Transcript chromatography

CHROMATOGRAPHY
Magnet Analytical Chemistry
Unit 4
CHROMATOGRAPHY
Physical separation method based on the differential migration of analytes
in a mobile phase as they move along a stationary phase. Separation occurs
because of differences in particle mass and attraction to the solvent.
Mechanisms of Separation:
•Partitioning
•Adsorption
•Size Exclusion
•Ion Exchange
•Affinity
Chromatographic Separations:
Based on the distribution (partitioning) of the solutes between the mobile
and stationary phases, described by a partition coefficient, K:
K = Cs/Cm
where Cs is the solute concentration in the stationary phase and Cm is its
concentration in the mobile phase.
Principle
The level of Interaction (adsorption) packing material & sample A,B
differs,
resulting in different speeds of travel of A & B in a media (paper,
column etc.).
Usually sample to be analyzed is injected into a carrier (gas or liquid).
Carrier is usually inert (does not react with packing materials).
The components in sample, being separated after chromatography,
are analyzed.
Types of chromatography
LC - Liquid (carrier & A,B)
Chromatography
GC - Gas (carrier & A,B) Chromatography
HPLC - High Pressure Liquid
Chromatography
TLC – Thin Layer Chromatography
Paper Chromatography
Chromatography Terms:
The analyte is the substance to be separated during chromatography.
Analytical chromatography is used to determine the existence and
possibly also the concentration of analyte(s) in a sample.
A bonded phase is a stationary phase that is covalently bonded to the
support particles or to the inside wall of the column tubing.
A chromatogram is the visual output of the chromatograph. In the
case of an optimal separation, different peaks or patterns on the
chromatogram correspond to different components of the separated
mixture.
Chromatography is a physical method of separation that distributes
components to separate between two phases, one stationary
(stationary phase), while the other (the mobile phase) moves in a
definite direction.
The eluate is the mobile phase leaving the column.
The eluent is the solvent that carries the analyte.
An immobilized phase is a stationary phase that is immobilized
on the support particles, or on the inner wall of the column
tubing.
The mobile phase is the phase that moves in a definite direction.
Preparative chromatography is used to purify sufficient
quantities of a substance for further use, rather than analysis.
The retention time is the characteristic time it takes for a particular
analyte to pass through the system (from the column inlet to the
detector) under set conditions. See also: Kovats' retention index
The sample is the matter analyzed in chromatography. It may consist
of a single component or it may be a mixture of components.
The solute refers to the sample components in partition
chromatography.
The solvent refers to any substance capable of solubilizing another
substance, and especially the liquid mobile phase in liquid
chromatography.
The stationary phase is the substance fixed in place for the
chromatography procedure.
Gel-filtration chromatography: (aka: size exclusion) particles are
separated based on size. The separation is carried out on the basis of
hyrdodynamic diameter of the molecules. [large molecules
exit (elute) first]
Ion-exchange chromatography: The mechanism of ion-exchange used
allows the separation of analytes based on charge. Separation of
charged compounds takes place through a charged stationary phase.
Least charged particles will elute first. (two different modes: column
and planar)
Affinity chromatography: particles are passed through a
column of beads containing a covalently bound high affinity
group for the particle of interest. Bound particles are eluted by
free high affinity group.
Adsorption chromatography: the analyte is passed over an adsorbent
bed. Different compounds present in the mixture get adsorbed onto
the bed at different rates. [large molecules
exit (elute) first]
Partition chromatography: Components of the analyte are separated
through the use of partitions of a solute between two solvents. In this
process one of the solvents is immobilized by the means of a
substance present in the filter paper or column.
High Performance Liquid chromatography: particles are separated on
the basis of their idiosyncratic polarities. Interaction of these particles
with the stationary phase of the column is also considered for
analysis. A pump is used to push the mobile phase through the
column. There is a detector following the stationary phase to analyze
the retention time of the various particles.
Gas chromatography: the analyte is moved through the
column via a high pressure gas (like helium). Flame
ionization detectors (mass spec.) and thermal
conductivity (pyrolysis) are used to evaluate the
separated particles.
Reverse-Phase chromatography: the stationary phase is
made of hydrophobic compounds; they attract the
hydrophobic compounds in the mobile phase allowing
the elution of the hydrophobic particles.
Thin Layer chromatography: particles are separated using
a stationary phase of paper, it involves a stationary phase
of a thin layer of adsorbent like silica gel, alumina, or
cellulose on a flat, inert substrate.
Paper chromatography: the analyte is moved
through the column via a paper and solvent.
This paper is made of cellulose, a polar
substance, and the compounds within the
mixture travel farther if they are non-polar.
More polar substances bond with the cellulose
paper more quickly, and therefore do not travel
as far.
Size Exclusion
chromatography:
Biochemists refer to a protein's
size in terms of its molecular
weight, in kDa
(a kilodalton, kD or kDa, is
1000 times the molecular mass
of hydrogen)
Each amino acid residue
counts for about 110 daltons,
that is, about 0.11 kDa.
Ion-exchange example with proteins:
Vertical Gel Electrophoresis: