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HISTORY
INTRODUCTION
Mikhail Semyonovich Tsvet (18721919)
was a Russian botanist who inventedadsorption chromatography technique in
1906 during his research on
plant pigments. Tsvet's work involved
the study of plant pigments, such as
chlorophylls and carotenoids. In 1901 he
developed the techniques of adsorption
chromatography. He passed solutions of
leaf colourants dissolved in a light petrol
mix through a column of powdered
chalk. The distinct colour bands formed
by the different pigments could then beand analysed.The method was described on 30 December 1901 at the XI
Congress of Naturalists and Physicians in St. Petersburg. The first printed
description was in 1905, in the Proceedings of the Warsaw Society of
Naturalists.
He first used the term "chromatography" in print in 1906 in his two papers
about chlorophyll in the German botanical journal, Berichte der Deutschen
botanischen Gesellschaft. In 1907 he demonstrated his chromatograph for
the German Botanical Society. However, scientists at the time could not
replicate his findings, believing that they were erroneous. However, 10
years after his death in 1919, others successfully repeated his results and
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The word chromatography is composed of two Greek roots, chroma
(colour) and graphein (to write), and its translation means colour
writing, which refers to visualizing the separated multicoloured rings onthe column. Another interpretation of this term links it to Tswetts
surname, which is colour in Russian. According to International Union of
Pure and Applied Chemistry (IUPAC) definition of chromatography in 1993,
chromatography is defined as the physical method of separation in which
the components to be separated are distributed between two phases, one
of which is stationary while the other moves in a definite direction.
Chromatography is the laboratory techniques for the separation of
mixtures. The mixture is dissolved in a fluid called the "mobile phase",
which carries it through a structure holding another material called the
"stationary phase". The various constituents of the mixture travel at
different speeds, causing them to separate. The separation is based on
differential partitioning between the mobile and stationary phases. Subtle
differences in a compound's partition coefficient result in differential
retention on the stationary phase and thus changing the separation. Any
Chromatography system is composed of three components, which are the
stationary phase, mobile phase and mixture to be separated. We can only
control stationary and mobile phase as mixtures are the problem we have
to deal with.
Stationary phase is a layer or coating on the supporting medium that
interacts with the analytes and is fixed in a place either in column or a
planar surface. It can be solid, liquid, gel or solid-liquid mixture. Its also
the part of the chromatographic system though which the mobile phase
flows where distribution of the solutes between the phases occurs. It
Mobile phase is the part of the chromatographic system which carries
the solutes through the stationary phase. It can be either liquids or
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Chromatography may be preparative or analytical. The purpose of
preparative chromatography is to separate the components of a mixture
for further use (and is thus a form of purification). Analyticalchromatography is done normally with smaller amounts of material and is
for measuring the relative proportions of analytes in a mixture.
There are many types of chromatography, types are as follows:
(a)Techniques by chromatographic bed shape
Column chromatography
Planar chromatography
Paper chromatography
Thin layer chromatography
(b)Techniques by physical state of mobile phase
Gas chromatography
Liquid chromatography
(c)Techniques by separation mechanism
Ion exchange chromatography
Size-exclusion chromatography
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COLUMN CHROMATOGRAPHY
Column chromatography is a method used to purify individual chemicalcompounds from mixtures of compounds. It is often used for preparative
applications on scales from micrograms up to kilograms. The main
advantage of column chromatography is the relatively low cost and
disposability of the stationary phase used in the process.
In column chromatography, the mobile phase is a solvent, and the
stationary phase is a finely divided solid, such as silica gel or alumina.
Chromatography columns vary in size and polarity. There is an element of
trial and error involved in selecting a suitable solvent and column for the
separation of the constituents of a particular mixture. A small volume of
the sample whose constituents are to be separated is placed on top of the
column. The choice of the eluting solvent should ensure that the sample is
soluble. However, if the sample was too soluble the mobile phase
(solvent) would move the solutes too quickly, resulting in the non-
separation of the different constituents.
