HL Chemistry - Option A : Modern Analytical Chemistry

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HL Chemistry - Option A Modern Analytical
Chemistry

• Chromatography

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CHROMATOGRAPHY
Chromatography basically involves the
separation of mixtures due to differences in
the distribution coefficient (equilibrium
distribution) of sample components between 2
different phases. One of these phases is a
mobile phase and the other is a stationary
phase.
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Stationary Phase Alumina
Acidic -Al-OH Neutral -Al-OH
-Al-O- Basic -Al-O-
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Stationary Phase Silica (SiO2)
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Distribution Coefficient (Equilibrium
Distribution )
Definition   Different affinity of these 2
components to stationary phase causes the
separation.
Concentration of component A in stationary phase
Concentration of component A in mobile phase
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Some Types of Chromatography

• Liquid Column Chromatography (Reverse Phase too)
• High Pressure (performance) Liquid Chromatograph
  (HPLC)
• Paper Chromatography
• Thin-layer Chromatography (TLC)
• Gas Liquid Chromatography
•  

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LIQUID COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column
packed with solid particles which may or may not
be coated with another liquid. With the proper
solvents, packing conditions, some components in
the sample will travel the column more slowly
than others resulting in the desired separation.
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Diagram of Simple Liquid Column Chromatography
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BASIC LIQUID CHROMATOGRAPHY
The 4 basic liquid chromatography modes are named
according to the mechanism involved  1. Liquid/S
olid Chromatography (adsorption
chromatography) A. Normal Phase LSC B. Reverse
Phase LSC  2. Liquid/Liquid Chromatography
(partition chromatography) A. Normal Phase
LLC B. Reverse Phase LLC  3. Ion Exchange
Chromatography  4. Gel Permeation Chromatography
(exclusion chromatography)
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LIQUID SOLID CHROMATOGRAPHY
The separation mechanism in LSC is based on the
competition of the components of the mixture
sample for the active sites on an absorbent such
as Silica Gel.
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LIQUID SOLID CHROMATOGRAPHY
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WATER-SOLUBLE VITAMINS
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WATER-SOLUBLE VITAMINS
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LIQUID-LIQUID CHROMATOGRAPHY
The stationary solid surface is coated with a 2nd
liquid (the Stationary Phase) which is immiscible
in the solvent (Mobile) phase. Partitioning of
the sample between 2 phases delays or retains
some components more than others to effect
separation.
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Chromatography Schematic
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ION-EXCHANGE CHROMATOGRAPHY
Separation in Ion-exchange Chromatography is
based on the competition of different ionic
compounds of the sample for the active sites on
the ion-exchange resin (column-packing).
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REMEMBER

• The stationary phase is POLAR
• The more polar component interacts more strongly
  with the stationary phase
• The more polar component moves more slowly.
• The non-polar component moves more rapidly.

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MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO
ACIDS
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Chromatography of Amino Acids
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GEL-PERMEATION CHROMATOGRAPHY
Gel-Permeation Chromatography is a mechanical
sorting of molecules based on the size of the
molecules in solution. Small molecules are able
to permeate more pores and are, therefore,
retained longer than large molecules.
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SOLVENTS
Polar Solvents Water gt Methanol gt Acetonitrile
gt Ethanol gt Oxydipropionitrile   Non-polar
Solvents N-Decane gt N-Hexane gt N-Pentane gt
Cyclohexane
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SELECTING AN OPERATING MODE
Sample Type LC Mode   Positional
isomers LSC or LLC Moderate Polarity
Molecules LSC or LLC Compounds with
Similar Functionality LSC or LLC Ionizable
Species IEC Compounds with Differing
Solubility LLC Mixture of Varying Sized
Molecules GCC
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1. Ultraviolet Detector 200-400nm 254
nm2. Reflective Index Detector Universal
Detector
Detectors
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Liquid Chromatography Set Up
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HPLC Chromatography

• Pump System. Mobil phase pressures up to 6000
  psi are necessary to achieve reasonable column
  elution times ( minutes). Typical flow rates
  are 0.1 to 10 mL/minute.
• Injection System. Used to introduce small
  samples (0.1 to 500 µL) into the carrier stream
  under high pressure.
• Reservoirs (Solvents). Multiple solvents are
  necessary for performing gradient elution's (i.e.
  changing the polarity of the mobil phase during a
  run).
• Chromatographic Column. Typically 10-30 cm in
  length containing a packing of 5-10 µm diameter.
  Many types of columns are available, depending on
  the type of liquid chromatography desired.
• Detector. Many types are available including UV,
  IR, refractive index, fluorescence, conductivity,
  mass spectrometry, and electrochemical. Diode
  array detectors are used when wavelength scans
  are desired.

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Schematic of an HPLC System
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HPLC System
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Pump System

• Desirable Features
• Must generate pressures up to 6,000 psi
• To allow for separation in reasonable time frames
• Flow-rates range from 0.1 to 10 mL/minute
• Limited pulsing in the system
• Many HPLC systems have a dual pump system to
  minimize pulsing
• Flow control and reproducibility lt 0.5
• Corrosion resistance

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Sample Injection System

• Used to introduce small samples (0.001 to 0.5 mL)
  into the carrier stream under high pressure

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HPLC Detectors

• No universal or versatile detector
• Types
• General respond to mobil phase bulk properties
  which vary in the presence of solutes (e.g.
  refractive index)
• Specific respond to some properties of the
  solute (not possessed by the mobil phase (e.g. UV
  adsorption)
• Hyphenated detector LC-MS

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Absorbance Detectors

• The UV/Vis source usually comes from a
  monochromator so the wavelength can be selected,
  or scanned.
• Absorbance increases as eluate passes through the
  cell.
• If wavelength scanning is desired, the flow is
  stopped long enough for the scan to take place.
• Its possible to have the same setup using IR
  light, although not as common since many useful
  solvents are not IR transparent.

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Diode Array Detector
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HPLC Detectors
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HPLC Column

• Must operate in high pressure
• Usually constructed of metals
• Typical dimensions
• 10-30 cm long
• 1-3 cm ID
• Contains packing material which holds the
  stationary phase
• Many types exist
• Typical packing materials are 5-10 µm in diameter
• Guard column used to extend life of main column

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Type of HPLC Depends on

• Molecular weight of solute
• Water solubility of the solute
• Polarity of the solute
• Ionic/non-ionic character of the solute

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Separation Principles in HPLC

• General Rule of Thumb
• Polarity of analytes polarity of stationary
  phase ? polarity of mobile phase
• To achieve good separation, the analytes should
  interact with the stationary phase, but not too
  strongly or the retention time will become very
  long

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Reversed order of elution
Increasing Mobil phase Polarity, Decreases
Elution Time
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Typical Applications of HPLC Chromatography
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HPLC of Orange Juice Compounds
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How to Increase HPLC Resolution
1. Increase column length 2. Decrease column
diameter 3. Decrease flow-rate 4. Pack column
uniformly 5. Use uniform stationary phase
(packing material) 6. Decrease sample
size 7. Select proper stationary phase 8. Select
proper mobile phase 9. Use proper
pressure 10. Use gradient elution
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Separating Proteins from Mixtures
In order to understand and study proteins it is
essential to separate them from the biological
fluid. Proteins can be separated from each
other based on differences in physical
properties Due to different amino acid sequences
proteins differ in solubility, size, charge, and
binding affinity and can be separated on either
of these properties.
The inside of a cell. White shapes are proteins
(several 10s of thousands per cell).
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Water, Chemical bonds and groups
Amino acids, pH dependence
Protein primary sequence, peptide bonds,
secondary structures
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Protein studies Understanding protein structure
and function relationships
All proteins have a distinctive 3D structural
conformation This unique structure enables its
function Amino acid sequence determines
structure A major goal of biochemistry is to
determine how amino acid sequences specify the 3D
conformations of proteins and to catalogue all
proteins in cells.
Characterization
cell
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Protein purification general experimental setup
Homogenize
Centrifugation
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Gel permeation chromatography separating on
basis of size
Mixture of proteins

