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Sci.chem FAQ - Part 5 of 7
Section - 23. Chromatographic Techniques

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Top Document: Sci.chem FAQ - Part 5 of 7
Previous Document: 22. Spectroscopic Techniques
Next Document: 25. Radiochemical Techniques
See reader questions & answers on this topic! - Help others by sharing your knowledge
There are chromatography mailing lists and WWW sites available that provide 
comprehensive introductions and access to chromatography experts. The
following are simple introductions to popular techniques. 

23.1  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, and the water adsorbed ( hydrogen bonded ) from 
air onto the hydroxyl groups of the cellulose becomes 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 both natural and artificial pigment 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]. 

23.2  What is Thin Layer Chromatography?

Thin layer chromatography involves the use of a particulate sorbant on an
inert sheet of glass, plastic, or metal. The solvent is allowed to travel
up the plate with the sample spotted on the sorbant just above the solvent.
Depending on the sorbant, 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 the 
extensive clean-up steps required 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 
be analysed 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].  

23.3  What is Gas Chromatography? 

Gas chromatography is the use of a carrier gas to convey the sample ( as a
vapour ) through a column consisting of an inert support and a stationary 
phase that interacts with sample components, thus it is usually partition 
chromatography. 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 hydrocarbon that had a high boiling point, eg 
squalane. 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 different polarity columns to 
obtain satisfactory peak resolution is what has made GC so popular. 

The column is placed in an oven that 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 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.

23.4  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
hold 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, hydroxyapatite) 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 
eluted solvent 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].

23.5  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 "reverse-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 and
Kirkland [12], however, unlike GC, you also need to search the journals 
( Journal of Chromatography, Journal of Liquid Chromatography  ) to find 
relevant examples to assist with method development. 
 
23.6  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 soften 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 eluant 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].
  
23.7  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.

23.8  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 
analytes migrate and separate according to their charge under the specific 
pH conditions, as also happens for electrophoresis. The capillary can also 
be used for isoelectric focusing of proteins. The use of salt or vacuum 
mobilisation is no longer required.  

23.9  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.
As water usually has a higher dissolved gas content, then a gradient 
programme may cause the gases to come out of solution as the mobile phase
components mix. 

There are three traditional 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, although maintaining a positive He pressure is even better.
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. Helium is preferred as the degassing solvent because it has
relatively low solubility in water, and the solubility is less affected by 
temperature.

The following data is from Kaye and Laby, 13th edition, and the units are 
the number of cm3 of gas at 0C and 760 mmHg which dissolve in 1 cm3 of water 
at the temperature stated ( when the gas is at 760 mmHg pressure and in 
equilibrium with the water ).

Temp.(C)   0        10       20       30       40       50      60
Helium   0.0098   0.0091   0.0086   0.0084   0.0084   0.0086   0.0090
Hydrogen 0.0214   0.0195   0.0182   0.0170   0.0164   0.0161   0.0160
Nitrogen 0.0230   0.0185   0.0152   0.0133   0.0119   0.0108   0.0100
Oxygen   0.047    0.037    0.030    0.026    0.022    0.020    0.019
Argon    0.054    0.041    0.032    0.028    0.025    0.024    0.023
CO2      1.676    1.163    0.848    0.652    0.518    0.424    0.360

I've no explanation for the aberrant trend for helium at higher temperatures,
but I assume it's real - but it's irrelevant for HPLC solvents that are 
usually stored at ambient temperature. Points to note - the lower solubility 
of helium over the range of concern, *and* the lower rate of change of 
decreasing solubility with increasing temperature. There is heat generated
in the compression of the solvent, along with friction in HPLC pump heads 
and, more importantly, HPLC columns are often heated - thus the solvent 
could outgas and form bubbles in UV detector cells that are at ambient. 
By using helium, there is less chance of that happening. For example, if the 
temperature increased from 10C to 40C, the undissolved gas volume would be 
0.0007 cm3 for helium, and 0.0066 cm3 for nitrogen.

Modern HPLCs are sold with a "solvent degassing module" that removes 
undissolved gases in the solvent automatically. These usually consist of
a tube made from gas-permeable membrane that passes through a vacuum
chamber. 
 
23.10  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 are 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
in reverse-phase systems. The subject is discussed in detail in Snyder 
and Kirkland [10].

