The two major kinds of chromatographic techniques are Adsorption and Partition

Chromatography.
Difference between Adsorption and Partition Chromatography
The difference is based on physical and chemical substance analyzed and other mobile and
stationary phases used.

Adsorption chromatography principle:

It is a process of separation of components in a mixture introduced into chromatography
system based on the relative differences in adsorption of components to the stationary phase
present in the chromatography column. Here the molecules or components of the mixture
travel with different rates due to differences in their affinity towards stationary phase.
Adsorption means a physical attachment between the compound and the particles of stationary
phase. Based on nature, polar compounds adsorb with stronger or greater intensity to the polar
stationary phase while non-polar compounds adsorb better to the non-polar stationary phase
than polar components. Hence during separation of components, when we use a polar
stationary phase, polar components elute out late due to greater adsorption and non-polar
components get out of the column or elute out first. This is exactly reverse on using a non-polar
stationary phase. This adsorption chromatography applies to only solid-liquid or solid-gas
chromatography. Because the adsorption phenomenon is an inherent property of solids and
hence it is used with only solid stationary phase chromatography.

Partition (coefficient) chromatography:

Partition chromatography is a process of separation whereby the components of the mixture
get distributed into two liquid phases due to differences in partition coefficients during the flow
of mobile phase in the chromatography column. Here the molecules get preferential separation
in between two phases. i.e., both stationary phase and mobile phase are liquid in nature. So
molecules get dispersed into either phases preferentially. Polar molecules get partitioned into
polar phase and vice-verse. This mode of partition chromatography applies to Liquid-liquid,
liquid-gas chromatography and not to solid-gas chromatography. Because partition is the
phenomenon in between a liquid and liquid or liquid and gas or gas and gas. But not in solid
involvement. In high performance liquid chromatography (HPLC), Paper chromatography, gas
chromatography, high performance thin layer chromatography (HPTLC), partition
chromatography is the principle of separation. While in column chromatography, Thin layer
chromatography etc. adsorption chromatography is used.
Efficiency and resolution in chromatography

Column efficiency:

The efficiency of a column is reported as the number of theoretical plates (plate number), N, a

concept Martin borrowed from his experience with fractional distillation:

where tr is the retention time measured from the instant of injection and w is the peak width
obtained by drawing tangents to the sides of the Gaussian curve at the inflection points
and extrapolating the tangents to intercept the baseline. The distance between the intercepts is the
peak width.. Poor chromatograms are those with early peaks (small tr) that are broad (large w),
hence giving small N values, while excellent chromatograms are those with late-appearing peaks
(large tr) that are still very narrow (small w), thereby producing a large N. The number of
theoretical plates is a measure of the “goodness” of the column. Plate numbers may range from
100 to 106. The peak width determined from the chromatogram includes contributions from the
sample-injection technique, extraneous tubing, and the detector. These are extra column
contributions to peak broadening.

Resolution
In general, resolution is the ability to separate two signals. In terms of chromatography, this is
the ability to separate two peaks. Resolution, R, is given by

where tr1 and tr2 and w1 and w2 are the times and widths, respectively, of the two immediately

adjacent peaks. If the peaks are sufficiently close, which is the pertinent problem, w is nearly the
same for both peaks and resolution may be expressed as

Band Broadening (increase in peak width)

Band broadening is a phenomenon that reduces the efficiency of the separation being carried out
–leading to poor resolution and chromatographic performance. This is problematical in terms of
both the quality of the separation obtained and the accuracy with which sample components can
be quantified.

It is well recognized now that column band broadening originates from three main sources:

1) multiple path of an analyte through the column packing; 2) molecular diffusion; 3) effect
of mass transfer between phases.
Along with band broadening, there is always a possibility of tailing, ie sticking of the compound
on to the stationary phases resulting in mixing with the samples coming after that, may also
result.

Types of stationary phases in chromatography:

Non-polar stationary phases, which are compounds, such as hydrocarbons (paraffin) or silicone
oils (polysiloxanes) without grafted polar groups. Examples of such stationary phases are:
squalane (C30H62 hydrocarbon), silicone oils metylsilicon type (names OV 1, SE-30). These
phases separate the analyzed compounds in order of increasing boiling points.

Polar stationary phases, which contain a high proportion of polar groups, i.e. the average
molecular weight polyethylene glycol (Carbowax 20M), silicone oils with cianopropil groups
(OV 225 cianopropyl-methyl-phenyl silicone) diethylene glycol succinate (DEGS),
nitrotereftalic ester of polyethylene glycol (FFAP) etc. They differentiate between the non-polar
and polar compounds, retaining only those which are polar, and are used especially to separate
polar compounds.

