Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Review
of
Chromatography
Presenting By:
Mr. Muhammad Kamran
&
Mr. Shaiq Ali
Muhammad Kamran

2
History
Mikhail Tswett, Russian, 18721919
Botanist
In 1906 Tswett used to chromatography to
separate plant pigments
He cal...
Milestones in Chromatography
1903 Tswett - plant pigments separated on chalk
columns
1931 Lederer & Kuhn - LC of carotenoi...
Introduction to Chromatography
Definition
Chromatography is a separation technique based on the different
interactions of ...
Chromatography
Is a technique used to separate
and identify the components
of a mixture.
Works by allowing the
molecules p...
Applications of Chromatography
Chromatography has application in every branch of the
physical and biological sciences
Nobe...
Applications of Chromatography
separating mixtures of
compounds

Forensics

Research

Pharmaceutical industry

identifyi...
Types of Chromatography…

Thin layer

Paper

9

HPLC

Gas

Column
Chromatographic
methods classification
Classification of chromatography according to ( 1)
:Phase involved
1.) The primary division of chromatographic techniques ...
2.) Further divisions can be made based on the type of
stationary phase used in the system:
Gas Chromatography
Name of GC ...
Liquid Chromatography
Name of LC Method
Adsorption chromatography
Partition chromatography
Ion-exchange chromatography
Siz...
Classification according to the packing of the( 2)
:stationary phase
In column chromatography the stationary phase is
cont...
(3) Classification according to the force of separation:
Mode or type

Stationary phase

Mobile
phase

Mechanism

Adsorpti...
Term

Definition

Solvent

Mobile liquid phase with no affinity to the stationary phase
.(i.e. inert towards it) & no effe...
SHAIQ ALI

17
Theory of Chromatography
1.) Typical response obtained by chromatography (i.e., a chromatogram):
chromatogram - concentrat...
Note: The separation of solutes in chromatography depends on two factors:
(a) a difference in the retention of solutes (i....
2.) Solute Retention:
A solute’s retention time or retention volume in chromatography is directly related to the
strength ...
3.) Efficiency:
Efficiency is related experimentally to a solute’s peak width.
- an efficient system will produce narrow p...
Number of theoretical plates (N):

compare efficiencies of a system for solutes that
have different retention times

N = (...
Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of
columns with different lengt...
3/18/2009

© RGR

24
a.) Eddy diffusion – a process that leads to peak (band) broadening due to the
presence of multiple flow paths through a p...
b.) Mobile phase mass transfer – a process of peak broadening caused by the
presence of different flow profile within chan...
c.) Stagnant mobile phase mass transfer – band-broadening due to differences in the
rate of diffusion of the solute molecu...
d.) Stationary phase mass transfer – band-broadening due to the movement of solute
between the stagnant phase and the stat...
e.) Longitudinal diffusion – band-broadening due to the diffusion of the solute along the
length of the column in the flow...
Van Deemter equation:

relates flow-rate or linear velocity to H:

H = A + B/µ + Cµ
where:

phase

µ = linear velocity (fl...
Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of the
mobile phase

µ optimum
Optim...
4.) Measures of Solute Separation:
separation factor (α) – parameter used to describe how well two solutes are separated b...
resolution (RS) – resolution between two peaks is a second measure of how well two
peaks are separated:
RS =

