Chapter 11
Column Liquid Chromatography
Types of Liquid Chromatography
•
•
•
•
Partition chromatography
Adsorption or liquid-solid
chromatography
Ion Exchange chromatography
Size exclusion or gel chromatography
Adsorption Chromatography
The stationary phase is solid. Separation is
due to adsorption/desorption steps
•Adsorbent can be
packed in a column
spread on a plate,
or impregnated in a
A porous paper
•Both solutes and
solvents will be
attracted to the
stationary phase
•If the solutes have
different degrees of
attraction
separation would be
achieved
Partition chromatography
Separation is based on solute partitioning
between two liquid phases
• More highly retained species have greater affinity
(solubility) for the stationary phase compared to the
mobile phase (solvent)
• Separation of solutes is based on the difference in
the relative solubilities
Modes of separation
• Normal phase partition chromatography
Polar stationary phase and nonpolar solvent
• Reverse phase partition chromatography
Nonpolar stationary phase and polar solvent
• Reverse phase is now more common
Changing the mode of separation will lead to a change
In the elution order of the solutes that could be almost
Reversed.
Ion exchange chromatography
• Stationary phase has charged surface opposite
that of the eluents
• Separation is based on the affinity of ions in solution for
oppositely charged ions on the stationary phase
Separation by ion exchange chromatography
Size exclusion chromatography
• Separation is based on molecular size. Stationary
phase is a material of controlled pore size. It is also
called Gel permeation chromatography
Size exclusion chromatography
• Columns are made to match the separation of
specific size ranges
• Larger species will elute first. They cannot pass
through many pores so their path is shorter
• Size exclusion liquid chromatog. Is useful for
determining size, size range and molecular weights
of polymers and proteins.
Separation by size exclusion chromatography
LC Solvents (Mobile phase)
• LC solvents depend upon the type of
chromatographic mode used:
* Normal Phase
* Reversed phase
Solvent selection
•
•
•
•
If the sample is water insoluble or nonpolar- normal phase mode is used
If the sample is water soluble or not soluble but polar- use the reverse phase
mode
It is seldom to find a single solvent does the job. Thus mixtures of two or more
solvents are used
Two factors are considered:
– Solvent strength, (o)
A measure of relative solvent polarity (ability to
displace a solute). It is the adsorption energy per unit area of
solvent. o for silica is about 0.8 of those on alumina
– Polarity index, (P’)
• Solvents that interact strongly with solutes are strong or polar solvents
• Polarity of solvents has been expressed by many terms, one of which is the
polarity index. Thus, the P’ value measures the relative polarity of various
solvents
Used for reversed phase methods
Solvent strength and polarity index
o
P’
Solvents used as mobile phase in liquid chromatography
Snyder classified solvents based on acidity and basicity,
dipole and chemical properties
Gradient elution
Advantages of gradient elution
Gradient elution
Gradient elution
Mobile Phases for adsorption chromatography
• Common mobile phases on alumina
hexane; chloroform; 2-propanol
Example: separation of amines
• Common mobile phases on Silica
hexane; chloroform; 2-propanol
Examples: separation of ethers, esters,
prophyrins, vitamins
Mobile Phases for partition chromatography
Ion exchange phases
High Performance Liquid
Chromatography
Column Liquid Chromatography
• LC techniques are: Classical LC and
HPLC or HSLC (S = speed)
• Both techniques have same basic
principle for separation but differ in
apparatus and practice used
• HPLC gives high speed, high
resolution, high sensitivity and
convenient for quantitative Analysis.
HPLC
originally refered to:
High Pressure Liquid Chromatography
currently refers to:
High Precision Liquid Chromatography
– the high pressure allows using small
particle size to allow proper separation at
reasonable flow rates
Features of HPLC compared to Classical LC
Particle size of the packing substance
• Classical LC utilises large porous particles that
make it difficult to speed up the flow rate by
pumping due to a decrease in resolution that results
from the mass transfer limitation in the deep pores.
These high capacity particles are good for
preparative chromatography
• Since the mass transfer coefficient is a function of
the square of dp (van deemter eq.) thus the HPLC
was based on using pellicular and porous
microparticles. Pellicular particles when packed into
narrow columns will lead to an increase in column
efficiency of 10 to 100 folds
• Pellicular particles have dense solid cores thus they
are easily packed
• Vs is significantly reduced and the sample capacity is
reduced to 0.05 to 0.1 of the totally porous packing
Effect of particle packing size on column efficiency
Column length
• Efficiency is very high due to packing thus
shorter columns are used (~ 20 cm)
• For difficult separations longer (50-100 cm)
are used with smallest available particles and
high pressure solvent feed
Effect of sample size on column efficiency
• Efficiency increases as the sample size
decreases
Effect of sample mass on column efficiency
Columns
• LC columns could be made of stainless steel, glass and glass
lined stainless steel that is used for extremely inert surfaces.
• In classical LC, elution takes place under gravity or low
pressure by using small pumps
• Columns can be thermostated by placing in an oven or using a
water jacket
• HPLC columns are mainly made of stainless steel packed with
the microparticles
• When same amount is injected in the HPLC column, narrower
and longer peaks are obtained that leads to greater detector
sensitivity
• In HPLC solvent consumption is reduced
• HPLC columns facilitate coupling to MS that requires flow rates
<50 L/min to avoid over pressuring the ion source in the MS
Components
Solvent Reservoir and Degassing System
Pumps
Precolumns
Sample Injection System
Columns
Temperature Control
Detectors
Readouts
Schematic diagram of a typical high performance liquid
chromatograph
Schematic of Liquid Chromatograph
Solvent Reservoir and Degassing System
– isocratic elution - single solvent
separation technique
– gradient elution - 2 or more solvents,
varied during separation
Improvement in separation
efficiency by gradient
elution. Column: 1m x
2.1mm id, precision-bore
stainless. Sample: 5L of
chlorinated benzenes in
isopropanol. Detector: UV
photometer (254 nm).
Conditions: temperature
60oC, pressure, 1200 psi.
Pumping systems
Requirements for pumping system
• Generation of pressure up to 6000 psi
• Pulse free output
• Flow rates of 0.1 to 10 ml/min
• Flow control and flow reproducibility of 0.5% relative or better
• Corrosion resistance components
Types of pumps:
• Direct pressure pumps (pneumatic pumps)
• Syringes (displacement pumps)
• Reciprocating pumps
They are limited to pressures less
Than 2000 psi
They are of limited solvent capacity (about 250 ml)
and inconvenience when the solvent is changed.
• They have small internal
volume (35-400 L)
• High output pressure (10000 psi)
• Readily adoptable to gradient
elution
• Constant flow rates that are
independent of column back
pressure and solvent viscosity
Widely used pumping; motor driven piston that pumps the solvent into
the chamber. The two ball check valves (f) Open and close alternatively
to control the solvent into and out of the chamber.
•It has the disadvantage of producing pulsed flow which must be damped
because its presence is manifested as baseline noise
Reciprocating pump
there
Sample Loop
Detectors
Properties of good detectors
Types of Detectors
• Absorbance (UV with Filters, UV with
Monochromators)
• IR Absorbance
• Fluorescence
• Refractive-Index
• Evaporative Light Scattering Detector
(ELSD)
• Electrochemical
• Mass-Spectrometric
• Photo-Diode Array
Ultraviolet detector
Refractive-Index Detector
Schematic of a differential refractive-index detector
Mass spectrometric detectors
• LC and MS appear to be incompatible.
HPLC
MS





Liquid phase operation
20-25 oC operation
Almost no sample limitation
Relatively inexpensive
Uses inorganic buffers
 Conventional flow rate
produce 550 ml/min gas at STP
Gas phase operation
100-350 oC operation
same volatility desired
Expensive
can’t tolerate inorganic
buffers
Accepts 10 ml/min
Features of LS-MS
• Its sensitivity approaches sub nanograms
• The significant problem here is 1 ml of hexane,
methanol, or water generates respectively, 180, 350
and 1250 ml/min gas
• Since MS operates under vacuum the sample vapor
must be removed without removing a substantial
amount of solute
LC-MS interface techniques
• Direct liquid inlet: small portion of LC effluent is directed into
the ion source
• Moving belt method: LC effluent is deposited onto a moving
belt, the solvent is evaporated, and the sample is volatilized
from the belt into the ion source
• Thermospray ionization: ions are created when aqueous
buffered mobile phase is passed through a heated stainless
steel capillary creating a supersonic jet of vapor with
subsequent evaporation of the mobile phase from charged
liquid droplets
Applications of Liquid Chromatography
Applications
•
•
•
•
•
•
Preparative HPLC -the process of isolation and purification of compounds.
analytical HPLC-obtain information about the sample compound.
