Chromatographic Techniques from FAQ SCI.CHEM Part 5
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What is Paper Chromatography?
Paper chromatography was the first analytical chromatographic technique
developed, allegedly using papyrus (Pliny). It was first published by Runge
in 1855, and consists of a solvent moving along filter or blotting paper.
The interaction between the components of the sample, the solvent and the
paper results in separation of the components. Most modern paper
chromatography is partition chromatography, where the cellulose of the
paper is the inert support, the water adsorbed ( hydrogen bonded ) from air
onto the hydroxyl groups of the cellulose is the stationary phase. If the
mobile phase is not saturated with water, then some of the stationary phase
water may be removed from the cellulose resulting in a separation that is
a mixture of partition and adsorption. Paper chromatography remains the
method of choice for a wide range of coloured compounds, and is used
extensively in flower colour research. The technique is suitable for any
molecules that are significantly less volatile than the solvent, and many
examples and references are provided in Heftmann [1].
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What is Thin Layer Chromatography?
Thin layer chromatography involves the use of a particulate sorbent on an
inert sheet of glass, plastic, or metal. The solvent is allowed to travel
up the plate with the sample spotted on the sorbent just above the solvent.
Depending on the sorbent, the separation can be either partition or
adsorption chromatography ( cellulose, silica gel and alumina are commonly
used ). The technique came to prominence during the late 1930s, however it
did not become popular until Merck and Desaga developed commercial plates
that provided reproducible separations. The major advantage of TLC is the
disposable nature of the plates. Samples do not have to undergo extensive
cleanups as they would for HPLC. The other major advantage is the ability
to detect a wide range of compounds cheaply using very reactive reagents
( iodine vapours, sulfuric acid ) or indicators. Non-destructive detection
( fluorescent indicators in the plates, examination under a UV lamp ) also
means that purified samples can be scraped off the plate and analyzed by
other techniques. There are special plates for such preparative separations,
and there are also high-performance plates that can approach HPLC resolution.
The technique is described in detail in Stahl [2] and Kirchner [3].
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What is Gas Chromatography?
Gas chromatography is the use of a gas to carry the sample through a column
consisting of an inert support and a stationary phase that interacts with
sample components, thus it is usually partition chromatography, however
there are also a range of materials, especially for permanent gas and
light hydrocarbon analysis that utilise adsorption. The simplest partition
systems consisted of a steel tube filled with crushed brick that had been
coated with a high boiling hydrocarbon. Today the technique uses very narrow
fused silica tubes ( 0.1 to 0.3mm ID ) that have sophisticated stationary
phase films ( 0.1 to 5um ) bonded to the surface and also cross-linked to
increase thermal stability. The ability of the film to retard specific
compounds is used to ascertain the "polarity" of the column. If benzene
elutes between normal alkanes where it is expected by boiling point ( midway
between n-hexane and n-heptane ), then the column is "non-polar" eg
squalane and methyl silicones. If the benzene is retarded until it elutes
after n-dodecane, then the column is "polar" eg OV-275 ( dicyanoallyl
silicone ) and 1,2,3-tris (2-cyanoethoxy) propane. In general polar columns
are less tolerant of oxygen and reactive sample components, but the ability
to select a select different polarity columns to obtain satisfactory peak
resolution is what made GC so popular.
The column is placed in an oven which has exceptional temperature control,
and the column can be slowly heated up to 350-450C ( sometimes starting at
-50C to enhance resolution of volatile compounds ) to provide separation of
wide-boiling range compounds. The carrier gas is usually hydrogen or helium,
and the eluting compounds can be detected several ways, including in flames
( flame ionisation detector ), by changes in properties of the carrier
( thermal conductivity detector ), or by mass spectrometry. The availability
of "universal" detectors such as the FID and MS, makes GC a popular tool in
laboratories handling organic compounds. There are also columns that have a
layer of 5-10 um porous particulate material (such as molecular sieve or
alumina ) bonded to the inner walls ( PLOT = Porous layer open tubular ),
and these are used for the separation of permanent gases and light
hydrocarbons. GC is restricted to molecules ( or derivatives ) that
are sufficiently stable and volatile to pass through the GC intact at the
temperatures required for the separation. Specialist books on the production
of derivatives for GC are available [4,5].
