

CHROMATOGRAPHY
By Dr Nur Zazarina Ramly
 Type of extractions

 Refers to the transfer of a solute from one liquid phase to another

 Integral to food analysis whether used for

 Preliminary sample cleanup
 Concentration of the component of interest
 As for analysis

 Extractions may be carried out by means

 Batch
 Continuous
 Countercurrent
 Batch extraction
 The solute is extracted from one solvent by shaking it with second,
immisicible solvent

 The solute partitions/distributes itself between the 2 phases

 The phases are allowed to separate and layer containing the desired
constituent is removed

 Disadvantages:
 Difficult to obtain clean separation of phases- emulsion formation
 single extraction usually incomplete
 Continuous extraction
 Continuous liquid-liquid extraction require special apparatus

 More efficient than batch extraction

 Example: Use Soxhlet extractor for extracting materials from solids

 Solvent is recycled so that the solid is repeatedly extracted with fresh

solvent
 Countercurrent extraction
 Refer to serial extraction process

 Separates 2 or more solutes with different partitions coeeficients from

each other by series partitions between 2 immiscible liquid phases
 Introduction
 Chromatography is a technique by which a mixture sample is
separated into components.

 Originally intended to separate and recover (isolate and purify)

the components of a sample

 Today, complete chromatography systems are often used to both

separate and quantify sample components.
 Basic principle

 The term, “chromatography" was coined by the Russian

botanist, Tswett

 He demonstrated that, when a plant extract was carried by

petroleum ether through a column consisting of a glass tube
packed with calcium carbonate powder, a number of dyes were
separated.

 He named this analysis method "Chromatographie" after

"chroma" and "graphos", which are Greek words meaning
"color" and “to draw," respectively.
 Figure 1

"Chromatography" represents a separation technique; whereas a "chromatograph" is a system for performing

chromatography. The chart displaying the time-dependent change in signal intensity as a result of the separation is
called a "chromatogram”.

Gas and liquid chromatography are common classifications that are based upon the mobile and stationary phases
utilized for the separation. Molecules that spend most of their time in the mobile phase are carried along faster
• Although the intended use of GC and LC are the same (i.e., separation and quantification), the
measurement subjects are different, as the sample conditions differ at separation.

• The stationary phase typically indicates a column (fillers), while the mobile phase, which is
referred to as the eluent in LC, indicates a vehicle to pour a sample into the column
 How is a sample separated into its
components in the column?

• The speed of a migrating sample component depends on whether the

component has an affinity for the stationary or mobile phase.

• This affinity appears via various actions: adsorption, partition, ion

exchange, etc.
• As shown, components that have a higher affinity for the mobile phase
compared with the stationary phase migrate more rapidly

• Whilst, components that have a higher affinity for the stationary phase are
eluted from the column later.

• The order and resolution of the components emerging from the column
depend on the type of selected stationary and mobile phases.
 Therefore……

 Chromatography is based on the principal that under the same

conditions, the time between the injection of a component into the
column and the elution of that component is constant.

 This characteristic is used to perform qualitative or quantitative

analysis.

 LC and GC systems are good at determining the content of a certain

component in a sample, rather than the types of the components of a
sample.
 SUCH ANALYSES ARE EXPLAINED USING THE MEASUREMENT OF
ASPARTAME, A SYNTHETIC SWEETENER CONTAINED IN BEVERAGES.

Qualitative analysis

A standard sample of aspartame was measured, yielding a peak at 12.5

minutes. This peak corresponds to aspartame, itself.

Next, a sample prepared from a beverage was measured under the same
conditions, yielding a number of peaks.

The peak appearing at 12.5 minutes can be regarded as that of aspartame

(Figure 3).

The following conditions were the same: the type of fillers, column sizes,
column temperature, composition of the mobile phase, and flow rate.
 Figure 3. Example of aspartame measurement

• Standard sample of aspartame was measured, yielding a peak at 12.5 minutes

• The peak appearing at 12.5 minutes can be regarded as that of aspartame

Quantitative analysis

•The height and area of a peak are proportional to the concentration of the corresponding
component.

