HPLC-GPC (1)

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HPLC GPC

• Sangita Rakshe

By, Sangita
Rakshe-Sandbhor Executive, Aquapharm
chemicals, Pune
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OBJECTIVE

• Whats it or what can be done by using GPC,
  HPLC
• How all they work ( basic principle , working
  mechanism )
• When to use this technics ( Application of this
  techniques )

3
Overview

• Chromatography and its principle
• Liquid Chromatography
• High performance Liquid Chromatography (HPLC)
• Instrumentation of HPLC
• Separation mechanism
• Applications
• Introduction gel permeation chromatography (GPC)
• Mechanism of GPC
• Instrumentation
• Method of analysing data
•  

4
History of Chromatography

• Chromatography, literally "color writing", was
  first employed by Russian botanist  Mikhail
  Tswett  in 1903
• He worked with chromatography primarily for the
  separation of plant pigments such
  as chlorophyll, carotenes, and xanthophylls.
  Since these components have different colors
  (green, orange, and yellow, respectively) they
  gave the technique its name.
• It is a physical separation method in which the
  components of a mixture are separated by
  differences in their distribution between two
  phases, one of which is stationary (stationary
  phase) while the other (mobile phase) moves
  through it in a definite direction. The
  substances must interact with the stationary
  phase to be retained and separated by it.

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CHROMATOGRAPHY TERMS

• Chromatogram
• It is the visual output of the chromatograph.
• Chromatograph
• It is equipment that enables a sophisticated
  Separation.
• Stationary phase (bounded phase)
• It is a phase that is bonded to the support
  particles or to the inside wall of the
• column tubing. It can be a solid, a gel, or a
  solid liquid combination
• Mobile phase/ Eluent
• It is the phase which moves in a definite
  direction. And carries mixture to be separated

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CHROMATOGRAPHY TERMS

• Analyte (Sample)
• It is the substance to be separated during
  chromatography.
• Eluate
• It is the mobile phase leaving the column.
• Retention time
• It is the characteristic time it takes for a
  particular analyte to pass through the system
  (from the column inlet to the detector) under set
  conditions.

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Three States of Matter and Chromatography Types
Mobile phase Mobile phase Mobile phase
Gas Liquid Solid
Stationary phase Gas
Stationary phase Liquid
Stationary phase Solid
Gaschromatography
Liquidchromatography
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Interaction Between Solutes, Stationary Phase,
and Mobile Phase

• Differences in the interactions between the
  solutes and stationary and mobile phases enable
  separation.

Solute
Degree of adsorption, solubility, ionicity,
affinity, hydrophobicity, etc.
Stationary phase
Mobile phase
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Classification of Chromatography A/c to physical
state of mobile phase
chromatography
Mobile phase Gas
Mobile phase liquid
Liquid chromatography
Gas chromatography
eg GLC GSC
Liquid -solid chromatography
Liquid-Liquid chromatography
eg column partition Paper partition
eg column adsorption TLC, HPLC
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Classification based on polarity of stationary
phase mobile phase
Normal phase chromatography
Reverse phase chromatography
Stationary phase polar(silica gel) Mobile phase
non-polar (n-hexane)
Stationary phase non polar (Octadecylsilane or
C18) Mobile phase polar (Acetonitrile, water)
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Chromatography
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Liquid Chromatography

• Chromatography in which the mobile phase is a
  liquid.
• Principle is same like chromatography i.e.
  differences in interaction of sample with mp sp
  leads to separation.
• The stationary phase is usually a solid or a
  liquid.
• In general, it is possible to analyze any
  substance that can be stably dissolved in the
  mobile phase.

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Separation Process and Chromatogram for Column
Chromatography

Chromatogram
Output concentration
Time
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From Liquid Chromatography to High Performance
Liquid Chromatography

• Higher degree of separation!? Refinement of
  packing material (3 to 10 µm)
• Reduction of analysis time!? Delivery of eluent
  by pump? Demand for special equipment that can
  withstand high pressures
• The arrival of high performance liquid
  chromatography!

15
HPLC

• Introduction to HPLC
• Instrumentation
• Mechanism / factors affecting HPLC
• Data analysing

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What is HPLC? (Principle)

• Originally referred to as High-Pressure Liquid
  Chromatography
• Now more commonly called High Performance Liquid
  Chromatography
• HPLC is really the automation of traditional
  liquid chromatography that involves
• injection of small volume of liquid sample
• in to column packed with small particles(3-5
  micron)
• where sample moved through the column with
  liquid (mobile phase) forced though the column by
  high pressure delivery pump
• The main principle of separation is adsorption
• The sample components have physical interaction
  with stationary phase
• The components bind at certain region on
  stationary phase (SP) based on their affinity
  towards SP. These bound molecules are then
  eluted with suitable mobile phase and get
  separated from one another.

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What is HPLC? (Principle)

• The mixture component travel according to their
  relative affinities towards the stationary
  phase, the component which have more affinity
  travels slower.
• The component which has less affinity travel
  faster
• Since no two component have same affinity towards
  SP hence the components get separated
• These separated components are detected by
  detector and output of detector called
  chromatogram
• In principle LC HPLC work with same way except
  the speed, efficiency, sensitivity ease of
  operation of HPLC is vastly superior.

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Types of HPLC

• Based on principle of separation
• Ion exchange chromatography
• Ion Pair chromatography
• Size exclusion chromatography (GPC)
• Based on Scale of operation
• Analytical HPLC
• Preparative HPLC
• Based on Stationary phase
• Normal Phase
• Reverse Phase

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Types of HPLC
Types of compounds Mode Stationary phase Mobile phase
Neutrals, Weak acids ,Weak bases Reversed phase C18, C8, C4 Water/Organic Modifiers
Ionics, Bases, Acids Ion Pair C18, C8 Water/Organic Ion-Pair reagent
Compounds not soluble in water Normal Phase Silica, Amino, Cyano, Diol Organics
Ionics/Inorganic ions Ion Exchange Anion or Cation Exchange Resin Organics
High Molecular Weight Compounds, Polymers Size Exclusion Polystyrene ,Silica Gel Filtration Aqueous Gel Permeation Organic
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Flow Channel Diagram for High Performance Liquid
Chromatograph
Detector
Column
Column oven (thermostatic column chamber)
Pump
Sample injection unit (injector)
Eluent (mobile phase)
Drain
Data processor
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Components of HPLC

1. Solvent Reservoir
2. Pumps
3. Sample Injection System
4. Columns
5. Detectors
6. Data Processing
7. Waste

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Components of HPLC

• Solvent Reservoir
• Mobile phase
• isocratic elution - single solvent separation
  technique
• gradient elution - 2 or more solvents, varied
  during separation
• Isocratic is most common
• Pump
• Role is to force a liquid (mobile phase) to the
  column with specific flow rate, expressed in
  ml/min
• A pump capable of pumping solvent up to a
  pressure of 4000 psi and at flows of up to 10
  ml/min
• Normal flow rate in HPLC is 1to 2 ml/min

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Components of HPLC

• Sample Injection System
• It serves to introduce liquid sample in to flow
  stream of mobile phase
• A fixed-volume loop of between 1 200 ?l (20 ?l
  is often used as standard)
• It can be carried out by using ,
• Syringe/injector (manual )
• Auto injector

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Components of HPLC

• Column
• The heart of a HPLC system is the column.
• The column contains the packing of fine particles
  that called as stationary phase.
• packing - silica gel, alumina, polymers
• Diameter - 3 mm to 50 mm
• Height - 5 cm to 30 cm
• The individual components are retained by the
  stationary phase differently and separate from
  each other with the eluent.
• Normally, columns are filled with silica gel
  because its particle shape, surface properties,
  and pore structure help to get a good separation.
• Silica is wetted by nearly every potential
  mobile phase, is inert to most compounds
• Silica can be used to separate a wide variety of
  chemical compounds, and its chromatographic
  behavior is generally reproducible.
• The pH stability range for silica gel is 2-7

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Picture of an HPLC column
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Types of HPLC based on Stationary phase (column)

• Normal Phase.
• Polar stationary phase and non-polar solvent,
  E.g. silica gel- Hexane
• Least polar compound comes out first
• Reverse Phase
• Non-polar stationary phase and a polar
  solvent, E.g. silica gel -C18- water, ACN
• Most polar compounds comes out first

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Representative HPLC Detectors

• UV-VIS absorbance detector
• Photodiode array-type UV-VIS absorbance detector
• Fluorescence detector
• Refractive index detector
• light scattering detector

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HPLC Detector

cont..

• Ultraviolet (UV)
• This type of detector responds to substances that
  absorb light.
• UV detectors are the most versatile, having the
  best sensitivity
• UV detectors cannot be used for testing
  substances that are low in chromophores
  (colorless ) as they cannot absorb light.
• The majority of organic compounds can be
  analyzed by UV/VIS detectors.
• Almost 70 of published HPLC analyses were
  performed with UV/VIS detectors.
• Due to ease of its operation, makes the UV
  detector the most useful and the most widely used
  detector. 

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UV-VIS Absorbance Detector
C Concentration
Detection cell
Ein
Eout
A
l
C
A eCl log (Eout / Ein)
(A absorbance, E absorption coefficient)
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Data Processing

• Using specific software that is connected to HPLC
  machine
• Receive the information from HPLC detector and
  present it as a graph
• The graph gives information about qualitative
  data (Retention time) and quantitative data (area
  under curve)

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Chromatogram
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Operation

• Equilibrate the column with mobile phase till the
  base line is stabilized
• Inject 20µl of mobile phase into the system and
  record the chromatogram(blank)
• Inject 20µl of standard solution into the system
  and record the chromatogram (make calibration
  curve)
• Inject the 20µl of sample solution into the
  system and record the chromatogram
• Wash the column with distilled water for 20
  minutes and with MeOH for 20 minutes

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Operation

cont
-Methanol (any solvent) for 10-30 minutes
-H20
(10-30 minutes) -Mobile Phase

-Standard
-Sample -H20
-MeOH
Washing
Analysis
Washing
Preparation of standard and samples is done by
PPM calculations (Parts per million) (1 PPM is 1
mg in 1 lt solution)
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Calibration Curve of standard for sample analysis
Area
Concentration
A1
Calibration curve
C1
A4
A2
A3
C2
Peak area
A2
A3
C3
A1
A4
C1
C2
C3
C4
C4
Concentration
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Chromatogram
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Normal Phase / Reversed Phase
Stationary phase Mobile phase
Normal phase High polarity (hydrophilic) Low polarity (hydrophobic)
Reversed phase Low polarity (hydrophobic) High polarity (hydrophilic)
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Comparison of Normal Phase and Reversed Phase

• Normal Phase
• Effective for separation of structural isomers
• Sp has short life
• Stabilizes slowly and is prone to fluctuations in
  retention time
• Eluents are expensive, toxic

• Reversed Phase
• Wide range of applications
• Stationary phase has long service life
• Stabilizes quickly
• Eluents are inexpensive and easy to use, less
  toxic

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Stationary Phase and Mobile Phase Used in Normal
/ Reversed Phase Mode

• Normal Phase
• Stationary Phase
• Silica gel -Si-OH
• Cyano type -Si-CH2CH2CH2CN
• Amino type -Si-CH2CH2CH2NH2
• Diol type -Si-CH2CH2CH2OCH(OH)-CH2OH
• Mobile Phase
• Basic solvents Aliphatic hydrocarbons, aromatic
  hydrocarbons,eg. Hexane, xylene, benzene etc.
• Reversed Phase
• Stationary phase Low polarity
• Octadecyl group-bonded silical gel (ODS)
• Mobile phase High polarity
• Water, methanol, acetonitrile
• buffer

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Separation Column for Reversed Phase
Chromatography

• C18 (ODS) type
• C8 (octyl) type
• C4 (butyl) type

• Phenyl type

Si
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
-O-Si
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
C18 (ODS)
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Effect of Chain Length of Stationary Phase in RP
C8
Medium
C18 (ODS)
Strong
C4
Weak
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RP Mechanism (Simple)
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Relationship Between Retention Time and Polarity
OH
C18 (ODS)
Weak
Strong
CH3
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Eluent Used in Reversed Phase Mode

• Eluent used for RP
• Water (buffer solution)
• Water soluble organic solvent e.g.Methanol,
  Acetonitrile, Tetrahydrofuran etc.
• The mixing ratio of the water (buffer solution)
  and organic solvent has the greatest influence on
  separation
• If a buffer solution is used, its pH value is an
  important separation parameter.

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What affect HPLC peak separation

• Column Parameters
• Stationary Phase Polymer, Silica gel, Purity of
  material
• Type of bonded phase C8,C18 etc
• Packing Pore Size/Structure, Particle size
• Column length
• Instrument Parameters
• Temperature
• Flow rate
• Detector
• Sample Parameters
• Concentration
• Matrix
• Solvent
• Mobile phase
• PH
• Buffer ( Eluent type)
• organic modifier( Eluent composition )

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Importance of pH

• Affects ionizable compounds
• organic acids
• organic bases
• In reversed phase we need to suppress ionization
  as much as possible (as Stationary phase is
  hydrophobic)
• May need very precise pH control
• Buffer Solutions Used for HPLC Eluent
• Requirements
• Does not adversely affect detection.
• Does not damage column or equipment.
• Inexpensive.

