Name: Laiba Arshad

Dep: BS Chemistry
Subject: Inorganic chemistry
Roll no: 14186
Submitted to: Sir Suleman

High performance liquid chromatography:

High Performance Liquid Chromatography (HPLC) is an analytical
technique used for the separation of compounds soluble in a
particular solvent.
History of HPLC

Liquid chromatography was initially discovered as an analytical

technique in the early twentieth century and was first used as a
method of separating colored compounds. This is where the
name chromatography chroma means color, graphy means
writing, was derived. A Russian botanist named Mikhail S. Tswett
used a rudimentary form of chromatographic separation to purify
mixtures of plant pigments into the pure constituents. He
separated the pigments based on their interaction with
a stationary phase, which is essential to any chromatographic
separation. The stationary phase he used was powdered chalk
and aluminia, the mobile phase in his separation was the solvent.
After the solid stationary phase was packed into a glass column
(essentially a long, hollow, glass tube) he poured the mixture of
plant pigments and solvent in the top of the column. He then
poured additional solvent into the column until the samples were
eluted at the bottom of the column. The result of this process
most crucial to his investigation was that the plant pigments
separated into bands of pure components as they passed through
the stationary phase. Modern high performance liquid
chromatography or HPLC has its roots in this separation, the first
form of liquid chromatography. The chromatographic process has
been significantly improved over the last hundred years, yielding
greater separation efficiency, versatility and speed.

Affinities for Mobile and Stationary Phases

All chromatographic separations, including HPLC operate under

the same basic principle; every compound interacts with other
chemical species in a characteristic manner. Chromatography
separates a sample into its constituent parts because of the
difference in the relative affinities of different molecules for the
mobile phase and the stationary phase used in the separation.

Distribution Constant

All chemical reactions have a characteristic equilibrium constant.

For the reaction

Aaq+Bs⇌Abs (1)(1)Aaq+Bs⇌ABs

There is a chemical equilibrium constant Keq that dictates what

percentage of compound A will be in solution and what
percentage will be bound to the stationary compound B. During a
chromatographic separation, there is similar relationship between
compound A and the solvent, or mobile phase, C. This will yield
an overall equilibrium equation which dictates the quantity
of A that will be associated with the stationary phase and the
quantity of A that will be associated with the mobile phase.
 Amobile⇌Astationary (2)(2)Amobile⇌Astationary

The equilibrium between the mobile phase and stationary phase

is given by the constant Kc.

Kc=(aA)S(aA)M≈cScM(3)(3)Kc=(aA)S(aA)M≈cScM

Where Kc, the distribution constant, is the ratio of the activity of

compound A in the stationary phase and activity of
compound A in the mobile phase. In most separations, which
contain low concentrations of the species to be separated, the
activity of A in each is approximately equal to the concentration
of A in that state. The distribution constant indicates the amount
of time that compound A spends adsorbed to the stationary
phase as the opposed to the amount of time A spends solvated
by the mobile phase. This relationship determines the amount of
time it will take for compound A to travel the length of the
column. The more time A spends adsorbed to the stationary
phase, the more time compound A will take to travel the length of
the column. The amount of time between the injection of a
sample and its elution from the column is known as the retention
time; it is given the symbol tR.
The amount of time required for a sample that does not interact
with the stationary phase, or has a Kc equal to zero, to travel the
length of the column is known as the void time, tM. No compound
can be eluted in less than the void time.

Retention Factor

Since Kc is a factor that is wholly dependent on a particular

column and solvent flow rate, a quantitative measure of the
affinity of a compound for a particular set of mobile and
stationary phases that does not depend on the column geometry
is useful. The retention factor, k, can be derived from Kc and is
independent of the column size and the solvent flow rate.

kC=KCVSVM(4)(4)kC=KCVSVM
The retention factor is calculated by multiplying the distribution
constant by the volume of stationary phase in the column and
dividing by the volume of mobile phase in the column.

Selectivity

In order to separate two compounds, their respective retention

factors must be different, otherwise both compounds would be
eluted simultaneously; the selectivity factor is the ratio of the
retention factors.

α=kBkA(5)(5)α=kBkA

Where B is the compound that is retained more strongly by the

column and A is the compound with the faster elution time.

Band Broadening

As a compound passes through the column it slowly diffuses

away from the initial injection band, which is the area of greatest
concentration. The initial, narrow, band that contained all of the
sample becomes broader the longer the analyte remains in the
column. This band broadening increases the time required for
complete elution of a particular compound and is generally
undesirable. It must be minimized so that overly broad elution
bands do not overlap with one another.

Separation Efficiency

The overriding purpose of a chromatographic separation is just

that, to separate two or more compounds contained in solution.
In analytical chemistry, a quantitative metric of every
experimental parameter is desired, and so separation efficiency is
measured in plates. The concept of plates as a separation metric
arose from the original method of fractional distillation, where
compounds were separated based on their volatilities through
many simultaneous simple distillations, each simple distillation
occurred on one of many distillation plates. In chromatography,
no actual plates are used, but the concept of a theoretical plate,
as a distinct region where a single equilibrium is maintained,
remains. In a particular liquid chromatographic separation, the
number of theoretical plates and the height equivalent to a
theoretical plate (HETP) are related simply by the length of the
column

N=LH(6)(6)N=LH

Where N is the number of theoretical plates, L is the length of the

column, and H is the height equivalent to a theoretical plate. The
plate height is given by the variance (standard deviation squared)
of an elution peak divided by the length of the column.

H=σ2L(7)(7)H=σ2L

The standard deviation of an elution peak can be approximated

by assuming that a Gaussian elution peak is roughly triangular, in
that case the plate height can be given by the width of the elution
peak squared times the length of the column over the retention
time of the that peak squared times 16.

H=LW216t2R(8)(8)H=LW216tR2

Using the relationship between plate height and number of

plates, the number of plates can also be found in terms of
retention time and peak width.

N=16(tRW)2(9)(9)N=16(tRW)2
In order to optimize separation efficiency, it is necessary in
maximize the number of theoretical plates, which requires
reducing the plate height. The plate height is related to the flow
rate of the mobile phase, so for a fixed set of mobile phase,
stationary phase, and analytes; separation efficiency can be
maximized by optimizing flow rate as dictated by the van
Deemter equation.

H=A+Bv+Cv(10)(10)H=A+Bv+Cv

The three constants in the van Deemter equation are factors that
describe possible causes of band broadening in a particular
separation. AA is a constant which represents the different
possible paths that can be taken by the analyte through the
stationary phase, it decreases if the packing of the column is kept
as small as possible. BB is a constant that describes the
longitudinal diffusion that occurs in the system. CC is a constant
that describes the rate of adsorption and desorption of the
analyte to the stationary phase. AA, BB and CC are constant for
any given system (with constant analyte, stationary phase, and
mobile phase), so flow rate must be optimized accordingly. If the
flow rate is too low, the longitudinal diffusion factor (BvBv) will
increase significantly, which will increase plate height. At low flow
rates, the analyte spends more time at rest in the column and
therefore longitudinal diffusion in a more significant problem. If
the flow rate is too high, the mass transfer term (CvCv) will
increase and reduce column efficiency. At high flow rates the
adsorption of the analyte to the stationary phase results in some
of the sample lagging behind, which also leads to band
broadening.

Resolution
The resolution of a elution is a quantitative measure of how well
two elution peaks can be differentiated in a chromatographic
separation. It is defined as the difference in retention times
between the two peaks, divided by the combined widths of the
elution peaks.

RS=2[(tR)B−(tR)A]WB+WA(11)(11)RS=2[(tR)B−(tR)A]WB+WA

Where B is the species with the longer retention time,

and tR and W are the retention time and elution peak width
respectively. If the resolution is greater than one, the peaks can
usually be differentiated successfully.
HPLC as a solution to efficiency problems
While all of these basic principles hold true for all
chromatographic separations, HPLC was developed as method to
solve some of the shortcomings of standard liquid
chromatography. Classic liquid chromatography has several
severe limitations as a separation method. When the solvent is
driven by gravity, the separation is very slow, and if the solvent is
driven by vacuum, in a standard packed column, the plate height
increases and the effect of the vacuum is negated. The limiting
factor in liquid chromatography was originally the size of the
column packing, once columns could be packed with particles as
small as 3 µm, faster separations could be performed in smaller,
narrower, columns. High pressure was required to force the
mobile phase and sample through these new columns, and
previously unneeded apparatus was required to maintain
reproducibility of results in this new instruments. The use of high
pressures in a narrow column allowed for a more effective
separation to be achieved in much less time than was required for
previous forms of liquid chromatography.

Apparatus

Specialized apparatus is required for an HPLC separation because

of the high pressures and low tolerances under which the
separation occurs. If the results are to be reproducible, then the
conditions of the separation must also be reproducible. Thus
HPLC equipment must be of high quality; it is therefore
expensive.

Solvent

The mobile phase, or solvent, in HPLC is usually a mixture of

polar and non-polar liquid components whose respective
concentrations are varied depending on the composition of the
sample. As the solvent is passed through a very narrow bore
column, any contaminants could at worst plug the column, or at
the very least add variability to the retention times during
repeated different trials. Therefore HPLC solvent must be kept
free of dissolved gases, which could come out of solution mid-
separation, and particulates.

