Chromatography

Chromatography refers to a set of techniques used to separate different compounds.  The word
comes from the Greek chromatos (color) and graphein (to write).  So, as one might guess,
chromatography involves separating chemicals and identifying them by color.

Chromatography has many uses.  It is commonly used in laboratories to isolate new compounds,
analyze subtle differences between different environmental samples, and even in the sequencing
of DNA.

To perform chromatography, one needs two things a matrix and a color indicator.  A matrix is
simply the material to which a sample is applied.  the material is often porous, acting as a filter
that affects the rate of flow of a sample.  Special beads with different pore sizes are commonly
used to separate proteins in biochemistry labs.

The matrix may interact with a sample, and depending on the chemical properties of both, affect
its flow.  Paper is one such matrix, and is commonly used to analyze mixtures of sugars.

The color indicator may be the compound itself.  Some organic compounds are bright yellow or
orange, which makes them easy to identify on a chromatogram.  Others need to react with other
chemicals to become colored.  Dyes specific for particular chemical properties can be used to
identify samples.  For example, rhodamine dye can be applied to visualize fats and oils.

HPLC
High-performance liquid chromatography (HPLC) is an advanced form of liquid
chromatography used in separating the complex mixture of molecules encountered in chemical
and biological systems, in order to understand better the role of individual molecules. In liquid
chromatography, a mixture of molecules dissolved in a solution (mobile phase) is separated into
its constituent parts by passing through a column of tightly packed solid particles (stationary
phase). The separation occurs because each component in the mixture interacts differently with
the stationary phase. Molecules that interact strongly with the stationary phase will move slowly
through the column, while the molecules that interact less strongly will move rapidly through the
column. This differential rate of migration facilitates the separation of the molecules.
 Fig.HPLC Apparatus

The advantages of HPLC over other forms of liquid chromatography are several. It allows
analysis to be done in a shorter time and achieves a higher degree of resolution, that is, the
separation of constituents is more complete. In addition, it allows stationary columns to be
reused a number of times without requiring that they be regenerated, and the results of analysis
are more highly reproducible. A further advantage of HPLC is that it permits both
instrumentation and quantitation to be automated.

Types of HPLC
There are several column types, according to their function, they can be classified as:

a) Normal phase:-
In this column type the retention is governed by the interaction of the polar parts of the stationary
phase and solute. For retention to occur in normal phase, the packing must be more polar than
the mobile phase with respect to the sample. Therefore, the stationary phase is usually silica and
typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of
these.

b) Reverse phase:-
In this column the packing material is relatively nonpolar and the solvent is polar with respect to
the sample. Retention is the result of the interaction of the nonpolar components of the solutes
and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy
liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-
organic mixtures such as methanol-water or acetonitrile-water.

c) Size exclusion:-
In this column type molecules are separated according to size. Small molecules penetrate into the
pores within the packing while larger molecules only partially penetrate the pores. The large
molecules elute before the smaller molecules.

d) Ion exchange:-
In this column type the sample components are separated based upon attractive ionic forces
between molecules carrying charged groups of opposite charge to those charges on the stationary
phase. Separations are made between a polar mobile liquid, usually water containing salts or
small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.
This HP 1090 Chromatograph is also equipped with an oven in the column compartment. The
function of the oven is to provide an homogeneus air-bath temperature when it is required for
some methods, such as the carbohydrate separation method which requires a constant
temperature of 85ºC.

Principle:
In isocratic HPLC the analyte is forced through a column of the stationary phase (usually a tube
packed with small round particles with a certain surface chemistry) by pumping a liquid (mobile
phase) at high pressure through the column. The sample to be analyzed is introduced in a 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 and acetonitrile). 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 reverse 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; whereas the more hydrophilic compounds will elute under conditions of relatively low
methanol. 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 optimum HPLC method for the analyte - the
method which gives the best separation of peaks.

Instrumentation for HPLC

Each contemporary hplc instrument consists of a pump for solvent delivery, an injector device
for sample introduction, column(s) for sample separation, detector(s) for visualization of the
separated components (solutes), and a computer for system control and data acquisition and
reduction, as depicted in Figure 1. Precise temperature control of columns and some other parts
of a chromatographic system becomes an important prerequisite for a successful separation.
Solvent Delivery
The function of the solvent delivery system is to deliver the mobile phase (eluent) through the
chromatograph, accurately and reproducibly. The most popular are reciprocating-piston pumps,
usually with two pump heads containing two sets of moving parts: the check valves and seal-
piston assembly. For analytical chromatography, such pumps can deliver liquid at a flow rate
from 100 μL/min up to 10 mL/min. Microscale capillary chromatography needs syringe (positive
displacement) pumps with capability to produce a flow rate as low as 1 μL/min, while
preparative chromatography utilizes flow rates up to 50 mL/min. Gradient pumping system is
capable of deliveringn more than one solventduring an analysis with variable mobile phase
composition. The blending of the solvents can occur in one of two ways: high pressure mixing or
low pressure mixing. In the former case the solvents are mixing on the injector side, while in the
latter case the mixing takes place at atmospheric pressure and a single high pressure solvent
delivery system is used to pump the mixture. Accurate and reproducible eluent flow rate and
composition are important prerequisites for successful separation and detection. This is achieved
in contemporary “intelligent” pumps through completely independent, digitally controlled piston
drives. The sophisticated, software-driven mechanism produces pulse-free eluent delivery,
compensates for changes in eluent viscosity, and automatically purges any gaseous mobile phase,
thereby enhancing performance for both isocratic and gradient applications (2). The quality of
solvents affects the accuracy of flow and baseline noise. On-line solvent filtering and degassing
become the necessary elements of any commercial hplc system.

Sample Introduction
To minimize the dispersion and broadening of peaks, sample solution must be injected as a sharp
plug with minimal interruption of flow. Typically, two-position six-port valves are used for
sample injection, which may be operated manually or automatically (such as in an autosampler,
where the sample is introduced from a sample vial held in an injection station). While the general
intention is to keep the injection volume as small as possible, the limits are imposed by column
dimensions, the sensitivity of detectors, and the nature of separation. An additional factor is the
concentration of the analyte, which for high molecular weight species may often be selected very
low to reduce solution viscosity and avoid intermolecular interaction.

Stationary and Mobile Phases

Chromatographic columns provide the means for polymer fractionation or separating mixtures
into components. The selectivity, capacity, and efficiency of the columns are all affected by the
nature of the packing material. Most modern hplc packings are porous spherical microparticles
with 3- to 10-μm size. The efficiency of a column increases with decreasing particle size, but so
does the backpressure, which usually cannot exceed 27.2 MPa (4000 psi) on traditional
instruments. The use of new generation of ultra-high pressure chromatographs with backpressure
up to 690 MPa (100,000 psi) can potentially reduce the particle size up to 1 μm or lower. In
general, silica-based columns can withstand higher pressure as compared with resin-based
columns. Pores (mean diameter usually from 6 to 100 nm) play a crucial role in hplc, as they
provide the surface where retention and hence separation occur. The chemical structure of the
surface determines the thermodynamic “environment” for the
analyte in thin surface layer (stationary phase) and in such a way determines the mechanism of
retention. Flexible macromolecules approaching the internal surface of a solid particle change
their spatial conformations because of steric interaction, which plays an important role in any
mode of polymer chromatography. This interaction restricts thefluctuation motion of a
macromolecule, decreases its conformation entropy, and effectively reduces the retention. In sec,
where particles with inert surface are used and the size of macromolecules is comparable with
the internal pore diameter, steric interaction is dominant and separation occurs according to size
of macromolecules. All other modes of polymer chromatography also exploit enthalpic
(attraction) specific interactions between macromolecules and the functional groups on the
surface. For this reason they are often lumped together as interaction polymer chromatography
(ipc). The name “adsorption polymer chromatography” is also used because the size of
macromolecules usually far exceeds the size of the surface functional groups participating in
interaction, and retention has the adsorption mechanism.