tr2 – tr1

(...
Importan Chromatographic formulae

34
THANK YOU
THANK YOU

35
Upcoming SlideShare
Loading in …5
×

Presentation of chromatography by Shaiq Ali

3,584 views

Published on

Published in: Education, Technology
  • Be the first to comment

Presentation of chromatography by Shaiq Ali

  1. 1. Review of Chromatography Presenting By: Mr. Muhammad Kamran & Mr. Shaiq Ali
  2. 2. Muhammad Kamran 2
  3. 3. History Mikhail Tswett, Russian, 18721919 Botanist In 1906 Tswett used to chromatography to separate plant pigments He called the new technique chromatography because the result of the analysis was 'written in color' along the length of the adsorbent column Chroma means “color” and graphein means to “write” 3
  4. 4. Milestones in Chromatography 1903 Tswett - plant pigments separated on chalk columns 1931 Lederer & Kuhn - LC of carotenoids 1938 TLC and ion exchange 1950 reverse phase LC 1954 Martin & Synge (Nobel Prize) 1959 Gel permeation 1965 instrumental LC (Waters) 4
  5. 5. Introduction to Chromatography Definition Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium. The chromatographic process occurs due to differences in the distribution constant of the individual sample components. Components: mobile phase: a solvent that flows through the supporting medium stationary phase: a layer or coating on the supporting medium that interacts with the analytes supporting medium: a solid surface on which the stationary phase is bound or coated 5
  6. 6. Chromatography Is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster.
  7. 7. Applications of Chromatography Chromatography has application in every branch of the physical and biological sciences Nobel prizes were awarded between 1937 and 1972 alone 12 for work in which chromatography played a vital role 7
  8. 8. Applications of Chromatography separating mixtures of compounds Forensics Research Pharmaceutical industry identifying unknown compounds establishing the purity or concentration of compounds monitoring product formation in the pharmaceutical and biotechnology industries 8
  9. 9. Types of Chromatography… Thin layer Paper 9 HPLC Gas Column
  10. 10. Chromatographic methods classification
  11. 11. Classification of chromatography according to ( 1) :Phase involved 1.) The primary division of chromatographic techniques is based on the type of mobile phase used in the system: Type of Chromatography   Gas chromatography (GC) Liquid chromatograph (LC) Type of Mobile Phase gas liquid 11
  12. 12. 2.) Further divisions can be made based on the type of stationary phase used in the system: Gas Chromatography Name of GC Method Type of Stationary Phase Gas-solid chromatography solid, underivatized support Gas-liquid chromatography liquid-coated support Bonded-phase gas chromatography chemically-derivatized support 12
  13. 13. Liquid Chromatography Name of LC Method Adsorption chromatography Partition chromatography Ion-exchange chromatography Size exclusion chromatography Affinity chromatography Type of Stationary Phase solid, underivatized support liquid-coated or derivatized support support containing fixed charges porous support support with immobilized ligand 13
  14. 14. Classification according to the packing of the( 2) :stationary phase In column chromatography the stationary phase is contained in a tube called the column. Planar chromatography. In this geometry the stationary phase is configured as a thin two-dimensional sheet. (i) In paper chromatography a sheet or a narrow strip of paper serves as the stationary phase. (ii) In thin-layer chromatography a thin film of a stationary phase of solid particles bound together for mechanical strength with a binder, such as calcium sulfate, is coated on a glass plate or plastic or metal sheet. 14
  15. 15. (3) Classification according to the force of separation: Mode or type Stationary phase Mobile phase Mechanism Adsorption Chromatography Solid that attracts the solutes Liquid or gas Solutes move at different rates according to the forces of attraction to the stationary .phase Partition Chromatography Thin film of liquid Liquid or formed on the gas surface of a solid inert support Ion Exchange Chromatography Solid resin that carries fixed ions & mobile couterions of opposite charge attached by covalent bonds Liquid Solute ions of charge opposite to the fixed containing ions are attracted to the resin by electrolytes electrostatic forces & replace the mobile .counterions Molecular Exclusion Chromatography Porous gel with no attractive action on solute molecules Liquid :Molecules separate according to their size 1.Smaller molecules enter the pores of the gel, and need a larger volume of eluent. 2.Larger molecules pass through the column at a faster rate. Affinity Chromatography Solid on which specific molecules are immobilized Liquid or gas Special kind of solute molecules interact 15 with those immobilized on the stationary phase Solutes equilibrate between the 2 phases according to their partition coefficients
  16. 16. Term Definition Solvent Mobile liquid phase with no affinity to the stationary phase .(i.e. inert towards it) & no effect on solutes Developer Any liquid with more affinity to the stationary phase than the solvent but less than solutes and just capable to move .them through the column Effluent .Any liquid that passes out of the column Eluent Any liquid that has lesser affinity to the stationary phase .than solutes but is capable to move them out of the column Eluate .Fraction of eluent containing a required specific substance Retention ) volume (VR or retardation volume): Volume of mobile phase that ( passes out of the column, before elution of a specific .substance 16
  17. 17. SHAIQ ALI 17
  18. 18. Theory of Chromatography 1.) Typical response obtained by chromatography (i.e., a chromatogram): chromatogram - concentration versus elution time Wh Wb Inject Where: tR = retention time tM = void time Wb = baseline width of the peak in time units Wh = half-height width of the peak in time units 18
  19. 19. Note: The separation of solutes in chromatography depends on two factors: (a) a difference in the retention of solutes (i.e., a difference in their time or volume of elution (b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation system) Peak width & peak position determine separation of peaks A similar plot can be made in terms of elution volume instead of elution time 19