- Specific stationary phases, which are used in certain cases. They contain compounds that
interact only with certain components of the mixture to be analyzed, for example AgNO3
dissolved in polyethylene glycol which forms adduct with olefins. –

Chiral stationary phases, containing chiral compounds interacting with only one optical
isomer of a pair of enantiomers. Such phases are based on cyclodextrin or certain amino acids. –

Factors affecting chromatography

1)Polarity of stationary phase and nature of compound:

Given the polarity of the stationary phase and possible interactions, organic compounds can be
grouped in terms of chromatographic separation in the following five classes: - Class I very polar
compounds, able to give hydrogen bonds: water, glycerin, glycols, hydroxyacids, and amino
acids. These compounds are difficult to separate by gas chromatography, due to high polarity.
Excepting water, they derivatized before separation. - Class II polar compounds, which have
active hydrogen atoms: alcohols, carboxylic acids, phenols, primary and secondary amines, nitro
and nitriles with a hydrogen atom in ┙ position. These compounds are separated in polar
stationary phases. - Class III intermediate polarity compounds, without active hydrogen: ethers,
esters, aldehydes, ketones, nitro and nitriles without hydrogen atom in ┙ position. These
compounds are separated on stationary phases of intermediate polarity. - Class IV compounds
with low polarity, but which have active hydrogen: aromatic hydrocarbons, alkenes, chloroform,
methylene chloride, dichloroethane, trichloroethane www.intechopen.com Stationary Phases 37
etc. These compounds are separated on stationary phases of intermediate polarity or non-polar. -
Class V non-polar compounds: alkanes, cycloalkanes. These compounds separate in non-polar
stationary phases.
2)Polarity of Solvent:The solvent polarity plays a crucial role in chromatographic separation.
Highly polar compounds are broughu out using high polar solvents and low polar solvents are
brought out by low polar solvents. Usually, all the technies uses hexane to start with and
gradually move on to higher polarity. A mixture of solvents depending on the nature of the
compounds is the best way for separation

3) Temperature: Increase in temperature results in increase in the rate of movement of solvent.

Decrease the adsorbtion of compound with stationary phase. Hence an optimum temperature is
required to maintain a proper column efficiency.

4) Pressure: High pressure is applied for gas an suprecritcal fluid chromatographic techniques
for maintaining the flow of the mobile phase. While using high grade silica or alumina as
stationary phase, the movement of solvent will be limited. So to increase the rate of the column,
pressure is applied. High pressure is required at times for suitable flow and desorbtion.

Supercritical fluid chromatography

A supercritical fluid (SCF) is any substance at a temperature and pressure above its critical point,
where distinct liquid and gas phases do not exist. It can effuse through solids like a gas, and
dissolve materials like a liquid.

Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a
supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and
purification of low to moderate molecular weight, thermally labile molecules and can also be
used for the separation of chiral compounds. Principles are similar to those of high performance
liquid chromatography (HPLC), however SFC typically utilizes carbon dioxide as the mobile
phase; therefore the entire chromatographic flow path must be pressurized. Because the
supercritical phase represents a state in which liquid and gas properties converge, supercritical
fluid chromatography is sometimes called convergence chromatography.

Some properties of supercritical fluids are:

 they have densities and other properties between liquids and gases
 they can dissolve a large number of compounds
 since they easily evaporate off, the analytes can be easily recovered
  they are non-toxic and inexpensive
 (instrumentation similar to gas chromatography)

.
Detectors

Characteristics of detectors

(1) it should response to all compounds in the mixture ( a general detector) or it should response
with known sensitivity (a specific detector).

(2) It should not response to mobile phase.

(3) It should give linear response to solute concentration.

(4) It should be unaffected by variation in temperature and flow rate.