Column

In the HPLC column, the components of the sample separate

based on their differing interactions with the column packing. If a
species interacts more strongly with the stationary phase in the
column, it will spend more time adsorbed to the column's
adsorbent and will therefore have a greater retention time.
Columns can be packed with solids such as silica or alumina;
these columns are called homogeneous columns. If stationary
phase in the column is a liquid, the column is deemed a bonded
column. Bonded columns contain a liquid stationary phase
bonded to a sold support, which is again usually silica or alumina.
The value of the constant C described in the van
Deemter equation is proportional, in HPLC, to the diameter of the
particles that constitute the column's packing material.

Pump
The HPLC pump drives the solvent and sample through the
column. To reduce variation in the elution, the pump must
maintain a constant, pulse free, flow rate; this is achieved
with multi-piston pumps. The presence of two pistons allows the
flow rate to be controlled by one piston as the other recharges.
A syringe pump can be used for even greater control of flow rate;
however, the syringe pump is unable to produce as much
pressure as a piston pump, so it cannot be used in all HPLC
applications.
Detector
The HPLC detector, located at the end of the column, must
register the presence of various components of the sample, but
must not detect the solvent. For that reason there is no universal
detector that works for all separations. A common HPLC detector
is a UV absorption detector, as most medium to large
molecules absorb UV radiation. Detectors that measure
fluorescence and refractive index are also used for special
applications. A relatively new development is the combination of
an HPLC separation with an NMR detector. This allows the pure
components of the sample to be identified and quantified by
nuclear magnetic resonance after having been separated by
HPLC, in one integrated process.

Technique

Normal Phase vs. Reverse Phase

If the stationary phase is more polar than the mobile phase, the
separation is deemed normal phase. If the stationary phase is
less polar than the mobile phase, the separation is reverse
phase. In reverse phase HPLC the retention time of a compound
increases with decreasing polarity of the particular species. The
key to an effective and efficient separation is to determine the
appropriate ratio between polar and non-polar components in the
mobile phase. The goal is for all the compounds to elute in as
short a time as possible, while still allowing for the resolution of
individual peaks. Typical columns for normal phase separation are
packed with alumina or silica. Alkyl, aliphatic or phenyl bonded
phases are typically used for reverse phase separation.

Gradient Elution vs. Isocratic Elution

If the composition of the mobile phase remains constant
throughout the HPLC separation, the separation is deemed
an isocratic elution. Often the only way to elute all of the
compounds in the sample in a reasonable amount of time, while
still maintaining peak resolution, is to change the ratio of polar to
non-polar compounds in the mobile phase during the sample run.
Known as gradient chromatography, this is the technique of
choice when a sample contains components of a wide range of
polarities. For a reverse phase gradient, the solvent starts out
relatively polar and slowly becomes more non-polar. The gradient
elution offers the most complete separation of the peaks, without
taking an inordinate amount of time. A sample containing
compounds of a wide range of polarities can be separated by a
gradient elution in a shorter time period without a loss of
resolution in the earlier peaks or excessive broadening of later
peaks. However, gradient elution requires more complex and
expensive equipment and it is more difficult to maintain a
constant flow rate while there are constant changes in mobile
phase composition. Gradient elution, especially at high speeds,
brings out the limitations of lower quality experimental apparatus,
making the results obtained less reproducible in equipment
already prone to variation. If the flow rate or mobile phase
composition fluctuates, the results will not be reproducible.

Applications
HPLC can be used in both qualitative and quantitative
applications, that is for both compound identification and
quantification. Normal phase HPLC is only rarely used now,
almost all HPLC separation can be performed in reverse phase.
Reverse phase HPLC (RPLC) is ineffective in for only a few
separation types; it cannot separate inorganic ions (they can be
separated by ion exchange chromatography). It cannot separate
polysaccharides (they are too hydrophilic for any solid phase
adsorption to occur), nor polynucleotides (they adsorb irreversibly
to the reverse phase packing).

Lastly, incredibly hydrophobic compounds cannot be separated

effectively by RPLC (there is little selectivity). Aside from these
few exceptions, RPLC is used for the separation of almost all
other compound varieties. RPLC can be used to effectively
separate similar simple and aromatic hydrocarbons, even those
that differ only by a single methylene group.

RPLC effectively separates simple amines, sugars, lipids, and

even pharmaceutically active compounds. RPLC is also used in the
separation of amino acids, peptides, and proteins. Finally RPLC is
used to separate molecules of biological origin. The determination
of caffeine content in coffee products is routinely done by RPLC in
commercial applications in order to guarantee purity and quality
of ground coffee. HPLC is a useful addition to an analytical
arsenal, especially for the separation of a sample before further
analysis.

The End