There are several manufacturers of GC instruments whose catalogues and
brochures provide good introduction to the technique. (eg Hewlett Packard,
Perkin Elmer, Carlo Erba ). The catalogues of suppliers of chromatography
consumables also contain explanations of the criteria for selection of the
correct columns and conditions for analyses, and they provide an excellent
indication of the range of applications available. Well-known suppliers
include Alltech Associates, Supelco, Chrompack, J&W, and Restek. They also
sell most of the standard GC texts, as do the instrument manufacturers.
Popular GC texts include "Basic Gas Chromatography" [6], "High-Resolution
Gas Chromatography" [7], and "Open Tubular Column Gas Chromatography" [8].
There are Standard Retention Index Libraries available [9], however they
really only complement unambiguous identification by mass spec. or
dual-column analysis.
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What is Column Chromatography?
Column chromatography consists of a column of particulate material such as
silica or alumina that has a solvent passed through it at atmospheric or low
pressure. The separation can be liquid/solid (adsorption) or liquid/liquid
(partition). The columns are usually glass or plastic with sinter frits to
hod the packing. Most systems rely on gravity to push the solvent through.
The sample is dissolved in solvent and applied to the front of the
column. The solvent elutes the sample though the column, allowing the
components to separate based on adsorption ( alumina, hydroxylapatite) or
partition ( cellulose, diatomaceous earth ). The mechanism for silica
depends on the hydration. Traditionally, the solvent was non-polar and the
surface polar, although today there are a wide range of packings including
bonded phase systems. Bonded phase systems usually utilise partition
mechanisms rather than adsorption. The solvent is usually changed stepwise,
and fractions are collected according to the separation required, with the
eluate usually monitored by TLC.
The technique is not efficient, with relatively large volumes of solvent
being used, and particle size is constrained by the need to have a flow of
several mls/min. The major advantage is that no pumps or expensive equipment
are required, and the technique can be scaled up to handle sample sizes
approaching a gram in the laboratory. The technique is discussed in detail
in Heftmann [1].
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What is High Pressure Liquid Chromatography?
HPLC is a development of column chromatography. it was long realised that
using particles with a small particle size ( 3,5,10um ) with a very narrow
size distribution would greatly improve resolution, especially if the flow
rate and column dimensions could be adjusted to minimise band-broadening.
Pumps were developed that could handle both the chemicals and pressures
required. Traditional column chromatography ( nonpolar solvent and
polar surface ) is described as "normal" and, as well as silica, there are
columns with amino, diol, and cyano groups. If the system uses a polar
solvent ( water, methanol, acetonitrile etc. ) and a non-polar surface it
is described as "reversed phase". Common surface treatments of silica include
octadecylsilane ( aka ODS or C18), and it has been the development of
reverse-phase HPLC that has experienced explosive growth. Reverse-phase HPLC
is the method of choice for larger non-volatile biomolecules, however it is
only recently that a replacement "universal" detector ( evaporative
light-scattering ) has emerged. The most popular detector (UV), places
constraints on the solvents that can be used, and the refractive index
detector can not easily be used with solvent gradients. There are several
excellent books introducing HPLC, including the classic "Introduction to
Modern Liquid Chromatography" [10]. HPLCs can be a pain to operate, and
novices should borrow "Troubleshooting LC Systems" by Dolan and Snyder [11].
There is also a handy basic primer on developing HPLC methods by Snyder [12],
however, unlike GC, you need to search the journals ( Journal of
Chromatography, Journal of Liquid Chromatography ) to find relevant examples
to assist method development.
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What is Ion Chromatography?
Ion chromatography has become the method of choice for measuring anions
( eg Cl-, SO4=, NO3- ) in aqueous solutions. It is effectively a development
from ion-exchange systems ( which were extensively developed to deionise
water and aqueous process streams ), and brings them down to HPLC size.
IC uses pellicular polymeric resins that are compatible with a wide pH range.