– identification,
– quantification,
– resolution of a compound.
Chemical Separations - using HPLC
– certain compounds have different migration rates given a particular
column and mobile phase.
Purification - 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.
– choose the conditions, such as the proper mobile phase, to allow
adequate separation to collect or extract the desired compound as it
elutes from the stationary phase.
Identification of compounds by HPLC is a crucial part of any HPLC assay.
– accomplished by researching the literature and by trial and error.
– Identification of compounds can be assured by combining two or more
detection methods.
Quantification - the process of determining the unknown concentration of
a compound in a known solution.
– inject a series of known concentrations of the standard compound
solution
– chromatograph of these known concentrations
– peaks that correlate to the concentration of the compound injected
Adsorption chromatography
Adsorption chromatography
• It is LSC and oldest chromatographic method
introduced by Tswett that became the HPLC
technique
• Silica and alumina are mostly used as the stationary
phases in thin layer or column
• How do adsorbents separate compounds?
• Consider the surface of the most widely adsorbent,
silica gel….
• Silica gel is a stable porous solid terminated at the
surface with silanol (Si-OH) or siloxane (Si-O-Si)
bonds
• The slightly acidic silanol groups are of importance
in separation however, siloxane bonds are of little or
no influence
Most acidic
Silanol groups
Silanol groups and interactions with solutes
• Silanol groups have varying degrees of acidity
• The most acidic ones are located at adjacent silicon atoms with
intermolecular H-bonding. These lead to undesirable effects
like chemisorption and peak tailing
• To avoid problems, polar modifier such as water is added in
order to deactivate the strongest adsorption sites
• Interactions between adsorbent surface and solute vary from
nonspecific (dispersion or vander Waal’s forces) to specific
ones (electrostatic interactions such as permanent dipoles or
electron donor acceptor interactions such as hydrogen
bonding)
• Retention on silica gel or alumina is governed mainly by
interactions with the polar functional groups of the solute
• Compounds of different chemical types (hydrocarbons and
alcohols) are easily separated by LSC
• Homologous or other mixtures differing
in the extent of aliphatic substitution
(no change in polarity) cannot be
differentiated
• LSC is unique in its ability to separate
polyfunctional compounds especially
positional isomers
• With a few exceptions, the order of
retention times on silica and alumina
is:
olefins < aromatic hydrocarbons <
halides, sulfides < ethers < nitro
compounds < esters ~ aldehydes ~
ketones < alcohols amines < sulfones <
sulfoxides < amides < carboxylic acids
Intramolecular hydrogen
bonding; i.e, less intermolecular
interaction with the surface
Solvent Selection for Adsorption Chromatography
• In liquid-solid chromatography, the
only variable available to optimize the
retention factor and the selectivity
factor is the composition of the mobile
phase (in contrast to partition
chromatography, where the column
packing has a pronounced effect on the
selectivity factor)
Influence of the Mobile Phase in LSC:
Gradient Elution
• Interactions in LSC involve a competition between the solute
molecules (X) and the molecules (S) of the mobile phase
adsorption sites. This equilibrium is illustrated by
• Thus, stronger adsorption of the mobile
phase decreases adsorption of the solute.
• Solvents can be classified according to their
strength of adsorption (solvent or eluent
strength, o).
• Such a quantitative classification is referred
to as an eluotropic series.
• An eluotropic series can be used to find an
optimum solvent strength for a particular
separation.
• Using a solvent of constant composition is
called isocratic elution.
Solvent Strength
• The polarity index, P', used in partition chromatography can also
serve as a rough guide to the strengths of solvents for adsorption
chromatography.
• Solvent (Eluent) strength °, which is the solvent adsorption energy
per unit surface area is a much better index.
• This parameter depends upon the adsorbent. ° values for silica are
about 0.8 of those on alumina.
