What is Chromatography?
is a physical method of separation in which
compounds are distributed between two phases, a stationary phase and
a mobile phase.
How does the chromatographic process occur?
What is the separation due to?
The chromatographic process occurs as a result of repeated sorption/desorption acts during the movement of the sample
components along the stationary bed.
The separation is due to
differences in affinity for the stationary and mobile phase
Explain the stationary phase of chromatography?
A solid or a liquid coated onto a solid. The stationary phase may be coated onto a flat surface (PLANAR chromatography e.g. TLC) or packed into a column (COLUMN chromatography e.g. HPLC, GC) or coated onto the inner walls of a very narrow column (OPEN TUBULAR chromatography e.g. CAPILLARY GC)
Explain the mobile phase of chromatography?
Liquid or a gas. The mobile phase is called the eluent.
When
the eluent emerges from the end of the column, it is called
the eluate.
The process of passing liquid or gas through the
column is called elution.
What happens during elution?
During elution, analyte molecules are continually
distributing themselves between the stationary phase
and the mobile phase.
While in the mobile phase, the
analytes are carried forward with it, but they remain at
a standstill in the stationary phase.
Different molecules
have different affinities for the stationary phase- those
with the greatest affinity remain on the column the
longest.
What is chromatography?
A physical method of separation in which compounds are
distributed between two phases; a stationary
(fixed) and a mobile
phase
phase (moving).
What does the chromatography process occur as a result of?
What does sorption mean?
The chromatography process occurs as a result of
repeated sorption/desorption acts during the movement
of the sample components along the stationary bed:
Sorption is the transfer of an analyte (solute) from the
mobile phase to the stationary phase.
How is separation achieved?
Separation is achieved due to differences in affinity of
the solute for the stationary and mobile phases and so
in different mobilities of solutes
Do each different type of chromatography differ in the kind of mobile and stationary phase used?
Yes
In the mobile phase for HPLC what is occuring?
Where do separations occur?
How does the detector respond?
Mobile phase continuously pumped
through the system at a fixed flow rate.
Specific volume of sample swept onto
column such that flow is uninterrupted and
no air is introduced
Separation occurs on column.
Longer column = better separation
Detector responds to a physico-chemical
property of the analyte
While moving through the chromatographic system, the collection of analyte molecules (ideally of one type if we are getting a good
separation) is known as a “???”.
When they are detected having left the column they are displayed as a “???” on a “???”
1- Band
2- Peak
3- Chromatogram
What is a chromatogram?
A chromatogram is a plot of detector response versus time. It tells us the time
taken for each analyte to pass through the system (retention time). The size of the
peaks also tells us the amount of each analyte present in the sample
What is peak area proportional to?
Peak area is proportional to analyte amount
What is reversed-phase chromatography?
Non-polar stationary phase and a polar (water containing) mobile phase.
Polar molecules retained less than non polar molecules
What are the modes of Chromatography based upon the type of
interaction of the analyte with the stationary phase? (5)
1: Adsorption
2: Partition
3: Ion Exchange
4: Size Exclusion
5: Affinity
Adsorption mode of chromatography, explain it: (4)
-Analytes “land” on surface - do
not penetrate
-Non-polar interactions between
analyte hydrophobic portion and
bonded phase
-Degree of interaction depends on
relative polarities of SP and MP
-Weak interactions
(Hydrogen bonding)
(Dipole forces)
(Solute is absorbed on the surface of the stationary phase)
Partition mode of chromatography, explain it: (3)
-Analyte distributes between
aqueous mobile phase and organic
stationary phase
-Based on relative solubility of
mobile phase
-Interactions
(Hydrophobic)
(Hydrogen Bonding)
(Solute is dissolved in a liquid phase coated on the surface of solid support)
Can adsorption and Partition occur simultaneously?
Adsorption and Partition can occur simultaneously eg. SiO2
Ion Exchange mode of chromatography, explain it: (2)
-SP contains fixed charged groups e.g.-SO3 or -N(CH3)3 and mobile counter groups
which exchange with analyte
-Interaction
(Electrostatic forces)
Size exclusion mode of chromatography, explain it (3)
-Separates molecules by size, molecules
separated by differences in diffusion
through pores
-Sieving, no real interaction
-Small molecules travel longer
Affinity mode of chromatography, explain it (1)
Specific interactions like
particular antibody to a protein
What’s the comparison between LC and GC?
GC (Gas Chromatography)
- High temperature – thermally labile samples
*Destructive detectors – FID
- Analyte must be volatile
- Most samples are under 500 Da (volatility)
*Samples must be in organic solvents (requiring extraction)
*Sample size usually 1-5 µL with detector sensitivity ng to pg on column
HPLC (High Performance Liquid Chromatography)
- Room temp.
*Non-destructive detectors
- No volatility issues but analyte must be soluble in mobile phase
- Analytes spanning a wide polarity range
*Samples can be aqueous
*Sample volume 1-50 µL (1-10 µg analyte per gram of stat. phase)
What’s the comparison between LC and GC? (4)
If the sample cannot be analysed by Gas Chromatography without lengthy sample preparation (indicating issues with volatility), then HPLC should be the technique of choice.
HPLC is the best choice for higher molecular weight analytes and analytes which may potentially degrade when heated.
HPLC separations involve both mobile phase and stationary phase.
In GC, mobile phase only carries analytes through the column.
In HPLC, the polarity of the mobile phase and chemical nature of the
stationary phase means that the separation is driven by selectivity.
In GC , there are less options to improve selectivity and the separation
Is mainly driven by much higher efficiency
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Topic 123
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Section 269
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Section 3 Richie Ryan Intermolecular Interactions75
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Section 4 Richie Ryan Chromatographic band broadening62
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Section 5 Richie Ryan HPLC Columns and Column selectivity for Reversed phase HPLC78
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Section 6 Analytical Method Development -Reversed phase HPLC for Neutral Analytes62
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Section 7 Richie Ryan Analytical Method Development Reversed phase HPLC for ionisable analytes65
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Section 8 Richie Ryan Gradient HPLC Method Development53
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Topic 1 Gas Chromatography Overview Wayne Cummins28
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GC Topic 2. Inlets and Sample Introduction Wayne Cummins130
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Topic 3. Column Formats and Stationary Phase Selection Wayne Cummins106
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Topic 4. Column Parameters Wayne Cummins133
