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The Chromatographic technique that being used is Gas chromatography (GC) and High
performance liquid chromatography (HPLC). Chromatography which is a Latins means colored
drawing was first used by Russia botanist, Mikhail Tswett in 1906 to describe the separation that
occurred when solutions of plant pigment pass through column of calcium carbonate/alumina
using petroleum ether.
Tswett conduct it by filled an open glass column with particles. Two specific materials
that he found useful were calcium carbonate and alumina. By pouring his sample into the
column, allowed it to pass into the column that the particle was placed and followed by pouring
pure solvent. As the sample passed down through the column by gravity, different colored bands
could be seen separating because some components were moving faster than others. He related
these separated, different-colored bands to the different compounds that were originally
contained in the sample. He had created an analytical separation of these compounds based on
the differing strength of each compounds chemical attraction to the particles. The compounds
that were more strongly attracted to the particles will slow down, while other compounds more
strongly attracted to the solvent moved faster. This process can be described as follows: the
compounds contained in the sample distribute, or partition differently between the moving
solvent, called the mobile phase, and the particles, called the stationary phase. This causes each
compound to move at a different speed, thus creating a separation of the compounds.
In 1941, Martin and Synge predicted that liquid partition chromatography mobile phase
can also be vapor not only liquid. In 1951, Martin and his co-worker A.T. James bring the
concept into reality practical when they published their paper describing the first GC and
demonstrated the technique by separating and quantitatively determining the component of C1C12 fatty acid mixture. Due to that, the importance of GC was recognized almost immediately
by petrochemical laboratory, which faced the challenge of analyzing complex hydrocarbon
mixture.
GC is conduct by placing a small amount of the sample into a hot injector port using a
syringe. The injector is set to a temperature higher than the components boiling points. So,
components of the mixture evaporate into the gas phase inside the injector. A carrier gas, such
as helium (inert gas), flows through the injector and push the gas components of the sample onto
the GC column. It is within the column that separation of the components takes
place. Molecules partition between the carrier gas (mobile phase) and the high boiling liquid
(the stationary phase) within the GC column. After components of the mixture move through the
GC column, they reach a detector. Ideally, components of the mixture will reach the detector at
varying times due to differences in the partitioning between mobile and stationary phases. The
detector sends a signal to the chart recorder which results in a peak on the chart paper. The area
of the peak is proportional to the number of molecules generating the signal. That is basically
how HPLC and GC was conducted.
Uses of HPLC
This technique is used for chemistry and biochemistry research analyzing complex
mixtures, purifying chemical compounds, developing processes for synthesizing chemical
compounds, isolating natural products, or predicting physical properties. It is also used in quality
control to ensure the purity of raw materials, to control and improve process yields, to quantify
assays of final products, or to evaluate product stability and monitor degradation.
In addition, it is used for analyzing air and water pollutants, for monitoring materials that
may jeopardize occupational safety or health, and for monitoring pesticide levels in the
environment. Federal and state regulatory agencies use HPLC to survey food and drug products,
for identifying confiscated narcotics or to check for adherence to label claims.
Principle of HPLC
The components of a basic high-performance liquid chromatography [HPLC] system are shown
in the simple diagram in figure.
Column
The column is one of the most important components of the HPLC chromatograph
because the separation of the sample components is achieved when those components pass
through the column. The High performance liquid chromatography apparatus is made out of
stainless steel tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30cm.
Normally, columns are filled with silica gel because its particle shape, surface properties, and
pore structure help to get a good separation. Silica is wetted by nearly every potential mobile
phase, is inert to most compounds and has a high surface activity which can be modified easily
with water and other agents. Silica can be used to separate a wide variety of chemical
compounds, and its chromatographic behavior is generally predictable and reproducible.
Pump
The role of the pump is to propel (force) a liquid (the mobile phase) through the chromatograph
at a specific flow rate, expressed in ml/min. Normal flow rates in HPLC are 1-2 ml/min and
typical pumps can reach pressures in the range of 2000 9000 psi but in applications covered
under UHPLC mode operating pressure can be as high as 15000-18000 psi. The normal flow rate
stability must be < 1%.
