Biological Spectroscopy

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Biological Spectroscopy

• Separation Science

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Chromatography

• A general term that describes the separation of
  mixtures into their individual components by
  causing them to pass through a column of solid or
  liquid.
• Chromatography involves a sample (or sample
  extract) being dissolved in a mobile phase (which
  may be a gas, a liquid or a supercritical fluid).
  
• The mobile phase is then forced through an
  immobile, immiscible stationary phase.

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Chromatography

• The phases are chosen such that components of the
  sample have differing solubilities in each phase.
• A component which is quite soluble in the
  stationary phase will take longer to travel
  through it than a component which is not very
  soluble in the stationary phase but very soluble
  in the mobile phase.
• As a result of these differences in mobilities,
  sample components will become separated from each
  other as they travel through the stationary phase.

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Chromatography

• The distribution of analytes between phases can
  often be described quite simply. An analyte is in
  equilibrium between the two phases.
• The equilibrium constant, K, is termed the
  partition coefficient defined as the molar
  concentration of analyte in the stationary phase
  divided by the molar concentration of the analyte
  in the mobile phase

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Chromatography

• The time between sample injection and an analyte
  peak reaching a detector at the end of the column
  is termed the retention time (tR ). Each analyte
  in a sample will have a different retention time.
  The time taken for the mobile phase to pass
  through the column is called tM.

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Fused silica capillary columns

• These have much thinner walls than the glass
  capillary columns, and are given strength by the
  polyimide coating.
• These columns are flexible and can be wound into
  coils. They have the advantages of physical
  strength, flexibility and low reactivity.

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Retention time
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Column temperature

• For precise work, column temperature must be
  controlled to within tenths of a degree.
• The optimum column temperature is dependant upon
  the boiling point of the sample. As a rule of
  thumb, a temperature slightly above the average
  boiling point of the sample results in an elution
  time of 2 - 30 minutes.

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Column temperature

• Minimal temperatures give good resolution, but
  increase elution times.
• If a sample has a wide boiling range, then
  temperature programming can be useful. The column
  temperature is increased (either continuously or
  in steps) as separation proceeds.

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Column types

• Packed
• Contain a finely divided, inert, solid support
  material.
• Coated with liquid stationary phase.
• Most packed columns are 1.5 - 10m in length and
  have an internal diameter of 2 - 4mm.

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Column types

• Capillary
• Capillary columns have an internal diameter of a
  few tenths of a millimeter.
• They can be one of two types wall-coated open
  tubular (WCOT) or support-coated open tubular
  (SCOT).
• Wall-coated columns consist of a capillary tube
  whose walls are coated with liquid stationary
  phase. In support-coated columns, the inner wall
  of the capillary is lined with a thin layer of
  support material onto which the stationary phase
  has been adsorbed. SCOT columns are generally
  less efficient than WCOT columns.
• Both types of capillary column are more efficient
  than packed

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Gas Chromatograph
Injection Port
Detector
Capillary Column
Data System or Recorder
Carrier Gas Supply
Oven
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Gas Chromatography
Gas Chromatograph
Sample mixture of volatile liquids (1?L)
Gas Chromatogram
B
E
C
A
Abundance
D
0
5
10
15
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Time (minutes)
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Liquid Chromatography

• High-performance liquid chromatography (HPLC) is
  a form of liquid chromatography to separate
  compounds that are dissolved in solution.
• HPLC instruments consist of a reservoir of mobile
  phase, a pump, an injector, a separation column,
  and a detector.
• Compounds are separated by injecting a plug of
  the sample mixture onto the column.
• The different components in the mixture pass
  through the column at different rates due to
  differences in their partitioning behavior
  between the mobile liquid phase and the
  stationary phase.

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Liquid Chromatography

• Temperature is not as important as in GC since
  volatility is not important.
• LC mobile phases are e.g. water, methanol,
  acetonitrile
• Single solvent referred to as isocratic.
• Mixtures of solvents may be used, start with one
  mixture, gradually change to a different mixture
  (gradient elution).

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Schematic of liquid chromatograph
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Liquid chromatography
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Liquid chromatography

• In isocratic HPLC the analyte is forced through a
  column of the stationary phase (usually a tube
  packed with small round particles with a certain
  surface chemistry) by pumping a liquid (mobile
  phase) at high pressure through the column. The
  sample to be analyzed is introduced in a small
  volume to the stream of mobile phase and is
  retarded by specific chemical or physical
  interactions with the stationary phase as it
  traverses the length of the column. The amount of
  retardation depends on the nature of the analyte,
  stationary phase and mobile phase composition.
  The time at which a specific analyte elutes
  (comes out of the end of the column) is called
  the retention time and is considered a reasonably
  unique identifying characteristic of a given
  analyte.

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Liquid chromatography

• The use of pressure increases the linear velocity
  (speed) giving the components less time to
  diffuse within the column, leading to improved
  resolution in the resulting chromatogram. Common
  solvents used include any miscible combinations
  of water or various organic liquids (the most
  common are methanol and acetonitrile).
• Water may contain buffers or salts to assist in
  the separation of the analyte components, or
  compounds such as Trifluoroacetic acid which acts
  as an ion pairing agent.