(5) It should not contribute to zone spreading

The various types of detectors used are

a) UV-Visible detector

b) Mass detector

c) Flame ionization detector

The operation of the FID is based on the detection of ions formed during combustion of organic
compounds in a hydrogen flame. The generation of these ions is proportional to the
concentration of organic species in the sample gas stream. Carrier gas from the column enters at
the bottom of the detector and is mixed with hydrogen combustion gas plus optional makeup gas
in the area below the flame jet. This mixture is then combined with air and burned just above the
jet tip. A negative polarizing voltage is applied between the jet tip and a collector electrode; as
electrons are formed, they are accelerated across the jet tip–collector gap by the electric field and
sent to an electrometer. Depending upon the FID design, either the collector or the jet tip is kept
at ground potential. FID measurements are usually reported as "as methane", meaning as the
quantity of methane which would produce the same response. Hydrocarbons generally have
molar response factors that are equal to the number of carbon atoms in their molecule, while
oxygenates and other species that contain heteroatoms tend to have a lower response factor.
Carbon monoxide and carbon dioxide are not detectable by FID. FID measurements are often
labelled "total hydrocarbons or "total hydrocarbon content" (THC)
Advantages and disadvantages

advantages.
a) Cost: Flame ionization detectors are relatively inexpensive to acquire and operate.
b) Low maintenance requirements: Apart from cleaning or replacing the FID jet, these detectors
require little maintenance.
c) Rugged construction: FIDs are relatively resistant to misuse.
d) Linearity and detection ranges: FIDs can measure organic substance concentration at very
low(10−13 g/s) and very high levels, having a linear response range of 107 g/s.

Disadvantages
Flame ionization detectors cannot detect inorganic substances and some highly oxygenated or
functionalized species like infrared and laser technology can. In some systems, CO and CO2 can
be detected in the FID using a methanizer, which is a bed of Ni catalyst that reduces CO and
CO2 to methane, which can be in turn detected by the FID. The methanizer is limited by its
inability to reduce compounds other than CO and CO2 and its tendency to be poisoned by a
number of chemicals commonly found in GC effluents.

Another important disadvantage is that the FID flame oxidizes all oxidizable compounds that
pass through it; all hydrocarbons and oxygenates are oxidized to carbon dioxide and water and
other heteroatoms are oxidized according to thermodynamics. For this reason, FIDs tend to be
the last in a detector train and also cannot be used for preparatory work.

d)  Electron capture detector (ECD) is a device for detecting atoms and molecules in a gas
through the attachment of electrons via electron capture ionization. is used in gas
chromatography to detect trace amounts of chemical compounds in a sample.
The electron capture detector is used for detecting electron-absorbing components (high
electronegativity) such as halogenated compounds in the output stream of a gas chromatograph.
The ECD uses a radioactive beta particle (electron) emitter in conjunction with a so-called
makeup gas flowing through the detector chamber. The electron emitter typically consists of a
metal foil holding 10 millicuriesof the radionuclide.Usually, nitrogen is used as makeup gas,
because it exhibits a low excitation energy, so it is easy to remove an electron from a nitrogen
molecule. The electrons emitted from the electron emitter collide with the molecules of the
makeup gas, resulting in many more free electrons. The electrons are accelerated towards a
positively charged anode, generating a current. There is therefore always a background signal
present in the chromatogram. As the sample is carried into the detector by the carrier gas,
electron-absorbing analyte molecules capture electrons and thereby reduce the current between
the collector anode and a cathode. The analyte concentration is thus proportional to the degree of
electron capture. ECD detectors are particularly sensitive to halogens, organometallic
compounds, nitriles, or nitro compounds.

e) Thermal Conductivity detector

he TCD consists of an electrically heated filament in a temperature-controlled cell. Under normal

conditions there is a stable heat flow from the filament to the detector body. When an analyte
elutes and the thermal conductivity of the column effluent is reduced, the filament heats up and
changes resistance. This resistance change is often sensed by a Wheatstone bridge circuit which
produces a measurable voltage change. The column effluent flows over one of the resistors while
the reference flow is over a second resistor in the four-resistor circuit. a signal. Since all
compounds, organic and inorganic, have a thermal conductivity different from helium, all
compounds can be detected by this detector. The TCD is often called a universal detector
because it responds to all compounds. Also, since the thermal conductivity of organic
compounds are similar and very different from helium, a TCD will respond similarly to similar
concentrations of analyte. Therefore, the TCD can be used without calibration and the
concentration of a sample component can be estimated by the ratio of the analyte peak area to all
components (peaks) in the sample. The TCD is a good general purpose detector for initial
investigations with an unknown sample. Since the TCD is less sensitive than the flame ionization
detector and has a larger dead volume it will not provide as good resolution as the FID.
However, in combination with thick film columns and correspondingly larger sample volumes,
the overall detection limit can be similar to that of an FID. The TCD is not as sensitive as other
detectors but it is non-specific and non-destructive. The TCD is also used in the analysis of
permanent gases (argon, oxygen, nitrogen, carbon dioxide) because it responds to all these pure
substances unlike the FID which cannot detect compounds which do not contain carbon-
hydrogen bonds.