• A mixture of proteins in a small volume is
  applied to a column filled with porous beads
• Because large proteins cannot enter the beads,
  they emerge sooner than do small ones
• A detector (e.g. UV) is used to detect protein
  fragments
• Fragments are collected separately

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Affinity Chromatography separating on the basis
of affinity

• To separate proteins that recognize a chemical
  group X
• X is covalently attached to beads that are packed
  in a column
• Sample of proteins is added
• Washed with buffer to remove non specifically
  bound protein
• Eluted with high concentration of soluble X

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Separation on the basis of charge
All proteins are charged. Their charges depend on
the relative number of acid and basic amino acids
in their primary structure. All proteins have a
pH value where they are uncharged the
isolelectic point (pI)
H2N- Met Ala Asn Cys His Glu Ser Thr Glu
Arg-COOH
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Ionic amino acids
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Separation on the basis of charge (continued)
H2N- Met Ala Asn Cys His Glu Ser Thr Glu
Arg-COOH
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Ion Exchange Chromatography separation on basis
of net charge

• Positive or negatively charged resin can be used
  for separation of positive or negatively charged
  proteins
• Sample of proteins is added
• Washed with buffer to remove non specifically
  bound protein
• Elute with increasing concentration of salt
• Proteins with highest net charge come of last

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Why hydrogels are used for protein separations
1. 2.
3.

• Correct protein folding in aqueous environment
• Proteins can denature on surfaces
• Hydrogels are gt90 water, good environment for
  proteins

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Compare Reverse Phase to Normal Phase Column
Chromatography
In Normal Phase Liquid Chromatography The
column packing in the column is very polar!
Polar compounds are going to be attracted to
the polar column packing by hydrogen bonding or
dipole-dipole attractions. Polar compounds are
going to move slowly! Non-polar compounds are
going to come off the column first, while the
polar compounds are going to come off column
last. Usually, one starts will a less polar
solvent to remove the less polar compounds, and
then you slowly increase the polarity of the
solvent to remove the more polar compounds.
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Reverse Phase Column Chromatography

• The stationary phase (column packing) is now
  NON-POLAR
• Non-polar compounds will move more slowly because
  they are attracted to the column packing.
• The more polar component moves more quickly down
  the column.
• Polar solvents, such as water and methanol are
  used in reverse phase chromatography
• Used mainly in columns, such as HPLC

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Reverse phase chromatography
Silica is alkylated with long chain hydrocarbon
groups, using 18 carbons long. This is usually
referred to as C-18 silica.
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Summary of Methodology

One of the main aims of biochemistry is to
characterize and catalogue all proteins in the
cell We have discussed some important tools for
separating proteins based on physical properties
such as size, affinity, charge. Chromatography
methods ion exchange, affinity, gel permeation
chromatography Electrophoresis iso electric
focusing, SDS PAGE, 2D gels (in the Biochemistry
lecture series)
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Overview of Paper Chromatography

• Works on principle of Partition.
• Separates dried liquid samples.
• Mobile phase is solvent used.
• Stationary phase is water bound to surface of
  paper.
• Advantage its cheap!

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Important Concepts in Paper Chromatography

• Capillary Action the movement of liquid within
  the spaces of a porous material due to the forces
  of adhesion, cohesion, and surface tension. The
  liquid is able to move up the filter paper
  because its attraction to itself is stronger than
  the force of gravity.
• Solubility the degree to which a material
  (solute) dissolves into a solvent. Solutes
  dissolve into solvents that have similar
  properties. (Like dissolves like) This allows
  different solutes to be separated by different
  combinations of solvents.
• Separation of components depends on both their
  solubility in the mobile phase and their
  differential affinity to the mobile phase and the
  stationary phase.

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Paper/TLC Chromatography Animation
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Simple Example of Paper Chromatography using
Sharpie Pens

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Dye Separation in a Black Sharpie

• 1. Dyes separated purple and black
• 2. Not soluble in low concentrations of
  isopropanol
• 3. Partially soluble in concentrations of
  isopropanol gt20

0
20
50
70
100
Concentration of Isopropanol
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Thin Layer Chromatography

• Works mainly on principle of adsorption.
• Mobile phase is the solvent
• Stationary phase is the solid on the plate.

