2. Liquid Chromatography
• LC is one of the most popular separation techniques used in labs for the
separation of a sample mixture on the basis of the interactions between the
individual molecules in the sample with the aforementioned stationary and
mobile phases. It can be carried out either in a column or on a sheet with a liquid
mobile phase and solid support as the stationary phase.
• The mobile phase travels down the stationary phase bringing along the
components of the sample separated during chromatography. In LC, the
interaction between sample molecules and the chromatography medium may be
based on several factors such as size, charge, affinity binding, or hydrophobicity.
• An advanced form of the LC technique that uses high pressure to force sample
through the column is called high performance liquid chromatography or high
pressure liquid chromatography (HPLC). It is currently the most extensively
used method of quantitative analysis in pharmaceutical analysis laboratories and
in the pharmaceutical industry as a whole.
3. LC, as used in the compendia, is synonymous with HPLC (both high-
pressure and high-performance). LC is a separation technique based on
a solid stationary phase and a liquid mobile phase.
Stationary phase Separations are achieved by partition, adsorption, or
ion-exchange processes, depending on the type of stationary phase
used. The most commonly used stationary phases are modified silica
or polymeric beads. The beads are modified by the addition of long-
chain hydrocarbons. The specific type of packing needed to complete
an analysis is indicated by the “L” designation in the individual
monograph (see also Chromatographic Columns). The size of the
beads is often described in the monograph as well. Changes in the
packing type and size are covered in System Suitability in this chapter.
Chromatographic column The term “column” includes stainless steel,
lined stainless steel, and polymeric columns, packed with a stationary
phase. The length and inner diameter of the column affects the
separation, and therefore typical column dimensions are included in
the individual monograph. Changes to column dimensions are
discussed in System Suitability.
4. Mobile phase The mobile phase is a solvent or a mixture of solvents, as defined
in the individual monograph.
Apparatus A liquid chromatograph consists of a reservoir containing the mobile
phase, a pump to force the mobile phase through the system at high pressure, an
injector to introduce the sample into the mobile phase, a chromatographic
column, a detector, and a data collection device.
Gradient elution The technique of continuously changing the solvent
composition during the chromatographic run is called gradient elution or
solvent programming. The gradient elution profile is presented in the individual
monograph as a gradient table, which lists the time and proportional
composition of the mobile phase at the stated time.
Procedure
1. Equilibrate the column and detector with mobile phase at the specified flow rate
until a constant signal is received.
2. Inject a sample through the injector, or use an autosampler.
3. Begin the gradient program.
4. Record the chromatogram.
5. Analyze as directed in the monograph
5. High performance liquid chromatography
(HPLC)
• High-performance liquid chromatography or commonly known as
HPLC, is an analytical technique used to separate, identify or
quantify each component in a mixture.
• The mixture is separated using the basic principle of
column chromatography and then identified and quantified by
spectroscopy.
• In the 1960s, the column chromatography LC with its low-pressure
suitable glass columns was further developed to the HPLC with its
high-pressure adapted metal columns.
• HPLC is thus basically a highly improved form of column liquid
chromatography. Instead of a solvent being allowed to drip
through a column under gravity, it is forced through under high
pressures of up to 400 atmospheres.
6. Principle
• The purification takes place in a separation column between a stationary and a
mobile phase.
• The stationary phase is a granular material with very small porous particles in a
separation column.
• The mobile phase, on the other hand, is a solvent or solvent mixture which is
forced at high pressure through the separation column.
• Via a valve with a connected sample loop, i.e. a small tube or a capillary made of
stainless steel, the sample is injected into the mobile phase flow from the pump to
the separation column using a syringe.
• Subsequently, the individual components of the sample migrate through the
column at different rates because they are retained to a varying degree by
interactions with the stationary phase.
• After leaving the column, the individual substances are detected by a suitable
detector and passed on as a signal to the HPLC software on the computer.
• At the end of this operation/run, a chromatogram in the HPLC software on the
computer is obtained.
• The chromatogram allows the identification and quantification of the different
substances.
7. Instrumentation
The Pump
• The development of HPLC led to the development of the pump system.
• The pump is positioned in the most upper stream of the liquid chromatography system and generates a flow of
eluent from the solvent reservoir into the system.
