Addis Ababa Science and Technology University

College of Applied Sciences

Department of Industrial Chemistry
Instrumental Analysis I, InCh2102
C h a p t e r 4 : H i g h Pe r f o r m a n c e L i q u i d
C h r o m a t o g r a p h y ( H P L C ) : Pa r t 2
July 06, 2022
K e b e d e N i g u s s i e M e k o n n e n ( P h . D. )

INCH2102 CHAPTER 4 PART 2 (KEBEDE NM) JULY 2022 1

…Chapter 4: High Performance Liquid
Chromatography (HPLC) -highlight
Introduction
High Performance Liquid Chromatography (HPLC): basics,
instrumentation, application, difference and similarity with
GC, classification
Ion exchange chromatography (IEC): basics, instrumentation,
classification, application
Size exclusion chromatography (SEC): basics, instrumentation,
application
Supercritical fluid chromatography (SFC): introduction to
supercritical fluids, basics, instrumentation, application,
difference and similarity with GC and HPLC

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 2

…Chapter objectives
At the end of this chapter, the students should be
able to:
Explain the basic principles of HPLC, IEC, SEC,
and SFC
Describe the instrumentation for HPLC, IEC, SEC,
and SFC
Describe the various types of liquid
chromatography techniques
Differentiate between the various types of
detectors used in HPLC, IEC, SEC, and SFC
Describe the application of HPLC, IEC, SEC, and
SFC

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 3

 … Chapter 4: High Performance Liquid
Chromatography (HPLC) –Part 2: section outline
Ion Exchange Chromatography
 Basics
 Classification of IEC
 Principles of IEC
 Resins in IEC
 Eluents in IEC
 Instrumentation of IEC
 Applications of IEC

Size Exclusion Chromatography (SEC)

 Basics
 Classification of SEC
 Principles of SEC
 Instrumentation of SEC
 Advantages and Disadvantages of SEC
 Applications of SEC

Supercritical Fluid Chromatography (SFC)

 Introduction
 Supercritical Fluids
 Instrumentation of SFC
 Applications of SFC
 SFC Compared with GC and HPLC: Advantages and Limitations

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 4

…Ion Exchange Chromatography (IEC)
Basics
Classification of IEC
Principles of IEC
Resins in IEC
Eluents in IEC
Instrumentation of IEC
Applications of IEC

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 5

…Ion Exchange Chromatography (IEC)
Basics
 Ion exchange (IEC) chromatography can separate molecules or
groups of molecules that have only slight differences in charge
 Separation is based on reversible interaction between charged
molecule and oppositely charged chromatography medium
 form of LC that uses ion exchange resins to separate atomic or
molecular ions based on their interaction with resin
 is based on selective exchange of ions in sample with counter-
ions in stationary phase
 used for separation of ionic materials by passage of solution
through column or across surface consisting of porous polymeric
resin incorporating exchangeable ions
 limited to samples containing ionized or partially ionized solutes
 column packing consists of ion-exchange resins bonded to inert
polymeric particles

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 6

…classification of IEC
anion exchange chromatography
 uses anion exchange column
 binding ions are negative, and immobilized functional group is positive
 In anion exchange column, packing is positively charged and
therefore retains negatively charged molecules by coulombic
interaction
 bound molecules are eluted with anion gradient
cation exchange chromatography
 uses cation exchange column
 positively charged ions bind to negatively charged resin
 media in cation exchange column is negatively charged, binding
positively charged molecules
 cations are used for elution of bound molecules (and therefore
needs cation gradient)

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 7

…principles of IEC
IEC relies on charge-charge interactions between ions in
sample and charges immobilized on resin of choice
can be subdivided into cation exchange chromatography
and anion exchange chromatography
Once solutes are bound, column is washed to equilibrate it
in starting buffer, which should be of low ionic strength, then
bound molecules are eluted off using gradient of second
buffer which steadily increases ionic strength of eluent
solution
Alternatively, pH of eluent buffer can be modified to give
matrix change at which they will not interact and molecule of
interest elutes from resin
If it is negatively charged at pH you wish, use anion exchanger;
if it is positive, use cation exchanger