------------------------------


Subject: 24. Extraction Techniques   
     
24.1  What is Solvent Extraction? 

Solvent extraction is usually used to recover a component from either a solid
or liquid. The sample is contacted with a solvent that will dissolve the
solutes of interest. Solvent extraction is of major commercial importance
to the chemical and biochemical industries, as it is often the most efficient
method of separation of valuable products from complex feedstocks or
reaction products. Some extraction techniques in involve partition between two 
immiscible liquids, others involve either continuous extractions or batch
extractions. Because of environmental concerns, many common liquid/liquid
processes have been modified to either utilise benign solvents, or move to
more frugal processes such as solid phase extraction. The solvent can be a 
vapour, supercritical fluid, or liquid, and the sample can be a gas, liquid 
or solid. There are a wide range of techniques used, and details can be found 
in Organic Vogel, Perry, and most textbooks on unit operations. 

24.2  What is Solid Phase Extraction? 

Solid Phase Extraction (SPE) is an alternative to liquid/liquid extraction,
and has become the method of choice for the separation and purification of
a wide range of samples in the laboratory. The sample is usually dissolved 
in an appropriate solvent and passed through a small bed of adsorbent of
very consistent particle size and shape to maximise separation efficiency. 
The compounds are eluted with step changes of small volumes of solvents. 
The major advantage is that solvent volumes are greatly reduced. There is 
a newer, modified technique that is used in analytical laboratories, called 
Solid Phase Micro Extraction. This immerses a fused silica fibre coated with 
a stationary phase into the sample solution for several minutes, The analytes
adsorb onto the stationary phase, which is subsequently pushed into a hot GC 
injector to rapidly desorb the sample for analysis.  

24.3  What is Supercritical Fluid Extraction? 

Refer to Section 19.3 for some critical data on common supercritical fluids. 
Supercritical fluids have been investigated since last century, with the 
strongest commercial interest initially focusing on the use of supercritical 
toluene in petroleum and shale oil refining during the 1970s. Supercritical 
water is also being investigated as a means of destroying toxic wastes, and
as an unusual synthesis medium [1]. The biggest interest for the last decade
has been the applications of supercritical carbon dioxide, because it has
a near-ambient critical temperature (31C), thus biological materials can
be processed at temperatures around 35C. The density of the supercritical
CO2 at around 200bar pressure is close to that of hexane, and the solvation 
characteristics are also similar to hexane, thus it acts as a non-polar 
solvent. Around the supercritical region CO2 can dissolve triglycerides at 
concentrations up to 1% mass. The major advantage is that a small reduction 
in temperature, or a slightly larger reduction in pressure, will result in 
almost all of the solute precipitating out as the supercritical conditions
are changed or made subcritical. Supercritical fluids can produce a 
product with no solvent residues. Examples of pilot and production scale 
products include decaffeinated coffee, cholesterol-free butter, low-fat meat, 
evening primrose oil, squalene from shark liver oil. The solvation 
characteristics of supercritical CO2 can be modified by the addition of an 
entrainer, such as ethanol, however some entrainer remains as a solvent 
residue in the product, negating some of the advantages of the "residue-free"
extraction.
 
There are other near-ambient temperature supercritical fluids, including 
nitrous oxide and propane, however there are safety issues with some of them. 
There are several introductory texts on supercritical fluid extraction, 
including some the ACS Symposium series [2-4]. There are also a large 
number of articles on applications of the technique, including processing [5],
extraction of natural products [6], and chemical synthesis [7]. The major 
concentration of information occurs in the various proceedings of the 
International Symposium on Supercritical Fluids [8]. There is also a Journal 
of Supercritical Fluids.

24.4  What traditional process extracted perfume from flower petals?

The traditional cold-fat extraction process is known as " enfleurage". 
It is a very interesting, historical process used to obtain the essential 
oils and perfume components from rose, jasmine, and other flowers. The
rose and jasmine flowers continue to produce perfume during the long 
process. Thus the technique can obtain more perfume from those flowers than 
if they were just macerated and extracted by hot fat, solvent or steam  
when they were picked - as happens to many other plant perfume sources.
The process uses a fat comprised of 40 parts of beef tallow and 60 parts
of lard. The two fats are melted together, and repeatedly beaten under
cold water and alum solutions to purify them. Benzoin is added to the
fat mixture to prevent biological degradation.

The fat is spread about 4mm thick on both sides of 0.5 x 0.5 metre glass 
plates in wooden frames. Flowers are pressed into the fat on one side of 
the frame only, and the frames stacked vertically so that the flowers are
very close to the layer of fat on the frame above. After 1-3 days, the 
flowers are stripped off and fresh flowers added to the other layer of fat
that had not been used, and the frame are again stacked. The cycle is 
repeated about 30 - 35 times, or until the fat is saturated with perfume.
The saturated fat is known as "pomade". The fat is removed from the frames
and extracted with alcohol to collect the perfume. the alcohol is cooled
and filtered to remove most of the dissolved fat. The alcohol solution
is called the "extract", and the residue after evaporation of the solvent
is known as the "enfleurage absolute".     

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