TERM PAPER

CHEMISTRY
Topic: Column Chromatography

Submitted to:
Dr Dwarika Prasad

Department of Chemistry

Date of submission: 11/11/10

Submitted
by:
Suhail Ahmad Khan

Section: B5001

Roll No: 61
 Registration No:
11010937
ACKNOWLEDGMENT:
First and foremost I thank my teacher who
assigned me this term paper to bring out
my creative capabilities and helped me a
lot to complete it.

I express my gratitude to my parents for

bringing a countinous source of
encouragement and for all the financial
aide they provided me.

Also would love to thank my friends for

helping me complete this assignment on
time.
 Suhail Ahmad Khan

Contents:
Definition
Procedure
System
Thin Layer Chromatography
Gas liquid Chromatography
High Performance Liquid Chromatography HPLC
Gas Chromatography-Mass Spectrometry
 Analysis
 Types of Ionization
Applications
References



 COLUMN
CHROMATOGRAPHY
Definition:
Column chromatography in chemistry is a method used to purify individual
chemical compounds from mixtures of compounds. It is often used for
preparative applications on scales from micrograms up to kilograms.

Procedure:
The classical Preparative
chromatography column is a
glass tube with a diameter
from 5 to 50 mm and a height
of 50 cm to 1 m with a tap at
the bottom. A slurry (a thick
suspension of solids in a
liquid) is prepared of the
eluant (mobile phase) with the
stationary phase powder and
then carefully poured into the
column. Care must be taken
to avoid air bubbles. A
solution of the organic
material is pipetted on top of
the stationary phase. This
layer is usually topped with a
small layer of sand or with
Column chromatography on a large scale in
cotton or glass wool to protect
the 1950s.
the shape of the organic layer
from the velocity of newly
added eluant. Eluant is slowly passed through the column to advance the
organic material. Often a spherical eluent reservoir or an eluent-filled and
stoppered separating funnel is put on top of the column.

The individual components are retained by the stationary phase differently and
separate from each other while they are running at different speeds through the
column with the eluant. At the end of the column they elute one at a time. During
the entire chromatography process the eluant is collected in a series of fractions.
The composition of the eluent flow can be monitored and each fraction is
analyzed for dissolved compounds, e.g. by analytical chromatography, UV
absorption, or fluorescence. Colored compounds can be seen through the glass
wall as moving bands.

Stationary Phase (Adsorbent)

The stationary phase or adsorbent in column chromatography is a solid. The

most common stationary phase for column chromatography is silica gel, followed
by alumina. Cellulose powder has often been used in the past. Also possible are
ion exchange chromatography, reversed-phase chromatography (RP), affinity
chromatography or expanded bed adsorption (EBA). The stationary phases are
usually finely ground powders or gels and/or are microporous for an increased
surface, though in EBA a fluidized bed is used.

Mobile Phase (Eluent)

The mobile phase or eluent is either a pure solvent or a mixture of different

solvents. It is chosen so that the retention factor value of the compound of
interest is roughly around 0.75 in order to minimize the time and the amount of
eluent to run the chromatography. The eluent has also been chosen so that the
different compounds can be separated effectively. The eluent is optimized in
small scale pretests, often using thin layer chromatography (TLC) with the same
stationary phase.

A faster flow rate of the eluent minimizes the time required to run a
column and thereby minimizes diffusion, resulting in a better separation. 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. Better flow rates can be
achieved by using a pump or by using compressed gas (e.g. air, nitrogen) to
push the solvent through the column (flash column chromatography)

Systems

Automated flash chromatography systems attempt to minimize human

involvement in the purification process. Automated systems may include
components normally found on HPLC systems (gradient pump, sample injection
apparatus, UV detector) and a fraction collector to collect the eluent.

The software controlling an automated system will coordinate the components

and help the user to find the resulting purified material within the fraction
collector. The software will also store results from the process for archival or later
recall purposes.