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 8

…
In cation-exchange chromatography, stationary phase, which
consists of large number of acid groups attached to polymeric
resin, is slurried with water and applied to column
mobile phase, which contains inorganic salt dissolved in suitable
solvent, is applied to the column
As mobile phase passes through column, exchange between H+
ions on polymeric ion-exchange resin of stationary phase and
cations of salt in mobile phase occur
solution which is collected at bottom of column contains acid form
of inorganic salt
newly formed acidic solution can then be titrated using
standardized base to determine number of moles present in
sample
molecular weight of substance and identity of unknown can then
be determined based on number of moles and weight of sample

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 9

…Resins for IEC
column packings for IEC consist of ion-exchange resins bonded to
inert polymeric particles (typically 10 µm diameter)
Here retention is based on attraction between solute ions and
charged sites bound to stationary phase
Ions of same charge are excluded - so column could be either
packed or open columns
Some of IEC column:
 Polystyrene ion-exchange resin of beaded particles < 10 µm -
contains substituted styrene ring as monomer, where commonly
used substituents (R group) are sulfonic acid, carboxylic acid,
quaternary amine, and methylammonium
 Silica-based ion-exchange resin - consists of substituted silica
group as monomer where commonly used substituents (R group)
are sulfonic acid, carboxylic acid, quaternary amine, and
methylammonium

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 10

…
 Polystyrene-type resins - allow cross linkage which increases
stability of chain. Higher cross linkage reduces swerving, which
increases equilibration time and ultimately improves selectivity.
Commonly four types are known, namely:
 DOWEX 50 – cation exchanger with sulfonic group
 IRC 50 – cation exchanger with carboxylic acid group
 DOWEX – anion exchanger with quaternary amine group
 IR 45 – anion exchanger with methylammonium group
 Polysaccharides-type resins: Cellulose and sephadex (cross-
linked dextran) ion exchangers (gels) - possess larger pore sizes
and low charge densities making them suitable for protein
separation. Commonly four types are known, namely:
 DEAE (DiEthylAminoEthane)-cellulose and DEAE-sephadex –
anion exchanger with diethylaminoethyl group
 CM-cellulose and CM-sephadex – cation exchanger with
carboxymethyl group
 Phospho-cellulose and phosphor-sephadex – cation exchanger
with a phosphoryl group

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 11

…
strong anion exchange matrix is quaternary ammonium, and is
designated Q, while weak anion exchange matrix is diethylaminoethyl, or
DEAE

strong cation exchange columns have functional group sulfonic acid,

which is designated S, while weak cation exchange columns usually have
functional group carboxymethyl and are designated CM

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 12

…Eluents for IEC
choice of eluent depends on many factors, namely:
 pH
 buffer capacity
 concentration of eluent
 nature of eluent’s reaction with column and packing material
Eluents in anion exchanger chromatography
 In non-suppressed anion chromatography, where eluent and
analyte are not altered between column and detector
 Aromatic carboxylic acids are used in conductivity detection because of
their low self-conductivity
 Aliphatic carboxylic acids are used for UV/visible detection because
they are UV transparent
 Inorganic acids can only be used in photometric detection
 In suppressed anion chromatography, where eluent and analyte
are treated between column and detection
 Only alkali hydroxides and carbonates, borates, hydrogen carbonates,
and amino acids can be used as eluents

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 13

…
Eluents in cation exchanger chromatography
 primary eluents used in cation chromatography of alkali
metals and ammoniums are mineral acids such as HNO3
 When cation is multivalent, organic bases such as
ethylenediamine serve as main eluents
 If both alkali metals and alkali earth metals are present,
HCl or 2,3-diaminopropionic acid is used in combination
with pH variation

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 14

…Instrumentation of IEC
Ions in solution can be detected by measuring conductivity of
solution
IEC is similar to HPLC in instrumentation by eluent delivery
(eluent reservoir and pump), sample injection, analytical
separation (both have analytical column and guard column),
detection, data acquisition, and instrument control
main difference between IEC and HPLC - presence of third
column in case of IEC, suppressor column (eluent suppressor
that converts ionic eluent into non-ionic species that doesn’t
interfere with detection)

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 15

…
suppressor column
short column (tube) that is inserted into flow stream just after
analytical column
packed with ion exchange resin itself - resin that removes
mobile phase ions from effluent, much like deionizing cartridge
removes ions in laboratory tap water, and replaces them with
molecular species
mixed-bed ion exchange resin is used, for example, in
deionizing cartridges, such that tap water ions (such as Ca2+
and CO32-) are exchanged for H+ and OH- ions, which in turn
react to form water - resulting water is thus deionized
Of course, in HPLC experiment, analyte ions must not be
removed in this process, and thus suppressors must be selective
only for mobile phase ions

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 16

…Applications of IEC
Ion exchange column chromatography is powerful separation
technique for mixtures of proteins and other biomolecules, finding
use in biopharmaceutical production, basic research, clinical
diagnostics, quality control, and other analytic applications
Cation exchange chromatography provide an easy, economical
step in protein purification and analysis protocols
Combined with optimized binding and elution conditions, anion
exchange chromatography can be used at all stages of
biomolecule isolation and analysis including initial cleanup,
purification, separation of analytes, and removal of contaminants
such as endotoxins, host cell proteins, or ionic compounds
IEC applicable for: polar or aqueous solution, separation of
inorganic ions, separation of biological molecules (proteins, amino
acids, etc), deionization of water, etc.

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 17

…Size Exclusion Chromatography (SEC)
Basics
Classification of SEC
Principles of SEC
Instrumentation of SEC
Advantages and Disadvantages of SEC
Applications of SEC

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 18

…Size Exclusion Chromatography (SEC)
also known as gel chromatography or molecular exclusion
chromatography
chromatographic method that uses porous particles to
separate molecules according to their molecular size, shape,
and weight
usually applied to large molecules or macromolecular
complexes such as proteins and industrial polymers
underlying principle of SEC is that particles of different sizes
will elute (filter) through a stationary phase at different rates
 if particles are loaded simultaneously or near-
simultaneously, particles of same size should elute together
 This is usually achieved with column, tightly packed with
extremely small porous polymer beads designed to have
pores of different sizes

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 19

…
As solution travels down column some particles enter pores
Larger particles cannot enter into as many pores - the larger
the particles, the less are the overall volume to traverse over
length of column, and the faster the elution
Molecules that are smaller than pore size can enter particles
and therefore have longer path and longer transit time than
larger molecules that cannot enter particles
stationary phase is hydrophilic column packing material and
aqueous mobile phase to separate, fractionate, or measure
molecular weight distribution of molecules soluble in water,
such as polysaccharides and proteins
Softs gels are used such as dextran (Sephadex), agarose
(sepharose), polyacrylamide (bio gel)

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 20

… Classification of SEC
Gel permeation chromatography (GPC)
 stationary phase is hydrophobic column packing material and non-
aqueous mobile phase (organic solvent), such as tetrahydrofuran (THF),
trichlorobenzene, toluene, and chloroform, to analyze for organic
polymers such as polystyrene
 Semi-rigid or rigid gels are used such as polystyrene, controlled
porosity glass beads, alkylated dextran
Gel filtration chromatography (GFC)
 GFC utilizes mobile phases that are water-based solutions and is used
to analyze for naturally occurring polymers, such as proteins and
nucleic acids
GPC stationary phases are rigid gels, such as silica gel, whereas
GFC stationary phases are soft gels
Neither technique utilizes gradient elution because stationary
phase pore sizes are sensitive to mobile phase changes

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 21

…Principles of SEC
mixture of molecules dissolved in liquid (mobile phase) is applied
to chromatography column which contains solid support in form of
microscopic spheres or beads (stationary phase)
mass of beads within column is often referred to as column bed
selection of molecules based on their molecular size and shape
utilizes molecular sieve properties of variety of porous materials
column of gel particles or porous glass granules is in equilibrium
with suitable solvent for molecules to be separated
Large molecules that are completely excluded from pores will
pass through interstitial spaces, while smaller molecules will be
distributed between solvent inside and outside molecular sieve and
will then pass through column at lower rate: the smaller the
molecules, the longer the retention time

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 22

…

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 23

…Instrumentation of SEC

Stationary Phase
 Gels that are commonly used include cross-linked dextran,
agarose, polyacrylamide, poly acryloyl morphine, and
Polystyrenes
 These are semi-permeable, porous beads with well-
defined range of pore sizes

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 24

…
three types of gel:
 Soft gel separation of proteins - e.g. dextran (Sephadex),
polyacrylamide gel
 Semi-rigid gels separation of non-polar polymers in non-polar
solvents - e.g. bio beats
 Highly rigid gels and glasses separation of polar solvents
Before column packing, these gels or stationary phases are
soaked overnight to prevent breaking or bursting of column due to
swelling of gel
Mobile phase
 liquid used to dissolve biomolecules to make mobile phase is
usually known as buffer - mixture of biomolecules (sample)
dissolved in buffer
 choice of mobile phase to be used in any separation will
depend on type of separation to be achieved and components
to be separated
 e.g. Tetrahydrofuran, chloroform, dimethylformamide

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 25

…
Column packing
 either porous silica or semi-rigid (highly cross-linked) organic
gels, in most cases copolymers of styrene and divinylbenzene
 hollow tube tightly packet with extremely small porous polymer
beads design to have force of different sizes
Pumps (syringe pump)
 used to maintain constant flow rate during entire chromatogram
(very important in SEC)
Chromatogram
 small molecules will enter all pores; intermediate molecules due
to velocity of mobile phase will not be able to diffuse into pores
thus will be retained less effectively
 initial peak contains totally excluded solute; final peak contains
totally included solute

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 26

…detectors
Three common spectroscopy detection techniques are refractive
index (RI), evaporative light scattering (ELS), and ultraviolet (UV)
Concentration sensitive detectors
 Solute property detectors - UV absorption detectors
 Bulk property detectors - Refractive index (R.I) detectors
 Evaporation detectors - Evaporation light scattering detectors
(ELSD)
Molecular weight-sensitive detectors
 Light scattering detectors
 Viscosity detectors
 Other detectors
 FID, MSD, or Fourier Transform Infrared Spectrometer (FTIR)

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 27

…Advantages and disadvantages of SEC
Advantages
 can be carried out at room temperature
 samples are not decomposed because there is no exposure
to high temperatures
 identify high mass components even in low concentrations
 absolute molecular weight can be obtained
 short analysis time and well-defined separation
 narrow bands, which leads to good sensitivity
 no sample loss as solute does not interact with stationary
phase
 small amount of mobile phase is required

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 28

…
Disadvantages
 filtration of sample should be done before injecting into
column to prevent dust and other particles from ruining
column and interfering with detector
 limited number of peaks that can be obtained within short
time scale of the GPC run
 bad response for very small molecular weights
 high investment costs may be required
 inapplicable to sample with similar sizes, such as isomers
 10% difference in molecular mass is required for reasonable
resolution

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 29

…Applications of SEC
SEC is a widely used technique for purification and analysis of
synthetic and biological polymers, such as proteins,
polysaccharides, and nucleic acids
generally used to separate biological molecules, and to determine
molecular weights and molecular weight distributions of polymers
mainly applied in separation and characterization of molecules of
different molecular weights
main application of SEC is as follows:
Purification
 Purification of biological macromolecules like viruses, proteins,
enzymes, hormones, nucleic acid, antibiotics, and polypeptides.
Molecular weight determination
Desalting
 separating large molecules of biological origin from inorganic
and ionizable

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 30

…
Solution concentrations
 solution of high molecular weight substance can be
concentrated while water and low molecular weight
substance remain in solution. After some time, gel is
removed by centrifugation, leaving high molecular material
in solution whose concentration has increased but pH and
ionic strength are unaltered
Protein binding studies
 commonly used to study reversible binding of ligand to
macromolecular such as proteins including receptor
proteins
 can determine quaternary structure of purified proteins
 also used for co-polymerization studies

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 31

…Supercritical Fluid Chromatography (SFC)
Introduction
Supercritical Fluids
Instrumentation of SFC
Applications of SFC
SFC Compared with GC and HPLC: Advantages and
Limitations

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 32

…Supercritical fluid chromatography (SFC)
Introduction
 Substances can be in solid, liquid, or gaseous state depending
on temperature and pressure conditions
 water is liquid at room temperature and pressure, but at
atmospheric pressure, it changes to vapor (gas) at 100 °C and
ice (solid) at 0 °C
 When water is placed inside sealed container and subjected to
vacuum, some of water evaporates and remainder remains in
liquid phase
 When water vapor pressure reaches certain value, evaporation
rate becomes equal to condensation rate - referred to as
saturated water vapor pressure, and depends on temperature
 As vessel is heated, liquid water expands and evaporates, so
that its density is reduced - leads to increase in density of vapor
phase

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 33

…
 If temperature exceeds 374 °C and pressure exceeds
22.06 MPa, density of liquid and vapor phases become
same, making it impossible to distinguish between liquid
water and water vapor
 water in this state will not become liquid even if pressure is
increased further - dense gas that doesn’t liquefy
 Such state is called supercritical state, and substance in
supercritical state is called supercritical fluid
 temperature and pressure at which substance becomes
supercritical are called critical point, temperature is called
critical temperature, and pressure is called critical pressure