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Common buffers
PH range
2-13
3-5
2-7
Useful buffering between pH 2-8.
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Characteristics of Phosphate Buffer Solution

• Advantages
• Three dissociation states (pKa 2.1, 7.2, 12.3)
• Possible to prepare buffer solutions of various
  pH values.
• No UV absorption
• Inexpensive

• Disadvantages
• No volatility
• Difficult to use for LCMS or evaporative light
  scattering detection.

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Relationship between Polarity of Eluent and
Retention Time in Reversed Phase Mode
Eluent Methanol / Water
60/40
70/30
80/20
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Replacement of Eluent

• Aqueous solutions containing salt and organic
  solvents must not be exchanged directly.

• Mutually insoluble solvents must not be exchanged
  directly.

Buffer solution
Water
Water-soluble organic solvent
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Mixing, Filtration of the Eluent
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Applications (HPLC)
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Applications (HPLC)

• HPLC is one of the most widely applied analytical
  separation techniques.
• Pharmaceutical
• Tablet dissolution of pharmaceutical dosages.
• Shelf life determinations of pharmaceutical
  products.
• Identification of drug products.
• Pharmaceutical quality control.
• Environmental
• chemicals in Drinking Water.
• Forensics
• identification and quantification of the drug
• Identification of steroids in serum, urine,
  sweat and hair.
• Forensic analysis of textile dyes.
• Clinical
• Analysis of antibiotics.
• Food and Flavor
• Ensuring soft drink consistency and quality.
• Analysis of vicinal diketones in beer.
• Sugar analysis in fruit juices.
• Chemicals in vegetables and fruits.

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Gel Permeation chromatography (GPC)

• Introduction gel permeation chromatography (GPC)
• Mechanism of GPC
• Instrumentation
• Method of analysing data

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Gel Permeation chromatography (GPC)

• Introduction gel permeation chromatography (GPC)
• Mechanism of GPC
• Instrumentation
• Method of analysing data

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GPC

• Introduction
• Types of Liquid Chromatography or HPLC
• Interactive adsorption, partition, ion
  exchange, etc
• Non-interactive GPC/SEC
• Size-exclusion chromatography (SEC) or GPC is a
  chromatographic method in which molecules in
  solution are separated by their size
• GPC have different names in different fields.
  Such as size exclusion chromatography (SEC), gel
  filtration chromatography (GFC) and variants of
  these such as HPSEC, HPGFC, HPGPC. There may be
  some differences ,but the instruments are
  basically all the same thing.
• It is usually applied to large molecules or
  macromolecular complexes such as proteins and
  industrial polymers

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GPC

• Things you should know about GPC/SEC
• Gel permeation chromatography/size exclusion
  chromatography is a type of liquid chromatography
  (LC).
• GPC/SEC can be performed in a wide range of
  solvents from non-polar organics to aqueous
  applications.
• GPC/SEC uses columns packed with very small,
  round, porous particles to separate molecules
  contained in the solvent that is passed through
  them.
• GPC/SEC separates molecules on the basis of
  their size, hence size exclusion.
• The first GPC/SEC columns were packed with
  materials referred to as gels, hence gel
  permeation. GPC/SEC is used to determine the
  molecular weight distributions of polymers.
• The particles in the columns are made from
  polymers that have been cross-linked SDB
  copolymer or other material such as spherical
  silicas

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GPC

• Why do GPC ?
• GPC is the only technique for characterizing
  polymer molecular weight distribution i.e. PDI
• As Mw/Mn (PDI) decreases the strength and
  toughness of the polymer increases
• However as Mw/Mn decreases the polymer becomes
  more difficult to process
• GPC provides key information to predict the
  processability and material properties of a
  polymer

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GPC

• Polymer Molecules in Solution
• Polymer molecules can be described as long chains
  of monomers linked together, they dont exist
  like that in solution.
• Once they have been dissolved, the molecules coil
  up on themselves to form a coil conformation,
  which resembles a ball of string.
• So although they are chains, when we analyze them
  by GPC/SEC they behave like tiny spheres, the
  size of the sphere dependent on the molecular
  weight higher molecular weight polymers coil up
  to form larger spheres.