• Note that solvent-to-solvent differences in ° roughly parallel those
for P'.
• For a given isocratic elution, the initial solvent is selected by
matching the relative polarity to that of sample components.
• Solvent is chosen to match the most polar functional group.
Alcohols for –OH group and amines for amino acids.
• If in an isocratic elution the k'-values for the solutes are too small
(sample elutes rapidly) then a weaker (low °, less polar) solvent is
selected
• On the other hand, if the sample does not elute in a reasonable time
because of high k’ values then a stronger (high °, more polar)
solvent would be selected.
Choice of Solvent Systems (binary mixture)
• Binary solvent mixtures may be used to find an
optimum value of the solvent-strength parameter  °
• Two compatible solvents are chosen, one of which is
too strong (° too large) and the other is too weak. A
suitable value for k' is then obtained by varying the
volume ratio of the two.
• a mixture of isooctane (° = 0.01) and methylene
chloride (° = 0.42) can be matched with an isocratic
solvent strength similar to that of carbon
tetrachloride (° = 0.18).
• Unfortunately ° does not vary linearly (because of
solvent-solvent and preferential solvent-surface
interactions) with volume ratios. Thus, calculating an
optimal mixture is more difficult.
General Elution Problem
• This problem appears with isocratic solvent systems
and multicomponent samples with widely differing
k'-values.
• If a strong isocratic mobile phase is selected that will
adequately elute strongly retained compounds, then
the weakly retained ones will be eluted too quickly
and will be poorly separated
• Conversely, if a weak mobile phase is chosen, so
that weakly retained sample components will be
retained and separated, then very strongly retained
solutes may not be eluted at all-or only very slowly
• The most common solution is using a technique
called solvent programming or gradient elution.
Here, elution is begun with a weak solvent and the
solvent strength is increased with time. The changes
are made either stepwise or continuously
Applications of Adsorption Chromatography
 Adsorption chromatography is best suited for
nonpolar compounds having molecular weights less
than perhaps 5000.
 Although some overlap exists between adsorption
and partition chromatography, the methods tend to
be complementary.
 Generally, liquid-solid chromatography is best suited
to samples that are soluble in nonpolar solvents and
correspondingly have limited solubility in aqueous
solvents such as those used in the reversed-phase
partition procedure.
 A particular strength of adsorption chromatography,
which is not shared by other methods, is its ability to
differentiate among the components of isomeric
mixtures.
Partition Chromatography
(Liquid-liquid and Bonded phase Chromatography)
Features of Partition Chromatography
 Most of the applications have been to nonionic , polar
compounds of low to moderate molecular weight (usually
<3000).
 Recently,
however,
methods
have
been
developed
(derivatization and ion pairing) for separations to ionic
compounds.
 Partition chromatography can be subdivided into liquid-liquid
and bonded-phase chromatography. The difference in these
techniques lies in the method by which the stationary phase is
held on the support.
 With liquid-liquid, a liquid stationary phase is retained on the
surface by physical adsorption.
 With bonded-phase, the stationary phase is bonded chemically
to the support surfaces.
•
• Liquid-liquid chromatography is limited to compounds
with comparatively low values of K (or k'), because the
stationary phase must be a good solvent for the
sample but a poor solvent for the mobile phase.
• In practice, increasing solvent strength in order to
elute compounds with high K- (or k'-) values will
increase the solubility of the stationary phase and
remove the stationary phase from its support.
• When the solvent strength is high enough to dissolve
an appreciable amount of stationary phase,
presaturation is made difficult.
– in conventional LLC solvent programming is ruled out.
• Even with its limitations, LLC is a very useful
technique because it can resolve minute differences in
the solubility of the solute.
• Many solvent pairs are available, and the choice of the
proper ones allows great selectivity to be achieved.
• Both Paper chromatography and TLC are examples of
LLC.
Liquid-liquid chromatography
• The stationary and mobile phases are selected so as to have
little or no mutual solubility.
• Therefore, they generally are quite different in their solvent
properties. For example one might choose water as the
stationary phase and pentane as the mobile phase for normal
LLC.
– However, water does have a finite (though very slight)
solubility in pentane.
• Using pentane will slowly remove the water and change the
nature of the separation mechanism.
