CHAPTER FOUR
4. HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
 Although gas chromatography is widely used, it is limited
to samples that are thermally stable and not easily
volatilized.
 Nonvolatile samples, such as peptides and carbohydrates,
can be analyzed by GC, but only after they have been
made more volatile by a suitable chemical derivatization.
 For this reason, the various techniques included within
the general scope of liquid chromatography are among the
most commonly used separation techniques.
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 Liquid chromatography is a separation technique which uses liquid as
mobile phase and that involves the placement (injection) of a small
volume of liquid sample into a tube packed with porous particles
(stationary phase) where individual components of the sample are
transported along the packed tube (column) by a liquid moved by
gravity.
 It includes all planar chromatographic techniques.
 The components of the sample are separated from one another by
the column packing that involves various chemical and/or physical
interactions between their molecules and the packing particles.
 The separated components are collected at the exit of this column
and identified by an external measurement technique, such as a
spectrophotometer that measures the intensity of the color, or by
another device that can measure their amount.
 HPLC is among the most popular liquid chromatographic techniques.
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 High-performance liquid chromatography, HPLC, is a type
of chromatography that employs a liquid mobile phase and
a very finely divided stationary phase.
 To obtain satisfactory flow rates, the liquid must be
pressurized to several hundred or more pounds per square
inch.
 Based on its usage of high pressure for pushing the mobile
phase as well as sample components it is sometimes
called as high pressure liquid chromatography.
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4.1. Principles of HPLC
 In high-performance liquid chromatography (HPLC), a liquid sample,
or a solid sample is dissolved in a suitable solvent and carried through
a chromatographic column by a liquid mobile phase.
 It is a technique for the separation of components of mixtures by
differential migration through a column containing a microparticulate
solid or liquid, which is coated on the wall of column, stationary
phase.
 Solutes are transported through the column by a pressurized flow of
liquid mobile phase, and are detected as they are eluted.
 Separation is determined by solute/stationary-phase interactions,
including liquid–solid adsorption, liquid–liquid partitioning, ion
exchange and size exclusion, and by solute/mobile-phase
interactions. In each case, however, the basic instrumentation is
essentially the same except nature of packing of column.
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4.2. Instruments for HPLC
A common high-performance liquid chromatography
consists of the following five major components:
 Solvent delivery system
 Sample injection valve
 Column
 Detection and recording system
 Microcomputer with control and data-processing software
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Figure 4.1. Schematic diagram of a high-performance liquid chromatograph (HPLC)
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A. Mobile Phases
 The mobile phase or eluent, is either a single solvent or a blend of two or
more having the appropriate eluting power and resolution for the sample
components.
 These are determined by its overall polarity, the polarity of the stationary
phase and the nature of the sample components
 Unlike a GC carrier gas, which plays no part in chromatographic retention
and selectivity, the composition of an HPLC mobile phase is crucial in both
respects.
 For normal-phase separations (stationary phase more polar than mobile
phase), eluting power increases with increasing solvent polarity, whilst for
reversed-phase separations (stationary phase less polar than mobile
phase), eluting power decreases with increasing solvent polarity.
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Choosing Mobile phase:
 It ranges from a nonpolar liquid to aqueous buffers mixed
with an organic solvent.
 The mobile phases are aqueous solutions containing
methanol, water-miscible organic solvents and ionic
species, in the form of a buffer.
 Solvent strength and selectivity are determined by kind
and concentration of added ingredients and ions in this
phase which compete with analyte ions for the active site
in the packing.
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Properties of the mobile phase must be
 Dissolve the sample.
 Have a strong solvent strength leads to reasonable retention times.
 Interact with solutes in such a way that leads to selectivity.
 Beyond these criteria selection of mobile phase depends on the
polarity index, which is a quantitative measure of solvents polarity.
 Table 4.1. provides the polarity index, P’, of several commonly used
mobile phases, in which large values of P’ correspond to more polar
solvents. Mobile phase of intermediate polarity can be fashioned by
mixing two or more solvents in table 4.1.
 For example, a binary mobile phase made by combining solvents A
and B has the polarity index P’AB, of
P’AB = P’AῳA+ P’B ῳB
 Where P’A and P’B are polarity indices of solvent A and B, and ῳA and
ῳB are the volume fractions of the two solvents, respectively.