There is an optimum flow rate for each particular separation. A faster flow
rate of the eluent minimizes the time required to run a column and
thereby minimizes diffusion, resulting in a better separation. However, the
maximum flow rate is limited because a finite time is required for analyte
to equilibrate between stationary phase and mobile phase, see Van
Deemter's equation. A simple laboratory column runs by gravity flow. The
flow rate of such a column can be increased by extending the fresh eluent
filled column above the top of the stationary phase or decreased by the
tap controls. Faster flow rates can be achieved by using a pump or by
using compressed gas (air, nitrogen, or argon) to push the solvent through
the column.
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The classical preparative chromatography column is a glass tube with a
diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and
some kind of a filter (a glass frit or glass wool plug to prevent the loss of
the stationary phase) at the bottom. Two methods are generally used toprepare a column: the dry method, and the wet method.
The preparations of the two methods are further discussed as follows:
a) Prepare the column.
The column is packed using a simple dry-pack method.
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Plug a Pasteur pipette with a
small amount of cotton; use a
wood applicator stick to tamp
it down lightly. Take care thatyou do not use either too
much cotton or pack it too
tightly. You just need enough
to prevent the adsorbent from
leaking out.
Add dry silica gel adsorbent,
230-400 mesh -- usually the
jar is labelled "for flash
chromatography." One wayto fill the column is to invert
it into the jar of silica gel and
scoop it out.
Then tamp it down before
scooping more out.
Another way to fill the
column is to pour the gel into
the column using a 10 mL
beaker.
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When properly packed, the
silica gel fills the column to
just below the indent on the
pipette. This leaves a space
of 45 cm on top of the
adsorbent for the addition of
solvent. Clamp the filledcolumn securely to a ring
stand.
Whichever method you use to
fill the column, you must
tamp it down on the bench
top to pack the silica gel. You
can also use a pipette bulb to
force air into the column and
pack the silica gel.
b) Pre-elute the column.
The procedure for the experiment that you are doing will probably
specify which solvent to use to pre-elute the column. A non-polar
solvent such as hexanes is a common choice.
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Add hexanes (or other specified
solvent) to the top of the silica
gel. The solvent flows slowly
down the column; on the
column above, it has flowed
down to the point marked by
the arrow.
Monitor the solvent level, both
as it flows through the silica gel
and the level at the top. If you
are not in a hurry (or busy
doing something else), you can
let the top level drop by gravity,
but make sure it does not gobelow the top of the silica.
Again, the arrow marks how far
the solvent has flowed down
the column.
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Speed up the process by using a
pipette bulb to force the solvent
through the silica gel. Place the
pipette bulb on top of thecolumn, squeeze the bulb, and
then remove the bulb while it is
still squeezed. You must be
careful not to allow the pipette
bulb to expand before you
remove it from the column, or
you will draw solvent and silica
gel into the bulb.
When the bottom solvent level
is at the bottom of the column,
the pre-elution process is
completed and the column isready to load.
If you are not ready to load your
sample onto the column, the
column can be left at this point.
Just make sure that it does not
go dry, therefore keep the topsolvent level above the top of
the silica (as shown in the
picture to the left) by adding
solvent as necessary.
c) Load the sample onto the silica gel column.
Two different methods are used to load the column: the wet method
and the dry method: wet and dry.
In the wet method, the sample to be purified (or separated into
components) is dissolved in a small amount of solvent, such as
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hexanes, acetone, or other solvent. This solution is loaded onto the
column.
Wet loading method
The column at the left is being
loaded by the wet method. Once
it's in the column, fresh eluting
solvent is added to the top and
you are ready to begin the
elution process.
Sometimes the solvent of choice to load the sample onto the column is
more polar than the eluting solvents. In this case, if you use the wet
method of column loading, it is critical that you only use a few drops of
solvent to load the sample. If you use too much solvent, the loading
solvent will interfere with the elution and hence the purification or
separation of the mixture. In such cases, the dry method of column
loading is recommended.