• For this reason, the mobile phase must be presaturated with
the stationary phase before it enters the column (or plate).
• Presaturation can be done by stirring the two phases together
until equilibration takes place; but, in LC, it is more
conveniently done by placing a precolumn before the injector
and the chromatographic column.
• The precolumn should contain a high-surface-area packing,
such as silica gel, coated with a high percentage (say 30 to 40%
by weight) of the stationary phase used in the analytical
column.
Drawbacks of LLC:
• finding immiscible solvent pairs,
• presaturing the mobile phase to avoid
removal of coated stationary phase,
• the impossibility of using gradient elution to
solve the general elution problem
Solution
• Use of chemically bonded stationary phases.
Bondedphase chromatography (BPC) now
dominates in use all modes of HPLC.
• Microparticulate silica gel is the base
material used for the synthesis of almost all
chemically bonded phases.
Bonded-Phase Chromatography
• The supports are prepared from rigid silica, or silica-based,
compositions.
• The surface of fully hydrolyzed silica (hydrolyzed by heat-ing
with 0.1 M HCl for a day or two) is made up of chemically
reactive silanol groups.
The most useful bonded-phase coatings are siloxanes
formed by reaction of the hydrolyzed surface with an
organochlorosilane
R:
alkyl group or a substituted alkyl group.
• The unreacted SiOH groups, unfortunately,
impart an undesirable polarity to the surface,
which may lead to tailing of chromatographic
peaks, particularly for basic solutes.
• To lessen this effect, siloxane packings are
frequently capped by further reaction with
chlorotrimethylsilane that, because of its
smaller size, can bond many of the unreacted
silanol groups.
• The -Si-O-Si-C bond is stable under most conditions used in LC
but is attacked by hydrolysis under basic conditions (pH > 7).
• Bonded phase stationary phase can be used with gradient
elution. This is a major advantage of BPC.
• Two main techniques can be classified, based on the relative
polarities of the stationary and mobile phases: (a) Normal
phase BPC, and (b) reversed-phase BPC.
• Normal-phase BPC is used when the stationary phase (e.g.,
aminopropyl) is more polar (as evidenced by the predominant
functional group) than the mobile phase (e.g., hexane).
• Reversed-phase BPC is used when the stationary phase is
nonpolar (e.g., octadecylsilane) and the mobile phase is polar
(e.g., water-methanol). Solute-elution order is often the reverse
of that observed with normal-phase BPC.
– The technique is ideally suited to substances insoluble or only
sparingly soluble in water but soluble in alcohols or other
water-miscible organic solvents.
– Because many organic compounds show this solubility behavior,
reversed-phase BPC is the most widely used mode of HPLC,
accounting for about 60%of the published applications.
Reversed-Phase and Normal-Phase Bonding
Phase Chromatography
 Based upon the relative polarities of the mobile and stationary
phases, two types of partition chromatography are
distinguishable.
normal-phase BP chromatography
 Highly polar stationary phase such as water or
triethyleneglycol supported on silica or alumina particles; a
relatively nonpolar solvent such as hexane serves as the
mobile phase.
 The least polar component is eluted first because in a relative
sense, it is the most soluble in the mobile phase
 Increasing the polarity of the mobile phase has the effect of
decreasing the elution time.
• Normal-phase BPC can replace LSC on silica gel in many
applications
Reversed-phase chromatography
• The stationary phase is nonpolar, often a
hydrocarbon, and the mobile phase is relatively
polar (such as water, methanol, or acetonitrile).
• The most polar component appears first, and
increasing the mobile phase polarity increases
the elution time.
• Perhaps three quarters of all high-performance
liquid chromatography is currently being
carried out in columns with reversed-phase
packings.
Reasons for the wide usage of RP-BPC
• Nonionic, ionic, and ionizable compounds can often be
separated, sometimes at the same time, using a single column
and mobile phase.
• Bonded-phase columns are relatively stable provided certain
precautions, especially pH control, are taken.
• The predominant mobile phase, water, is inexpensive and
plentiful.
• The most frequently used organic modifier, methanol, can be
obtained at a reasonable price and of sufficient purity in most
places in the world.
• The elution order is often predictable because retention time
usually increases as the hydrophobic character of the solute
increases.
