Chromatography Part-III

Dr. BASAVARAJAIAH S. M.
Assistant Professor and Coordinator
P.G. Department of Chemistry
Vijaya College
Bangalore-560 004
CHROMATOGRAPHY-III
(HPLC, and GAS)

CONTENTS:
High Performance Liquid Chromatography (HPLC)
Gas Chromatography
Theory of column efficiency in chromatography

High Performance Liquid Chromatography
(HPLC)
 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 with its low-pressure
suitable glass columns was further developed to the HPLC with
its high-pressure adapted metal columns.
 HPLC is 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.

TYPES OF HPLC
 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.

PRINCIPLE OF HPLC
 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.
At the end of each run, a chromatogram in the HPLC software on the
computer is obtained.
The chromatogram allows the identification and quantification of the
different substances.

INSTRUMENTATION OF HPLC
Pump (Solvent Manager)
Injector (Auto sampler)
HPLC Column (Stationary phase)
Detector
Sample
Chromatogram
Computer Data Station
Waste
Solvent
Reservoir
(Mobile Phase)

 As shown in the Figure, HPLC instrumentation includes a
pump, injector, column, detector, and integrator or acquisition and
display system.
 The heart of the system is the column where separation
occurs. Since the stationary phase may be composed of micron-
sized porous particles, a high-pressure pump is required to move
the mobile phase through the column.
 The chromatographic process begins by injecting the solute
into the injector at the end of the column.
 Separation of components occurs as the analytes and mobile
phase are pumped through the column.

Eventually, each component elutes from the column as a peak
on the data display.
Detection of the eluting components is important, and the
method used for detection is dependent upon the detector used.
The response of the detector to each component is displayed on
a chart recorder or computer screen and is known as a
chromatogram.
To collect, store, and analyze the chromatographic data,
integrators, and other data-processing equipment are frequently
used.

Mobile Phase and Reservoir
The type and composition of the mobile phase affect the separation of
the components.
Different solvents are used for different types of HPLC. For normal-
phase HPLC, the solvent is usually nonpolar, and, in reverse-phase
HPLC, the solvent is normally a mixture of water and a polar organic
solvent.
The purity of solvents and inorganic salts used to make the mobile
phase is paramount.
A general rule of thumb is to use the highest purity of solvent that is
available and practical depending on the particular application.
The most common solvent reservoirs are as simple as glass bottles
with tubing connecting them to the pump inlet.

Pumps
 High-pressure pumps are needed to push the mobile phase
through the packed stationary phase.
 A steady pump pressure (usually about 1000–2000 psi) is
needed to ensure reproducibility and accuracy. Pumps are
typically known to be robust, but adequate maintenance must be
performed to maintain that characteristic.
 Inability to build pressure, high pressure or leakage could
indicate that the pump is not functioning correctly. Proper
maintenance of the pump system will minimize downtime.

Injectors
 The injector can be a single injection or an automated injection
system.
 An injector for an HPLC system should provide an injection of
the liquid sample within the range of 0.1-100 ml of volume with
high reproducibility and under high pressure (up to 4000 psi).
 For liquid chromatography, liquid samples can be directly
injected and solid samples need only to be diluted in the
appropriate solvent.

Column
The separation is performed inside the column.
The recent columns are often prepared in a stainless steel,
instead of glass columns.
The packing material generally used is silica or polymer gels.
A variety of column dimensions are available including
preparative, normal-bore, micro-and mini-bore, and capillary
columns.
The most widely used packing materials for HPLC separations
are silica-based. The most popular material is octadecyl-silica
(ODS-silica), which contains C18 coating, but materials with C1,
C2, C4, C6, C8, and C22 coatings are also available.

DETECTORS
There are many different types of detectors that can be used
for HPLC.
The detector is used to sense the presence of a compound
passing through and to provide an electronic signal to a data-
acquisition device.
The main types of detectors used in HPLC are refractive index
(RI), ultraviolet (UV-Vis), and fluorescence, but there are also
diode array, electrochemical, and conductivity detectors. Each
detector has its assets, limitations, and sample types for which it
is most effective.