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 34

…Supercritical fluid (SCF)
 is phase of material at critical temperature and critical
pressure of material
 Critical temperature is temperature at which gas cannot
become liquid as long as there is no extra pressure; and,
critical pressure is minimum amount of pressure to liquefy
gas at its critical temperature
 SCFs combine useful properties of gas and liquid phases,
as it can behave like both gas and liquid in terms of
different aspects
 SCF provides gas-like characteristic when it fills container
and it takes shape of container
 Motion of molecules are quite similar to gas molecules
 SCF behaves like liquid because its density property is
near liquid and, thus, SCF shows similarity to dissolving
effect of liquid

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 35

…
After CP (critical point), no
matter how much pressure or
temperature is increased,
material cannot transform
from gas to liquid or from
liquid to gas phase -
supercritical fluid form
Increasing temperature
cannot result in turning to gas,
and increasing pressure
cannot result in turning to
liquid at this point
In phase diagram, field
above Tc and Pc values is
defined as supercritical
region
Gas Supercritical fluid Liquid
Density (g/cm3) 0.6×10-3-2.0×10-3 0.2-0.5 0.6-2.0
Diffusivity (cm2/s) 0.1-0.4 10-3-10-4 0.2×10-5-2.0×10-5
Viscosity (cm/s) 1×10-4-3×10-4 1×10-4-3×10-4 0.2×10-2-3.0×10-2

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 36

…
Critical temperature and critical pressure for various
compounds
Compound Critical temperature Critical pressure
(℃) (MPa)
NH2 132 110
CO2 31 72
N2O 36 70.6
H2O 374 215
C3H8 97 41.5
C6H14 234 28.98
CH3OH 239 78.9
C2H5OH 243 62.2
C6H5CH3 318 40.1
InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 37
…instrumentation of SFC

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 38

…
SFC has similar setup to HPLC instrument
both use similar stationary phases with similar column
types
However, there are some differences
Temperature is critical for SCFs, so there should be
heat control tool in system similar to that of GC
there should be pressure control mechanism,
restrictor, because pressure is another essential
parameter in order for SCF materials to be kept at
required level
Microprocessor mechanism is placed in instrument for
SFC - collects data for pressure, oven temperature,
and detector performance to control related pieces of
instrument
InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 39
…
Stationary phase
 SFC columns are similar to HPLC columns in terms of coating
materials
Mobile phases
 mobile phase can be selected from solvent groups of inorganic
solvents, hydrocarbons, alcohols, ethers, halides; or can be
acetone, acetonitrile, pyridine, etc.
 most common SCF which is used in SFC is CO2 because its critical
temperature and pressure are easy to reach
 Additionally, CO2 is low-cost, easy to obtain, inert towards UV,
non-poisonous and good solvent for non-polar molecules
 Other than CO2, ethane, n-butane, N2O,
dichlorodifluoromethane, diethyl ether, ammonia,
tetrahydrofuran can be used

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 40

…
CO2 is commonly used SCF since its critical temperature is
just 31.1 °C and its critical pressure is only 72 bar
Advantages of CO2 as SCF
 It is chemically inert and nontoxic
 It is non-flammable
 It is non-polar and dissolves oils and fats well
 Since CO2 is released as gas at normal temperatures and
pressures, solvent removal is easy, allowing more accurate
component concentrations to be determined
 High-purity CO2 can be obtained at low price, so low running
costs can be realized
 Since CO2 emitted from petrochemical factories is collected,
refined, and used, it does not increase CO2 emissions

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 41

 …
Properties of some solvents as mobile phase at the critical point
Solvent Critical Critical Pressure
Temperature (°C) (bar)
Carbon dioxide (CO2) 31.1 72
Nitrous oxide (N2O) 36.5 70.6
Ammonia (NH3) 132.5 109.8
Ethane (C2H6) 32.3 47.6
n-Butane (C4H10) 152 70.6
Diethyl ether (Et2O) 193.6 63.8
Tetrahydrofuran (THF, C4H8O) 267 50.5
Dichlorodifluoromethane 111.7 109.8
(CCl2F2)
InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 42
…
Detectors
One of biggest advantage of SFC over HPLC is range
of detectors
FID - which is normally present in GC setup, can also
be applied to SFC
Such detector can contribute to quality of analyses
of SFC since FID is highly sensitive detector
SFC can be coupled with mass spectrometer, UV-
visible spectrometer, or IR spectrometer more easily
than can be done with an HPLC
Some other detectors which are used with HPLC can
be attached to SFC such as fluorescence emission
spectrometer or thermionic detectors