The nonsteric interactions in ipc depend on the chemical structure of the analyte, and also on
nature of stationary and mobile phases. In normal- or reversedphase hplc, neutral solutes are
separated on the basis of their polarity. In the former case, polar stationary phases are employed
(eg, bare silica with polar silanol groups) and less polar mobile phases based on nonpolar
hydrocarbons are used for elution of the analytes. Solvent selectivity is
 Fig. Instrumentation of HPLC

controlled by adding a small amount of a more polar solvent, such as 2-propanol or acetonitrile
or other additives with large dipole moments (methylene chloride and 1,2-dichloroethane),
proton donors (chloroform, ethyl acetate, and water), or proton acceptors (alcohols, ethers, and
amines). Correspondingly, the more polar the solute, the greater is its retention on the column,
yet increasing the polarity of the mobile phase results in decreased solute retention. The opposite
situation takes place for reversed-phase separation, when nonpolar stationary phase is
accompanied by a polar mobile phase, typically water, to which a less polar solvent such as
acetonitrile or methanol is added, and solvent selectivity is controlled by the nature and amount
of the added solvent. As a result, a decrease in the polarity of the mobile phase leads to a
decrease in solute retention. Many organic polymers are not soluble in water, and other polar
solvents can
be used. Thus, the combination of acetonitrile as an initial solvent with addition of
tetrahydrofuran (THF) can be used in reversed-phase separation of many styrenebased
copolymers and acrylic polymers. Modern reversed-phase chromatography typically refers to the
use of chemically bonded stationary phases, where functional groups, such as alkyl ( CH3,
C4H9, C8H17, and C18H37) groups, phenyl ( C6H5) groups, cyano [ (CH2)3CN] groups, and
amino [ (CH2)3NH2)] groups, are bonded to the silica. Note that some of these bonded packings,
such as those with cyano- and amino-groups, as well as diol groups
[ (CH2)3OCH2CH(OH)CH2OH], can also be used in normal-phase separations, as they contain
moderately polar moieties. The same stationary phases are also used for separation of
biomolecules in hydrophobic-interaction chromatography. Weak nonionic interactions between
hydrophobic portions of proteins and nonpolar moieties of the stationary phase have an entropic
nature and are highly sensitive to salt concentration in a totally aqueous, buffered mobile phase
of high ionic strength. In ion-exchange chromatography, the ionic species, such as proteins or
synthetic polyelectrolytes, are separated on the basis of differences in electric charge. The
stationary phases with ionic groups (fixed ions) are employed, and the mechanism of retention is
the electrostatic attraction of ionic solutes in solution to opposite charged ions (anions or cations)
on the stationary phase support. The stationary phase for ion-exchange chromatography is called
ion exchanger and is classified as an anion-exchange material when the fixed ion carries a
positive charge, and as a cation exchanger in the opposite case. There are strong and weak ion
exchangers depending on the pH range over which the stationary phase can retain the charge on
the fixed ion. Acrylic-based copolymers treated with an appropriate reagent to produce the
desired functional group are the most used ion exchanger because of wider pH range (pH 1–13)
and higher ion-exchange capacity as compared with silica-based columns (pH 2–8). Eluents for
ion-exchange chromatography are aqueous solutions of salts with small addition of organic
solvent.
Selectivity in the separation of polyelectrolytes may be varied by changing either the pH of the
mobile phase or the nature or concentration of the displacing (competing) ions.
Chromatography, SEC is used for the separation of biomolecules on the basis of the lock and-key
mechanism prevalent in biological systems.