Most applications in drug analysis use detectors that respond to the
absorption of ultraviolet radiation (or visible light) by the solute as it
passes through the flow-cell inside the detector.
The recent development of the so-called hyphenated techniques has
improved the ability to separate and identify multiple entities within a
mixture.
These techniques include liquid chromatography-mass spectrometry
(LC-MS), liquid chromatography-mass spectrometry (LC-MS), liquid
chromatography-infrared spectroscopy (LC-IR), and liquid
chromatography-nuclear magnetic resonance (LCNMR).
These techniques usually involve chromatographic separation followed
by peak identification with a traditional detector such as UV, combined
with further identification of the compound with the MS, IR, or NMR.

Data Acquisition/Display Systems
Since the detector signal is electronic, the use of modern data
acquisition techniques can aid in the signal analysis.
The data acquisition system of most HPLC systems is a
computer.
The computer integrates the response of the detector to each
component and places it into a chromatograph that is easy to read
and interpret.
Other more advanced features can also be applied to a
chromatographic system.
These features include computer-controlled automatic injectors,
multi-pump gradient controllers, and sample fraction collectors.

ISOCRATIC Vs GRADIENT ELUTION
Elution techniques are methods of pumping the mobile phase
through a column.
In the isocratic method, the composition of the mobile phase
remains constant, whereas in the gradient method the
composition changes during the separation process.
The isocratic method is the simplest technique and should be
the first choice when developing a separation.
Eluent gradients are usually generated by combining the
pressurized flows from two pumps and changing their individual
flow rates with an electronic controller or data system while
maintaining the overall flow rate constant.

ADVANTAGES OF HPLC
Speed
Efficiency
Accuracy
Versatile and extremely precise when it comes to identifying
and quantifying chemical components.

Applications of HPLC:
Pharmaceutical /Biopharmaceutical
Pharmaceutical quality control.
Shelf-life determinations of pharmaceutical products.
Identification of counterfeit drug products.
Complex molecules separation.
Environmental
Biomonitoring of pollutants.
Water monitoring-Phenol content and toxic components
checking.

Clinical
Analysis of antibiotics and blood substances.
Detection of endogenous neuropeptides in brain extracellular
fluid.
Food and Flavor
Sugar analysis in fruit juices.
Ensuring soft drink consistency and quality.

GAS CHROMATOGRAPHY
Gas chromatography is a term used to describe the group of
analytical separation techniques used to analyze volatile
substances in the gas phase.
In gas chromatography, the components of a sample are
dissolved in a solvent and vaporized in order to separate the
analytes by distributing the sample between two phases: a
stationary phase and a mobile phase.
The mobile phase is a chemically inert gas that serves to carry
the molecules of the analyte through the heated column.

Gas chromatography is one of the sole forms of
chromatography that does not utilize the mobile phase for
interacting with the analyte.
The stationary phase is either a solid adsorbent, termed gas-
solid chromatography (GSC), or a liquid on an inert support,
termed gas-liquid chromatography (GLC).
Since GC is a gas-based separation technique, it is limited to
components that have sufficient volatility and thermal stability.

 A compound is vaporized, introduced into the carrier gas and
then carried onto the column.
 The sample is then partitioned between the gas, and the
stationary phase.
 The compounds in a sample are slowed down to varying
degrees due to the sorption and desorption on the stationary
phase.
 The elution of the compound is characterized by the partition
ratio kD′, which is a dimensionless quantity also called the
capacity factor.
Practical Aspects of Gas Chromatography-Theory:

 The partition ratio can also be thought of as the ratio of the
time required for the compound to flow through the column (the
retention time) to the elution time of an unretained compound.
 The value of the capacity factor is dependent on several
elements of the chromatographic system, including the chemical
nature of the compound; the nature, amount, and surface area of
the stationary phase; the column temperature; and the gas flow
rate.
 Capacity factor is essential for separation by GC because
separation is only possible if the compounds in the sample have
different capacity factors.