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Isocratic Versus Gradient elution
Isocratic elution
 mobile phase polarity stays constant throughout
elution process.
 use of a constant mobile phase composition to elute
solutes.
Gradient Elutions
 mobile phase composition varies throughout elution
time.
 mobile phase polarity varies throughout elution process
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 The elution order of solutes in HPLC is governed by
polarity.
 In a normal-phase separation the least polar solute
spends proportionally less time in the polar stationary
phase and is the first solute to elute from the column.
 Retention times are controlled by selecting the mobile
phase, with a less polar mobile phase leading to longer
retention times
 If, for example, a separation is poor because the solutes
are eluting too quickly, switching to a less polar mobile
phase leads to longer retention times and more
opportunity for an acceptable separation
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 When two solutes are adequately resolved, switching to a
more polar mobile phase may provide an acceptable
separation with a shorter analysis time.
 In a reverse-phase separation the order of elution is
reversed, with the most polar solute being the first to
elute.
 Increasing the polarity of the mobile phase leads to
longer retention times, whereas shorter retention times
require a mobile phase of lower polarity.
 An eluotropic series of solvents, which lists them in order
of increasing polarity, is a useful guide to solvent selection
for HPLC separations.
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Table 4.1. An eluotropic series of solvents for HPLC
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B. HPLC Columns
 HPLC typically includes two columns which are an
analytical column responsible for the separation and a
guard column.
 The guard column is placed before the analytical column,
protecting it from contamination.
 Guard Columns: Two problems tend to shorten the
lifetime of an analytical column.
 First, solutes binding irreversibly to the stationary phase
degrade the column’s performance by decreasing the
available stationary phase.
 Second, particulate material injected with the sample
may clog the analytical column.
 To minimize these problems, a guard column is placed
before the analytical column.
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 It is also used to remove particulate matter and
contamination, protect analytical column and control the
temperature to be: < 150 °C.
 Guard columns usually contain the same particulate
packing material and stationary phase as the analytical
column, but are significantly shorter and less expensive;
a length of 7.5 mm and a cost one-tenth of that for the
corresponding analytical column.
 Because they are intended to be sacrificial, guard
columns are replaced regularly.
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 Analytical Columns: This is a column that true separation
is takes place.
 The most commonly used columns for HPLC are
constructed from stainless steel with internal diameters
between 2.1 mm and 4.6 mm, and lengths ranging from
approximately 30 mm to 300 mm.
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Stationary Phases
 In liquid–liquid chromatography the stationary phase is a
liquid film coated on a packing material consisting of 3–10
µm porous silica particles.
 HPLC stationary phases are predominantly chemically-
modified silicas, unmodified silica or cross-linked co-
polymers of styrene and divinyl benzene.
 The surface of silica is polar and slightly acidic due to the
presence of silanol (Si-OH) groups.
 It can be chemically modified with reagents, such as
chlorosilanes, which react with the silanol groups
replacing them with a range of other functionalities.
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 The resulting bonded phases, which are hydrolytically
stable through the formation of siloxane (Si-O-Si-R)
bonds, have different chromatographic characteristics
and selectivities to unmodified silica.
 The properties of a stationary phase are determined by the nature of
the organosilane’s alkyl group.
 If R is a polar functional group, then the stationary phase will be
polar. Examples of polar stationary phases include those for which R
contains a cyano (–C2H4CN), diol (–C3H6OCH2CHOHCH2OH), or amino (–
C3H6NH2) functional group.
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 The combination of a polar stationary phase and a
nonpolar mobile phase is called normal-phase
chromatography
 In reverse-phase chromatography, which is the more
commonly encountered form of HPLC, the stationary
phase is nonpolar and the mobile phase is polar.
 The most common nonpolar stationary phases use an
organochlorosilane for which the R group is an n-octyl (C8)
or n-octyldecyl (C18) hydrocarbon chain.
 Most reverse phase separations are carried out using a
buffered aqueous solution as a polar mobile phase.
 Liquid chromatography using a nonpolar stationary phase
and a polar mobile phase is called reverse-phase
chromatography.
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Table 4.2. Stationary phases for HPLC
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C. HPLC Detectors
 Detection systems are used to detect solutes.
 Unlike gas chromatography, there is no universal
detector, a detector which responds to most types of
solutes.
