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Semester one
Block 2 - Respiratory
Chromatography
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Cards (25)
What are the 2 phases in chromatography?
Stationary
&
mobile phase
.
What is a stationary phase?
A stationary phase is a solid or liquid material that is used in
chromatography
to separate and analyze different components of a mixture.
What is a mobile phase?
The liquid or gas that
carries
the sample through a chromatography system.
What will the mobile phase do?
Carry the sample through the
stationary phase
.
List the common types of chromatographic techniques
Column
chromatography
Thin
layer
chromatography (TLC)
Gel
filtration
Ion
exchange
What does normal phase column chromatography do?
Separates compounds based on their
polarity
.
Explain the process of normal phase column chromatography
Column is tightly packed with
stationary phase
Silica
interacts with drug (more strong with polar compounds)
Solvent is carried over to a detector which measures
UV rays
and displays
peaks
based on the components in the solvent
The peaks can be studied later to determine their structure
What is the most common stationary phase in chromatography?
Silica gel
Will
polar
or non-polar compounds interact more strongly with the
stationary phase
?
Polar
What does elution mean?
Movement of compounds through the
column
.
List the difference in elution & binding to stationary phase between non-polar & polar compounds
Polar - bind
strongly
& elute
slowly
Non-polar - bind
weakly
& elute
quickly
Give an example of a high and low polar solvent
High -
water
Low -
n-hexane
How does elution link to retention time?
Fast elution =
short
retention time (
weaker
interactions with stationary phase)
Slow elution =
long
retention time (
stronger
interactions with stationary phase)
Column chromatography is often used as a purification tool - what does this mean?
It
isolates
the
desired
compounds in their
pure
form from the rest of the mixture (into
fractions
).
How does thin layer chromatography work?
Thin layer of
stationary
phase (silica gel) spread on a flat surface
Small amount of sample applied at
bottom
of TLC plate
Place placed in shallow container with a solvent (
mobile
phase) at the bottom - this moves up the plate by
capillary
action
As solvent rises, compounds will move at different rates depending on their
polarity
More polar = interact more
strongly
with stationary phase = move
slower
Less polar = interact more
weakly
with stationary phase = move
faster
After solvent reached top, plate is removed & compounds visualised using
UV
light
What does retention factor measure?
How far the
substance
has travelled.
What can TLC be used for?
Assessing
purity
of a
compounds
Identify compounds based on their
RF values
What is gel filtration chromatography used for?
To purify large
molecules
Remove salts from large molecules e.g.
proteins
Determine
molecular weight
of unknown proteins
How does gel filtration chromatography work?
Column packed with porous beads (
stationary
phase)
Sample loaded onto column & a buffer (
mobile
phase) is used
Large
molecules can't enter pores of beads & pass through column faster (elute
faster
)
Smaller
molecules enter pores & take longer to pass through (elute
slower
)
Components get
separated
due to
elution
times
Give examples of the stationary phases used in gel filtration chromatography
Dextran
&
agarose
How is the stationary phase in ion exchange chromatography different to other methods of chromatography?
They stationary phase must have a charge (must have
ionisable
groups on it).
How does ion exchange chromatography work?
It has the same principle as
column chromatography
but
stationary phase
will be charged & attract the
opposite charge
.
Molecules will
weak ionic interactions
will elute first.
How can we elute the solvent in ion exchange chromatography?
By changing the
pH
or
ionic strength
.
What will the charge be on anionic and cationic exchangers
Anionic =
+ve
Cationic =
-ve
When is ion exchange chromatography used?
To separate charges on
amino
acids
Water
purification