•A calibration curve is created using the standard sample. The concentration of aspartame
in the beverage can be determined from the peak area of the detected aspartame.

• Attention should be given to the fact that a qualitative analysis includes

many uncertainties. Other components may have been eluted together
with aspartame.
• Performance of a quantitative analysis requires the preparation of a calibration
curve.

• It is very difficult to perform the qualitative or quantitative analysis of a

component for which a standard sample is not available.
 GAS CHROMATOGRAPHY
 GC is a commonly used analytic technique in many research and
industrial laboratories.

 A broad variety of samples can be analyzed as long as the compounds

are sufficiently thermal stable and volatile

 Compounds
 Gases, liquid, dissolved solids
 Organic and inorganic materials
 MW ranging from 2 to > 1000 Da

Stainless steel capillary

column (up to 400°C)
•Coupling of liquid chromatography (LC) or gas chromatography (GC)
separations to a mass spectrometer provides physical separation of
metabolites, introducing different compounds into the mass spectrometer at
different times.

•Chromatography decreases analysis speed but offers two main benefits:

• metabolites with identical masses but different retention times can be
distinguished

• And the separation of metabolites from interfering substances allows

for improved quantitative accuracy.
 The principle of GC
 Like for all other chromatographic techniques, a mobile and a stationary
phase are required

 The mobile phase (=carrier gas) is comprised of an inert gas e.g. helium,
argon, nitrogen, etc.

 The stationary phase consists of a packed column where the packing or

solid support itself acts as stationary phase, or is coated with the liquid
stationary phase (=high boiling polymer).

 More commonly used in many instruments are capillary columns, where

the stationary phase coats the walls of a small-diameter tube (e.g. 0.25 mm
film in a 0.32 mm tube).

 The stronger the interaction is the longer the compound remains attached
to the stationary phase, and the more time it takes to go through the
column (=longer retention time).

 Sample preparation

 High temp of the injection port will result degradation of the non volatile
constituents

 Thus, create false GC peaks corresponding to the volatile degradation products

formed.

 Do some type of sample preparation, component isolation and concentration

prior to analysis

 Sample preparation often involves

 Grinding
 Homogenization
 Reducing particle size
 Enzymes could alter composition of the food product- Inactivate the enzyme
via
 High temperature-short time thermal processing
 Frozen conditions
 Drying
 Homogenization with alcohol

 Microbial growth/chemical reaction may also occur in food during sample

preparation-produce volatile compound thus generates false peak

 Microbes often inhibited by

 Chemical means
 Thermal processing
 Drying
 Frozen storage
 HOW DOES GC/LC WORKS???

 The sample extract is injected onto a GC or LC column.

 For GC, chemical conversion of metabolites to less polar forms, more

volatile (derivatization) allows for detection of more metabolites.

 Flow (velocity) of gas (GC) or liquid (LC) carries compounds through the
column.

 The column retains metabolites based on physical properties, and

different metabolites reach the detector at different times.

 Ion abundances for individual masses are measured relative to the time
the extract was introduced to the chromatography column (retention
time). – mass spec detector
• Measurement of the area under the chromatographic peak for a particular mass and
retention time provides a measure of the amount of the metabolite that elutes at
that time.

• For targeted metabolic profiling analyses, two stages of mass spectrometry (MS/MS)
filter out interfering signals after molecular ions are broken into fragments between
mass-analysis steps.

• Reductions in background interference by the MS/MS technique allow for more rapid
and accurate metabolite analyses, which are needed for large-scale metabolite-
profiling HPLC, high-pressure liquid chromatography.
Diagram of GC
Metabolic profiling analyses
 WHAT INFLUENCE THE SEPARATION??
1. Polarity of the stationary phase

•Polar compounds interact strongly with a polar stationary phase, hence

have a longer retention time than non-polar columns.

•Chiral stationary phases based on amino acid derivatives, cyclodextrins,

chiral silanes, etc are capable to separate enantiomers, because one form is
slightly stronger bonded than the other one, often due to steric effects.

2. Temperature

•The higher the temperature, the more of the compound is in the gas phase.

•It does interact less with the stationary phase, hence the retention time is
3. Carrier gas flow

•If the carrier gas flow is high, the molecules do not have a chance to interact
with the stationary phase.