 Many types of detectors have been investigated, and the
most widely used detectors for HPLC are:
 Absorbance (UV with Filters, UV with Monochromators and
Photo-Diode Array), IR Absorbance, Fluorescence,
Refractive-Index, Evaporative Light Scattering Detector
(ELSD), Electrochemical, Mass-Spectrometric detector.
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Ideally, detectors should have the following characteristics:
 a rapid and reproducible response to solutes that is
independent on flow rate;
 high sensitivity, i.e. able to detect very low levels of
solutes;
 Good stability in operation;
 a signal directly proportional to solute concentration o
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 insensitivity to changes in temperature and flow rate;
 high reliability and ease of use and
 non-destructive for the sample.
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Table 4.3. Common Liquid Chromatographic Detectors
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4.3. Modes of HPLC
 Modes of HPLC are defined by the nature of the stationary
phase, the mechanism of interaction with solutes, and the
relative polarities of the stationary and mobile phases.
 Almost any type of solute mixture can be separated by
HPLC because of the wide range of stationary phases
available, and the additional selectivity provided by
varying the mobile phase composition.
 Both normal-and reversed phase separations are possible,
depending on the relative polarities of the two phases.
 Although these are sometimes referred to as modes of
HPLC, the nature of the stationary phase and/or the
solute sorption mechanism provide a more specific means
of classification, and modes based on these and the types
of solutes to which they are best suited are summarized
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4.3.1. Adsorption HPLC
 In adsorption chromatography, the stationary phase is the
surface of a finely divided polar solid.
 With such a packing, the analyte competes with the
mobile phase for sites on the surface of the packing, and
retention is the result of adsorption forces.
 Separations are usually normal-phase with a silica gel
stationary phase and a mobile phase of a nonpolar solvent
blended with additions of a more polar solvent to adjust
the overall polarity or eluting power, e.g. n-hexane +
dichloromethane or di-ethyl ether.
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 Solutes are retained by surface adsorption; they compete with
solvent molecules for active silanol sites (Si-OH), and are eluted in
order of increasing polarity.
 In general, the polarities of common organic functional groups in
increasing order are:
 Aliphatic hydrocarbons < olefins < aromatic hydrocarbons < halides <
sulfides < ethers < nitro compounds < esters = aldehydes = ketones <
alcohols = amines < sulfones < sulfoxides < amides < carboxylic acids <
water.
 This mode is not used extensively, but is suitable for mixtures of
structural isomers and solutes with differing functional groups.
 Members of a homologous series cannot be separated by adsorption
chromatography because the nonpolar parts of a solute do not
interact with the polar adsorbent surface.
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4.3.2. Partition HPLC (Partition chromatography)
 Partition chromatography is the most widely used type of HPLC.
 In partition chromatography, liquids are used as the stationary phase
which is immiscible with the liquid mobile phase.
 The stationary phase may be adsorbed on the surface of a silica or
alumina support materials or chemically bonded with silica.
Partition chromatography can be classified based on two criteria:
A. the means by which the stationary phase is attached with support
materials
B. the polarities of the mobile and stationary phases
 A. Based on the first criteria partition chromatography can be divided
into two:
 1. Liquid-liquid partition chromatography
 2. Modified or bonded phase partition chromatography (liquid-
bonded phase chromatography
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 In Liquid-liquid partition chromatography, the liquid
stationary phase is adsorbed on the surface of the support
materials, silica or alumina.
 In Modified partition or bonded-phase chromatography
(BPC) that includes, most HPLC stationary phases that are
chemically-modified silicas, or bonded phases, by far the
most widely used being those modified with nonpolar
hydrocarbons.
 The liquid stationary phase is chemically bonded with
silanol group.
 The solute sorption mechanism is described as modified
partition, because, although the bonded hydrocarbons are
not true liquids, organic solvent molecules from the
mobile phase form a liquid layer on the surface
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 The preparation of bonded phase stationary phase
involves the following step:
 Si-OH is the silanol group i.e. support material.
 The R group will determine the polarity of the stationary
phase.
 The most popular phase is octadecyl (C18 or ODS), and
most separations are reversed-phase, the mobile phase
being a blend of methanol or acetonitrile with water or
an aqueous buffer.
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B. Based on the polarities of the mobile and stationary phase
 Partition chromatography can be divided into two based
on the polarities of the mobile and stationary phase.
