Lecture 13

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12 Νοε 2013 (πριν από 3 χρόνια και 9 μήνες)

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THIN LAYER
CHROMATOGRAPHY

Thin layer chromatography (TLC) is a simple,
inexpensive method which requires a minimum
of instrumentation and can be used for
separation of simple mixtures. It is possible to
separate up to 70 samples and standards on a
single plate, which makes analysis quick and
inexpensive.

TLC

TLC is performed using thin sheets of glass,
aluminum or plastic coated with a layer of
stationary phase, usually silica gel. However,
other coatings such as alumina,
Florisil
,
polyamide, cellulose, C
-
18, C
-
8, ion exchangers,

and chemically bonded amino,
cyano
,
diol
, or
phenol layers can also be used.

In TLC, the sample must be carefully applied to
the plate to minimize spreading. Sample sizes
from 0.1 mg to 50 mg are the best for TLC.
Smaller amounts are difficult to visualize, while
larger spots result in overloading and variable
results. Samples should be dissolved in relatively

volatile solvents (0.5 to 5mL) so that the spots do
not spread excessively(3
-
4 mm diameter).

The spotted plate is placed in a development tank
with its lower side immersed in solvent to a level
below the applied sample spots. The solvent
rises due to capillary flow in a process called
development. Development times can range from
3
-
60 minutes.

Retardation factors
R
f

are calculated by dividing
the distance moved by an
analyte

by the distance
moved by the solvent.

1) Visible
-

the bands or spots can be seen immediately.

2) Fluorescence
-

observed under UV light.

3) Absorb UV
-

plate has an indicator that when irradiated
will show
analytes

as dark spots on a bright
background.

4) Reaction with a chromogenic reagent
-

These reagents
may be general (reacting with many different
compounds) or selected (reacting only with certain
functional groups.

5) Organic Materials
-

treat plates with concentrated
sulfuric acid


and then heat at 200
o
C.
Analytes

show up as dark
spots.

Viewing Chamber

using UV light

Visualizing Spots on the Plate

More complete separation of sample components can be achieved

by two
-
dimensional development. In this process, the plate is

developed normally and following complete drying, it is turned 90
o


and the development of the plate is continued. This second

development is performed using a different mobile phase with

very different selectivity (otherwise little further separation

would result).

Two
-
Dimensional Development

Schematic representation of two
-
dimensional TLC separation of complex lipids
from A. thaliana. Abbreviations, MGDG,
monogalactosyldiacylglycerols
; DGDG,
digalactosyldiacylglycerol
; SQDG,
sulfoquinovosyldiacylglycerol
; DPG,
diphosphatidylglycerol
; PG,
phosphatidylglycerol
; PE,
phosphatidylethanolamine
;

PI, phosphatidylinositol; PS,
phosphatidylserine
; and PC,
phosphatidylcholine

For example, plant phospholipids and glycolipids can be separated by first
developing the plate in chloroform
-
methanol
-
water (75:25:2.5, by volume) in the first
direction. After allowing sufficient time for drying, the plate is developed, at right
angles to the first development, in chloroform
-
methanol
-
acetic acid
-
water (80:9:12:2,
by volume).

Quantification can be performed by manually
scraping off the selected spots (
analytes

and
standards) and analyzing them with GC, HPLC or
other quantitative method.

Quantitative Analysis

Analytes

can also be measured by scanning the
plates using an optical
densitometric

scanner
with
automatic sample application, and using
HPTLC plates, quantification with a precision
ranging from 1% to 3% can be achieved.

Rod Thin
-
Layer Chromatography

Instead of thin plates this
method uses a thin layer of
stationary phase coated onto
small diameter quartz rods.
The rods are spotted and
developed in a similar manner
to regular TLC plates

After development and drying,
the rods are automatically
passed through a specially

constructed flame ionization
detector (FID) at a constant
speed. The result is a
chromatogram similar to a
normal HPLC chromatogram.

SIZE EXCLUSION
CHROMATOGRAPHY

Molecules that are too large to enter into the
pores of the beads are
unretained

by the column
and are not separated.

Molecules that are very small in relation to the
pore size all behave similarly and these small
molecules are also not separated.

Medium sized
molecules are
separated based on
how far they penetrate
into the gel beads.

One of the earliest materials developed for size
exclusion is still in use and is called
Sephadex
.
Sephadex

is made from dextran cross
-
linked
(polymerized) with
epichlorohydrin
.

Structure of

Sephadex

The gels can be
manufactured

with different amounts of
cross
-
linking which will
produce gels of different
porosities and different
ranges of molecular size
separation.

Large molecules elute after a volume of mobile
phase equal to the void volume V
o

has passed
through the column. Small molecules will elute
with a volume of mobile phase equal to the void
volume and pore volume (V
o

+ V
i

). Medium size
molecules will elute between V
o

and (V
o

+ V
i

).


Proteins are often separated using
size exclusion.

Molecular Shape has an effect on the
relationship between the molecular
weight and retention
.

Cationic adsorption effects can be reduced by
the addition of mobile phases with an ionic
strength of 0.05
-
0.1 M. Low pH (around 3) will
also help reduce some stationary phase
interactions. Methanol can be added to the
mobile phase to reduce hydrophobic retention,
and ethylene glycol may help reduce the
adsorption of proteins.

Interactions between the
stationary phase and the
analytes

should be avoided.

Chiral

-

adjective
: not superimposable on its
mirror image: used to describe a molecule whose
arrangement of atoms is such that it cannot be
superimposed on its mirror image.

Enantiomers

-

noun
: mirror
-
image molecule:
either of a pair of molecules that are a mirror
image of each other in structure but cannot be
superimposed.

Chiral Chromatography

Chiral Chromatography

Because of their chemical and physical similarity,
enantiomers can be difficult to separate.
However, they can be separated by
chromatography, provided the system is also
chiral. This can be achieved by the use of a
chiral mobile phase, by a chiral liquid stationary
phase or a chiral solid stationary phase.

Chiral Chromatography

Mobile Phase

The addition of chiral atoms to the mobile phase
is one way to perform separations. If the chiral
reagent form a complex of some type with one
type of molecule of a pair of enantiomers,

chiral separation may result.

Chiral Chromatography

Brush Type

Stationary

Phases.

There are many solid stationary phases available.

Brush type phases, cavity phases (such as
cyclodextrins
, crown ethers and
macrocyclic

glycopeptide

antibiotics), protein phases, and
ligand
-
exchange phases are common examples.

Affinity Chromatography

Affinity Chromatography….

Is based on the interactions between two
components that are ideally suited to each other
both electrostatically and spatially. One
component is bonded to a solid support. The
bonded component interacts with the
analyte

and
the
analytes

is adsorbed from the solution.

Molecules such as these

do not match the ligand

and are not adsorbed

Affinity chromatography is the most specific
chromatographic method and the separation in
based on biochemical interactions such as:

Affinity Chromatography

antigen


antibody

enzyme


inhibitor


hormone


carrier

Affinity Chromatography

Elution