•The retention time is shorter, but the quality of separation deteriorates.

4. Column length

•The longer the column is the better the separation usually is.

•The trade-off is that the retention time increases proportionally to the

column length.
5. Amount of material injected

•If too much of the sample is injected, the peaks show a significant tailing, which
causes a poorer separation.

•Most detectors are relatively sensitive and do not need a lot of material.
Overall, high temperatures and high flow rates decrease the retention time,
but also deteriorate the quality of the separation.
 WIDELY USED

 Petroleum analysis

 Environmental analysis- NO2, CO2, CO

 Analysis of pharmaceutical products

 Biomedical analysis

 Food analysis
 GC-Food Analysis

 Determination of FA, TAG, cholesterols and other sterols

 Determination of simple sugar and oligosaccharides

 Determination of amino acids and peptides

 Determination of vitamins

 Determination of pesticides, herbicides and food additive

 Determination of flavour compound

 ADVANTAGES OF GC

 Fast analysis- typically minutes, can be automated

 Small samples (μl and μg)

 High resolution

 Reliable, relatively simple and cheap

 Non-destructive
 Allow online coupling e.g. MS

 Sensitive detector

 Highly accurate quantification

 DISADVANTAGES OF GC
 Limited to volatile samples

 Some samples may require intensive preparation

 Samples must be soluble and not react with column

 Requires spectroscopy (usually MS) to confirm the peak identity

 TYPICAL GC DETECTORS

1. Mass Spectrometer (GC/MS)

Many GC instruments are coupled with a mass spectrometer,

which is a very good combination.

The GC separates the compounds from each other, while the

2. Flame Ionization Detector (FID)

Operating principle:

 As compounds elute from the analytical column, they are burned in H2

flame

 A potentiol ~300 V is applied across the flame

 Flame carry a current across potential which is proportional to the organic

ions present in the flame from the burning organic compound

 The current flowing across flame is amplified and recorded

FID responds to organics on a weight basis

FID gives no response to H2O, NO2, CO2, H2S

 Responds is best with compounds containing C-C and C-H bonds

 Food analyst often work with organic compound which FID respond well to-
good sensitivity

 Flavour studies

 FA analysis

 Carbohydrate analysis

 Sterols

 Contaminants in foods

 Antioxidants
3. Thermal Conductivity Detector (TCD)

Operating principle:

 As carrier gas passes over hot filament (tungsten), it cools the filament

 The cooling process depending on carrier gas velocity and composition

 The temperature of filament determines its resistance to electrical current

 As compound elutes with carrier gas, the cooling effect on the filament
decrease resulting rise in temperature in the filament

 This increase the resistance and then monitored by GC electronics

 Choice of carrier gas is important since the differences between its thermal
properties and the analytes determine the response
 Carrier gas

 H2 is the best but flammable

 Helium is most commonly used

 TCD is universal in response and nondestructive to the sample- widely used in

food applications
4. Electron Capture Detector (ECD)

Operating principle:

 ECD contains radioactive foil coating that emits electrons as it undergo

decay

 Electrons are collected on an anode

 Standing current is monitored by instrument electronics

 As analyte elutes from GC column, it passes between the radioactive foil

and the anode

 Compounds that capture electrons reduce the standing current and thereby
give a measurable response
 Halogenated compounds or those with conjugated double bonds give the great
detector responds

 Disadvantages: ECD easily becomes saturated and thus, has very limited linear
response range

 In food applications, ECD is greatest in determining pesticides residues

 HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
 HPLC is basically is a highly improved form of column liquid
chromatography.

 Instead of a solvent being allowed to drip through a column under

gravity, it is forced through under high pressures of up to 400
atmospheres. That make it much faster.

 HPLC allow using a much smaller particle size for the column packing
material which gives much better surface area for interaction between
stationary phase and the molecules flowing past it.

