Chromatographic Methods & Instrumentation
Selections from Chapters 27-28 & 30
Chapter 27 – Gas chromatography
1st commercial instrument in 1955, now 106 GCs worldwide.
Gaseous analyte transported through the column by a gaseous
mobile phase or carrier gas.
 Injector port hot for rapid evaporation of entire sample
 Column hot enough for samples to have significant vapor
pressure
 Detector hot enough for all analytes to be completely
vaporized during detection.
Stationary Phases
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Most common columns are now FSWC. Capillary columns in
general:
Capillary columns advantages compared to packed columns
 higher resolution
 shorter analysis times
 greater sensitivity
Capillary columns disadvantage compared to packed columns
 smaller sample capacity
In GC separations, there are two variables most manipulated to
facilitate a separation.
1. Type of stationary phase (i.e. column type)
2. Column temperature
Choice of stationary phase: “like
dissolves like”
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Matching of analyte and column polarity results in elution times in
order of increasing boiling point.
Column Temperature – GC solution to general elution problem
(Ch. 26) where a sample has analytes with a very broad range of
boiling points.
GC detection systems – the perfect detector
 Extremely sensitive
 Universal
 Large linear range
 Robust
 Qualitative analysis
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2 most common GC detectors:
1. Thermal conductivity detector (TCD)
2. Flame ionization detector (FID)
TCD – universal, robust, large linear range, relatively insensitive.
Measures the ability of a gas to transport heat from a hot  cold
region.
Flame ionization detector – large linear range, sensitive, robust, not
universal.
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Carbon atoms when burned produce cations and electrons in flame.
Potential of +250V between flame tip and positive collector
electrode causes current flow.
Other more niche detectors exist…+ an important one – MS!
End of Chapter 27 questions/problems for GC:
1, 3, 5-7 for TCD and FID, 11-18, 23,24, 29, 30a-f
Chapter 28 – Liquid Chromatography
For compounds which are non-volatile, GC is not an option. Must
perform chromatography in the liquid phase (LC). Same for
thermally labile solutes.
LC is the most widely used of all separation methods.
Almost all LC (partition chromatography) is now HPLC (Higher
Performance Liquid Chromatography), because of small particle
size stationary phase for separation efficiency (Ch. 26).
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 Small particles decrease multipath flow term
 Small particles decrease C term since solute must not flow as
far in the mobile phase to equilibrate with stationary phase
(same reason no capillary columns in LC).
The penalty: column pressure P =
In LC separations, there are two variables most manipulated to
facilitate a separation:
 Type of stationary phase (column)
 Nature of mobile phase (contrast GC)
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R = aminopropyl, aminocyano, (polar)
R = C18, C8, phenyl (non-polar)
Choice of stationary phase in LC more complex than in GC
Analyte/stationary phase interactions + Analyte mobile phase
interactions. In general – stationary phase polarity should match
that of analytes, mobile phase polarity should be different.
Normal phase chromatography = polar stationary phase + nonpolar mobile phase.
Reversed phase chromatography = non-polar stationary phase +
polar mobile phase. (Most common type of partition HPLC).
Elution:
7
The stronger the solvent the shorter the retention time. Solvent
strength can be varied by solvent
gradients.
LC injection systems enable reproducible sample volumes to be
manually or robotically injected. Manual injection system:
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HPLC pumps must generate high pressures, pay for pulse free
output.
Columns – discussed to some extent previously.
Analytical columns = 10-15 cm, 40 – 70K theoretical plates
Recently UHPLC – very small particles ultra high pressures
All columns are expensive – guard columns
Detectors – the ideal LC detector has same characteristics as ideal
GC detector:
 Extremely sensitive
 Universal
 Large linear range
 Robust
 Qualitative analysis
Two most common LC detectors:
 UV-Vis absorbance detector
 Refractive index detector
UV-Vis absorbance detectors look like this:
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3 flavors of UV-Vis absorbance detectors, increasing order of cost
and power.
1. Single UV wavelength (254 nm Hg emission), since most
analytes absorb at this wavelength
2. Since 254 nm absorbance is not useful for all analytes, use
a detector to monitor absorbance at any single desired wavelength.
3. Acquire a full spectrum as a function of time, instead of
absorbance at a single wavelength.
Another “smart” detector common in GC and LC both, is mass
spectrometry (topic after chromatography).
Second most common detector in LC is the refractive index
(universal) detector.
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Advantage: Universal detector. Responds to any analyte with a
refractive index different from mobile phase.
Disadvantages: Much higher detection limit than most other
detectors, useless in gradient elution since mobile phase refractive
index is always changing.
The text describes method development procedures for RP-HPLC
(the most common type of partition chromatography) in Section
D2, these are details not to be discussed in class.
Note: The discussions above with respect to solvent strength,
reversed phase/normal phase, is all about partition chromatography
only. There are other types of liquid chromatography, some
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mentioned at the very beginning of Ch. 26 intro to
chromatography.
These include
 Ion Chromatography (28F)
 Size Exclusion Chromatography (28G)
 Affinity Chromatography (28H)
 Thin-Layer Chromatography (28I)
A quick discussion of the fundamentals of each.