Detectors
When separated macromolecules leave the column, they are detected by one or more on-line
electrical devices with signals proportional to the concentration of the analyte. In all hplc
detectors, the eluent flows through a measuring cell, where the change of a physical or chemical
property with elution time is detected. Each monitored trace represents a chromatogram: a set of
chromatographic peaks separated by baseline regions. The sensitivity (the ratio of peak height to
the sample concentration), signal-to-noise ratio (the amount of signal visible above the baseline
noise), as well as linear dynamic range (maximum linear response divided by the detector noise)
are the most important characteristics of any detector. Ultraviolet (uv) absorption photometers
capable of absorbing light at a selected wavelength (in the range 190–350 nm for uv detectors, or
190–700 nm for uv/Visible detectors) are the most popular detectors in hplc of both synthetic
and biological polymers. In case of proteins, wavelengths that are typically monitored include
the regions of absorbance of peptide bonds (210–220 nm), and tyrosine and tryptophan side
chains (ca 280 nm). Many synthetic polymers also contain chromophoric groups. Variable-
wavelength and photodiode array detectors have the advantage that more than one wavelength
can be monitored during a single run. Photodiode array detectors allow for simultaneous
measurement of an entire uv spectrum at any point of the chromatogram, which can aid in peak
identification. Among other selective (solute property) detectors used in hplc of polymers are
fluorescence and IR photometers. For example, fluorescence detectors can provide higher
sensitivity, as well as greater selectivity, in the detection of proteins containing tyrosine and
tryptophan residues that have intrinsic fluorescence. Both types of detectors are limited to certain
mobile phases. A good alternative is offline coupling of ftir to hplc system using an evaporative
interface (3). In such a system, the eluent is sprayed onto a Germanium disc, which can be
transferred to any ftir spectrometer to yield the full spectrum information over any peak of the
chromatogram. The same principle is applied also to the matrix-assisted laser
desorption/ionization mass spectrometer, which otherwise is difficult to couple to the hplc
system (4). A few universal (bulk property) detectors are of frequent use in hplc of polymers.
Refractive index detector is limited to isocratic elution and is utilized primarily in sec. The same
is true for the recently introduced density detector based on mechanical oscillator principle (B.
Trathnigg in Ref. 4). Evaporative light scattering detector (ELSD) becomes very popular in
nonaqueous gradient elution of synthetic copolymers and polymer blends (3). The eluent is
nebulized, and the solvent is evaporated from the droplets. Each droplet containing nonvolatile
material will form a particle, which scatters the light while crossing a light beam. This detector
has significant advantages over the uv detector for ability to work with aliphatic polymers
without chromophore groups, and also for its applicability to practically any mobile phase which
does not contain a nonvolatile buffer. Despite these obvious merits, ELSD cannot be considered
as an ultimate choice for quantitation because of nonlinear concentration response, which
depends on numerous factors such as chemical nature of the sample including its molar mass and
chemical composition, the eluent composition, viscosity and surface tension, and operating
conditions. Coupling ELSD to another detector can help to solve these problems with
quantitation.
The quadrupole mass spectrometers, single-quadrupole (MS) or triplequadrupole (MS–MS),
have proved to be the detectors of choice for interfacing with chromatographs. The resulting
combinations LC–MS or LC–MS–MS have become very popular among protein scientists. Soft
ionization methods, such as electrospray ionization and matrix-assisted laser desorption, are used
extensively for biopolymers and some ionic synthetic polymers (5). The interface usually
requires the use of very low flow rates (below 10 μL/min) and therefore necessitates capillary
chromatographic equipment, or a sample splitter. Miniaturization of separation with laboratory-
on-a-chip technology and microfluidics with nanoscale columns can additionally improve the
compatibility of column chromatography with mass spectrometry detection. In addition to the
widespread use of mass spectrometry as a detection technique, scientists report investigations of
other spectroscopic techniques such as nuclear magnetic resonance (nmr) and inductively
coupled plasma mass spectrometry. These emerging technologies in a few years may have a
major impact as online detection methods for characterizing both synthetic and biological
polymers. Miniaturization of separation makes these detectors more feasible for on-line
coupling. Using internal reflectance spectroscopy to study molecular interactions at phase
surfaces has helped researchers to understand retention mechanisms and to confirm or expand
chromatographic data.