 This allow a better separation of the components of the mixtures

 Sample components need not be volatile

 ADVANTAGES OF HPLC

 Speed –30 min or less (under high pressure up to 400 atm)

 Improved resolution (wide variety of stationary phase)

 Greater sensitivity (wide variety of detectors)

 Reusable columns

 Ideal for ionic species and large molecules (substance low volatility)

 Easy sample recovery

 DISADVANTAGES OF HPLC
 Complexity

 Low sensitivity for some compounds

 Cost

 Irreversibly absorbed compounds not detected

 Coelution difficult to detect

 INSTRUMENTATION

An HPLC system consists of a pumping unit, sample-injection unit, column, detection

unit, and data-processing unit. Each of these units is essential for performing the
analysis.
 Pumping unit
 Role is to delivery mobile phase through the system
 Typically at a flow rate 1ml/min

 It is necessary to pump the eluent at a constant flow rate and

pressure.

 The pump is selected to suit the purpose of the analysis.

 Analyses were first performed using isocratic (which resolves a

solute using a solvent system that does not change composition
during the run) separations in which the eluent composition remains
unchanged during the analysis.

 This techniques is adequate for simple separations.

 Pumping unit
 When a sample contains many components, such as a sample for
amino-acid analysis is analyzed, it is very difficult to separate all of
the components effectively using only one eluent.

 A gradient analysis allows the composition of the eluent to be

changed during the analysis.

 This often indicates that the concentration gradient is to be

generated in a linear manner.
 Pumping unit
 Gradient systems utilize
 Low pressure mixing- mobile phases components are mixed prior
to entering the high pressure pump
 High-pressure mixing- 2/more independent programmable
pumps are used

 However, if the eluent composition is changed in a stepwise fashion,

this is called a step gradient.
Figure 4 illustrates the merits of gradient analysis.

• If eluent A is used, the components eluted earlier are clearly separated, but the
components eluted later show broad peaks or may not elute from the column.

• In contrast, if eluent B is used, the former are insufficiently separated, while the
latter show sharp peaks.

• In this case, a gradient analysis in which the eluent composition is changed from the
A to B during the analysis can be used to improve the separation over time.
 Sample injection unit

 Role to place the sample into the flowing mobile phase for introduction into the
column

 This can be accomplished via a manual injector or an autosampler (large

numbers of sample).

 Each type is equipped with six-port valves, so that a sample can be injected into
the flow path at continuous pressure.

 For the manual injector, the knob is manually operated to deliver the sample to
the column
 Column
 A column is selected to suit both the sample and the purpose of
separation.

 The oven is used to maintain a constant column temperature.

 An analysis temperature is between 25℃ and 50℃ is often

selected.
 Detection unit
 The components eluted from the column are detected, and the
detection data are converted into an electrical signal.

 The detector is selected to suit the sample.

 In addition, the LC-MS system, in which the components separated by

HPLC are further analyzed using a mass spectrometer, is becoming
widely used because of its high sensitivity and the possibility of specific
detection.
 Data processing unit
 The concentration of each detected component is calculated from the
area or height of the corresponding peak, and reported.

 Although previously, easy-to-use integrators were mainly used, systems

in which a PC performs both the operation of the units and the analysis
of the results have recently played a central role.
 Are your friends alike or completely opposite?

 Everyone has his/her own taste. In the world of matter,

substances which are more similar have closer relationships
with one another and desire to exist together.

 Let's observe what occurs within a column.

•When entering the column, a sample delivered by the eluent is first
retained by the fillers.

•The extent to which each component of the sample is retained depends on

its properties.

•A component having properties which differ more from those of the filler
dissolves earlier into the eluent to migrate in the column.

•Meanwhile, a component having a higher affinity for the filler is retained

for a longer time, thus migrating more slowly in the column.

•Thus, the speed at which each component migrates in the column depends
on whether the component prefers (is similar in properties to) the filler or
the eluent.
 Separation methods
 Separation methods are classified into the four modes:
 Adsorption
 Partition
 Ion exchange
 Size exclusion

 Actually, some of these modes appear to cause separation in

combination.
Adsorption mode

•Silica gel or alumina is employed as a filler to separate substances that can

be adsorbed directly on the surface of the fillers.

•Due to its high selectivity, the silica gel column is still used for the
separation of isomers, etc.