 A variety of sample types can be successfully analyzed by GC. Unlike
HPLC, which is used to separate larger molecules, GC is best suited for
the analysis of samples with smaller molecules.
 Another important characteristic of samples for GC analysis is that
they must be volatile. If a compound is not volatile a technique called
derivatization can be used to increase its solubility and add to its
volatility.
 Derivatization of samples involves a chemical reaction that alters the
molecular structure of the analyte of interest to improve detection.
 Different types of compounds called derivatizing agents can be used
to increase the volatility of sample components. However, if possible it is
best to avoid derivatization to keep the separation simpler.
SAMPLES AND SAMPLE PREPARATION

CARRIER GAS
In the early days of GC experiments, the carrier gas was seen
merely as the mass transport system.
However, it is becoming clearer that carrier gas is integral to the
chromatographic process.
Several inert gases can be used as the carrier gas or mobile
phase of GC. Hydrogen, helium, and nitrogen are all common
carrier gases.
Each carrier gas has its benefits and systems for which it is best
suited.
For example, helium is the most common gas used with
GC/mass spectrometry systems.

 Before the carrier gas can be used, it is important to ensure
that it contains no oxygen because oxygen can have detrimental
effects on the stationary phase of GC.
 Also, the chemical nature of the carrier gas has an effect on
the efficiency of the GC column.
 The pressure at which the carrier gas is moving influences the
retention time of samples on the column. Increasing the pressure
decreases the retention time.
 Varying both the carrier gas and the pressure at which the gas
is exerted on the column can ensure that the sample has ample
time to interact with the stationary phase and improve the
separation.

INJECTORS
The introduction of the sample into a GC system is a critical
step in separation.
The reproducibility of the amount of sample injected is important
to ensure the reproducibility of results.
A sample can be injected manually into the system or by using
an autosampler system.
A major source of precision errors in GC is a poor injection
technique.
Autosamplers are very effective and help ensure that precisely
the same sample volume is injected every time, thereby
eliminating injection errors.

The injector temperature is also important for separation.
The temperature of the injector is used to rapidly vaporize the
liquid sample into a gaseous phase that can be carried to the
column for separation.
The temperature of the injector site can be varied to help
optimize separation.
Different sample components will dictate what temperature is
necessary for vaporization.

COLUMN/STATIONARY PHASE
 The column and stationary phase are responsible for the majority
of the separation of sample components.
 Interaction between the mobile phase, stationary phase, and
sample components determines how components are separated, so
a selection of columns and stationary packing material is critical.
 There is a great deal of variation in commercially available
columns and packing material.
 Depending on the components to be separated, the mobile phase
being used and the desired degree of separation, different
combinations of column type, column length and packing material
can be used to achieve optimal results.

Columns can be classified by column diameter and packing
material.
The three main types of GC columns are (1) conventional, (2)
preparative, and (3) capillary.
Columns can be either packed or open. Packed columns can
contain either a porous or nonporous stationary phase.
A multitude of different materials are used to pack columns. Each
material has its own properties, limitations, and effective separation
parameters.
The capillary column is the most frequently used column for GC
separations.
Both conventional and capillary columns have advantages and
disadvantages.