The sample is eluted through an ion-exchange column using a dilute sodium
hydroxide solution. The eluent is passed through self-regenerating
suppressors that neutralise eluant conductance, ensuring electrochemical
detectors ( conductivity or pulsed amperometric ) can detect the ions down
to sub-ppm concentrations. The major manufacturer of such systems is Dionex,
who hold several patents on column, suppression, and detection technology.
There are several books covering various aspects of the technique [13,14].
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What is Gel Permeation Chromatography
?
Gel Permeation chromatography ( aka Size Exclusion chromatography ) is based
on the ability of molecules to move through a column of gel that has pores of
clearly-defined sizes. The larger molecules can not enter the pores, thus
they pass quickly through the column and elute first. Slightly smaller
molecules can enter some pores, and so take longer to elute, and small
molecules can be delayed further. The great advantage of the technique is
simplicity, it is isocratic ( single solvent - no gradient programming ),
and large molecules rapidly elute. The technique can be used to determine
the molecular weight of large biomolecules and polymers, as well as
separating them from salts and small molecules. The columns are very
expensive and sensitive to contamination, consequently they are mainly used
in applications where alternative separation techniques are not available,
and sample are fairly clean. The best known columns are the Shodex
cross-linked polystyrene-divinylbenzene columns for use with organic solvents,
and polyhydroxymethacrylate gel filtration columns for use with aqueous
solvents. "Modern Size Exclusion Chromatography" [15], and Heftmann [1],
provide good overviews, and there are some good introductory booklets from
Pharmacia.
What is Capillary Electrophoresis?
Capillary electrophoresis uses a small fused silica capillary that has been
coated with a hydrophilic or hydrophobic phase to separate biomolecules,
pharmaceuticals and small inorganic ions. A voltage is applied and the
materials migrate and separates according to charge under the specific
pH conditions,as happen for electrophoresis.The capillary can also be used
for isoelectric focusing of proteins. The use of salt or vacuum mobilization
is no longer required.
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How do I degas chromatographic solvents?
One major problem with pressurising chromatography systems using liquid
solvents is that pressure reductions can cause dissolved gases to come out
of solution. The two locations where this occurs are the suction side of the
pump ( which is not self-priming, consequently a gas bubble can sit in the
pump and flow is reduced ), and at the column outlet ( where the bubbles
then pass through the detector causing spurious signals).Note that the
problem is usually restricted to solvents that have relatively high gas
solubilities - usually involving an aqueous component, especially if a
gradient is involved where the water/organic solvent ratio is changing.
There are three strategies used to remove problem dissolved gases from
chromatographic eluants. Often they are used in combination to lower the
dissolved gases.
a. Subject the solvent to vacuum for 5-10 mins. to remove the gases.
b. Subject the solvent to ultrasonics for 10-15 mins. to remove the gases.
c. Sparge the solvent with a gas that has a very low solubility compared
to the oxygen and nitrogen from the atmosphere. Helium is the preferred
choice - 5 minutes of gentle bubbling from a 7um sinter is usually
sufficient.
Note that most aqueous-based solvents usually have to be degassed every
24 hours. Also remember that solubility of gases increases as temperature
decreases, so ensure eluants are at instrument temperature prior to
degassing.
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What is chromatographic solvent "polarity"?
There are four major intermolecular interactions between sample and solvent
molecules in liquid chromatography, dispersion, dipole, hydrogen-bonding,
and dielectric. Dispersion interactions is the attraction between each pair
of adjacent molecules, and are stronger for sample and solvent molecules
with large refractive indices. Strong dipole interactions occur when both
sample and solvent have permanent dipole moments that are aligned. Strong
hydrogen-bonding interactions occur between proton donors and proton
acceptors. Dielectric interactions favour the dissolution of ionic
molecules in polar solvents. The total interaction of the solvent and
sample is the sum of the four interactions. The total interaction for a
sample or solvent molecule in all four ways is known as the "polarity" of
the molecule. Polar solvents dissolve polar molecules, and for normal
phase partition chromatography solvent strength increases with solvent
polarity, whereas solvent strength decreases with increasing polarity.
The subject is discussed in detail in Snyder and Kirkland [10].
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What is Paper Chromatography?