A representative example of column chromatography as part of an

undergraduate laboratory exercise is the separation of three components (out of
28) in the oil of spearmint: carvone, limonene and dehydrocarveol. A microscale
setup consisting of a Pasteur pipette as column with silica gel stationary phase
can suffice. The starting eluent is hexane and solvent polarity is increased during
the process by adding ethyl acetate.
THIN LAYER CHROMATOGRAPHY

Thin layer chromatography (TLC) is a

chromatography technique used to separate
mixtures. It involves a stationary phase
consisting of a thin layer of adsorbent
material, usually silica gel, aluminium oxide,
or cellulose immobilized onto a flat, inert
carrier sheet. A liquid phase consisting of
the solution to be separated is then
dissolved in an appropriate solvent and is
drawn up the plate viacapillary action,
separating the experimental solution based
on the polarity of the components of the
compound in question.

Its wide range of uses include;

Separation of black ink on a TLC plate
o assaying radiochemical purity
of radiopharmaceuticals
o determination of the pigments a plant contains
o detection of pesticides or insecticides in food
o analysing the dye composition of fibers in forensics, or
o identifying compounds present in a given substance
o monitoring organic reactions

number of enhancements can be made to the original method to automate

some steps, to increase the resolution achieved with TLC and to allow
more accurate quantitation. This method is referred to as HPTLC, or "High
Performance Tlc".

Plate Preparation

TLC plates are made by mixing the adsorbent, such as silica gel, with a small
amount of inert binder like calcium sulfate (gypsum) and water. This mixture is
spread as a thick slurry on an unreactive carrier sheet, usually glass, thick
aluminum foil, or plastic, and the resultant plate is dried and activated by heating
in an oven for thirty minutes at 110 °C. The thickness of the adsorbent layer is
typically around 0.1–0.25 mm for analytical purposes and around 1–2 mm for
preparative TLC. Every type of chromatography contains a mobile phase and a
stationary phase.

Technique

The process is similar to paper chromatography with the advantage of

faster runs, better separations, and the choice between different stationary
phases. Because of its simplicity and speed TLC is often used for
monitoring chemical reactions and for the qualitative analysis of reaction
products. A small spot of solution containing the sample is applied to a
plate, about one centimeter from the base.

The plate is then dipped in to a suitable solvent, such as ethanol or water,

and placed in a sealed container. The solvent moves up the plate by
capillary action and meets the sample mixture, which is dissolved and is
carried up the plate by the solvent. Different compounds in the sample
mixture travel at different rates due to the differences in their attraction to
the stationary phase, and because of differences in solubility in the solvent.

Separation of compounds is based on the competition of the solute and the

mobile phase for binding places on the stationary phase. For instance, if
normal phase silica gel is used as the stationary phase it can be
considered polar. Given two compounds which differ in polarity, the most
polar compound has a stronger interaction with the silica and is therefore
more capable to dispel the mobile phase from the binding places.
Consequently, the less polar compound moves higher up the plate
(resulting in a higher Rf value).

If the mobile phase is changed to a more polar solvent or

mixture of solvents, it is more capable of dispelling solutes from the silica
binding places and all compounds on the TLC plate will move higher up the
plate. Practically this means that if you use a mixture of ethyl acetate and
heptane as the mobile phase, adding more ethyl acetate results in higher
Rf values for all compounds on the TLC plate. Changing the polarity of the
mobile phase will not result in reversed order of running of the compounds
on the TLC plate. If a reversed order of running of the compounds is
desired, an apolar stationary phase should be used, such as C18-
functionalized silica.
 The appropriate solvent in context of Thin layer
chromatography will be one which differs from the stationary phase
material in polarity. If polar solvent is used to dissolve the sample and spot
is applied over polar stationary phase TLC, the sample spot will grow
radially due to capillary action, which is not advisable as one spot may mix
with the other. Hence, to restrict the radial growth of sample-spot, the
solvent used for dissolving samples in order to apply them on plates should
be as non-polar or semi-polar as possible when the stationary phase is
polar, and vice-versa.