The column resides in an oven, and temperature, which greatly
affects the effectiveness of the chromatographic separation, is an
extremely important factor used in controlling GC.
In many cases, isothermal (constant temperature) is not the
most effective temperature mode for sample separation; in such
cases, a temperature program can be used.
Most GC temperature programs have an initial temperature, a
ramp (degree increase per minute), and a final temperature.
OVEN

Detector
The detector is used to sense the presence of a compound passing
through and to provide an electronic signal to an integrator.
A variety of detectors are commercially available to be used with GC,
each having its own limitations and advantages:
Electron captures (ECD). The ECD is used with organic compounds
and has many environmental applications.
Flame ionization (FID). The most commonly used detector in GC,
FID is typically used with organic compounds and is widely used in the
quality-control analysis of pharmaceutical compounds.
Mass spectrometry (MSD). The MSD can be coupled with GC as a
powerful qualitative component for the identification of compounds.

Nitrogen phosphorous (NID). This detector is used most
commonly for drug analysis in tissues and body fluids.
Thermal conductivity (TCD). This detector is considered a
universal detector and is non-destructive to analytes.
RECORDER
Just as it is with HPLC, the recorder in a GC system serves to
convert the information collected by the detector into a format that
is understandable.
Since the detector signal is electronic, the use of modern data
acquisition can aid in signal analysis.

Applications of Gas Chromatography
Determination of volatile organic compounds in water or solid-
Environmental laboratories.
In the Food industry i.e., quality check for additives and flavoring
agents (i.e., identifying flavoring agent in wine: linalool, citronellol,
nerol, geraniol).
Analysis of fragrances in cosmetics.
Identification of drugs and trace components like toxins in Forensics.
Purity test for intermediates and final products in the pharmaceutical
industry.
Detection of sulfur compounds in gasoline and natural gas in the
petroleum industry.

THEORY OF COLUMN EFFICIENCY
PLATE THEORY
Developed by Martin & Synge.
The separation efficiency of a column can be expressed in
terms of the number of theoretical plates in the column.
The distribution of analytes between phases can often be
described quite simply.
An analyte is in equilibrium between the two phases;
 The equilibrium constant, K, is termed the partition coefficient;
defined as the molar concentration of analyte in the stationary phase
divided by the molar concentration of the analyte in the mobile phase.
Amobile Astationary
K

The time between sample injection and an analyte peak
reaching a detector at the end of the column is termed
the retention time (tR ). Each analyte in a sample will have a
different retention time.
The time taken for the mobile phase to pass through the
column is called tM.
When an analytes retention factor is less than one, elution is so fast that accurate
determination of the retention time is very difficult.
High retention factors (greater than 20) mean that elution takes a very long time.
Ideally, the retention factor for an analyte is between one and five.
tR and tM are easily
obtained from a
chromatogram.

 The plate model supposes that the chromatographic column
contains a large number of separate layers, called theoretical
plates.
 Separate equilibrations of the sample between the stationary
and mobile phase occur in these "plates".
 The analyte moves down the column by transfer of
equilibrated mobile phase from one plate to the next.
It is important to remember that the plates do not really exist;
they are a figment of the imagination that helps us understand the
processes at work in the column.

 They also serve as a way of measuring column efficiency,
either by stating the number of theoretical plates in a column, N (the more plates
the better),
or by stating the plate height; the Height Equivalent to a Theoretical Plate (the
smaller the better).
 If the length of the column is L, then the HETP is
 HETP = L / N
 The number of theoretical plates that a real column possesses can be found by
examining a chromatographic peak after elution;
where w1/2 is the peak width at half-height.
As it can be seen from this equation,
columns behave as if they have different numbers of plates for different
solutes in a mixture.
w is peak width w is peak height

Calculate the number of plates in the column resulting in the
chromatographic peak i.e. tR=52.3 mm and wb=9 mm.
Ans:
N= 16 (52.3/9)2
N= 540
Calculate the number of plates in the column resulting in the
chromatographic peak i.e. tR=40 mm and wb=5 mm.

