1. BY:- SWAPNIL NIGAM

8.  thermodynamically
rigorous


9. 

Distribution Ratio(D)”,
Where,
CA denotes
concentration of „A‟ in
all its form as
determined
analytically.

10. 


“Separation factor(or
coefficient)”

12. 
For example, suppose we have
Amount of solute in aqueous phase(xo)=300g
Volume of aqueous phase = 100ml
Volume of organic phase to be added = 200ml
Distribution ratio of the particles = 0.5

13. NOW,
The total amount of solute (x n) left non-
extracted in the aqueous phase („V‟ ml) on
adding the extractive organic phase
(„v‟ ml) can be calculated using the
formulae,

14. WHERE,
xo = amount of solute present before adding
extractive solvent
D = Distribution ratio of the solute particles
n = Number of times the solvent is added

15. CASE 1:
If n=1, i.e., the extractive solvent is added
in one complete go, then, from the
formulae,
we find that, x1 = 60g, that means 240g of
the solute gets extracted.
CASE 2:
If n=2, i.e., if we add the extractive solvent
in two parts each of 100 ml, then,

16. v=100ml, and using the formulae, we find
x2 to be 33g, which implies that, 267g of the
solute has been extracted.

17. 

18. 
 By formation of a neutral metal
chelate complex, or
 By ion association.

19. 
INNER COMPLEXES


21. 
 Basic strength of the chelating group
 Nature of the donor atoms of chelating
agent

22.  Size of the ring
 Resonance & steric effect

23. 


24.  The reagent and the metal
complex exist as undissociated
molecules in both phases.
 Solvation plays no significant part
in the extraction process.
 The solutes are uncharged particle
& their concentrations are so low
that the solutions do not deviate
much from ideality.

25. 


26. 



27. w w
w

28. w
o

29. 
o
w w

30. o
w
*
*

31. *
w

32. 



33. 


34. 



35.  Those formed from the reagents yielding
large organic ions, eg.
Tetraphenylarsonium[(C6H5)4As+], which
tend to form large ionic clusters with
oppositely charged ions, like ReO4- . They
do not have a hydration shell & thus,
disrupt the water structure, due to which
the tend to be pushed into the organic
phase.
 Those involving a cationic or anionic

36. chelate complex of the metal ion. Thus,
the chelating reagent consists of two
uncharged donor atoms. Eg. 1:10
phenanthroline forms cationic complexes.
 Those in which solvent molecules are
directly involved in the formation of ion-
association complex. Eg. ethers, esters
etc.

37. NAME FORMULAE REMARKS
ACETYLACETO- CH3COCHCOCH3
PHENONE
DIMETHYL- CH3COCHCOCH3
GLYOXIME

38. Partition Chromatography II
 Reverse Phase Chromatography
– Nonpolar Stationary Phase
– Polar Mobile Phase
 Normal Phase Chromatography
– Polar Stationary Phase
– Nonpolar Mobile Phase
 Column Selection
 Mobile-Phase Selection

39. Partition Chromatography III
 Research Applications
– Parathion in Insecticides:
O
– CH3CH2O P O NO2
CH3CH2O
– Cocaine in Fruit Flies: A Study of
Neurotransmission by Prof. Jay Hirsh, UVa

41. Adsorption Chromatography
 Classic
 Solvent Selection
 Non-polar Isomeric Mixtures
 Advantages/ Disadvantages
 Applications

42. What is Ion Chromatography?
 Modern methods of separating and determining ions
based on ion-exchange resins
 Mid 1970s
 Anion or cation mixtures readily resolved on HPLC
column
 Applied to a variety of organic & biochemical systems
including drugs, their metabolites, serums, food
preservatives, vitamin mixtures, sugars,
pharmaceutical preparations

43. The Mobile Phases are...
 Aqueous solutions
– containing methanol, water-miscible organic solvents
– also contain ionic species, in the form of a buffer
– solvent strength & selectivity are determined by kind
and concentration of added ingredients
– ions in this phase compete with analyte ions for the
active site in the packing

44. Properties of the Mobile Phase
 Must
– dissolve the sample
– have a strong solvent strength leads to reasonable
retention times
– interact with solutes in such a way as to lead to
selectivity

45. Ion-Exchange Packings
 Types of packings
– pellicular bead packing
• large (30-40 µm) nonporous, spherical, glass,
polymer bead
• coated with synthetic ion-exchange resin
• sample capacity of these particles is less
– coating porous microparticles of silica with a thin film
of the exchanger
• faster diffusion leads to enhanced efficiency

46. Ion-Exchange Equilibria
 Exchange equilibria between ions in solution and ions on
the surface of an insoluble, high molecular-weight solid
 Cation exchange resins
– sulfonic acid group, carboxylic acid group
 Anion exchange resins
– quaternary amine group, primary amine group
CM Cellulose DEAE Cellulose
Cation Exchanger Anion Exchanger

