High performance liquid chromatography

(HPLC)
An illustration of an HPLC instrument

Guard column

Pump Column Detector

(Stationary Phase)
Sample
Injection
Port

Solvent container Chart recorder

(Mobile Phase) or data processing
system

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 HPLC column
 Are made from stainless steel
tubing which is chemically inert
and stable to high pressure
 Typical length 30-300 mm and
inside diameter of 2.1-4.6 mm
 Internal surface should be smooth
through the length to prevent
irregularities which cause tunneling
of mobile phase and band
broadening
 Typical packing range in size from
3-10µ m porous silica particles
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Most common type of HPLC
 Partition chromatography
 Liquid-liquid partition chromatography
 Adsorption chromatography
 Liquid-solid adsorption chromatography

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Partition Chromatography
 Partition chromatography procedures can be subdivided
into two:
 liquid-liquid chromatography
 bonded-phase chromatography
 In liquid-liquid chromatography, the stationary phase is
a thin film coated on a packing material consisting of 3-
10 µm porous silica particles
 The stationary phase may be partially soluble in the
mobile phase causing it to be eluted from the column
and exposing some of the hydroxyl group on the silica
particles

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Liquid-liquid partitioned chromatography
 SP is coated on solid support of silica particles by
physical adsorption but this faced with numerous
problems:

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Bonded phase
 The loss of stationary phase can be prevented
by bonding it to the silica particles
 In the bonded-phase, the stationary phase is
bonded chemically to the support surfaces or
silica particles
 The common chemical used is an
organochlorosilane of the general formula
Si(CH3)2RCl

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A fully hydrolyzed surface of silica particle

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Column for bonded-phase chromatography

Coating with siloxanes

CH3 CH3
  
Si—OH + Cl—Si—R → Si—O— Si—R + HCl
  
CH3 CH3

Silica surface Organo-chlorosilane Siloxane

(With –OH)

R – alkyl group

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Silanization with dimethylchlorosilane
(DMCS) of support material

Organo-chlorosilane
(DMCS)

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The support is washed with methanol to convert 2nd Cl
to methoxy group

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Bonded-phase
 The properties of a stationary phase are
determined by the nature of the R group on the
organosilane
 Bonded phase is stable to pH 2-9 and
temperature to 80oC
 Fairly common bonded phase is made of linear
C18 hydrocarbon

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Normal-phase and Reversed-phase
and
 If the R group in the siloxane structure is
a polar functional group such as the
cyano (-C2H4CN), diol, amino and
dimethylamino groups the stationary
phase will be polar
 The combination of a polar stationary
phase with a non-polar mobile phase is
called normal phase chromatography
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Reversed-phase and normal-phase
 In reversed-phase packing, the stationary phase is non-
polar
 Most common nonpolar stationary phases use an
organochlorosilane in which the R group is an n-octyl
(C8) or n-octylydecyl (C18)
 The mobile phase used in the reversed phase is polar
(such as water, methanol or acetonitrile)
 About 75% of all HPLCs use columns packed with
reversed-phase stationary phase

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Bonded phase (C18)

HPLC column

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Liquid-solid adsorption chromatography
 Retention of liquid-solid chromatography is due to
adsorption of solute molecules on a solid SP
 Polar molecules are held more strongly and eluted
more slowly than do non-polar molecules
 Polarity of organic molecules is determined by
nature of their functional groups
 Other factor such as molecular structure and steric
factors are also play important role

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Principal substances used as SP are:
 Silica & Alumina : Silica has a higher sample
capacity than alumina and can be prepared in several
forms
 Porous microparticles: most popular form for
analytical work
 Small particles 3-10 µ m diameter
 Contains high concentration of surface hydroxyl groups
which are responsible for solute retention
 The surface may be coated with thin organic films as
SP
 Large surface area 100-900 m2 /g

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Supporting particles for SP

Silica Powder

Porous microparticles

Alumina Powder
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Porous-layer or pellicular particles -
consists of spherical glass beads
 30-40 µ m diameter covered with porous layer of
adsorbent 1- 3 µ m
 Adsorbent may be silica or a collection of small spherical
particles bonded to the larger glass beads or a bonded
organic polymer

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Ion Exchange Chromatography (IEC
or IC)
 IC is used for the separation of inorganic ions (both
cations and anions)
 The separation is based on exchange of ions in the
stationary phase
 It is also useful for the separation of amino acids or
other charged organic molecules
 The stationary phase in IC consists of beads made of
polystyrene polymer cross-linked with divinylbenzene
 The cross-linked polymer (resin) has free phenyl
groups attached to the chain, which can easily be
treated to add ionic functional groups.