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• TLC vs. Column Chromatography
• Thin-layer chromatography and column
  chromatography and are different types of liquid
  chromatography.
• The mobile (moving) phase is a liquid. The
  stationary phase is usually silica or alumina.
  This phase is very polar.
• The principle of operation is the same!

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Thin Layer Chromatography
The surface of a plate consists of a very thin
layer of silica on a plastic or aluminum
backing. The silica is very polar. This is the
stationary phase. Material is spotted at the
origin (bottom) of the TLC plate. The plate is
placed into a glass jar with a small amount of a
solvent in a glass jar. This solvent acts as the
moving phase. The plate is removed from the
bottle when the solvent is close to the top of
the plate. The spots are visualized (explanation
to follow). Non-polar compounds will be less
strongly attracted to the plate and will
spend more time in the moving phase. This
compound will move faster and will appear closer
to the top of the plate. Polar compounds will
be more strongly attracted to the plate and will
spend less time in the moving phase and appear
lower on the plate.
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Thin-Layer Chromatography A Two-Component
Mixture
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More polar!
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mixture
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Increasing Development Time
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Thin-Layer Chromatography Determination of Rf
Values
Rf of component A dA dS Rf
of component B dB dS
The Rf value is a decimal fraction, generally
only reported to two decimal places
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Thin-Layer Chromatography Qualitative Analysis
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Visualization Method

• The previous slide shows colored spots. Most of
  the time, the spots wont show unless they are
  visualized!
• Vizualization is a method that is used to render
  the TLC spots visible.
• A visualization method can be
• Ultraviolet light
• Iodine vapors to stain spots
• Colored reagents to stain spots
• Reagents that selectively stain spots while
  leaving others unaffected.

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TLC Advantages

• Advantages over paper
• Its faster
• It gives a better separation.
• It is more versatile as the solid on the plate
  can be varied.

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Uses of TLC

• To determine how many components there are in a
  mixture (is it really pure?)
• To determine the best solvent conditions for
  separation on a column
• To identify the substances being studied
• To monitor the composition of fractions collected
  from column chromatography
• To monitor the progress of a reaction

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Gas-Liquid Chromatography

• Works on principle of Partition.
• Mobile phase is the carrier gas.
• Stationary phase is oil bound to surface of beads
  within the column.

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Most Common Stationary Phases

• 1. Separation of mixture of polar compounds
• Carbowax 20M (polyethylene glycol)
• 2. Separation of mixtures of non-polar compounds
• OV101 or SE-30 (polymer of methylsilicone)
• Methylester of fatty acids
• DEGS (diethylene glycol succinate)

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Gas-Liquid Chromatography
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Gas-Liquid Chromatography

• Retention time is used to identify a component of
  a mixture. It depends on-
• The temperature of the column.
• The length of the column.
• The material used to pack the column.
• The gas pressure.

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Gas Liquid Chromatography

• The area under a peak is proportional to the
  amount of substance present.

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Gas Chromatography
Filters/Traps
Data system
Syringe/Sampler
Regulators
Inlets
Detectors

• gas system
• inlet
• column
• detector
• data system

Column
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Schematic Diagram of Flame Ionization Detector
Collector
Detector electronics
? - 220 volts
Flame
Chassis ground
Jet
Signal output
Column
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Gas-Liquid Chromatography

• Gas-Liquid Chromatography is often combined with
  mass spectroscopy. The GC separates the
  components then the MS analyses them.

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One possible Use of GC SEMI- QUANTITATIVE
ANALYSIS OF FATTY ACIDS
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Gas Chromatogram of Methyl Esters of Fatty Acids
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Another GC Use TENTATIVE IDENTIFICATION OF
UNKNOWN COMPOUNDS
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GLC ADVANTAGES
1. Very good separation 2. Time (analysis is
short) 3. Small sample is needed - ml 4. Good
detection system 5. Quantitatively analyzed
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DISADVANTAGES OF GAS CHROMATOGRAPHY
Material has to be volatilized at 250º C without
decomposition!
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Summary of Some Chromatographic Techniques