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Dry loading method
First dissolve the sample to be
analyzed in the minimum amount
of solvent and add about 100 mgof silica gel. Swirl the mixture
until the solvent evaporates and
only a dry powder remains. Place
the dry powder on a folded piece
of weighing paper and transfer it
to the top of the prepared
column. Add fresh eluting solvent
to the top, the elution process
are ready.
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d) Elute the column.
Force the solvent through the column by pressing on the top of the
Pasteur pipette with a pipette bulb. Only force the solvent to the very
top of the silica: do not let the silica go dry. Add fresh solvent as
necessary.
The solvent being forced
through the column with a
pipette bulb. The series of 5
photos below show the colored
compound as it moves through
the column after successive
applications of the pipette bulb
process. The last two photos
illustrate collection of the
colored sample. Note that the
collection beaker is changed as
soon as the colored compound
begins to elute. The process is
complicated if the compound is
not colored. In such
experiments, equal sized
fractions are collected
sequentially and carefully
labelled for later analysis.
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e) Elute the column with the second elution solvent.
If you are separating a mixture of one or more compounds, at this point
you would change the eluting solvent to a more polar system.
f) Analyze the fractions.
If the fractions are colored, you can simply combine like-colored
fractions, although TLC before combination is usually advisable. If the
fractions are not colored, they are analyzed by TLC (usually). Once the
composition of each fraction is known, the fractions containing the
desired compound(s) are combined.
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PLANAR CHROMATOGRAPHY
Planar chromatography is a separation technique in which the stationary
phase is present as or on a plane. The plane can be a paper, serving as
such or impregnated by a substance as the stationary bed (paper
chromatography) or a layer of solid particles spread on a support such as
a glass plate (thin layer chromatography).
Different compounds in the sample mixture travel different distances
according to how strongly they interact with the stationary phase as
compared to the mobile phase. The specific Retention factor (Rf) of each
chemical can be used to aid in the identification of an unknown substance.
The retention factor(R) may be defined as the ratio of the distance
travelled by the substance to the distance travelled by the solvent.
Rvalues are usually expressed as a fraction of two decimal places but it
was suggested by Smith that a percentage figure should be used instead.
If Rvalue of a solution is zero, the solute remains in the stationary phase
and thus it is immobile. If Rvalue = 1 then the solute has no affinity for
the stationary phase and travels with the solvent front. To calculate the
Rvalue, take the distance travelled by the substance divided by the
distance travelled by the solvent (as mentioned earlier in terms of ratios).
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PLANAR
CHROMATOGRAPHY
PAPER
CHROMATOGRAPHY
THIN LAYER
CHROMATOGRAPHY
For example, if a compound travels 2.1 cm and the solvent front travels
2.8 cm, (2.1/2.8) the Rvalue = 0.75
Example of retention factor (R) calculation
Planar chromatography is divided into:
PAPER CHROMATOGRAPHY
Paper chromatography is an analytical chemistry technique for separating
and identifying mixtures that are or can be colored, especially pigments. It
is also used for testing the purity of compounds, identifying substances or
used in secondary or primary colors in ink experiments. The stationary
phase is usually a piece of high quality filter paper. The mobile phase is a
developing solution that travels up the stationary phase, carrying the
samples with it. Components of the sample will separate readily according
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to how strongly they adsorb on the stationary phase versus how readily
they dissolve in the mobile phase.
When a colored chemical sample is placed on a filter paper, the colorsseparate from the sample by placing one end of the paper in a solvent.
The solvent diffuses up the paper, dissolving the various molecules in the
sample according to the polarities of the molecules and the solvent. If the
sample contains more than one color, that means it must have more than
one kind of molecule. Because of the different chemical structures of each
kind of molecule, the chances are very high that each molecule will have
at least a slightly different polarity, giving each molecule a different
solubility in the solvent. The unequal solubilities cause the various color
molecules to leave solution at different places as the solvent continues to
move up the paper. The more soluble a molecule is, the higher it will
migrate up the paper. If a chemical is very nonpolar it will not dissolve at
all in a very polar solvent. This is the same for a very polar chemical and a
very nonpolar solvent.