RATE THEORY (BAND BROADENING)
A more realistic description of the processes at work inside a column
takes account of the time taken for the solute to equilibrate between the
stationary and mobile phase (unlike the plate model, which assumes that
equilibration is infinitely fast).
The resulting band shape of a chromatographic peak is therefore
affected by the rate of elution.
It is also affected by the different paths available to solute molecules as
they travel between particles of stationary phase.
If we consider the various mechanisms which contribute to band
broadening, we arrive at the Van Deemter equation for plate height;
HETP = A + B / µ + C µ
where u is the average velocity of the
mobile phase. A, B, and C are factors
which contribute to band broadening.

Van Deemter plots
A plot of plate height vs average linear velocity of mobile phase.

The Van Deemter equation is an empirical formula describing the
relationship between plate height (H, the length needed for one
theoretical plate) which is a measure of column efficiency, and linear
velocity (µ). µ= L/tM
Smaller plate height values corresponds to greater column efficiencies.
The Van Deemter equation is governed by three cumulative terms: (A)
eddy diffusion, (B) longitudinal diffusion, and (C) mass transfer.
A loss in peak efficiency can be observed as a wider analyte band, and
therefore, these three terms can also be viewed as factors that contribute
to band broadening.
Van Deemter plot illustrates the effect of these terms, both individually
and cumulatively.

Eddy diffusion, the A term, is caused by a turbulence in the solute
flow path and is mainly unaffected by flow rate.
Eddy diffusion: A process that leads to peak (band) broadening due to
the presence of multiple flow paths through a packed column.
As solute molecules travel through the column, some arrive at the end
sooner then others simply due to the different path traveled around the
support particles in the column that result in different travel distances.

The B-term in the van Deemter equation, also known as longitudinal diffusion,
refers to the diffusion of individual analyte molecules in the mobile phase along the
longitudinal direction of a column. Longitudinal diffusion contributes to peak
broadening only at very low flow rates below the minimum (optimum) plate height.
Molecular diffusion takes place independent of the longitudinal (= axial) flow
direction. Longitudinal diffusion is the result of concentration differences in the
mobile phase. In the center of the peak zone the concentration is at its maximum. The
concentration before and after the peak zone is lower. This results in diffusion, both
in the direction of the mobile phase flow as well as in the opposite direction.
Some molecules move faster and others will move slower relative to the average
velocity, resulting in peak broadening. This effect will be relatively great at long
residence times in the column, which is the case at low flow rates. As the flow rate
increases, this effect will contribute less to the total peak broadening. In practice, it is
best to select flow rates that minimize the effect of longitudinal diffusion on column
efficiency.

MASS TRANSFER
The analyte takes a certain amount of time to equilibrate between the
stationary and mobile phase.
If the velocity of the mobile phase is high, and the analyte has a
strong affinity for the stationary phase, then the analyte in the mobile
phase will move ahead of the analyte in the stationary phase.
The band of analyte is broadened. The higher the velocity of mobile
phase, the worse the broadening becomes.

RETENTION FACTOR
The retention factor k for a sample peak is defined by;
k= tR-tM/tM
tR= Retention time, tM=time of unretained solute
EFFECTIVE PLATE NUMBER
The effective plate number is related to the retention factor and
plate number.
Neff= N (k/k+1)2

RESOLUTION IN CHROMATOGRAPHY
SEPARATION FACTOR
The separation factor, a, is a thermodynamic quantity that is a
measure of the relative retention of analytes, and is given by:
a= k2/k1
The resolution of two chromatographic peaks is defined by;
or

Comparison of Plate number, Retention factor and
Separation factor

1. Calculate the retention factor the chromatographic peak
obtained of tR is 52.3 mm and tM is 8 mm.
Ans:
k= tR-tM/tm = 52.3-8/8= 5.54
2. Calculate the retention factor and effective plate no of the
chromatographic peak obtained of tR is 45 mm,tM is 5 mm and
plate no is 940.
Ans:
k= tR-tM/tm
Neff= N (k/k+1)2

Chromatography Part-III

More Related Content

What's hot

Similar to Chromatography Part-III

More from DrBasavarajaiahSm

Recently uploaded

Chromatography Part-III