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GPC

• GPC Separation Mechanism
• Polymer is prepared as a dilute solution in the
  eluent and injected into the system
• The GPC column is packed with porous beads of
  controlled porosity and particle size
• Large molecules are not able to permeate all of
  the pores and have a shorter residence time in
  the column
• Small molecules permeate deep into the porous
  matrix and have a long residence time in the
  column
• Polymer molecules are separated according to
  molecular size, eluting largest first, smallest
  last

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GPC Separation Mechanism
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GPC instrument part
pump delivers eluent from reservoir at a
constant flow rate. injection valve permits
introduction of sample solution without
interrupting solvent flow. GPC tends to use
larger injection volumes (typically up to
200ul) GPC columns perform a separation based
on the molecular size of polymer molecules in
solution. Resolution and/or resolving range is
increased by use of multiple column systems
Detector responds to concentration of polymer
molecules eluting. Differential refractometer
(RI) commonly used detector  
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GPC instrument part
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GPC columns and detector

• Columns
• Column are packed with porous particles, having
  controlled pore size and particle size, typically
  polymer or silica
• Column dimensions typically 7-8 mm i.d. and
  250-300mm length
• Columns are usually employed in combinations of
  two or three columns to improve the resolution of
  the system. Guard columns are often used before
  the main column. As its name implies, the guard
  column protects the main column by stopping
  insoluble particles or contaminants that could
  block the main column set
• Detector
• Differential refractive index (DRI) is most
  common GPC detector is based on the principle
• of refractive index
• These detectors work by assessing the difference
  in refractive index between the sample solution
  and the pure solvent, so they are known as
  differential refractive index detectors. DRIs are
  sometimes referred to as universal detectors,
  as they tend to give a usable response for all
  types of polymer.

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GPC columns and detector
cont

• RI detector
• This detector that measure RI of analyte
  relative to solvent
• When a beam of light passes from one medium into
  another, it bends or direction changes is called
  refraction. The refractive index of material is
  measure of how much light bends when it enters to
  other medium
• DRI contains a flow cell with two parts one for
  sample other for reference solvent. Detector
  measure RI of both component
• When only solvent passing through sample cell,
  both RI are same
• But when analyte (sample solution ) pass through
  it , then there is difference in RI and these
  difference appears as a peak in chromatogram.

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GPC Sample preparation

• To prepare a sample for analysis it is first
  dissolved in an appropriate solvent, such as
  tetrahydrofuran (THF) for organic GPC or water
  based buffers for aqueous SEC.
• It is important that sample are allowed to swell
  and then fully dissolve in the solvent before
  being put through the chromatograph, which may
  take up to 12-24 hours.
• The eluent used to prepare the samples should be
  the same as the solvent running through the
  system i.e. mobile phase.
• Sample concentration employed during analysis is
  dependent on the molecular weight and the
  viscosity of the sample under investigation.
  Table gives some common sample concentrations
  and corresponding to mw for analysis on GPC/SEC.
•  

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GPC

• Additional detector Used in GPC
• Concentration detectors
• Differential refractometer (RI)
• Ultraviolet absorbance (UV)
• Evaporative light scattering or mass detector
  (ELS, EMD)
• Infra-red (IR)
• Molecular weight sensitive detectors
• Viscometry
• Light scattering

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GPC Data analysis

• Elution Profiles
• As a result of the GPC separation mechanism,
  polymer molecules elute from the column in order
  of size in solution
• Largest elute first, smallest elute last
• The separation is purely a physical, there is no
  interaction or binding
• If polymer molecules have the same molecular
  dimensions, they will co-elute by GPC and may not
  be separated by this technique
• The calibration curve describes how different
  size molecules elute from the column

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Calibration of GPC Columns Using Standards

• Chromatograph a series of well characterised,
  narrow polydispersity polymer standards
• Plot of peak retention time (RT) versus peak log
  molecular weight (logM)
• The calibration curve will be characteristic of
  the GPC column set used

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Determination of Polymer Molecular Weight
Distribution by GPC

• Produce a GPC calibration curve for the column
  set relating log M to retention time (RT)
• Chromatograph the polymer sample
• Normalise and integrate the GPC response versus
  retention time plot for the polymer sample
• Convert retention time to logM via the GPC
  calibration curve
• Present a logM distribution plot and calculate
  molecular weight averages (Mn, Mw) for the
  distribution

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Molecular Weight Distribution

• Mp is the molecular weight of the peak maxima
• For any polydisperse peak MnltMwltMz

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Polymer Calibrants for GPC

• Mn - number average molecular weight
• Mw - weight average molecular weight
• Mv - viscosity average molecular weight
• Mp - peak molecular weight
• Mw/Mn - polydispersity by GPC
• Most commonly used polymer calibrants
• Polystyrene - THF, toluene, chloroform
• Polymethyl methacrylate - ethyl acetate,
  acetone, DMF
• Polyethylene oxide/glycol - aqueous eluents, DMF

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HPLC GPC

Thank you
73
HPLC GPC