Two-way paper chromatography, also calledtwo-dimensional
chromatography, involves using two solvents and rotating the paper 90
in between. This is useful for separating complex mixtures of similar
compounds, for example, amino acids. Paper chromatography is a useful
technique because it is relatively quick and requires small quantities of
material. This method has been largely replaced by thin layer
chromatography, however it is still a powerful teaching tool.
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Example of paper chromatography
Example of two-way paper chromatography
THIN LAYER CHROMATOGRAPHY
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Thin layer chromatography is a chromatography technique used to
separate mixture performed on a sheet of glass, plastic, or aluminium foil,
which is coated with a thin layer ofadsorbent material, usually silica
gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbentis known as the stationary phase. After the sample has been applied on
the plate, a solvent or solvent mixture (known as the mobile phase) is
drawn up the plate via capillary action because different analytes ascend
the TLC plate at different rates, separation is achieved
Compared to paper, it has the advantage of faster runs, better
separations, and the choice between different adsorbents. For even better
resolution and to allow for quantification, high-performance TLC can be
used.
Thin layer chromatography can be used to monitor the progress of a
reaction identify compounds present in a given mixture and determine the
purity of a substance. Examples of thin layer chromatography are
analyzing ceramides and fatty acids and detection of pesticides or
insecticides in food and water, analyzing the dye composition of fibers in
forensics and assaying the radiochemical purity ofradiopharmaceuticals,
or identification ofmedicinal plants and their constituents.
Example separation of black ink on a TLC plate
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Example of thin layer chromatography of an extract of green
leaves (spinach) in 7 stages of development. Carotene elutes
quickly and is only visible until step 2. Chlorophyll A and B are
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halfway in the final step and lutein the first compound staining
yellow.
G AS CHROMATOGRAPHY
Gas chromatography al known specifically as gas-liquid chromatography, it is
an analytical technique for separating compounds based primarily on their
volatilities. Gas chromatography provides both qualitative and
quantitative information for individual compounds present in a sample.
Compounds move through a GC column as gases, either because the
compounds are normally gases or they can be heated and vaporized into
a gaseous state. The compounds partition between a stationary phase,
which can be either solid or liquid, and a mobile phase (gas). The
differential partitioning into the stationary phase allows the compounds to
be separated in time and space.
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Gas Chromatographic System
The schematic diagram of a gas chromatograph:
Instrumental components
Carrier gas
The carrier gas must be chemically inert. Commonly used gases include
nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is
often dependent upon the type of detector which is used. The carrier gas
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system also contains a molecular sieve to remove water and other
impurities.
Sample injection port
For optimum column efficiency, the sample should not be too large, and
should be introduced onto the column as a "plug" of vapour - slow
injection of large samples causes band broadening and loss of resolution.
The most common injection method is where a microsyringe is used to
inject sample through a rubber septum into a flash vapouriser port at the
head of the column. The temperature of the sample port is usually about
50C higher than the boiling point of the least volatile component of the
sample. For packed columns, sample size ranges from tenths of a
microliter up to 20 microliters. Capillary columns, on the other hand, need
much less sample, typically around 10-3 mL. For capillary GC, split/splitless
injection is used. Have a look at this diagram of a split/splitless injector;
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The injector can be used in one of two modes; split or splitless. The
injector contains a heated chamber containing a glass liner into which the
sample is injected through the septum. The carrier gas enters the
chamber and can leave by three routes (when the injector is in split
mode). The sample vapourises to form a mixture of carrier gas,
vapourised solvent and vapourised solutes. A proportion of this mixture
passes onto the column, but most exits through the split outlet. The
septum purge outlet prevents septum bleed components from entering
the column.