•However, this type of filler has the disadvantages of early deterioration and
poor reproducibility.
• The phenomenon of adsorption is utilized in deodorants etc. because of
having the property that retained substances are hard to remove.

• Thus, many adsorbed components remain uneluted, which reduces the

number of adsorption sites, leading to a gradual acceleration in elution.

• This mode causes no difficulties in analyses in which one column is used

once or twice for fractionation, such as in column chromatography.

• However, this mode is not very suitable for analyses in which one column
is used a number of times to treat many samples, as in today's HPLC.

• The partition mode was therefore created as another separation method.

Partition mode

•The term "ODS" or "C18" and the term "reverse phase" or "partition" are
often heard as the names for a column and separation method, respectively.

•All of these terms indicate the identical separation mode.

•As a filler in the partition system, silica gel bound chemically by ODS
(Octadesylsilane, which is a compound that contains a chain of 18 carbon
atoms, displaying oily properties)

•Most often used to make up for the disadvantage of the adsorption mode.
•This filler is stable when used, as one part of its silica gel surface is bound
by ODS, and the other adsorptive part of the surface is also treated.

•The filler can be regarded as a silica gel coated with ODS.

•A sample is separated between an ODS phase and an elute phase.

To understand this situation, image a separated dressing.

• The vinegar and salad oil are separately present in the two layers of
watery and oily phases, respectively.

• When this dressing is vigorously shaken, these two substances are

mixed.

• However, if left for a while, the two substances will again become
separated
• They are originally "water and oil“ that are incompatible with each
other.
To understand this situation, image a separated dressing.

• A sample added to a solution in such a condition is divided into the

water-soluble and oil-soluble components according to their properties
between the two phases.

• The components more soluble in the eluent are eluted earlier from the
column.

• Adsorption mode: "oily" eluent (hexane, octane, etc.) flows on "watery"

fillers.

• Partition mode: "watery" eluent (methanol, acetonitrile, water, etc.)

flows on "oily" fillers.
•When the same sample is separated by each of these modes, the components
are eluted in the reverse order.

•Therefore, the combination of "highly-polar fillers and low-polar eluent" in the

traditional adsorption mode – normal phase

•That of "low-polar fillers and highly-polar eluent" in the partition mode referred
to as reverse phase
 Normal phase HPLC

 The column is filled with tiny silica particles (polar absorbent), and the
solvent is non-polar - hexane, for example

 Polar compounds in the mixture being passed through the column will
stick longer to the polar silica than non-polar compounds will.

 The non-polar ones will therefore pass more quickly through the
column.

 Normal phase HPLC best applied to the separation of compounds that

are highly soluble in organic solvents such as
 Fat-soluble vitamins
 Phospholipids
 Reverse phase HPLC

 More than 70 % all HPLC separations are carried out by reverse

phase method.

 Utilize non polar stationary phase and polar mobile phase.

 The column size is the same, but the silica is modified to make it
non-polar by attaching long hydrocarbon chains to its surface -
typically with either 8 or 18 carbon atoms.

 A polar solvent is used - for example, a mixture of water and an

alcohol such as methanol.

 There will be a strong attraction between the polar solvent and

polar molecules in the mixture being passed through the column.
 There won't be as much attraction between the hydrocarbon chains
attached to the silica (the stationary phase) and the polar molecules in
the solution.

 Polar molecules in the mixture will therefore spend most of their time
moving with the solvent.

 Non-polar compounds in the mixture will tend to form attractions with

the hydrocarbon groups because of van der Waals dispersion forces.

 They will also be less soluble in the solvent because of the need to
break hydrogen bonds as they squeeze in between the water or
methanol molecules, for example.
 Therefore, they spend less time in solution in the solvent and this will
slow them down on their way through the column.

 Reversed phase HPLC is used for

 Analysis of plant proteins
 Cereal proteins
 Water and fat-soluble vitamins
Ion exchange mode

•Ionic substances, including amino acids and inorganic ions pass through an ODS column
without being separated by ODS.

•Such substances can be separated with an ion exchange resin.