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 43

…
Sample
 Generally speaking, samples need little preparation
 only major requirement is that it dissolves in solvent less
polar than methanol: it must have dielectric constant lower
than 33, since CO2 has low polarity and cannot easily
elute polar samples
 To combat this, modifiers are added to mobile phase

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 44

…
Modifiers
 added to mobile phase to play with its properties
 CO2 SCF lacks polarity - in order to add polarity to fluid
(without causing reactivity), polar modifier will often be
added
 Modifiers usually raise critical pressure and temperature
of mobile phase a little, but in return add polarity to
phase and result in fully resolved sample
 Unfortunately, with too much modifier, higher temperatures
and pressures are needed and reactivity increases (which
is dangerous and bad for operator)
 Modifiers, such as ethanol or methanol, are used in small
amounts as needed for mobile phase in order to create
more polar fluid
InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 45
…SFC compared with GC and HPLC –
advantages and limitations
SFC bring advantages and strong aspects of HPLC and GC
together
SFC can be more advantageous than HPLC and GC when
compounds which decompose at high temperatures with GC
and do not have functional groups to be detected by HPLC
detection systems are analyzed
Advantages over GC
 Able to analyze many solutes with no derivatization since there
is no need to convert most polar groups into nonpolar ones
 Can analyze thermally labile compounds more easily with high
resolution since it can provide faster analysis at lower
temperatures
 Can analyze solutes with high molecular weight due to their
greater solubizing power

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 46

…
Advantages over HPLC
 Because SCFs have low viscosities analysis is faster, there is
much lower pressure drop across column, and open tubular
columns can be used
 Shorter column lengths are needed (10-20 m for SFC
versus 15-60 m for HPLC) due to high diffusivity of SCF -
more interactions can occur in shorter span of
time/distance
 Resolving power is much greater (5×) than HPLC due to
high diffusivity of SCF - more interactions result in better
separation of components in shorter amount of time

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…
 HPLC has better selectivity than SFC owing to changeable
mobile phases (especially during particular experimental
run) and wide range of stationary phases
 Although SFC does not have selectivity of HPLC, it has
good quality in terms of sensitivity and efficiency
 SFC enables change of some properties during
chromatographic process - this tuning ability allows
optimization of analysis
 SFC has broader range of detectors than HPLC
General disadvantages of SFC
 Cannot analyze extremely polar solutes due to relatively
nonpolar mobile phase, CO2

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…
physical properties of SCFs between liquids and gases enables
SFC technique to combine with best aspects of HPLC and GC, as
lower viscosity of SCFs makes SFC faster method than HPLC -
lower viscosity leads to high flow speed for mobile phase
Thanks to critical pressure of SCFs, some fragile materials that are
sensitive to high temperature can be analyzed through SFC
 These materials can be compounds which decompose at high
temperatures or materials which have low vapor pressure/volatility
such as polymers and large biological molecules
 High pressure conditions provide chance to work with lower
temperature than normally needed - temperature-sensitive components
can be analyzed via SFC
In addition, diffusion of components flowing through SCF is higher
than observed in HPLC due to higher diffusivity of SCFs over
traditional liquids mobile phases - results in better distribution into
mobile phase and better separation

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 49

…applications of SFC
applications of SFC range from food to environmental to
pharmaceutical industries
In this manner, pesticides, herbicides, polymers, explosives and
fossil fuels are all classes of compounds that can be analyzed
SFC can be used to analyze wide variety of drug compounds such
as antibiotics, prostaglandins, steroids, taxol, vitamins,
barbiturates, non-steroidal anti-inflammatory agents, etc.
Chiral separations can be performed for many pharmaceutical
compounds
SFC is dominantly used for non-polar compounds because of low
efficiency of CO2 for dissolving polar solutes
SFC is used in petroleum industry for determination of total
aromatic content analysis as well as other hydrocarbon separations

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…

The End of Chapter 4. High Performance

Liquid Chromatography (HPLC) Part 2

Next

Chapter 5. Electrophoresis

InCh2102 Chapter 4 Part 2 (Kebede NM) July 2022 51