Columns
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There are two general types of column,packedand capillary(also known
as open tubular). Packed columns contain a finely divided, inert, solid
support material (commonly based on diatomaceous earth) coated with
liquid stationary phase. Most packed columns are 1.5 - 10m in length and
have an internal diameter of 2 - 4mm.
Capillary columns have an internal diameter of a few tenths of a
millimeter. They can be one of two types; wall-coated open tubular
(WCOT) or support-coated open tubular (SCOT). Wall-coated columns
consist of a capillary tube whose walls are coated with liquid stationary
phase. In support-coated columns, the inner wall of the capillary is lined
with a thin layer of support material such as diatomaceous earth, onto
which the stationary phase has been adsorbed. SCOT columns are
generally less efficient than WCOT columns. Both types of capillary
column are more efficient than packed columns.
In 1979, a new type of WCOT column was devised - the Fused Silica Open
Tubular(FSOT) column;
These have much thinner walls than the glass capillary columns, and are
given strength by the polyimide coating. These columns are flexible and
can be wound into coils. They have the advantages of physical strength,
flexibility and low reactivity.
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Oven
The column is placed in an oven where the temperature can be controlled
very accurately over a wide range of temperatures. For precise work,
column temperature must be controlled to within tenths of a degree. The
optimum column temperature is dependent upon the boiling point of the
sample. As a rule of thumb, a temperature slightly above the average
boiling point of the sample results in an elution time of 2 - 30 minutes.
Minimal temperatures give good resolution, but increase elution times. If a
sample has a wide boiling range, then temperature programming can be
useful. The column temperature is increased (either continuously or in
steps) as separation proceeds.
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Detectors
There are many detectors which can be used in gas chromatography.
Different detectors will give different types of selectivity. A non-selective
detector responds to all compounds except the carrier gas, a selective
detectorresponds to a range of compounds with a common physical or
chemical property and a specific detectorresponds to a single chemical
compound. Detectors can also be grouped into concentration dependant
detectors and mass flow dependant detectors. The signal from a
concentration dependant detector is related to the concentration of solute
in the detector, and does not usually destroy the sample Dilution of with
make-up gas will lower the detectors response. Mass flow dependant
detectors usually destroy the sample, and the signal is related to the rate
at which solute molecules enter the detector. The response of a mass flow
dependant detector is unaffected by make-up gas. Have a look at this
tabular summary of common GC detectors:
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Detector TypeSupport
gasesSelectivity
Detectabi
lity
Dynam
ic
rangeFlame
ionization
(FID)
Mass flowHydrogen
and airMost organic cpds. 100 pg 107
Thermal
conductivity
(TCD)
Concentrati
onReference Universal 1 ng 107
Electron
capture
(ECD)
Concentrati
onMake-up
Halides, nitrates,
nitriles, peroxides,
anhydrides,
organometallics
50 fg 105
Nitrogen-
phosphorusMass flow
Hydrogen
and air
Nitrogen,
phosphorus10 pg 106
Flame
photometric
(FPD)
Mass flow
Hydrogen
and air
possiblyoxygen
Sulphur,
phosphorus, tin,
boron, arsenic,
germanium,
selenium, chromium
100 pg 103
Photo-
ionization
(PID)
Concentrati
onMake-up
Aliphatics,
aromatics, ketones,
esters, aldehydes,
amines,
heterocyclics,
organosulphurs,
some
organometallics
2 pg 107
Hall
electrolytic
conductivity
Mass flowHydrogen,
oxygen
Halide, nitrogen,
nitrosamine, sulphur
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The effluent from the column is mixed with hydrogen and air, and ignited.
Organic compounds burning in the flame produce ions and electrons
which can conduct electricity through the flame. A large electrical
potential is applied at the burner tip, and a collector electrode is located
above the flame. The current resulting from the pyrolysis of any organic
compounds is measured. FIDs are mass sensitive rather than
concentration sensitive; this gives the advantage that changes in mobile
phase flow rate do not affect the detector's response. The FID is a useful
general detector for the analysis of organic compounds; it has high
sensitivity, a large linear response range, and low noise. It is also robust
and easy to use, but unfortunately, it destroys the sample.