  20. 20. 2.) Solute Retention: A solute’s retention time or retention volume in chromatography is directly related to the strength of the solute’s interaction with the mobile and stationary phases. Retention on a given column pertain to the particulars of that system: - size of the column - flow rate of the mobile phase Capacity factor (k’): more universal measure of retention, determined from t R or VR. k’ = (tR –tM)/tM or k’ = (VR –VM)/VM capacity factor is useful for comparing results obtained on different systems since it is independent on column length and flow-rate. 20
  21. 21. 3.) Efficiency: Efficiency is related experimentally to a solute’s peak width. - an efficient system will produce narrow peaks - narrow peaks  smaller difference in interactions in order to separate two solutes Efficiency is related theoretically to the various kinetic processes that are involved in solute retention and transport in the column - determine the width or standard deviation (σ) of peaks Estimate σ from peak widths, assuming Gaussian shaped peak: Wh Wb = 4σ Wh = 2.354σ Dependent on the amount of time that a solute spends in the column (k’ or tR) 21
  22. 22. Number of theoretical plates (N): compare efficiencies of a system for solutes that have different retention times N = (tR/σ)2 or for a Gaussian shaped peak N = 16 (tR/Wb)2 N = 5.54 (tR/Wh)2 The larger the value of N is for a column, the better the column will be able to separate two compounds. - the better the ability to resolve solutes that have small differences in retention - N is independent of solute retention - N is dependent on the length of the column 22
  23. 23. Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of columns with different lengths: H = L/N where: L = column length N = number of theoretical plates for the column Note: H simply gives the length of the column that corresponds to one theoretical plate H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size, etc.) to the kinetic processes that give rise to peak broadening: Why Do Bands Spread? a. Eddy diffusion b. Mobile phase mass transfer c. Stagnant mobile phase mass transfer (The fraction of the mobile phase contained within the pores of the particle) d. Stationary phase mass transfer e. Longitudinal diffusion © RGR 23
  24. 24. 3/18/2009 © RGR 24
  25. 25. a.) Eddy diffusion – a process that leads to peak (band) broadening due to the presence of multiple flow paths through a packed column. As solute molecules travel through the column, some arrive at the end sooner then others simply due to the different path traveled around the support particles in the column that result in different travel distances. Longer path arrives at end of column after (1). 25
  26. 26. b.) Mobile phase mass transfer – a process of peak broadening caused by the presence of different flow profile within channels or between particles of the support in the column. A solute in the center of the channel moves more quickly than solute at the edges, it will tend to reach the end of the channel first leading to bandbroadening The degree of band-broadening due to eddy diffusion and mobile phase mass transfer depends mainly on: 1) the size of the packing material 2) the diffusion rate of the solute 26
  27. 27. c.) Stagnant mobile phase mass transfer – band-broadening due to differences in the rate of diffusion of the solute molecules between the mobile phase outside the pores of the support (flowing mobile phase) to the mobile phase within the pores of the support (stagnant mobile phase). Since a solute does not travel down the column when it is in the stagnant mobile phase, it spends a longer time in the column than solute that remains in the flowing mobile phase. The degree of band-broadening due to stagnant mobile phase mass transfer depends on: 1) the size, shape and pore structure of the packing material 2) the diffusion and retention of the solute 3) the flow-rate of the solute through the column 27
  28. 28. d.) Stationary phase mass transfer – band-broadening due to the movement of solute between the stagnant phase and the stationary phase. Since different solute molecules spend different lengths of time in the stationary phase, they also spend different amounts of time on the column, giving rise to bandbroadening. The degree of band-broadening due to stationary phase mass transfer depends on: 1) the retention and diffusion of the solute 2) the flow-rate of the solute through the column 3) the kinetics of interaction between the solute and the stationary phase 28
  29. 29. e.) Longitudinal diffusion – band-broadening due to the diffusion of the solute along the length of the column in the flowing mobile phase. The degree of band-broadening due to longitudinal diffusion depends on: 1) the diffusion of the solute 2) the flow-rate of the solute through the column 29
  30. 30. Van Deemter equation: relates flow-rate or linear velocity to H: H = A + B/µ + Cµ where: phase µ = linear velocity (flow-rate x Vm/L) H = total plate height of the column A = constant representing eddy diffusion & mobile phase mass transfer B = constant representing longitudinal diffusion C = constant representing stagnant mobile & stationary phase mass transfer One use of plate height (H) is to relate these kinetic process to band broadening to a parameter of the chromatographic system (e.g., flow-rate). This relationship is used to predict what the resulting effect would be of varying this parameter on the overall efficiency of the chromatographic system. Number of theoretical plates(N) (N) = 5.54 (tR/Wh)2peak width (Wh) H = L/N 30
  31. 31. Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of the mobile phase µ optimum Optimum linear velocity (µopt) - where H has a minimum value and the point of maximum column efficiency: 31
  32. 32. 4.) Measures of Solute Separation: separation factor (α) – parameter used to describe how well two solutes are separated by a chromatographic system: α = k’2/k’1 k’ = (tR –tM)/tM where: k’1 = the capacity factor of the first solute k’2 = the capacity factor of the second solute, with k’2  k’1 A value of α  1.1 is usually indicative of a good separation Does not consider the effect of column efficiency or peak widths, only retention. 32
  33. 33. resolution (RS) – resolution between two peaks is a second measure of how well two peaks are separated: RS = tr2 – tr1 (Wb2 + Wb1)/2 where: tr1, Wb1 = retention time and baseline width for the first eluting peak tr2, Wb2 = retention time and baseline width for the second eluting peak Rs is preferred over α since both retention (tr) and column efficiency (Wb) are considered in defining peak separation. Rs  1.5 represents baseline resolution, or complete separation of two neighboring solutes  ideal case. 3/18/2009 © RGR Rs  1.0 considered adequate for33 most separations.
  34. 34. Importan Chromatographic formulae 34
  35. 35. THANK YOU THANK YOU 35

×