Data Treatment
The traditional HPLC method involves two types of data analysis: qualitative (peak
identification) and quantitative (determining the analyte concentration). Both analyses require
specific data manipulations, such as matching retention times, peak integration and peak
calibration. The use of spectroscopic detectors (photodiode array, ftir, and mass spectrometry)
suggests additional mathematical procedures because of the necessity to manipulate with uv-,
ftir-, and MS-spectra and corresponding library matching (6). Most commercial chromatographic
software packages contain necessary functionalities and can be applied to hplc of proteins,
peptides, and DNAs. The characterization of other natural and all synthetic polymers is a much
more difficult problem, which includes calculating the distributions of different macromolecular
parameters, such as molecular mass, chemical composition, branching frequency and end group
functionality, from raw chromatograms. This involves additional calibration procedures (column
calibration), calculation of statistical moments of distributions, etc. Two-dimensional
chromatographic cross-fractionation of synthetic copolymers requires the construction of two-
dimensional distributions to characterize the copolymer heterogeneity (7).

Advantages
Quick Results
HPLC, a modern tool used to separate compounds based on their polarity, works much quicker
than its predecessors. Traditional column chromatography methods rely on the work of gravity to
separate compounds. With the advent of HPLC, compounds can be separated using a pressure
pump that forces compounds to separate extremely quickly--normally within a few minutes.

High-Resolution Results
Because HPLC works through the use of a pump that produces high pressures, the compounds
are well separated and easy to read--meaning that they have a high resolution. Other separation
methods that rely on gravity often result in low separation or lack of separation in some cases---
making reading results difficult.

Other advantages include:

 HPLC columns can be reused without repacking or regeneration

 Greater reproducibility due to close control of the parameters affecting the efficiency of
separation

 Easy automation of instrument operation and data analysis

 Adaptability to large-scale, preparative procedures

Disadvantages
It is difficult to detect coelution (two compounds escaping from the tubing at once) with HPLC,
which may lend to inaccurate compound categorization. There is a high cost for equipment
needed to conduct HPLC. Its operation can be complex, requiring a trained technician to operate.
Because of the speed of the process, the equipment has low sensitivity to some compounds.

Applications for HPLC

 Purification refers to the process of separating or extracting the target compound from
other (possibly structurally related) compounds or contaminants. Each compound should
have a characteristic peak under certain chromatographic conditions. Depending on what
needs to be separated and how closely related the samples are, the chromatographer may
choose the conditions, such as the proper mobile phase, to allow adequate separation in
order to collect or extract the desired compound as it elutes from the stationary phase.
The migration of the compounds and contaminants through the column need to differ
enough so that the pure desired compound can be collected or extracted without incurring
any other undesired compound.
--HPLC of Proteins and Polynucleotides

 Chemical Separations can be accomplished using HPLC by utilizing the fact that certain
compounds have different migration rates given a particular column and mobile phase.
Thus, the chromatographer can separate compounds (more on chiral separations) from
each other using HPLC; the extent or degree of separation is mostly determined by the
choice of stationary phase and mobile phase.

 Quantification of compounds by HPLC is the process of determining the unknown

concentration of a compound in a known solution. It involves injecting a series of known
concentrations of the standard compound solution onto the HPLC for detection. The
chromatograph of these known concentrations will give a series of peaks that correlate to
the concentration of the compound injected.

 Preparative HPLC refers to the process of isolation and purification of compounds.

Important is the degree of solute purity and the throughput, which is the amount of
compound produced per unit time. This differs from analytical HPLC, where the focus is
to obtain information about the sample compound. The information that can be obtained
includes identification, quantification, and resolution of a compound.

 Identification of compounds by HPLC is a crucial part of any HPLC assay. In order to

identify any compound by HPLC a detector must first be selected. Once the detector is
selected and is set to optimal detection settings, a separation assay must be developed.
The parameters of this assay should be such that a clean peak of the known sample is
observed from the chromatograph. The identifying peak should have a reasonable
retention time and should be well separated from extraneous peaks at the detection levels
which the assay will be performed. To alter the retention time of a compound, several
 parameters can be manipulated. The first is the choice of column, another is the choice of
mobile phase, and last is the choice in flow rate. All of these topics are reviewed in detail
in this document.

 Identifying a compound by HPLC is accomplished by researching the literature and by

trial and error. A sample of a known compound must be utilized in order to assure
identification of the unknown compound. Identification of compounds can be assured by
combining two or more detection methods.