Top of Form
Bottom of Form
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Data Recorder System
The data recorder plots the signal from
the detector over time. This plot is
called a chromatogram. The
retention time, which is when the
component elutes from the GC system,
is qualitatively indicative of the type
of compound. The data recorder also
has an integrator component to calculate the area under the peaks or the
height of the peak. The area or height is indicative of the amount of each
component.
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LIQUID CHROMATOGRAPHY
Liquid chromatography is an analytical chromatographic technique that isuseful for separating ions or molecules that are dissolved in a solvent. If
the sample solution is in contact with a second solid or liquid phase, the
different solutes will interact with the other phase to differing degrees due
to differences in adsorption, ion-exchange, partitioning, or size. These
differences allow the mixture components to be separated from each
other by using these differences to determine the transit time of the
solutes through a column.
Simple liquid chromatography consists of a column with a fritted bottom
that holds a stationary phase in equilibrium with a solvent. Typical
stationary phases (and their interactions with the solutes) are: solids
(adsorption), ionic groups on a resin (ion-exchange), liquids on an inert
solid support (partitioning), and porous inert particles (size-exclusion). The
mixture to be separated is loaded onto the top of the column followed by
more solvent. The different components in the sample mixture pass
through the column at different rates due to differences in their partioning
behavior between the mobile liquid phase and the stationary phase. The
compounds are separated by collecting aliquots of the column effuent as
a function of time.
Schematic of a simple liquid chromatographic separation
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Conventional Liquid chromatography is most commonly used in
preparative scale work to purify and isolate some components of a
mixture. It is also used in ultratrace separations where small disposable
columns are used once and then discarded. Analytical separations of
solutions for detection or quantification typically use more sophisticated
high-performance liquid chromatography instruments. HPLC instruments
use a pump to force the mobile phase through and provide higher
resolution and faster analysis time.
High-Performance Liquid Chromatography (HPLC)
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High-Performance Liquid Chromatographic System
High-performance liquid chromatography (HPLC) is a form of liquid
chromatography to separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile phase, a pump, an
injector, a separation column, and a detector. HPLC is used in drug
analysis, toxicology, explosives analysis, ink analysis, fibers, and plastics
to name a few forensic applications.
Schematic of an HPLC instrument:
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Like all chromatography, HPLC is based on selective partitioning of the
molecules of interest between two different phases. Here, the mobile
phase is a solvent or solvent mix that flows under high pressure over
beads coated with the solid stationary phase. While travelling through the
column, molecules in the sample partition selectively between the mobile
phase and the stationary phase. Those that interact more with the
stationary phase will lag behind those molecules that partition
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preferentially with the mobile phase. As a result, the sample introduced at
the front of the column will emerge in separate bands (called peaks), with
the bands emerging first being the components that interacted least with
the stationary phase and as a result moved quicker through the column.The components that emerge last will be the ones that interacted most
with the stationary phase and thus moved the slowest through the
column. A detector is placed at the end of the column to identify the
components that elute. Occasionally, the eluting solvent is collected at
specific times correlating to specific components. This provides a pure or
nearly pure sample of the component of interest. This technique is
sometimes referred to as preparative chromatography.
Many different types of detectors are available for HPLC. The simplest and
least expensive is the refractive index detector (RI). Although this detector
is a universal detector, meaning it will respond to any compound that
elutes, it does not respond well to very low concentrations and as a result
is not widely used. On the other hand, detectors based on the absorption
of light in the ultraviolet and visible ranges (UV/VIS detectors and UV/VIS
spectrophotometers) are the most commonly used, responding to a wide
variety of compounds of forensic interest with good to excellent
sensitivity. The photodiode array detector (PDA) is especially useful since
it can produce not only a peak-based output (a chromatogram) but also a
UV/VIS scan of every component. In many ways, the ideal detector for
HPLC is a mass spectrometer (MS), which provides both quantitative
information and in most cases a definitive identification of each
component (qualitative information). However, HPLC-MS systems are
relatively complex and expensive and are not readily available in all labs.