 Normal phase partition chromatography
 Reversed phase partition chromatography
 In normal phase partition chromatography, the mobile
phase is nonpolar and the stationary phase is polar.
Nonpolar solutes have weak interaction and polar solutes
have strong interaction with stationary phase.
 The elution order is that nonpolar solutes elute first and
polar solutes elute late.
 Gradient elution can be carried out by increasing the
polarity of the mobile phase.
.
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 The elution order is that nonpolar solutes elute first and
polar solutes elute late.
 Gradient elution can be carried out by increasing the
polarity of the mobile phase.
 In reversed phase partition chromatography, the mobile
phase is polar and stationary phase is nonpolar.
 Polar solutes have weak interaction with the stationary
phase while nonpolar solutes have strong interaction with
stationary phase.
 The elution order is that polar solutes elute first and
nonpolar solutes elute late.
 Gradient elution can be carried out by decreasing the
polarity of the mobile phase.
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4.3.3 Ion-exchange chromatography
 Ion-exchange resins are used as stationary phases for
liquid chromatography (ion-exchange chromatography) to
separate charged species.
 Ion-exchange chromatography (IEC) stationary phases
for the separation of mixtures of ionic solutes, such as
inorganic cations and anions, amino acids and proteins,
are based on either microparticulate ion-exchange resins,
which are cross-linked co-polymers of styrene and divinyl
benzene, or on bonded phase silicas.
 Both types have either sulfonic acid cation-exchange sites
(-SO3
-H+) or quaternary ammonium anion-exchange sites (-
N+R3OH-) incorporated into their structures.
 In most cases, conductivity measurements are used to
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 In this column or chromatography 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.
 Ion exchange chromatography involves the separation
of ionizable molecules based on their total charge
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 This technique enables the separation of similar types of
molecules that would be difficult to separate by other
techniques because the charge carried by the molecule of
interest can be readily manipulated by changing buffer pH.
 Strong ion exchangers are often preferred resins for many
applications because their performance is unaffected by
pH.
 However, weak ion exchangers can be powerful separation
tools in cases where strong ion exchangers fail because
the selectivities of weak and strong ion exchangers often
differ .
 Depending on the pH of their environment, proteins may
carry a net positive charge, a net negative charge, or no
charge.
 The pH at which a molecule has no net charge is called its
isoelectric point, or pI.
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Figure 4.2. Charges in exchanger resign and solutes of interest
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 The counter ions to these fixed charges are mobile and
can be displaced by ions that compete more favorably for
the exchange sites.
 Ion-exchange resigns are divided into four categories:
strong acid cation exchangers, weak acid cation
exchangers, strong base anion exchangers and weak base
anion exchangers.
 The ion-exchange reaction of a monovalent cation, M+, at
a strong acid exchanger site is:
-SO3
-H+
(s) + M+
(aq) -SO3
- M+
(s) + H+
(s)
Al3+ > Ba2+ > Pb2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+ > Ag+ > K+ > NH4
+ > Na+ > H+ > Li+
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 Note that highly charged ions bind more strongly than ions
of lower charge.
 Within a group of ions of similar charge, those ions with
small hydrated radius or those that are more polarizable
bind more strongly.
 For a strong base anion exchanger the general order is:
 SO4
2- > I- > HSO4
- > NO3
- > Br- > NO2
- > Cl- > HCO3
- > CH3COO-
> OH- > F-
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4,3.4.Molecular (Size) Exclusion Chromatography
 Size- exclusion, or gel, chromatography is the newest of
the liquid chromatographic procedures.
 This is suitable for mixtures of solutes with relative
molecular masses (RMM) in the range 102–108 Da.
 Stationary phases are either microparticulate cross-linked
co-polymers of styrene and divinyl benzene with a narrow
distribution of pore sizes, or controlled-porosity silica
gels, usually end-capped with a short alkyl chain reagent
to prevent adsorptive interactions with solutes.
 Exclusion is not a true sorption mechanism because
solutes do not interact with the stationary phase
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They can be divided into three groups:
 Those larger than the largest pores are excluded
completely, and are eluted in the same volume as the
interstitial space in the column, Vo.
 Those smaller than the smallest pores, can diffuse
throughout the entire network and are eluted in a total
volume, Vtot.
 Those of an intermediate size separate according to the
extent to which they diffuse through the network of
pores, of volume Vp and are eluted in volumes between Vo
and Vtot.