An ideal pump should have the following characteristics:
The main types of pumps used in HPLC (or in LC) are the following:
Constant Pressure Pumps: Provide consistent continuous flow rate through the column with
the use of pressure from a gas cylinder.
Constant Flow Pumps: a) Reciprocating Piston pumps deliver solvents through reciprocating
motion of a piston in a hydraulic chamber. The main drawback of a reciprocating pump is that it
produces a pulsing flow. With a flow-sensitive detector, such as micro-adsorption detector, a
pulse damping system must be used and detector sensitivity is reduced. |
b) Syringe type pumps are suitable for small bore columns. Constant flow rate is delivered to
column by a motorized screw arrangement.
The pump should be inert to solvents, buffer salts and solutes. They are made of stainless steel,
titanium and resistant minerals.
Function of GC
Gas chromatography - specifically gas-liquid chromatography - involves a sample being
vaporized and injected onto the head of the chromatographic column. The sample is transported
through the column by the flow of inert, gaseous mobile phase. The column itself contains a
liquid stationary phase which is adsorbed onto the surface of an inert solid.
Typical uses of GC include testing the purity of a particular substance, or separating the different
components of a mixture (the relative amounts of such components can also be determined). In
some situations, GC may help in identifying a compound. In preparative chromatography, GC
can be used to prepare pure compounds from a mixture.
In gas chromatography, the mobile phase (or "moving phase") is a carrier gas, usually
an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a
microscopic layer of liquid or polymer on an inert solid support, inside a piece of
glass or metal tubing called a column (a homage to the fractionating column used in distillation).
The instrument used to perform gas chromatography is called a gas chromatograph (or
"aerograph", "gas separator").
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;
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
There are two general types of column, packed and 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.
Column temperature
For precise work, column temperature must be controlled to within tenths of a degree.
The optimum column temperature is dependant 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.
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, aselective detector responds to a range of compounds with a common
physical or chemical property and a specific detector responds to a single chemical compound.
Detectors can also be grouped intoconcentration 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.
Sample Preparation
Fundamental theory
The sample should be dissolved in the eluent to be used and filtrated with 0.45m
disposable filter. Then, it should be injected into HPLC/GC system about 10 to 50L. When the
sample is dissolved in the eluent, good separation can be expected even for the sample whose
peaks appear near Vo because the system peak will not appear near Vo. (When the column size is
4.6mmI.D. x 150 to 250mm length). Avoid overloading, not too much sample should not be
injected. If peak shape changes by diluting the sample 10 to 100 folds or by decreasing the
injection volume to one fifth, there is a possibility of sample overloading.
The injection volume of standards and that of actual sample should be the same. If they
are different, retention times may be different. Some troubles may happen when the actual
sample is analyzed though the troubles never happen when standards are analyzed. In most cases,
they are caused by inappropriate selection of the solvent in which the sample is dissolved.
Troubles
(a) Leading or tailing of peaks.
(b) Instability of retention time.
(c) Split of peak top.
(d) Very broad ghost peak.
(e) Increase of column pressure.
(f) The above troubles happen just for a specific sample (or peak).
Normal phase
Consider the situation that 15% dichloromethane/hexsane is used as the eluent and sample is
easy to be dissolved in dichloromethane. The best way is to dissolve the sample in the eluent.
If the sample cannot be dissolved in the eluent and 100% dichloromethane is selected as the
solvent in which sample is dissolved, some trouble may happen.
In reversed phase or normal phase chromatography, usually, a mixture of some solvents is used
as the eluent. The solvent in which the sample dissolves more easily advances the elution time.
Before selecting a solvent in which sample is dissolved, it is recommended to change the eluent
composition by 5 to 10% and check the elution time. Of course, it is neccesssary that the solvent
can be dissolved in the eluent.
