepson, 1953)
APPLICATIONS
Its applications covers all types of analytes, including:
— proteins,
— peptides,
— amino acids,
— poly-, oligo-, di- and monosaccharides,
— natural products,
— sterols,
— steroids,
— bile acids,
— pigment,
— dyes and
— inorganic species
— CLINICAL AND BIOCHEMICAL :
Pemisahan asam amino, peptide à investigasi struktur
protein
Urin, cairan biologis à asam amino, gula à diagnosis
kondisi patologi
— GENERAL ANALYTICAL
Analisis polimer, penetapan logam di tanah dan
specimen geologi, fenol dalam tanaman, alkaloid, dan
pemisahan seny. Yg dilabeli radioisotop
DAFTAR PUSTAKA
— Richard J. Block, Emmett L. Durrum, Gunter Zweig.
1995. A Manual of Paper Chromatography and
Paper Electrophoresis