Analysis

As the chemicals being separated may be colorless, several methods

exist to visualize the spots:

o Often a small amount of a fluorescent compound, usually

manganese-activated zinc silicate, is added to the adsorbent
that allows the visualization of spots under a blacklight (UV254).
The adsorbent layer will thus fluoresce light green by itself, but
spots of analyte quench this fluorescence.
o Iodine vapors are a general unspecific color reagent
o Specific color reagents exist into which the TLC plate is dipped
or which are sprayed onto the plate
o Once visible, the Rf value , or Retention factor, of each spot can
be determined by dividing the distance traveled by the product
by the total distance traveled
by the solvent (the solvent
front). These values depend
on the solvent used, and the
type of TLC plate, and are not
physical constants.

Gas -Liquid
Chromatography

Gas-liquid chromatography (GLC), or

simply gas chromatography (GC), is a
type of chromatography in which the
mobile phase is a carrier gas, usually an
inert gas such as helium or an unreactive

Gas Liquid Chromatography

gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or
polymer on an inert solid support, inside glass or metal tubing, called a column.
The instrument used to perform gas chromatographic separations is called a gas
chromatograph.

Gas Chromatography is different from other forms of chromatography because

the solutions travel through the column in a gas state. The interactions of these
gaseous analytes with the walls of the column causes different compounds to
elute at different times called retention time. The comparison of these retention
times is the analytical power of GC. This makes it very similar to HPLC.

GC Analysis

A gas chromatograph is a chemical analysis instrument for separating

chemicals in a complex sample. A gas chromatograph uses a flow-through
narrow tube known as the column, through which different chemical
constituents of a sample pass in a gas stream (carrier gas, mobile phase)
at different rates depending on their various chemical and physical
properties and their interaction with a specific column filling, called the
stationary phase. As the chemicals exit the end of the column, they are
detected and identified electronically. The function of the stationary phase
in the column is to separate different components, causing each one to exit
the column at a different time (retention time). Other parameters that can
be used to alter the order or time of retention are the carrier gas flow rate,
and the temperature.

In a GC analysis, a known volume of gaseous or liquid analyte is injected

into the "entrance" (head) of the column, usually using a microsyringe (or,
solid phase microextraction fibers, or a gas source switching system). As
the carrier gas sweeps the analyte molecules through the column, this
motion is inhibited by the adsorption of the analyte molecules either onto
the column walls or onto packing materials in the column. The rate at which
the molecules progress along the column depends on the strength of
adsorption, which in turn depends on the type of molecule and on the
stationary phase materials.
Since each type of molecule has a different rate of progression, the various
components of the analyte mixture are separated as they progress along
the column and reach the end of the column at different times (retention
time). A detector is used to monitor the outlet stream from the column; thus,
the time at which each component reaches the outlet and the amount of
that component can be determined. Generally, substances are identified
(qualitatively) by the order in which they emerge (elute) from the column
and by the retention time of the analyte in the column.

Data Reduction And Analysis

Qualitative analysis: Generally chromatographic data is presented as a graph

of detector response (y-axis) against retention time (x-axis). This provides a
spectrum of peaks for a sample representing the analytes present in a sample
eluting from the column at different times. Retention time can be used to identify
analytes if the method conditions are constant. Also, the pattern of peaks will be
constant for a sample under constant conditions and can identify complex
mixtures of analytes. In most modern applications however the GC is connected
to a mass spectrometer or similar detector that is capable of identifying the
analytes represented by the peaks.

Quantitive analysis: The area under a peak is proportional to the amount of

analyte present. By calculating the area of the peak using the mathematical
function of integration, the concentration of an analyte in the original sample can
be determined. Concentration can be calculated using a calibration curve created
by finding the response for a series of concentrations of analyte, or by
determining the relative response factor of an analyte. The relative response
factor is the expected ratio of an analyte to an internal standard (or external
standard) and is calculated by finding the response of a known amount of analyte
and a constant amount of internal standard (a chemical added to the sample at a
constant concentration, with a distinct retention time to the analyte).

