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Lecture 6: Analytical Separations

Analytical separations may blur the line


between analysis and purification
You can safely say your separation is
analytical when the it tells you something
specific
Examples about your analyte
of sometimes analytical
separations include:
Chromatographi
c: Thin Layer (TLC) / Paper
Size Exclusion (SEC) / Affinity
Electrophoretic:
Agarose/Polyacrylamide
Gel
Capillary
Electrophoresis
Analytical (CE)
ultracentrifugation
The Beginning: Paper
Chromatography
The unifying aspect of chromatographic
methods is that the separation is not with an
applied
The veryelectric field
first type of chromatography was
the adsorption variety, which includes
paper and TLC
Separated
chlorophylls and
karatenoids using
Mikhail calcium carbonate
Tsvet
1872-1919 as the stationary
About 20 phase
years later,
Richard L.M. Synge and
Archer Martin develop
paper chromatography and
win the nobel prize
Adsorption Chromatography
Mechanism
Adsorption chromatography uses a stationary
phase and a mobile phase
The stationary phase is usually a solid sheet
either cellulose (paper), silica gel (standard
TLC) or aluminum oxide
The stationary phase must have moderately
charged functional groups:
N C

Cellulo Silica Al2O


se Gel 3
Adsorption Chromatography
Mechanism
To move the analytes over the stationary
phase, we use a non-polar solvent which is
drawn up via capillary
Some popular actionsolvents are:
mobile phase
Non-polar: Alkanes (up to octane),
Diethyl ether
Moderate polarity: Alcohols, Ketones
(not acetone)
Polar: H2O, Weak Salt Buffers
TLC and Paper Visualization

Once youve run the molecule, how do you


see it?
If youre very lucky, then your analytes are
chromophoric

If youre somewhat lucky, then your analytes


absorb UV light

If youre unlucky then you have to use a stain


(like iodine)
TLC and Paper Chromatography
Nowadays
In Biochemistry, TLC and paper
chromatography had their heyday in the 50s
(Remember Sanger).
Nonetheless they are still routinely used as
basic analytical tools, particularly in organic
chemistry (TLC)
We dont separate proteins this way
electrophoretic separations work much
better
Paper Chromatography 2000-2007: 175
results 1960-1967: 720
TLC 2000-2007: 420 results
results
1980-1987: 394
results
Column Chromatography: Size
Exclusion
Adsorption chromatography can also be done
on a column, but more for separation than
analysis
Probably the most common chromatography
technique for proteins is size exclusion

It is also called gel


permeation
chromatography
Column is usually
agarose beads (i.e.
Sephadex)
Size Exclusion Examples

Analytical Size exclusion is usually used to


distinguish the oligomeric state of proteins
Chromatogr
am C-CIC-3

Standards
Mol. Wt.

Standards
Biochemistry (2007), 46 (51): 14996-15008 Stokes
Radius
C-CIC-3 is about 20.5 kDa but
the analysis shows 23kDa. J. Biochem. (2006), 139 (5): 813-820
Loose structure?
Affinity Chromatography

Affinity chromatography comes in a few


flavors: Immuno-, Immobilized Metal Ion
Affinity (IMAC) and just plain affinity (e.g.
GST)
The difference between this and adsorption
is that here the analyte actually sticks to the
column until it is washed off
Cu2+ / His6 and GST
/ Glutathione are
very common
ways of purifying
proteins
Nonetheless,
there are some
examples of
analytical affinity
Affinity Chromatography Examples

Uses the affinity of glucose for boronate to


detect the extent of glucosylation of Ig2
antibodies

Anal. Chem. (2007) 79 (24): 9403-9413


TAP Tagging

Tandem Affinity Purification (TAP) is a powerful


purification technique that analyzes
protein/protein interactions

Methods (2001) 24, 218229


Chromatography Instrumentation

The Analytical Chromatography instrument is


the FPLC

The Agilent 1200


The GE System
KTA
Electrophoresis Theory

Electrophoresis uses an electric current or


field to push analytes through a medium
In the simplest case, the electrophoretic
mobility of an analyte e is proportional to
v
e
the electric field E. E

In reality, though, electrophoretic mobility


has to take into account the double layer -
solvent ions of oposite charge that cluster
around the analyte.
Electrophoresis Theory

The effect of the double layer for a given


solvent and analyte are incorporated into the
. 0
Where is the dielectric constant
zeta potential term
e
of the solvent, 0 is the
permitivity of the vaccum
(8.85*10-12) and is the dynamic
So far we have assumed that we are dealing
vicocity
with particles whose size is on the order of
the double layer. But our analytes are much
bigger. In this case, we are more concerned
with the stokes radius relative to the amount
of charge. z Where z is the charge, is
ue
6r the viscosity and r is the
Stokes radius
Electrophoresis of Nucleic Acids