•Negative ions such as chloride (Cl-) and sulfide (SO42-) ions, and positive ions such as
Na+ and Ca2+ are separated with anion- and cation-exchange resins, respectively.
 Applications of Ion-Exchange
HPLC

 Ranging from detection of simple inorganic ions to analysis of

carbohydrates and amino acids to the preparative purification of
proteins

 Food analysis
 Determination of organic and inorganic ions in milk
 Organic acids in coffee extract and wine
 Choline in infant formula
 Trace metals, phosphates and sulfites in foods
 Applications of Ion-Exchange
HPLC

 Use a relatively low-capacity stationary phase (anion/cation exchange)

 Use a conductivity detector- all ions conduct electric current

 Mobile phase contain ions- b/ground conductivity high

 Use much lower capacity ion-exchange packing materials- more
dilute eluents could be employed
 Applications of Ion-Exchange
HPLC

 2 types ion chromatography

 Non-suppressed/single-column ion chromatography
 Detector is placed right after the column outlet
 Eluent are carefully chosen to maximize changes in conductivity as
sample components elute from the column
 Suppressed ion chromatography
 Utilizes an eluent that can be selectively removed
 Permits the use of more concentrated mobile phase and gradient
elution
 Adding second suppressor column/ion exchange membranes between
analytical column and conductivity detector
Size exclusion mode

•Macromolecular components such as synthetic resins and proteins can be separated

according to their size, with sieve-like fillers.

•The filler is spherical and contains many holes like a mesh.

•Small molecules can pass through the filler via these holes, but spend much time
running through mazes in the fillers.

•Meanwhile, large molecules unable to enter the holes migrate among the fillers,
arriving earlier at the outlet of the column.

•Molecules small enough to enter the cavity spend different times passing through the
column, according to their sizes.
• Calibration curve must be prepared using a standard sample with known molecular
weight
• vertical axis represents logarithmic molecular weight

• the horizontal axis represents elution time (volume).

• Then, the molecular-weight distribution and mean molecular weight of an unknown

sample can be determined.

• This separation mode is referred to as size exclusion (SEC: Size Exclusion

Chromatography)
 AFFINITY CHROMATOGRAPHY

 Affinity Chromatography is a relatively simple, yet quite effective

molecular technique

 This technique isolates antibodies, antigens, hormones, or other

proteins by taking advantage of their binding affinity for their
respective ligands.
 a receptor and ligand
 An antigen and an antibody
 An enzyme and substrate or substrate mimic

 Can offer high selectivity and high recovery of the target protein
 CONT…..

 Protein can conveniently tagged with a fusion construct to aid

purification to allow them separated by affinity

 Commonly used tag is His6 that non covalently binds with high affinity
to nickel ions

 This metal is bound to chromatographic matrix and is used to capture

the His-tag

 Generally matrices used with nickel have NTA or nitriloacetic acid

covalently attached to the matrix
 MATERIALS

• The major materials required for an affinity chromatography procedure

are

1. a bead matrix

1. a ligand

1. a solution containing the substrate to be isolated

1. a wash to elute the non-bound impurities in the solution

1. a final wash to elute the bound substrate from its ligand

• The bead matrix is an agarose gel loaded into an elution column.

• Sepharose is the most widely used matrix- the hydroxyl groups on the
sugar residues can be easily manipulated to accept a ligand.

• The ligand is then selected according to the desired isolate.

• If you wanted to isolate antibodies specific for antigen A from an

antiserum, you would choose antigen A as your ligand.

• If you wanted to isolate a specific enzyme, you could use either its
substrate, an inhibitor, or even a cofactor.
• Two factors are required for the ligand:

1. First, that it bind specifically and reversibly to the isolate.

• Ideally, the ligand should have an affinity for its substrate between the
range of 10-4 and 10-8M in free solution.

1. Secondly, that the ligand is capable of covalent bonding to the matrix without
disrupting its binding activity.

• This is usually facilitated by the placement of spacer arms between the

ligand and the matrix- incase the active site is buried deep within the
ligand.
• Next, one simply needs a mixture containing the desired isolate.

• The solution is usually a protein rich mixture such as antiserum, which is

poured into the elution column and allowed to run through the gel, at a
controlled rate.

