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CHROMATOGRAPHY
By Dr Nur Zazarina Ramly
Type of extractions
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
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?
• 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……
Qualitative analysis
Next, a sample prepared from a beverage was measured under the same
conditions, yielding a number of peaks.
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
•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.
Compounds
Gases, liquid, dissolved solids
Organic and inorganic materials
MW ranging from 2 to > 1000 Da
The mobile phase (=carrier gas) is comprised of an inert gas e.g. helium,
argon, nitrogen, etc.
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
Flow (velocity) of gas (GC) or liquid (LC) carries compounds through the
column.
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
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.
4. Column length
•The longer the column is the better the separation usually is.
•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
Biomedical analysis
Food analysis
GC-Food Analysis
Determination of vitamins
High resolution
Non-destructive
Allow online coupling e.g. MS
Sensitive detector
Operating principle:
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
As compound elutes with carrier gas, the cooling effect on the filament
decrease resulting rise in temperature in the filament
Choice of carrier gas is important since the differences between its thermal
properties and the analytes determine the response
Carrier gas
Operating principle:
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
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.
Reusable columns
Ideal for ionic species and large molecules (substance low volatility)
Cost
• 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
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.
•A component having properties which differ more from those of the filler
dissolves earlier into the eluent to migrate 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
•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.
• 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 term "ODS" or "C18" and the term "reverse phase" or "partition" are
often heard as the names for a column and separation method, respectively.
•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 vinegar and salad oil are separately present in the two layers of
watery and oily phases, respectively.
• 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.
• The components more soluble in the eluent are eluted earlier from the
column.
•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.
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.
Polar molecules in the mixture will therefore spend most of their time
moving with the solvent.
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.
•Ionic substances, including amino acids and inorganic ions pass through an ODS column
without being separated by ODS.
•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
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
•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
Can offer high selectivity and high recovery of the target protein
CONT…..
Commonly used tag is His6 that non covalently binds with high affinity
to nickel ions
1. a bead matrix
1. a ligand
• Sepharose is the most widely used matrix- the hydroxyl groups on the
sugar residues can be easily manipulated to accept a 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:
• 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.
• 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).
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.
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
• 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 )
3. Other advantages;
•Inexpensive
Carbon dioxide
•As it’s a small and linear molecule, it diffuses faster than conventional
liquid solvents.
• However, SF- CO2 because its low polarity, can be less effective to
extract the polar compounds in natural matrices.
• 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
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.
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
• 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.
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