• Columns equilibrate rapidly, thereby permitting faster method
development and sample turnaround after gradient elution.
Relationship between polarity and elution times for
normal phase and reversed phase chromatography
Effect of chain length of the alkyl group of the bonded
phase upon performance
•
• Longer chains produce packings that are more
retentive. In ad-dition, longer chain lengths permit
the use of larger samples.
• In
most
applications
of
reversed-phase
chromatog-raphy, elution is carried out with a highly
polar mobile phase such as an aqueous solution
containing various concentrations of such solvents
as methanol, acetoni-trile, or tetrahydrofuran.
• In this mode, care must be taken to avoid pH values
greater than about 7.5 because hydrolysis of the
siloxane takes place, which leads to degradation or
destruction of the packing.
•
Applications of Partition Chromatography
• Reversed-phase bonded packings, when
used in con-junction with highly polar
solvents (often aqueous), approach the ideal,
universal system for liquid chro-matography.
• Because of their wide range of applicability,
their convenience, and the ease with which k'
and a can be altered by manipulation of
aqueous mobile phases, these packings are
frequently applied before all others.
• Typical applications of bonded-phase
chromatography. (a) Soft-drink addi-tives.
Column: 4.6 x 250 mm packed with polar
(nitrile) bonded-phase packings. Isocratic
solvent: 6% HOAC/94% HZO. Flow rate: 1.0
cm3/min. (b) Organophosphate insecticides.
Column: 4.5 x 250 mm packed with 5-wm, C8,
bonded-phase par-ticles. Gradient: 67%
CH30H/33% H20 to 80 CH3/20% H20. Flow
rate 2 mL/min. Both used 254-nm UV
detectors.
Derivative Formation
• In some instances, the components of a
sample are converted to a derivative before,
or sometimes after, chromatographic
separation is undertaken for the following
reasons:
• Reduce the polarity of the species so that
partition rather than adsorption or
ion-exchange columns can be used
• Increase the detector response and thus
sensitivity, for all of the sample components
• Enhance the detector response to certain
components of the sample.
Use of derivatives to reduce polarity and enhance sensitivity
Ion-Pair Chromatography
• Ion-pair (or paired-ion) chromatography is a type of
reversed-phase partition chromatography that is used for the
separation and determination of ionic species.
• The mobile phase in ion-pair chromatography consists of an
aqueous buffer containing an organic solvent such as
methanol or acetonitrile and an ionic compound containing a
counter ion of opposite charge to the analyte.
• A counter ion is an ion that combines with the analyte ion to
form an ion pair, which is a neutral species that is retained by a
reversed-phase packing.
• Most of the counter ions contain alkyl groups to enhance
retention of the resulting ion pair on the nonpolar stationary
phase.
• Elution of the ion pairs is then accomplished with an aqueous
solution of methanol or other water soluble organic solvent.
• Applications of ion-pair chromatography frequently overlap
those of ion-exchange chromatography.
• An example of where the ion-pair method provides better
separations is for analyzing mixtures of chlorate and nitrate
ion. For this pair of solutes, selectivity with an ion-exchange
packing is poor.
ION-EXCHANGE CHROMATOGRAPHY
• Ion-exchange chromatography (IC), which is often
shortened to ion chromatography refers to modern
and efficient methods of separating and determining
ions based upon ion-exchange resins.
• Ion chromatography was first developed in the
mid-1970s when it was shown that anion or cation
mixtures can be readily resolved on HPLC columns
packed with anion-exchange or cation-exchange
resins.
• At that time, detection was generally performed with
conductivity measurements. Currently, other
detectors are also available for ion chromatography.
• Ion chromatography was an outgrowth of
ion-ex-change chromatography,
•
Ion-Exchange Equilibria
 Ion-exchange processes are based upon exchange equilibria
between ions in solution and ions of like sign on the surface of
an essentially insoluble, high-molecular weight solid.
 Natural ion-exchangers, such as clays and zeolites, have been
recognized and used for several decades.
 Synthetic ion-exchange resins were first produced in the
mid-1930s for water softening, water deionization, and solution
purification.
 The most common active sites for cation-exchange resins are
the sulfonic acid group -SO3- H+, a strong acid, and the
carboxylic acid group -COO- H+, a weak acid.