Applications

In general, substances that vaporize below ca. 300 °C (and therefore

are stable up to that temperature) can be measured quantitatively.
 The samples are also required to be salt-free; they should not contain
ions. Very minute amounts of a substance can be measured, but it is
often required that the sample must be measured in comparison to a
sample containing the pure, suspected substance.

Various temperature programs can be used to make the readings

more meaningful; for example to differentiate between substances
that behave similarly during the GC process.

Professionals working with GC analyze the content of a chemical

product, for example in assuring the quality of products in the
chemical industry; or measuring toxic substances in soil, air or water.
GC is very accurate if used properly and can measure picomoles of a
substance in a 1 ml liquid sample, or parts-per-billion concentrations
in gaseous samples

High Performance Liquid Chromatography (HPLC):

High-performance liquid chromatography (or High pressure liquid

chromatography, HPLC) is a form of column chromatography used frequently in
biochemistry and analytical chemistry to separate, identify, and quantify
compounds. HPLC utilizes a column that holds chromatographic packing material
(stationary phase), a pump that moves the mobile phase(s) through the column,
and a detector that shows the retention times of the molecules. Retention time
varies depending on the interactions between the stationary phase, the
molecules being analyzed, and the solvent(s) used.
Operation

The sample to be analyzed is introduced in small volume to the stream of mobile

phase and is retarded by specific chemical or physical interactions with the
stationary phase as it traverses the length of the column. The amount of
retardation depends on the nature of the analyte, stationary phase and mobile
phase composition. The time at which a specific analyte elutes (comes out of the
end of the column) is called the retention time and is considered a reasonably
unique identifying characteristic of a given analyte.

The use of pressure increases the linear velocity (speed) giving the
components less time to diffuse within the column, leading to improved resolution
in the resulting chromatogram. Common solvents used include any miscible
combinations of water or various organic liquids (the most common are methanol
). Water may contain buffers or salts to assist in the separation of the analyte
components, or compounds such as trifluoroacetic acid which acts as an ion
pairing agent.

A further refinement to HPLC has been to vary the mobile phase composition
during the analysis; this is known as gradient elution. A normal gradient for
reversed phase chromatography might start at 5% methanol and progress
linearly to 50% methanol over 25 minutes, depending on how hydrophobic the
analyte is. The gradient separates the analyte mixtures as a function of the
affinity of the analyte for the current mobile phase composition relative to the
stationary phase.

This partitioning process is similar to that which occurs during a liquid-liquid

extraction but is continuous, not step-wise. In this example, using a
water/methanol gradient, the more hydrophobic components will elute (come off
the column) under conditions of relatively high methanol (which is hydrophobic);
whereas the more hydrophilic compounds will elute under conditions of relatively
low methanol/high water. The choice of solvents, additives and gradient depend
on the nature of the stationary phase and the analyte. Often a series of tests are
performed on the analyte and a number of generic runs may be processed in
order to find the HPLC method which gives the best separation of peaks.

Types of HPLC
 o Normal phase chromatography
o Reversed phase chromatography
o Size exclusion chromatography
o Ion exchange chromatography
o Bioaffinity chromatography
o Isocratic flow and gradient elution

(A composition of the mobile phase that remains constant throughout the

procedure is termed Isocratic.)

PARAMETERS OF HPLC

Internal diameter: The internal diameter (ID) of an HPLC column is a critical

aspect that influences sensitivity and determines the quantity of analyte that can
be loaded onto the column. Larger columns are usually seen in industrial
applications such as the purification of a drug product for later use. Low ID
columns have improved sensitivity and lower solvent consumption at the
expense of loading capacity.

Particle size: Most traditional HPLC is performed with the stationary phase
attached to the outside of small spherical silica particles (very small beads).
These particles come in a variety of sizes with 5 μm beads being the most
common. Smaller particles generally provide more surface area and better
separations, but the pressure required for optimum linear velocity increases by
the inverse of the particle diameter squared.This means that changing to
particles that are half as big, keeping the size of the column the same, will double
the performance, but increase the required pressure by a factor of four.