• High-pressure generation is a “standard” requirement of pumps besides which, it should also to be able to provide a
consistent pressure at any condition and a controllable and reproducible flow rate.
• Most pumps used in current LC systems generate the flow by back-and-forth motion of a motor-driven piston
(reciprocating pumps). Because of this piston motion, it produces “pulses”.
Injector
• An injector is placed next to the pump.
• The simplest method is to use a syringe, and the sample is introduced to the flow of eluent.
• The most widely used injection method is based on sampling loops.
• The use of the autosampler (auto-injector) system is also widely used that allows repeated injections in a set
scheduled-timing.
Column
• The separation is performed inside the column.
• The recent columns are often prepared in a stainless steel housing, instead of glass columns.
• The packing material generally used is silica or polymer gels compared to calcium carbonate.
The eluent used for LC varies from acidic to basic solvents.
• Most column housing is made of stainless steel since stainless is tolerant towards a large variety of solvents.
Detector
• Separation of analytes is performed inside the column, whereas a detector is used to observe the obtained
separation.
• The composition of the eluent is consistent when no analyte is present. While the presence of analyte changes the
composition of the eluent. What detector does is to measure these differences.
• This difference is monitored as a form of an electronic signal. There are different types of detectors available.
8. Recorder
• The change in eluent detected by a detector is in the form of an electronic signal, and thus it is still
not visible to our eyes.
• In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a computer-based data
processor (integrator) is more common.
• There are various types of data processors; from a simple system consisting of the in-built printer
and word processor while those with software that are specifically designed for an LC system which
not only data acquisition but features like peak-fitting, baseline correction, automatic concentration
calculation, molecular weight determination, etc.
Degasser
• The eluent used for LC analysis may contain gases such as oxygen that are non-visible to our eyes.
• When gas is present in the eluent, this is detected as noise and causes an unstable baseline.
• Degasser uses special polymer membrane tubing to remove gases.
• The numerous very small pores on the surface of the polymer tube allow the air to go through while
preventing any liquid to go through the pore.
Column Heater
• The LC separation is often largely influenced by the column temperature.
• In order to obtain repeatable results, it is important to keep consistent temperature conditions.
• Also for some analysis, such as sugar and organic acid, better resolutions can be obtained at
elevated temperatures (50 to 80°C).
• Thus columns are generally kept inside the column oven (column heater).
9. Types
Normal phase:
• Column packing is polar (e.g silica) and the mobile phase is non-
polar. It is used for water-sensitive compounds, geometric isomers,
cis-trans isomers, and chiral compounds.
Reverse phase:
• The column packing is non-polar (e.g C18), the mobile phase is
water+ miscible solvent (e.g methanol). It can be used for polar,
non-polar, ionizable, and ionic samples.
Ion exchange:
• Column packing contains ionic groups and the mobile phase is
buffer. It is used to separate anions and cations.
Size exclusion:
• Molecules diffuse into pores of a porous medium and are separated
according to their relative size to the pore size. Large molecules
elute first and smaller molecules elute later.
10. Applications
• The HPLC has developed into a universally applicable method so that it finds its
use in almost all areas of chemistry, biochemistry, and pharmacy.
• Analysis of drugs
• Analysis of synthetic polymers
• Analysis of pollutants in environmental analytics
• Determination of drugs in biological matrices
• Isolation of valuable products
• Product purity and quality control of industrial products and fine chemicals
• Separation and purification of biopolymers such as enzymes or nucleic acids
• Water purification
• Pre-concentration of trace components
• Ligand-exchange chromatography
• Ion-exchange chromatography of proteins
• High-pH anion-exchange chromatography of carbohydrates and
oligosaccharides
11. Advantages
• Speed
• Efficiency
• Accuracy
• Versatile and extremely precise when it comes to identifying and
quantifying chemical components
Limitations
• Cost: Despite its advantages, HPLC can be costly, requiring large quantities of
expensive organics.
• Complexity
• HPLC does have low sensitivity for certain compounds, and some cannot be
detected as they are irreversibly adsorbed.
• Volatile substances are better separated by gas chromatography.
12. Gas Chromatography
• GC is another widely used chromatography technique. Here the mobile phase is
usually an inert gas such as helium or argon (also known as the carrier gas),
while the technique itself is performed in a capillary or packed column made up
of inert materials. Even though packed columns are cheaper and user-friendly,
capillary columns provide greater resolution and are relatively expensive.