If were trying to separate nucleic acids, we


have a problem because:
They are always negatively charged
at All
neutral pH. will travel in the same
molecules
direction
Each additional nucleoside confers an
additional charge, so charge is directly
proportional to size.
All molecules will have the same e

The solution is to use a gel which consists of


pores surrounded by cross-linked fibers
This will make e EM image
dependent on the of Agarose
Stokes radius Gel
DNA/RNA Electrophoresis

Double stranded DNA or RNA are molecules


that repel themselves. They will all form rod-
like structures.
So now we can separate our nucleic acids on
the basis of size. We can visualize with a
fluorescent dye (usually ethidium bromide)
and compare to a standard to get a relatively
good quantitative size:
Electropherogra
m

Single stranded DNA or RNA may interact


with itself forming a supercoil. e for
supercoiled DNA or RNA is a mite
unpredictable.
Protein Electrophoresis

Proteins are even trickier than DNA/RNA:


They are all effectively supercoiled (3
Structure)
They can be either positively or negatively
charged
The number of charges depends on the
amino acid sequence and is not proportional
to size. The solution is to
expose the protein
so a detergent
(usually sodium
dodecyl sulphate -
SDS)

- +
ve ve
Protein Electrophoresis

Proteins will bind an amount of SDS roughly


proportional to their size (1.4g/g
polypeptide), so we have effectively created
the same situation as for DNA/RNA
The gel of choice for
The
electrophoresis.
SDS-page protein
Standard
electrophoresis is
ladder
polyacrylamide. Danger!
The Over- Acrylamide monomers
Expressed are potent neurotoxins
Sometimes proteins are
protein of pre-boiled in a reducing
interest? agent to eliminate S-S.
Proteins are visualized using a dye
(coomassie blue) or other stain (silver)
2D Electrophoresis

If you want to analyze the whole protein


content of a cell, a 1D separation just isnt
gonna do it.The solution is to do a 2D
separation using a gel that has a pH
gradient.will run on this gel (in both
Proteins
directions) until they hit their
isoelectric point where they
aggregate

Then you can soak


the gel in SDS, flip
the voltage 90 and - +
separate by size. ve ve
2D Gels

2D Electrophoresis is a powerful tool for


analyzing the protein complement of simple
cells
Capillary Electrophoresis

You can also make molecules pass through a


narrow (usually fused silica) capillary
Typically, your analyte is in a buffer with net
+ve charge, which generates an
electroosmotic flow (EOF) towards the
cathode
EOF overpowers , thus all analytes move
e
towards the cathode at rates dependent on
their e
Capillary Electrophoresis

The advantages of CE are:


Really good separation of slow diffusing
analytes
Where: e is the
eV difference in apparent
1 e
D
Rsep e
electrophoretic mobility,
4 (e o )



is the average
electrophoretic mobility,
Resolution is limited
V is thebyapplied
Diffusion
voltage
coefficient andand
flowDrate
is the diffusion
coefficient
Very, very low sample consumption
Analytical Ultracentrifugation

In analytical ultracentrifugation, analytes are


separated on the basis of their sedimentation
when they experience a centripetal force.
Usually carried out at speeds
around 60,000
Analytical Ultracentrifugation

Or, described mathematically


angular radius from center
mass of a single velocity of spin
M 2
molecule Fs m 2 r r
N

This is counteracted by the Boyant force (due


to displacement of solute) and the frictional
force coefficient of
sedimentation
mass of solute velocity
displaced friction
Fb m0 r 2
Solvent
F f f s
M density in
Sedimentati
m0 g/mL
M (1 ) s on
N 2 s coefficient
Volume occupied by 1g Nf r
of solute
Sedimentation Velocity Experiments

The most basic type of ultracentrifugation


experiment is to measure the rate at which
the analyte moves away from the center of
rotation
What is actually measured is the
movement of the boundary
between dissolvedc 1analyte
c and 2 2
rD
empty buffer t r r r s r c
M (1 ) s
2 s
Nf r
RT
f
ND
RTs
Mr
D(1 )
Sedimentation Equilibrium

In sedimentation equilibrium, an equilibrium


is established between sedimentation away
from the center of rotation and diffusion
towards the center of rotation.
( r 2 r02 ) / 2
C A ( r ) C A, 0 e
M (1 ) 2

RT
Density Gradient Centrifugation

Density gradient is similar to equilibrium


sedimentation except there is a permanent
solvent density gradient. This sharpens the
contrast between the sedimentation
equilibria of samples with the same shape,
but slightly different masses:

Meselson and Stahl,


P.N.A.S., 44, (1958), 671-
682)