Expanded bed adsorption Chromatography

It is a preparative chromatographic technique which makes processing of viscous and particulate
liquids possible. In EBA chromatography, the settled bed is first expanded by upward flow of
equilibration buffer. The crude feed, a mixture of soluble proteins, contaminants, cells, and cell
debris, is then passed upward through the expanded bed. Target proteins are captured on the
adsorbent, while particulates and contaminants pass through. A change to elution buffer while
maintaining upward flow results in desorption of the target protein in expanded-bed mode.
Alternatively, if the flow is reversed, the adsorbed particles will quickly settle and the proteins
can be desorbed by an elution buffer. The mode used for elution (expanded-bed versus settled-
bed) depends on the characteristics of the feed. After elution, the adsorbent is cleaned with a
predefined cleaning-in-place (CIP) solution, with cleaning followed by either column
regeneration (for further use) or storage.

Principle
The protein binding principles in EBA are the same as in classical column chromatography and
the common ion-exchange, hydrophobic interaction and affinity chromatography ligands can be
used.After the adsorption step is complete, the fluidized bed is washed to flush out any
remaining particulates. Elution of the adsorbed proteins was commonly performed with the
eluent flow in the reverse direction; that is, as a conventional packed bed, in order to recover the
adsorbed solutes in a smaller volume of eluent. However, a new generation of EBA columns has
been developed, which maintain the bed in the expanded state during this phase, producing high-
purity, high yields of e.g. MAbs [monoclonal antibodies] in even smaller volumes of eluent.
Process duration at manufacturing scale has also been cut considerably (under 7 hours in some
cases).EBA may be considered to combine both the "Removal of Insolubles" and the "Isolation"
steps of the 4-step downstream processing heuristic. The major limitations associated with EBA
technology is biomass interactions and aggregations onto adsorbent during processingWhere
classical column chromatography uses a solid phase made by a packed bed, EBA uses particles
in a fluidized state, ideally expanded by a factor of 2. Expanded bed adsorption is, however,
different from fluidised bed chromatography in essentially two ways: one, the EBA resin
contains particles of varying size and density which results in a gradient of particle size when
expanded; and two, when the bed is in its expanded state, local loops are formed. Particles such
as whole cells or cell debris, which would clog a packed bed column, readily pass through a
fluidized bed.[3] EBA can therefore be used on crude culture broths or slurries of broken cells,
thereby bypassing initial clearing steps such as centrifugation and filtration, which is mandatory
when packed beds are used. In older EBA column designs, the feed flow rate is kept low enough
that the solid packing remains stratified and does not fluidize completely. Hence EBA can be
modelled as frontal adsorption in a packed bed, rather than as a well-mixed, continuous-flow
adsorber.

Size exclusion chromatography

Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is

a chromatographic method in which molecules in solution are separated by their size, and in
some cases molecular weight. It is usually applied to large molecules or macromolecular
complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used
to transport the sample through the column, the technique is known as gel-filtration
chromatography, versus the name gel permeation chromatography, which is used when an
organic solvent is used as a mobile phase. The chromatography column is packed with fine,
porous beads which are composed of dextran polymers (Sephadex), agarose (Sepharose), or
polyacrylamide (Sephacryl or BioGel P). The pore sizes of these beads are used to estimate the
dimensions of macromolecules. SEC is a widely used polymer characterization method because
of its ability to provide good molar mass distribution (Mw) results for polymers.

The advantages of this method include good separation of large molecules from the small
molecules with a minimal volume of eluate and that various solutions can be applied without
interfering with the filtration process, all while preserving the biological activity of the particles
to separate. The technique is generally combined with others that further separate molecules by
other characteristics, such as acidity, basicity, charge, and affinity for certain compounds. With
size exclusion chromatography, there are short and well-defined separation times and narrow
bands, which lead to good sensitivity. There is also no sample loss because solutes do not
interact with the stationary phase.The other advantage to this experimental method is that in
certain cases, it is feasible to determine the approximate molecular weight of a compound. The
shape and size of the compound (eluent) determine how the compound interacts with the gel
(stationary phase).

The main application of gel-filtration chromatography is the fractionation of proteins and other
water-soluble polymers, while gel permeation chromatography is used to analyze the molecular
weight distribution of organic-soluble polymers. nother use of size exclusion chromatography is to
examine the stability and characteristics of natural organic matter in water