• The first wash must be of sufficient salt concentration, pH, or temperature to

elutes all unbound impurities from the solution, but not so extreme that it
causes the isolate to dissociate from the ligand.

• The second wash however, must elute the isolate, therefore, it must be
sufficiently extreme in concentration, pH, or temperature.
PROCEDURE
 First…..
 Binding of the selected ligand to the matrix requires that a covalent bond to be
formed between the two (Fig. 1, Top panel).

 This is facilitated by derivitization of the sugar residues' hydroxyl groups.

 It is important to realize that the substrate might not be able to reach the ligand
active site if it is hidden deep within the ligand.

 Therefore, most ligands are attached first to spacer arms which are then bonded
to the matrix.

 The ligand-matrix gel is then loaded into an elution column.

PROCEDURE
 Second…..
 Once the column has been prepared, the mixture containing your favorite isolate
is poured into the elution column (Fig. 1, middle panel).

 Once in the column, gravity pulls the solution through the gel, because most of
the proteins do not bind to the ligand-matrix complex.

 However, when the ligand's recognized substrate passes through the gel, it binds
to the ligand-matrix complex, halting its passage through the gel.

 Some of the impurities flow through the gel due to gravity, but most remain,
unbound, in the gel column.
PROCEDURE
 Third…..
 In order to remove these unbound impurities, a wash of extreme pH, salt
concentration, or temperature is run through the gel (Fig. 1, middle panel).

 It is important to use a strong wash so that all the impurities are removed, but it
is also just as crucial that the wash be not so strong that it removes the bound
isolates.

 Once the impurities are washed-out, the only remaining part of the protein
mixture should be the desired isolates.
PROCEDURE
 Final steps…..
 Finally to collect your favorite isolate, which is still bound to the ligand-matrix in
the gel, a stronger second wash is run through the column (Fig. 1, bottom panel).

 This second wash relies on the reversible binding properties of the ligand, which
allows the bound protein to dissociate from its ligand in the presence of this
stronger wash.

 The protein is then free to run through the gel and be collected.
 Supercritical fluid

 When a certain fluid is forced to a pressure (Pc) and

temperature (Tc) higher than its critical point, it becomes a
supercritical fluid.

 Under these conditions, the different properties of the fluid are

placed between those of a gas and those of a liquid.
• Supercritical fluid can be formed from

• A conventional gas by increasing the pressure

• A conventional liquid by raising the temperatures

• Commonly used fluid is CO2- purchased as liquefied gases

• Not a good solvent for polar and high molecular weight compounds

• Although a supercritical fluid density is similar to a liquid and its

viscosity is similar to a gas, its diffusivity is intermediate.
• Thus, the supercritical state of a fluid has been defined as a state in
which liquid and gas are indistinguishable to each other,

• Or as a state in which the fluid is compressible (i.e. similar behavior to a

gas) even though posses a density similar of a liquid and, therefore, has
its solvating power.

• In other word, SF have densities and dissolving capacities similar to

those of certain liquids but lower viscosities and better diffusion
properties.
• In the vicinity of the critical point, the physical properties of the liquid
and the vapor change dramatically, with both phases becoming ever
more similar.

• For instance, liquid water under normal conditions is nearly

incompressible, has a low thermal expansion coefficient, has a high
dielectric constant, and is an excellent solvent for electrolytes.

• Near the critical point, all these properties change into the exact
opposite: water becomes compressible, expandable, a poor dielectric, a
bad solvent for electrolytes, and prefers to mix with nonpolar gases and
organic molecules.
 IMPORTANT PROPERTIES OF
SUPERCRITICAL FLUIDS
1. SFs have high densities (0.2-0.5gm/cm 3 )

• Have a remarkable ability to dissolve large, non-volatile molecules,

• For example- CO2 (mobile phase) readily dissolves n-alkanes containing 5

to 30 carbon atoms, di-n-alkyl phthalates with dialkyl group containing 4-
16 carbon atoms and several polycyclic and aromatic compounds with
many rings.

• Solvation strength of SF is directly related to the fluid density.