Pore size: Many stationary phases are porous to provide greater surface area.
Small pores provide greater surface area while larger pore size has better
kinetics, especially for larger analytes. For example a protein which is only
slightly smaller than a pore might enter the pore but not easily leave once inside.

Pump pressure:

Pumps vary in pressure capacity, but their performance is measured on their

ability to yield a consistent and reproducible flow rate. Pressure may reach as
high as 40 MPa , or about 400 atmospheres. Modern HPLC systems have been
improved to work at much higher pressures, and therefore are able to use much
smaller particle sizes in the columns (<2 μm). These "Ultra High Performance
Liquid Chromatography" systems or UHPLCs can work at up to 100 MPa
(15,000 lbf/in²), or about 1000 atmospheres. The term "UPLC", though
sometimes used is a trademark of Waters Corporation and not the name for the
technique in general.

Gas Chromatography-Mass Spectrometry:

Gas chromatography-mass spectrometry (GC-MS) is a method that combines
the features of gas-liquid chromatography and mass spectrometry to identify
different substances within a test sample. Applications of GC-MS include drug
detection, fire investigation, environmental analysis, explosives investigation, and
identification of unknown samples. GC/MS can also be used in airport security to
detect substances in luggage or on human beings. Additionally, it can identify
trace elements in materials that were previously thought to have disintegrated
beyond identification.

The GC-MS has been widely heralded as a "gold standard" for forensic
substance identification because it is used to perform a specific test. A specific
test positively identifies the actual presence of a particular substance in a given
sample. A non-specific test, however, merely indicates that a substance falls
into a category of substances. Although a non-specific test could statistically
suggest the identity of the substance, this could lead to false positive
identification.

INSTRUMENTATION

The GC-MS is composed of two major building blocks: the gas chromatograph
and the mass spectrometer. The gas chromatograph utilizes a capillary column
which depends on the column's dimensions (length, diameter, film thickness) as
well as the phase properties (e.g. 5% phenyl polysiloxane). The difference in the
chemical properties between different molecules in a mixture will separate the
molecules as the sample travels the length of the column. The molecules take
different amounts of time (called the retention time) to come out of (elute from)
the gas chromatograph, and this allows the mass spectrometer downstream to
capture, ionize, accelerate, deflect, and detect the ionized molecules separately.
The mass spectrometer does this by breaking each molecule into ionized
fragments and detecting these fragments using their mass to charge ratio.

These two components, used together, allow a much finer degree of

substance identification than either unit used separately. It is not possible to
make an accurate identification of a particular molecule by gas chromatography
or mass spectrometry alone. The mass spectrometry process normally requires a
very pure sample while gas chromatography using a traditional detector (e.g.
Flame Ionization Detector) detects multiple molecules that happen to take the
same amount of time to travel through the column (i.e. have the same retention
time) which results in two or more molecules to co-elute.

Sometimes two different molecules can also have a similar pattern of

ionized fragments in a mass spectrometer (mass spectrum). Combining the two
processes makes it extremely unlikely that two different molecules will behave in
the same way in both a gas chromatograph and a mass spectrometer. Therefore
when an identifying mass spectrum appears at a characteristic retention time in a
GC-MS analysis, it typically lends to increased certainty that the analyte of
interest is in the sample

ANALYSIS:
A mass spectrometer is typically utilized in one of two ways: Full
Scan or Selective Ion Monitoring (SIM). The typical GC/MS
instrument is capable of performing both functions either individually
or concomitantly, depending on the setup of the particular instrument.

Full Scan MS

When collecting data in the full scan mode, a target range of mass fragments is
determined and inputed into the instrument's method. An example of a typical
broad range of mass fragments to monitor would be m/z 50 to m/z 400. The
determination of what range to use is largely dictated by what one anticipates in
being in the sample while being cognizant of the solvent and other possible
interferences. A MS should not be set to look for mass fragments too low or else
one may detect air (found as m/z 28 due to nitrogen), carbon dioxide (m/z 44) or
other possible interferences. Additionally if one is to use a very large scan range
then sensitivity of the instrument is decreased due to performing fewer scans per
second since each scan will have to detect a wide range of mass fragments.