 Anionic exchangers contain tertiary amine groups
-N(CH3)3OH- or primary amine groups -NH3+OH-;
• the former is a strong base and the latter a weak one.
 When a sulfonic acid ion-exchanger is brought in contact with
an aqueous solvent containing a cation Mx+ :
Cation exchanger
Similarly a strong base exchanger interacts with the
anion Ax- as shown by the reaction
Anion exchanger
Influences on Distribution Coefficients and Selectivity
• Ion-exchange chromatography involves more
variables than other forms of chromatography.
• Distribution coefficients and selectivities are
functions of:
 pH,
 solute charge and radius,
 resin porosity,
 ionic strength and type of buffer,
 type of solvent,
 temperature, and so forth.
• The number of experimental variables makes
ion-exchange chromatography a very versatile
technique, since each may be used to effect a better
separation, but a difficult one because of the time
needed to optimize a separation.
 By selecting a common reference ion such as H+,
distribution ratios for different ions on a given type
of resin can be experimentally compared.
 Such experiments reveal that polyvalent ions are
much more strongly held than singly charged
species. Within a given charge group, however,
differences appear that are related to the size of the
hydrated ion as well as to other properties.
 Thus, for a typical sulfonated cation-exchange resin,
values for Kex decrease in the order
• For anions, Kex for a strong base resin decreases in the order
Ion Chromatography
• Ion chromatography (IC) is an ion-exchange
technique that uses, most popularly, a low-capacity
column combined with a conductivity detector
• Its most frequent practical application is the
determination of trace anions in aqueous solution.
• The low-capacity column allows the use of a buffer
with a low ionic strength.
• There are two forms of IC practiced today:
(a) suppressed or dual-column IC,
(b) nonsuppressed or single column IC.
Ion Chromatography with Eluent
Suppressor Columns
 The widespread application of ion chromatography
for the determination of inorganic species was
inhibited by the lack of a good general detector.
 Conductivity detectors are an obvious choice for this
task.
 They can be highly sensitive, they are universal for
charged species, and, as a general rule, they
respond in a predictable way to concentration
changes.
 The only limitation arises from the high electrolyte
concentration required to elute most analyte ions in
a reasonable time.
 As a consequence, the conductivity from the
mobile-phase components tends to swamp that from
analyte ions, thus greatly reducing the detector
sensitivity.
 The problem of high eluent conductance was solved
by the introduction of a so-called eluent suppressor
column immediately following the analytical
ion-exchange column.
 The suppressor column is packed with second
ion-exchange resin that effectively converts the ions
of the solvent to a molecular species of limited
ionization without affecting the analyte ions.
 For example, when cations are being separated and
determined, hydrochloric acid is often chosen as the eluting
reagent, and the suppressor col-umn is an anion-exchange
resin in the hydroxide form. The product of the reaction in the
suppressor is water. That is,
 H+(aq) + Cl-(aq) + Resin+OH-(s) ---> Resin+Cl-(s) + H2O
 The analyte cations are, of course, not retained by this second
column.
 For anion separations, the suppressor packing is the acid form
of a cation-exchange resin. Sodium bi-carbonate or carbonate
may serve as the eluting agent. The reaction in the suppressor
is then
Na+(aq) + HCO 3- (aq) + Resin-H+(s) -> Resin-Na+(s) + H2CO3(aq)
 Here, the largely undissociated carbonic acid does not
contribute significantly to the conductivity.
 An inconvenience associated with the original suppressor
columns was the need to regenerate them periodically
(typically, every 8 to 10 hr) in order to convert their packings
back to the original acid or base form.
Single-Column Ion Chrornatography
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Equipment has also become available commercially for ion
chromatography in which no suppressor column is used.
This approach depends upon the small differences in conductivity
between the eluted sample ions and the prevailing eluent ions.
To amplify these differences, low-capacity exchangers are used, which
make possible elution with species having low equivalent
conductances.
Single-column chromatography tends to be somewhat less sensitive
and to have a more limited range than ion chromatography with a
suppressor column.
An indirect photometric method that permits the separation and
detection of nonabsorbing anions and cations without a suppressor
column has recently been described.