Other detectors that are sometimes used include fluorescence detectors
(which are very sensitive) and electrochemical detectors.
Unlike in gas chromatography (GC) in which the mobile phase is an inert
gas, the mobile phase in HPLC can be one of many different solvents or
combinations of solvents. This imparts to HPLC a greater flexibility and
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range of application than has GC. Because the sample does not have to be
converted to the gas phase, compounds such as explosives that break
down at high temperatures are much more amenable to HPLC than GC.
For HPLC, all that is required is that the sample be soluble in the solventsselected for the analysis. In addition, there are several types of HPLC
defined by the type of mobile phase and stationary phase that is used. For
forensic applications, one of the most commonly used types of HPLC is
referred to as reversed phase. In this type of HPLC, the mobile phase is
a solvent or mix of solvents that are polar, meaning that different parts
of the individual solvent molecules carry a partial positive or negative
charge. Water, methanol (methyl alcohol), ethanol (ethyl alcohol), and
acetone are examples of polar solvents. The stationary phase in reverse
phase HPLC is a non-polar material such as a long chain hydrocarbon
molecule. In this type of HPLC, components in the sample will partition
and separate based on their degree of interaction with the stationary
phase relative to the mobile phase. In other words, the separation is
based primarily on the relative polarity of the sample molecules. Reverse
phase HPLC is used in drug analysis (LSD for example), analysis of cutting
agents such as sugars, explosives, and gunshot residue (GSR), and
forensic toxicology.
Normal phase HPLC uses a polar stationary phase and a non-polar mobile
phase, but this is not widely used in forensic applications. Size exclusion
chromatography (SEC) is more common and separates compounds based
on relative sizes. The stationary phase in SEC is composed of a gel with
different sizes of microscopic pores through it. The larger the molecule,
the longer it takes for it to navigate through the pores and reach the
detector. SEC is useful for the analysis of large molecules that come in a
range of sizes such as plastic polymers, proteins, and nitrocellulose, a
component of GSR. Chiral chromatography, a relatively recent
development, is making inroads into forensic science since it is capable of
separating enantiomers, molecules that are mirror images of each other.
This capability is particularly valuable in forensic toxicology and drug
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analysis. Finally, ion exchange chromatography is available for detection
species such as nitrate (NO,3-) and other ions.
ION - EXCHANGE CHROMATOGRAPHY
The most popular method for the purification of proteins and other
charged molecules is ion exchange chromatography. It uses an ion
exchange mechanism to separate analytes based on their respective
charges. It is usually performed in columns but can also be useful in
planar mode. Ion exchange chromatography uses a charged stationary
phase to separate charged compounds including anions, cations, amino
acids, peptides, and proteins.
In cation exchange chromatography, positively charged molecules are
attracted to a negatively charged solid support. Conversely, in anion
exchange chromatography, negatively charged molecules are attracted to
a positively charged solid support. In conventional methods the
stationary phase is an ion exchange resin that carries charged functional
http://en.wikipedia.org/wiki/Anionhttp://en.wikipedia.org/wiki/Cationhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Peptidehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Ion_exchange_resinhttp://en.wikipedia.org/wiki/Functional_grouphttp://en.wikipedia.org/wiki/Anionhttp://en.wikipedia.org/wiki/Cationhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Peptidehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Ion_exchange_resinhttp://en.wikipedia.org/wiki/Functional_group8/3/2019 Laboratory Instrumentation Chromatography
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groups which interact with oppositely charged groups of the compound to
be retained.