1. Ion chromatography – based on ion exchange equilibria between
analyte and stationary phase.
Consider the competition for sites on the cation exchange resin:
The equilibrium (distribution) constant, or selectivity coefficient,
describes the relative selectivity for different cations for the
stationary phase.
In general ion exchangers more strongly bond ions of higher
charge, increased polarizability, and decreased hydrated radius.
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2. Size exclusion chromatography – small molecules penetrate
porous stationary phase, large molecules do not. Large molecules
are eluted first since they pass through a shorter volume. No
intermolecular forces involved. Used to purify macromolecules in
biochemistry.
Calibrate using standard standard curve of log molecular mass vs.
retention volume.
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3. Affinity chromatography used to isolate a single compound from
a complex mixture by selective binding to the stationary phase,
usually using enzyme/substrate, antibody/antigen, or
receptor/hormone interactions.
4. Thin-layer chromatography. Seems different but really just
partition chromatography in 2 dimensions rather than 3.
Since x-axis is distance, not time, define retardation factor:
RF = dR/dM
The retention factor by analogy to what has been done before:
k = (dM – dR)/dR
Chapter 28 Questions & Problems
1 (a,d,e,f) 2, 3, 4, 6, 7(a,b,d,e,g), 8, 10, 11, 13, 14, 20
Capillary Electrophoresis Basics – Chapter 30.
Separation based on differential migration rates of charged species
in an electric field – excellent for biological molecules and
inorganic ions.
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This is the instrumental version of slab gel electrophoresis where
the sample is introduced as a spot on the gel, and when separation
is thought to be complete turn off power supply and separated
species visualized by staining.
Capillary electrophoresis affords superior separations to slab
electrophoresis (and chromatographic methods for that matter) for
many reasons.
Since there is no stationary phase in capillary electrophoresis:
H = A + B/u + Cu
So how does separation occur?
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The solute migration velocity is related to the electric field strength
and the solute’s electrophoretic mobility.
Since separations based on diffential solute migration velocity, and
all solutes experience the same electric field strength, what effects
the electrophoretic mobility?
The greater the charge to size ratio, the higher the electrophoretic
mobility and the higher the solute migration velocity.
Although it’s not really analogous (no equilibria), theoretical plates
can be determined from an electropherogram.
The higher the voltage the greater the number of theoretical plates.
Compare capillary to slab gel electrophoresis:
1. Faster separations (higher voltages obtainable in
capillary)
2. More efficient separations (narrower peaks)
HPLC – N = 5,000 – 20,000
Capillary GC – N < 150,000
CE – N = 100,000 – 1,000,000
A primary reason for such efficient separations is due to
electroosmotic flow, which also explains why both cations and
anions flow in the same direction towards the negative cathode.
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The electroosmotic flow velocity is given by an equation
analogous to that of solute migration velocity earlier
In the presence of electroosmotic flow the velocity of an ion is the
sum of its migration velocity and the electoosmotic flow velocity.
Since electroosmotic flow velocity > solute migration velocity, all
solutes, even anions, are swept to the cathode.
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Simplest: capillary zone electrophoresis (CZE) where the buffer
composition is constant throughout, and the electric field causes
the species to migrate and separate into zones
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There are other variants of capillary electrophoresis including
 Capillary gel electrophoresis (30C-2), used for DNA
sequencing. Combines an electrophoretic separation
mechanism with that of size exclusion chromatography by
filling a capillary with a porous gel polymer matrix to “sieve”
based on molecular size.
 Capillary Isotachophoresis (30C-3), where the sample is
injected between 2 buffers resulting in sharp bands.
 Capillary Isoelectric focusing (3-C-4) for the separation of
amphiprotic species, especially amino acids/proteins. Here
the buffer composition varies throughout to form a pH
gradient. A convenient excuse to revisit some acid/base
equilibria.
At the isoelectric point the solute no longer migrates in the
electric field, and is focused in a narrow region in the capillary.
(Separation is not achieved by differences in migration rates,
but differences in Ka and Kb values.)
The focused bands are then moved by pressure or altering the
pH gradient by changing the solution in the electrode
compartment.
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None of the above can separate neutral molecules well…
 Micellar Electrokinetic chromatography (30C-5) where
molecules are dissolved in a negatively charged SDS micelle
(made of surfactant molecules). Molecules equilibrate to
varying extents with the micelle and buffer, resulting in
different migration (retention?) times. This is really a true
chromatographic separation based on partition between 2
phases within the electrophoretic capillary.
The micelles have a much slower electrophoretic mobility than
the buffer. Solutes which interact more with the micelles (nonpolar molecules) migrate more slowly than solutes which
partition more in the buffer. Thus this is a partition between 2
phases, one moving faster than the other.
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 Capillary Electrochromatography. Separation mechanisms
based on both CE and LC partition chromatography since the
capillary is packed with an HPLC stationary phase.
No discussion of field flow fractionation (section 30E)
Instrumentation for CE (section 30B-4) not specifically addressed.
Same detector issues as LC.
Chapter 30 Questions/Problems
1-8
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