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 In size- exclusion chromatography, fractionation is based on size of
the molecules.
 There are two commonly known packings for size- exclusion
chromatography:
 Gel filtration is a type of size- exclusion chromatography in which
the packing is hydrophilic and is used to separate polar species.
 Gel permeation is a type of size- exclusion chromatography in which
the packing is hydrophobic. It is used to separate nonpolar species.
 In size exclusion the HPLC column is consisted of substances which
have controlled pore sizes and is able to be filtered in an ordinarily
phase according to its molecular size.
 Small molecules penetrate into the pores within the packing while
larger molecules only partially penetrate the pores (Figure 4.3).
 The large molecules elute before the smaller molecules.
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 Size exclusion chromatography (SEC), also called gel
filtration chromatography, separates molecules based on
their sizes.
 SEC resins are gels that contain beads with a known pore
size.
 When dissolved molecules of various sizes flow into
the column, smaller dissolved molecules flow more
slowly through the column because they penetrate
deep into the pores, whereas large dissolved molecules
flow quickly through the column because they do not
enter the pores.
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Figure 4.3. Working principle of molecular (size) exclusion chromatography
SEC is of particular value in characterizing polymer mixtures and in separating biological
macromolecules such as peptides and proteins. It is also used for preliminary separations prior
to further analysis by other more efficient modes of HPLC.
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 To summarize, the chromatographer will choose the best
combination of a mobile phase and particle stationary
phase with appropriately opposite polarities.
 Then, as the sample analytes move through the column,
the rule like attracts like will determine which analytes slow
down and which proceed at a faster speed.
 After selection of an appropriate mode, column and detector
for the solutes to be separated, the composition of the mobile
phase must be optimized to achieve the required separation.
 A trial and error approach or a computer aided investigation
can be adopted.
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Summary of major components of HPLC and their function
1. Pump: The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate, expressed in milliliters
per min (mL/min).
 Normal flow rates in HPLC are in the 1- to 2-mL/min range.
 Typical pumps can reach pressures in the range of 6000-9000 psi (400- to 600-
bar).
 During the chromatographic experiment, a pump can deliver a constant
mobile phase composition (isocratic) or an increasing mobile phase
composition (gradient).
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 2. Injector: The injector serves to introduce the liquid sample into the flow
stream of the mobile phase.
 Typical sample volumes are 5- to 20-microliters (µL).
 The injector must also be able to withstand the high pressures of the liquid
system.
 An autosampler is the automatic version for when the user has many samples
to analyze or when manual injection is not practical
3. Column: Considered the “heart of the chromatograph” the column’s
stationary phase separates the sample components of interest using various
physical and chemical parameters.
 The small particles inside the column are what cause the high backpressure at
normal flow rates.
 The pump must push hard to move the mobile phase through the column and
this resistance causes a high pressure within the chromatograph.
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 4. Detector: The detector can see (detect) the individual
molecules that come out (elute) from the column.
 A detector serves to measure the amount of those
molecules so that the chemist can quantitatively analyze
the sample components.
 The detector provides an output to a recorder or
computer that results in the liquid chromatogram (i.e.,
the graph of the detector response).
 5. Computer: Frequently called the data system, the
computer not only controls all the modules of the HPLC
instrument but it takes the signal from the detector and
uses it to determine the time of elution (retention time)
of the sample components (qualitative analysis) and the
amount of sample (quantitative analysis).
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Application of HPLC
 High performance liquid chromatography is now one of
the most powerful tools in analytical chemistry.
 It has the ability to separate, identify, and quantitate the
compounds that are present in any sample that can be
dissolved in a liquid.
 Today, compounds in trace concentrations as low as parts
per trillion (ppt) may easily be identified.
Fields in which HPLC is used
 HPLC can be, and has been, applied to just about any
sample, such as pharmaceuticals, food, cosmetics,
environmental matrices, forensic samples, and industrial
chemicals.
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 Biogenic substances like; Sugars, lipids, nucleic acids,
amino acids, proteins, peptides, steroids, amines, etc.
 Medical products for drugs, antibiotics, etc.
 Food products like; vitamins, food additives, sugars,
organic acids, amino acids, etc.
 Environmental samples; for analysis of inorganic ions,
hazardous organic substances, etc.
 Organic industrial products like; synthetic polymers,
additives, surfactants, etc.
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