2. A second important property of SFs is that dissolved analytes can be
easily recovered by simply allowing the solutions to equilibrate with the
atmosphere at low temperatures,

•Example an analyte dissolved in the SF- CO2 can be recovered by simply

reducing the pressure and allowing to evaporate under ambient laboratory
conditions.

•This property is particularly useful with thermally unstable analytes.

3. Other advantages;

•Inexpensive

•Ecofriendly and non-toxic

•Higher diffusion constants

•Lower viscosities relative to liquid solvents

 SUPERCRITICAL FLUIDS: CARBON DIOXIDE

Carbon dioxide

•Non-flammable, nontoxic and eco-friendly solvent with low critical

temperature (Tc) of 304K and moderate critical pressure (Pc) of 73bar.

•It is miscible with variety of organic solvents and is readily recovered

after processing.

•As it’s a small and linear molecule, it diffuses faster than conventional
liquid solvents.

• It is often used to replace freon and certain organic solvents.

• CO2 is a gas at room temperature, so once the extraction is
completed, and the system decompressed

• A complete elimination of CO2 is achieved without residues and the

extract obtained remains free in the solvent.

• At industrial scale, when CO2 consumption is high, the operation can

be controlled to recycle it.

• However, SF- CO2 because its low polarity, can be less effective to
extract the polar compounds in natural matrices.

• To overcome this shortcoming, modifiers (also called co-solvents like

methanol) are commonly used.

• Modifier can enhance solute solubility, improve peak shape and alter
selectivity
• Other SF that have been used in food applications include

• Nitrous oxide

• Trifluoromethane

• Sulfur hexafluoride

• Pentane

• Ammonia
 SUPERCRITICAL FLUID
CHROMATOGRAPHY
 Chromatography is an analytical technique used for the separation of
complex chemical mixtures into individual components.
 Primarily nonpolar compound- fats, oils and other lipids

 In SFC, the sample is carried through a separating column by a SF.

 Then, the mixture is divided into unique bands based on the amount of
interaction between the individual analytes and the stationary phase in
the column (packed/capillary).

 As these bands leave the column, their identities and quantities are
determined by a detector
 SF used as mobile phase act as both substance carries like mobile phase
in GC and dissolve these substance like solvents in LC.

 SFC is a hybrid of gas and liquid chromatography

 When the mobile phase is below its critical temperature and above
its critical pressure, it acts as a liquid, so the technique is liquid
chromatography (LC)

 Whilst, when the mobile phase is above its critical temperature and
below its critical pressure, it acts as a gas so the technique is gas
chromatography (GC)

 Thus SFC combines some of the best features of each, LC as well as GC.
 SFC is important because it permits separation and determination of
group of compounds that are not conveniently handled by either GC or
LC

 GC is inapplicable for nonvolatile or thermally unstable compounds.

 LC cannot be employed for compounds with those functional groups

that cannot be detected by either spectroscopic or electrochemical
detectors used in LC.
 SFC FLOW CHART

• In SFC, the mobile phase is initially pumped as a liquid and is brought into the
supercritical region by heating it above its supercritical temperature prior to enter the
column.

• It passes through an injection valve (oven coloum) where the sample is introduced into
the supercritical stream and then into the analytical column.

• It is maintained supercritical as it passes through the column into the detector by a

pressure restrictor placed either after the detector or at the end of the column
SFC has been applied to wide variety of materials

Pesticides

Supercritical fluid extraction and chromatography has been used for the
analysis of pesticide residues in canned foods, fruits and vegetables wherein
pyrethroids, herbicides, fungicides and carbamates have been tested.

Lipids

SFC has also been applied to analyze phospholipids after conversion to

diacylglycerol derivatives.

Separation of fatty acid methyl esters, biosynthetic polyunsaturated fatty

acids (PUFA), nonsaponifiable lipids, cholesterol and its esters in human
serum and food samples, mono-, di- and triglycerides in pharmaceutical has
been successfully carried out by SFC.
Natural products

SFC has been successfully utilized for the separation of underivatized

triterpene acids, estimation of caffeine from tea and conjugated bile acids.

Capillary-SFC has been used for analysis of panaxadiol / panaxatriol in

ginseng and its preparations, vegetable carotenoids and pyrolizidine
alkaloids