1974, Edward Randall made a major improvement in the extraction technique that
reduced the extraction time dramatically. In his method, the sample was totally immersed in the
boiling solvent. Compared to the classical method where the condensed extracting solvents
temperature is slightly below the boiling point, the Randall method was faster because analytes
are more soluble in hot solvent than in warm solvent. First, the thimble is lowered into the
boiling solvent until the appropriate extraction takes place. Then, to flush residual extract from
the sample, a rinse step follows. In this second stage, the thimble is raised above the boiling
solvent for a period of time until residual extract is
removed from the solid material by the condensed
solvent, just as is performed in the original method.
Finally, the drying step removes the solvent from the
solvent flask and concentrates the analytes for further
processing. In this step, by closing a solvent return
valve, the condensed solvent is redirected away from
the sample and boiling solvent and collected in a
reservoir for possible re-use or disposal. In some
systems, there is a fourth step where the sample cup
is lifted from the heat source and allowed to
evaporate further without the chance of sample
overheating, boiling dry, or potential oxidation.
samples for introduction into the gas chromatographic instrument and occasionally into HPLC
systems.
A sample preparation procedure would be a modification or treatment of the sample prior
to sample introduction for the purpose of improving the introduction process into the GC. For
example, performing a dynamic headspace process with an intermediate cold adsorbent trap to
concentrate the analytes of interest prior to sample introduction into the GC would constitute a
sample preparation procedure. Derivatizing an adsorbed polar compound on a solid material,
thereby releasing it into vapor phase for subsequent sampling, would be a sample preparation
process. Whatever you call it, it is most important that the components of interest be transferred
from the point of collection to the GC column without being lost or modified.
Headspace Technique
Headspace refers to the vapors that form over liquids and solids. If the sample is in
thermodynamic equilibrium with the gas phase in a closed thermostatic vessel, this method of
analysis is referred to static headspace sampling. If an inert gas is passed through or over the
sample and the stripped sample volatiles accumulated in an adsorbent trap or cryogenic trap, then
the method is referred to as dynamic headspace or purge and trap. Since only the volatiles are
sampled, headspace analysis is ideal for dirty samples, solid materials, samples with high boilers
of no interest, samples with high water content, or samples that are difficult to handle by
conventional chromatographic methods. These matrices remain behind since only the volatile
portion of the sample is in the headspace. In headspace sampling, calibrations are performed by
preparing a solution of the analytes in a volatile solvent then injecting a small known amount
into the closed headspace container so that the entire solution is vaporized.
Dynamic headspace sampling continuously removes headspace vapors above a liquid or
solid sample. The flow of gas over the sample (purging) will further volatilize analytes that can
be trapped by an adsorbent or by cryogenic means. The trapping process will refocus
(concentrate) the volatiles which are then re-volatilized into the gas chromatograph by thermal
desorption. The dynamic process of purge and trap is particularly useful for analytes that are too
low in concentration to be measured by static headspace methods. Purge and trap, or gas phase
stripping, generally refers to the process where urging gas is bubbled below the surface of a
liquid sample using a fritted orifice to produce finely dispersed bubbles.
The proper selection of a trap is an important consideration in ensuring that the desired
analytes are quantitatively recovered. An adsorbent trap (typically a glass tube) is filled with
porous sorbent material (typically 50 mg to one gram) that could be a polymer, carbon, silica gel,
or alumina. Polymeric materials sometimes need to be cleaned prior to use in order to remove
residual monomer. Tenax is especially useful since it is hydrophobic, water is un-retained, and it
has a high sample capacity. Carbon materials are excellent, high capacity traps, but sometimes
irreversibly adsorb certain classes of analytes. Silica gel and alumina have high capacity but take
up water. These traps containing adsorbent may also be used off-line to collect volatile organic
samples from air and then transported back to the laboratory for subsequent analysis. As
mentioned above, analytes are transferred to the GC by thermal desorption, perhaps coupled with
cold trapping via cryogenics to refocus the analytes. An especially important parameter of a
particular trap is the breakthrough capacity, which determines how much organic material is
trapped from the gaseous sample before it elutes from the opposite end of the trap and is no
longer retained. Volatile organics may pass through a trap very quickly while semi- or nonvolatile organics may adsorb strongly and never elute. For volatile organics, the trap may be
cryogenically cooled or a different adsorbent material may be used that has a higher
breakthrough volume for the analytes.