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Liquid chromatography

• A further refinement to HPLC has been to vary the
  mobile phase composition during the analysis,
  this is known as gradient elution. A normal
  gradient for reverse phase chromatography might
  start at 5 methanol and progress linearly to 50
  methanol over 25 minutes, depending on how
  hydrophobic the analyte is. The gradient
  separates the analyte mixtures as a function of
  the affinity of the analyte for the current
  mobile phase composition relative to the
  stationary phase. This partitioning process is
  similar to that which occurs during a
  liquid-liquid extraction but is continuous, not
  step-wise.

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Liquid chromatography

• In an example, using a water/methanol gradient,
  the more hydrophobic components will elute (come
  off the column) under conditions of relatively
  high methanol whereas the more hydrophilic
  compounds will elute under conditions of
  relatively low methanol.
• The choice of solvents, additives and gradient
  depend on the nature of the stationary phase and
  the analyte. Often a series of tests are
  performed on the analyte and a number of generic
  runs may be processed in order to find the
  optimum HPLC method for the analyte - the method
  which gives the best separation of peaks.

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Types of liquid chromatography

• Normal phase chromatography
• Normal phase HPLC was the first kind of HPLC
  chemistry used, and separates analytes based on
  polarity. This method uses a polar stationary
  phase and a non-polar mobile phase, and is used
  when the analyte of interest is fairly polar in
  nature.
• The polar analyte associates with and is retained
  by the polar stationary phase. Adsorption
  strengths increase with increase in analyte
  polarity, and the interaction between the polar
  analyte and the polar stationary phase (relative
  to the mobile phase) increases the elution time.
• The interaction strength not only depends on the
  functional groups in the analyte molecule, but
  also on steric factors and structural isomers are
  often resolved from one another.
• Use of more polar solvents in the mobile phase
  will decrease the retention time of the analytes
  while more hydrophobic solvents tend to increase
  retention times.

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Types of liquid chromatography

• Reversed phase HPLC
• Consists of a non-polar stationary phase and a
  moderately polar mobile phase. One common
  stationary phase is a silica which has been
  treated with RMe2SiCl, where R is a straight
  chain alkyl group such as C18H37 or C8H17. The
  retention time is therefore longer for molecules
  which are more non-polar in nature, allowing
  polar molecules to elute more readily. Retention
  time is increased by the addition of polar
  solvent to the mobile phase and decreased by the
  addition of more hydrophobic solvent. Reversed
  phase chromatography is so commonly used that it
  is not uncommon for it to be incorrectly referred
  to as HPLC without further specification.
• It operates on the principle of hydrophobic
  interactions which result from repulsive forces
  between a relatively polar solvent, the
  relatively non-polar analyte, and the non-polar
  stationary phase.

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Types of liquid chromatography

• Reversed phase HPLC
• The driving force in the binding of the analyte
  to the stationary phase is the decrease in the
  area of the non-polar segment of the analyte
  molecule exposed to the solvent. This hydrophobic
  effect is dominated by the decrease in free
  energy from entropy associated with the
  minimization of the ordered molecule-polar
  solvent interface. The hydrophobic effect is
  decreased by adding more non-polar solvent into
  the mobile phase. This shifts the partition
  coefficient such that the analyte spends some
  portion of time moving down the column in the
  mobile phase, eventually eluting from the column.

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Types of liquid chromatography

• Size exclusion chromatography
• Ion exchange chromatography
• Bioaffinity chromatography

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Chromatography summary

• The derivatization must be highly reproducible
  and usually proceed to completion in order to
  maintain adequate accuracy.
• The capillary columns in GC can have much higher
  efficiencies than their LC counterpart and thus
  GC can more easily handle multicomponent mixtures
  such as essential oils.
• On the other hand, only LC can separate the
  peptides, polypeptides, proteins and other large
  biopolymers that are important in biotechnology

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Capillary electrophoresis
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Capillary electrophoresis

• Has very high efficiencies, meaning hundreds of
  components can be separated at the same time
• Requires minute amounts of sample
• Can be easily automated
• Can be used quantitatively
• Consumes limited amounts of reagents

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Capillary electrophoresis

• Capillary electrophoresis (CE) encompasses a
  family of related separation techniques that use
  narrow-bore fused-silica capillaries to separate
  a complex array of large and small molecules.
• High electric field strengths are used to
  separate molecules based on differences in
  charge, size and hydrophobicity.
• Sample introduction is accomplished by immersing
  the end of the capillary into a sample vial and
  applying pressure, vacuum or voltage.
• Depending on the types of capillary and
  electrolytes used, the technology of CE can be
  segmented into several separation techniques.

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Detection
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Chromatography summary

• Gas chromatography has an entirely different
  field of applications to that of liquid
  chromatography.
• In general, gas chromatography is used for the
  separation of volatile materials and liquid
  chromatography for the separation of involatile
  liquids and solids.
• There are certain compounds, however, that can be
  separated with either techniques, and more
  importantly, many involatile substances such as
  amino acids, steroids and high molecular eight
  fatty acids can be derivatized to form volatile
  substances that can be separated by GC.