• GC is another widely used chromatography technique. Here the mobile phase is
usually an inert gas such as helium or argon (also known as the carrier gas),
while the technique itself is performed in a capillary or packed column made up
of inert materials. Even though packed columns are cheaper and user-friendly,
capillary columns provide greater resolution and are relatively expensive.
• The stationary phase is either a granular solid (i.e. gas-solid chromatography),
or a granular solid coated with a thin film of nonvolatile liquid (i.e. gas-liquid
chromatography). A majority of analytical gas chromatographs employ capillary
columns, where the stationary phase directly coats the walls of a tube with a
small diameter.
13. • GC is usually used to separate vaporizable or volatile compounds
and test their purity. It is also used to quantify the different
components in a mixture. Although volatility of a sample is a
prerequisite for GC analysis, the modification of the functional
group of a certain molecule by a process known as derivatization
enables the analysis of compounds that otherwise could not be
easily monitored by GC.
• In GC, the separation of components of a mixture depends on the
length and temperature of the column, as well as on the flow rate
of the carrier gas. These conditions must be optimized for a
particular analysis.
• In both GC and LC, detection of the individual components in the
sample can be carried out by several methods. The most common
and sensitive detection method is mass spectrometry, which
identifies compounds based on the atomic sample organization of
the molecules and their charge state.
14. Liquid stationary phase This type of phase is available in packed or capillary
columns.
Packed column gas chromatography The liquid stationary phase is
deposited on a finely divided, inert solid support, such as diatomaceous earth,
porous polymer, or graphitized carbon, which is packed into a column that is
typically 2–4 mm in internal diameter and 1–3 m in length.
Capillary column gas chromatography In capillary columns, which
contain no packed solid support, the liquid stationary phase is deposited
on the inner surface of the column and may be chemically bonded to it.
Stationary phase This type of phase is available only in packed columns. In
these columns the solid phase is an active adsorbent, such as alumina, silica, or
carbon, packed into a column. Polyaromatic porous resins, which are sometimes
used in packed columns, are not coated with a liquid phase
15. Apparatus A gas chromatograph consists of a carrier gas source, injection port,
column, detector, and recording device. The injection port, column, and detector are
temperature controlled and may be varied as part of the analysis. The typical carrier
gas is helium, nitrogen, or hydrogen, depending on the column and detector in use.
The type of detector used depends on the nature of the compounds analyzed and is
specified in the individual monograph. Detector output is recorded as a function of
time, and the instrument response, measured as peak area or peak height, is a
function of the amount present.
Temperature program The length and quality of a GC separation can be
controlled by altering the temperature of the chromatographic column. When a
temperature program is necessary, the individual monograph indicates the conditions
in table format. The table indicates the initial temperature, rate of temperature
change (ramp), final temperature, and hold time at the final temperature.
Procedure
1. Equilibrate the column, injector, and detector with flowing carrier gas until a
constant signal is received.
2. Inject a sample through the injector septum, or use an autosampler.
3. Begin the temperature program.
4. Record the chromatogram.
5. Analyze as indicated in the monograph.
16. Differences between of LC and GC
Liquid Chromatography Gas Chromatography
Mobile phase is a liquid Mobile phase is a gas
Separation is based on interaction of solute with the
chromatography medium
Separation is primarily based on the boiling points of
solute molecules
Can be performed in a sheet or a column Can be carried out only in a column
Can be used to separate any soluble compound, e.g. amino
acids, proteins, drugs, nucleic acids, lipids, antioxidants,
carbohydrates, and natural and artificial polymers
Can be applied in the separation of volatile compounds
and gaseous mixtures
Usually carried out at room temperature so heat-sensitive
compounds can be safely analyzed using the technique
Performed at higher temperatures so thermally labile
substances might get denatured
Solute retention here is based on the interaction of solutes
with the mobile and stationary phases so it is easy to
optimize results
Separation is based on the boiling points of the solute
molecules so it is not very flexible in terms of optimizing
separation
This is a relatively slower technique The analysis is faster and usually measured in minutes,
although it can take as little as a couple of seconds
Usually gives a greater peak or broader band resulting in
lower resolution
Provides comparatively better resolution
Uses polar solvents like water or methanol Uses any solvent that vaporizes