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Structure of cross-linked polystyrene ion-exchange resin

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There are 4 types of ion exchange resins used in analytical
chemistry as shown below:

Type of Exchange Functional Exchange group

Cation exchanger (-ve)

i )Strong acid Sulfonic acid --SO3 – H+
ii) Weak acid Carboxylic acid --CO2- H+
Anion exchanger (+ve)
i ) Strong base Quaternary Ammonium Ion--NR3+OH-
Amine Group --NH3+OH-
ii) Weak base

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Cation exchange resins
 These resins contain acidic functional groups
added to the aromatic ring of the resin
 The strong-acid cation exchange has a
sulphonic acid group --- SO3 - H+, which are
strong acids much like sulfuric acid
 The weak-acid cation exchange has a carboxylic
acid group, --CO2 - H+, which are only
partially ionized
 The protons (H+) on these groups can be
exchanged with other cations
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e.g.
--- SO3 - H+ + Na+ Cl- → --- SO3 - Na+ + H+ Cl-
cation exchange dil. Solution effuent

 In the above example, a dilute solution of sodium

chloride is passed through a cation exchange
column in the hydrogen ion form
 The H+ is exchanged for Na+ and effluent
composition includes dilute solution of HCl

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Anion Exchange resins
 Basic groups on the resin on which the hydroxyl anion can be
exchanged with other anion make up the anion exchange resins
 There are strong-base groups (quaternary ammonium group) and
weak-base groups (amine group)
 The exchange reactions can be represented by :

--NR3+OH- + Na+ Cl- → --NR3+Cl- + Na+ OH-

Anion Exchange dil. Sol. Effluent

 When a dilute solution of NaCl is passed through an anion

exchange, the composition of the effluent include a dilution solution
of NaOH

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Ion chromatography with eluent
suppressor column
 The presence of high concentration of
electrolyte required to elute most sample ions
supersede analyte ion signal
 This difficulty can be overcome by having a
second column to neutralise the electrolyte ions
 This column is placed immediately after ion-
exchange column
 The column is packed with second ion-
exchange resin to convert the ions of the mobile
phase to a molecular species of limited
ionisation without affecting the analyte ions
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Ion suppressor column

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e.g. HCl as mobile phase

R+OH- + (H+ + Cl-)aq + → R+Cl- + H2O

e.g. NaHCO-3 as mobile phase

R-H+ + (Na+ + HCO-3)aq → R-Na+ + H2CO3(aq)

 The products such as H2O and H2CO3 of ions

in the mobile phase permit detection of
analyte ions by conductivity detector
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The role of suppression in ion chromatography
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Mobile phases (M.P.)
 The elution order of solutes in HPLC is governed by polarity
 In normal phase separation:
 Least polar solute is the first to elute from the column
 Retention times are controlled by selecting the mobile phase
 A less polar mobile phase lead to longer retention times
 In reverse phase, the order of elution is reversed:
 Most polar solute is the first to elute from the column
 Increasing polarity of mobile phase lead to longer retention times
 The MP interact with solute components and have direct
influence on partition coefficients and solute retention on the
column

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Selection of the mobile phase
 Predicting the proper solvent is important and can be
done experimentally
 Both partition and adsorption chromatography
processes are based on differences in polarity of solute
 Partition process is sensitive to small molecular weight
differences in solutes and can be used to separate
members of homologous series
 Adsorption process is sensitive to steric effects and is
used for separation of similar compounds having
different steric configuration
 As a general rule, highly polar materials are best
separated by partition chromatography while very non-
polar material are separated by adsorption
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 Polarity of compounds

 Increasing of orders of polarity of compounds

are as follow:

Increasing polarity
Hydrocarbon
oxygenated hydrocarbon
proton donors
ionic compounds

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Some commonly used solvents used in HPLC in order of
increasing polarity
Mobile phase Polarity Index
n-Hexane 0.04
Carbon tetrachloride 1.6
Toluene 2.4
Diethyl ether 2.8 Increasing polarity
Tetrahydrofuran 4.0
Ethanol 4.3
Ethyl acetate 4.4
Methanol 5.1
Acetonitrile 5.8
Water 10.2
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 Supercritical fluid chromatography
(SFC)
 Basic principles are similar to GC or LC
 In this case the mobile phase is a
supercritical fluid
 It permits separation and determination of
group of compounds that are not
conveniently handled by either gas or
liquid chromatography

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Supercritical fluid chromatography
(SFC)
 These compounds :
i) Non-volatile or thermally labile
ii) No functional groups - detection by
spectroscopy or electrochemical not possible.
 Most common fluid used is CO2
 Supercritical fluid chromatography has been applied to a
wide variety of materials including natural products,
drugs, food, polymer etc

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Illustration of components for SFC

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Phase diagram for
CO2 supercritical fluid

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