A sample is introduced, either manually or with an autosampler, into asample loop of known volume. A buffered aqueous solution known as the
mobile phase carries the sample from the loop onto a column that
contains some form of stationary phase material. This is typically a resin
or gel matrix consisting of agarose or cellulose beads with covalently
bonded charged functional groups. The target analytes (anions or cations)
are retained on the stationary phase but can be eluted by increasing the
concentration of a similarly charged species that will displace the analyte
ions from the stationary phase.
http://en.wikipedia.org/wiki/Functional_grouphttp://en.wikipedia.org/w/index.php?title=Autosampler&action=edit&redlink=1http://en.wikipedia.org/wiki/Buffer_solutionhttp://en.wikipedia.org/wiki/Agarosehttp://en.wikipedia.org/wiki/Cellulosehttp://en.wikipedia.org/wiki/Covalent_bondhttp://en.wikipedia.org/wiki/Functional_grouphttp://en.wikipedia.org/w/index.php?title=Autosampler&action=edit&redlink=1http://en.wikipedia.org/wiki/Buffer_solutionhttp://en.wikipedia.org/wiki/Agarosehttp://en.wikipedia.org/wiki/Cellulosehttp://en.wikipedia.org/wiki/Covalent_bond8/3/2019 Laboratory Instrumentation Chromatography
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S IZE-EXCLUSION CHROMATOGRAPHY
Size-exclusion chromatography (SEC) is also known as gel permeation
chromatography (GPC) or gel filtration chromatography. It separates
molecules according to their size or more accurately according to their
hydrodynamic diameter or hydrodynamic volume. Smaller molecules are
able to enter the pores of the media and, therefore, molecules are
trapped and removed from the flow of the mobile phase. The average
residence time in the pores depends upon the effective size of the analyte
molecules. However, molecules that are larger than the average pore size
of the packing are excluded and thus suffer essentially no retention; such
species are the first to be eluted. It is generally a low-resolution
chromatography technique and thus it is often reserved for the final,
"polishing" step of purification. It is also useful for determining the tertiary
structure and quaternary structure of purified proteins, especially since it
can be carried out under native solution conditions.
http://en.wikipedia.org/wiki/Tertiary_structurehttp://en.wikipedia.org/wiki/Tertiary_structurehttp://en.wikipedia.org/wiki/Quaternary_structurehttp://en.wikipedia.org/wiki/Solutionhttp://en.wikipedia.org/wiki/Tertiary_structurehttp://en.wikipedia.org/wiki/Tertiary_structurehttp://en.wikipedia.org/wiki/Quaternary_structurehttp://en.wikipedia.org/wiki/Solution8/3/2019 Laboratory Instrumentation Chromatography
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Size-exclusion chromatography is a widely used technique for the
purification and analysis of synthetic and biological polymers, such as
proteins, polysaccharides and nucleic acids.
The advantages of this method include good separation of large molecules
from the small molecules with a minimal volume of elute, and that various
solutions can be applied without interfering with the filtration process, all
while preserving the biological activity of the particles to be separated.
With size exclusion chromatography, there are short and well-defined
separation times and narrow bands, which lead to good sensitivity. There
is also no sample loss because solutes do not interact with the stationary
phase. Disadvantages are that only a limited number of bands can be
accommodated because the time scale of the chromatogram is short, and,
in general, there has to be a 10% difference in molecular mass to have a
good resolution
http://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Polysaccharideshttp://en.wikipedia.org/wiki/Nucleic_acidshttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Polysaccharideshttp://en.wikipedia.org/wiki/Nucleic_acids8/3/2019 Laboratory Instrumentation Chromatography
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Exclusion chromatography separates molecules on the basis of size. A
column is filled with semi-solid beads of a polymeric gel that will admit
ions and small molecules (blue) into their interior but not large ones
(shown in red). When a mixture of molecules and ions dissolved in a
solvent is applied to the top of the column, the smaller molecules (and
ions) are distributed through a larger volume of solvent than is available
to the large molecules. Consequently, the large molecules move more
rapidly through the column, and in this way the mixture can be separated
(fractionated) into its components.
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