Here also, no suppressor column is used, but instead, anions or
cations that absorb Uv or Vis radiation are used to displace the
analyte ions from the column.
When the analyte ions are displaced from the exchanger, their place is
taken by an equal number of eluent ions (provided, of course, that the
charge on the analyte and eluent ions is the same).
Size-exclusion Chromatography
 Gel-permeation or gel-filtration chromatography
applicable to high molecular weight species
 Packing is small silica or polymer particles
containing a network of uniform pores into which
solute and solvent molecules can diffuse.
 The average residence time of analyte molecules in
the pores depends upon the effective size of these
molecules
 Molecules that are larger than the average pore size
will not be retained
 Molecules with sizes smaller than those of the pores
will be retained
 Intermediate size molecules will penetrate according
to their sizes. Thus fractionation occurs.
• The process is almost always carried out in a
column, but it also has been performed on a thin
layer.
• Column packing materials with pores of different
(controlled) sizes are generally used.
• The materials can be soft gels, semirigid gels, or
rigid materials.
• The soft and semirigid gels can change their pore
sizes, depending on the solvent used as a mobile
phase.
• The soft gels, of the polydextran or agarose type,
can swell to many times their dry volume, whereas
the semirigid gels of the polyvinylacetate or
polystyrene type swell to 1.1 to 1.8 times their dry
volume. Rigid materials, such as porous glass or
porous silica beads, have fixed pore sizes and do
not swell at all.
Theory of Size-Exclusion Chromatography
• The total volume Vt of a column packed with a porous polymer
or silica gel is given by
 Vt = Vg + Vi + Vo
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Vg is the volume occupied by the solid matrix of the gel,
Vi is the volume of solvent held in its pores,
Vo is the free volume outside the gel particles.
Assuming no mixing or diffusion, Vo also represents the
theoretical volume of solvent required to transport through the
column those components too large to enter the pores of the
gel.
 In fact, however, some mixing and diffusion will occur, and as a
consequence the nonretained components will appear in a
Gaussian-shaped band with a concentration maximum at Vo.
 For components small enough to enter freely into the pores of
the gel, band maxima will appear at the end of the column at an
eluent volume corresponding to (Vi + Vo).
 Molecules of intermediate size are able to transfer
into some fraction K of the solvent held in the pores;
the elution volume Ve for these retained molecules is
Ve = Vo + KCVi
(1)
• Equation 1 applies to all of the solutes on the
column.
 For molecules too large to enter the gel pores, KC = 0
and Ve = Vo; for molecules that can enter the pores
unhindered, KC = 1 and Ve = (Vo + Vi).
 In deriving Eq. 1, the assumption was made that no
interaction, such as adsorption, occurs between the
solute molecules and the gel surfaces. With
adsorption, the amount of interstitially held solute
will increase; with small molecules, KC will then be
greater than unity.
 Eq. 1 rearranges to
•
KC = (Ve - Vo)/Vi = CslCm
(2)
 where KC is the distribution constant
for the solute. Values of KC range from
zero for totally excluded large
molecules to unity for small molecules.
 The useful molecular weight range for a
size-exclusion packing is conveniently illustrated by
means of a calibration curve such as that shown in
the upper part of the Figure.
 Molecular weight, which is directly related to the size
of solute molecules, is plotted against retention
volume VR. Note that the ordinate scale is
logarithmic.
 The exclusion limit defines the molecular weight of a
species beyond which no retention occurs. All
species having greater molecular weight than the
exclusion limit are so large that they are not retained
and elute together to give peak A in the
chromatogram shown.
 The permeation limit is the molecular weight
below which the solute molecules can
penetrate into the pores completely.
 All molecules below this molecular weight
are so small that they elute as the single
band labeled D.
 As molecular weights decrease from the
exclusion limit, solute molecules spend more
and more time, on the average, in the particle
pores and thus move progressively more
slowly.
 It is in the selective permeation region that
fractionation occurs, yielding individual
solute peaks such as B and C in the
chromatogram.
Vo
Vi
Limit below which solute
molecules can penetrate
completely into the pores
No retention
beyond this MW
Permeation limit
Molecules below this
MW are so small that
they elute as the single
band D
A
Unretained
large
molecules
B
C
D
Such calibration curves are supplied by manufacturers
of packing materials