Full Scan is useful in determining unknown compounds in a sample.

It provides more information than SIM when it comes to confirming or resolving
compounds in a sample. During instrument method development it may be
common to first analyze test solutions in full scan mode to determine the
retention time and the mass fragment fingerprint before moving to a SIM
instrument method.

Selective Ion Monitoring

In Selective Ion Monitoring (SIM) certain ion fragments are entered into the
instrument method and only those mass fragments are detected by the mass
spectrometer. The advantages of SIM are that the detection limit is lower since
the instrument is only looking at a small number of fragments (e.g. three
fragments) during each scan. More scans can take place each second. Since
only a few mass fragments of interest are being monitored, matrix interferences
are typically lower. To additionally confirm the likelihood of a potentially positive
result, it is relatively important to be sure that the ion ratios of the various mass
fragments are comparable to a known reference standard.

Types of Ionization:
After the molecules travel the length of the column, pass through the transfer line
and enter into the mass spectrometer they are ionized by various methods with
typically only one method being used as any given time. Once the sample is
fragmented it will then be detected, usually by an electron multiplier diode, which
essentially turns the ionized mass fragment into an electrical signal that is then
detected.

The ionization technique chosen is independent of using Full Scan or

SIM.

Electron Ionization

By far the most common and perhaps standard form of ionization is electron
ionization (EI). The molecules enter into the MS (the source is a quadrupole or
the ion trap itself in an ion trap MS) where they are bombarded with free
electrons emitted from a filament, not much unlike the filament one would find in
a standard light bulb. The electrons bombard the molecules causing a hard
ionization that fragments the molecule, and the way in which a molecule
fragment is usually typical for all EI techniques.

Chemical Ionization

In chemical ionization a reagent gas, typically methane or ammonia is introduced

into the mass spectrometer. Depending on the technique (positive CI or negative
CI) chosen, this reagent gas will interact with the electrons and analyte and
cause a 'soft' ionization of the molecule of interest. A softer ionization fragments
the molecule to a lower degree than the hard ionization of EI. One of the main
benefits of using chemical ionization is that a mass fragment closely
corresponding to the molecular weight of the analyte of interest is produced.

Positive Chemical Ionization

In Positive Chemical Ionization (PCI) the reagent gas interacts with the target
molecule, most often with a proton exchange. This produces the species in
relatively high amounts.

Negative Chemical Ionization

In Negative Chemical Ionization (NCI) the reagent gas decreases the impact of
the free electrons on the target analyte. This decreased energy typically leaves
the fragment in great supply.

APPLICATIONS:
Law Enforcement

GC-MS is increasingly used for detection of illegal narcotics, and may eventually
supplant drug-sniffing dogs. It is also commonly used in forensic toxicology to
find drugs and/or poisons in biological specimens of suspects, victims, or the
deceased.
Medicine

In combination with isotopic labeling of metabolic compounds, the GC-MS is

used for determining metabolic activity. Most applications are based on the use
of 13C as the labeling and the measurement of 13C/12C ratios with an isotope
ratio mass spectrometer (IRMS); an MS with a detector designed to measure a
few select ions and return values as ratios.

Criminal Forensics

GC-MS can analyze the particles from a human body in order to help link a
criminal to a crime. The analysis of fire debris using GC-MS is well established,
and there is even an established American Society for Testing Materials (ASTM)
standard for fire debris analysis. GCMS/MS is especially useful here as samples
often contain very complex matrices and results, used in court, need to be highly
accurate.
References:

i. http://orgchem.colorado.edu/hndbksupport/colchrom/colchrom.ht
ml
ii. http://www.chem.ubc.ca/courseware/121/tutorials/exp3A/columnc
hrom/
iii. http://en.wikipedia.org/wiki/Column_chromatography
iv. http://www.chemguide.co.uk/analysis/chromatography/column.ht
ml
v. http://www.absoluteastronomy.com/topics/Column_chromatograp
hy
vi. http://www.wfu.edu/chemistry/courses/organic/CC/index.htm