48. Eluent Suppressor Technique
 Made possible the conductometric detection of eluted
ions.
 Introduction of a eluent suppressor column
immediately following the ion-exchange column.
 Suppressor column
– packed with a second ion-exchange resin
 Cation analysis
 Anion analysis

50. Size Exclusion
Chromatography(SEC)
 Gel permeation(GPC), gel filtration(GFC)
chromatography
 Technique applicable to separation of high-molecular
weight species
 Rapid determination of the molecular weight or
molecular-weight distribution of larger polymers or
natural products
 Solute and solvent molecules can diffuse into pores --
trapped and removed from the flow of the mobile
phase

51. SEC(continued)
 Specific pore sizes.average residence time in the pores
depends on the effective size of the analyte molecules
– larger molecules
– smaller molecules
– intermediate size molecules

52. SEC Column Packing
 Small (~10 µm) silica or polymer particles containing
a network of uniform pores
 Two types (diameters of 5 ~ 10 µm)
– Polymer beads
– silica-based particles

53. Advantages of Size Exclusion
Chromatography
 Short & well-defined separation times
 Narrow bands--> good sensitivity
 Freedom from sample loss, solutes do not interact
with the stationary phase
 Absence of column deactivation brought about by
interaction of solute with the packing

54. Disadvantages
 Only limited number of bands can be accommodated
because the time scale of the chromatogram is short
 Inapplicability to samples of similar size, such as
isomers.
– At least 10% difference in molecular weight is required
for reasonable resolution

56. Instrumentation
 Instruments required:
– Mobile phase reservoir
– Pump
– Injector
– Column
– Detector
– Data system

57. Schematic of liquid
chromatograph

58. Mobile phase reservoir
 Glass/stainless steel reservoir
 Removal of dissolved gases by degassers
– vacuum pumping system
– heating/stirring of solvents
– sparging
– vacuum filtration

59. Elution methods
 Isocratic elution
– single solvent of constant composition
 Gradient elution
– 2 or more solvents of differing polarity used

61. Pumping System I
 Provide a continuous constant flow of the
solvent through the injector
 Requirements
– pressure outputs up to 6000 psi
– pulse-free output
– flow rates ranging from .1-10 mL/min
– flow control and flow reproducibility of
.5% or better
– corrosion-resistant components

62. Pumping System II
 Two types:
– constant-pressure
– constant-flow
 Reciprocating pumps
– motor-driven piston
– disadvantage: pulsed flow creates noise
– advantages: small internal volume (35-400 L), high
output pressures (up to 10,000 psi), ready adaptability
to gradient elution, constant flow rates

63. Pumping System III
 Displacement pumps
– syringe-like chambers activated by screw-driven
mechanism powered by a stepper motor
– advantages: output is pulse free
– disadvantage: limited solvent capacity (~20 mL) and
inconvenience when solvents need to be changed
 Flow control and programming system
– computer-controlled devices
– measure flow rate
– increase/decrease speed of pump motor

64. Sample Injection Systems
 For injecting the solvent through the column
 Minimize possible flow disturbances
 Limiting factor in precision of liquid chromatographic
measurement
 Volumes must be small
 .1-500 L
 Sampling loops
– interchangeable loops (5-500 L at pressures up to
7000 psi)

66. LC column
LC injector

67. Liquid Chromatographic Column
 Smooth-bore stainless steel or heavy-walled glass
tubing
 Hundreds of packed columns differing in size and
packing are available from manufacturers ($200-
$500)
 Add columns together to increase length

68. Liquid Chromatographic
Columns II
 Column thermostats
– maintaining column temperatures constant to a few
tenths degree centigrade
– column heaters control column temperatures (from
ambient to 150oC)
– columns fitted with water jackets fed from a constant
temperature bath

69. Detector
 Mostly optical
 Equipped with a flow cell
 Focus light beam at the center for
maximum energy transmission
 Cell ensures that the separated
bands do not widen

70. Some Properties of Detector
 Adequate sensitivity
 Stability and reproducibility
 Wide linear dynamic range
 Short response time
 Minimum volume for reducing zone broadening

71. More Properties of Detector
 High reliability and ease of use
 Similarity in response toward all analytes
 Selective response toward one or more classes of
analytes
 Non-destructive

72. Types of Detector
 Refractive index
 UV/Visible
 Fluorescence
 Conductivity
 Evaporative light scattering
 Electrochemical

73. Refractive Index I
 Measure displacement of beam with respect to
photosensitive surface of dectector

74. Refractive Index II
 Advantages
– universal respond to nearly all solutes
– reliable
– unaffected by flow rate
– low sensitive to dirt and air bubbles in the flow cell

75. Refractive Index III
 Disadvantages
– expensive
– highly temperature sensitive
– moderate sensitivity
– cannot be used with gradient elution

76. UV/Visible I
 Mercury lamp
 = 254nm
 = 250, 313, 334 and 365nm with filters
 Photocell measures absorbance
 Modern UV detector has filter wheels for rapidly
switching filters; used for repetitive and quantitative
analysis

77. UV/Visible II

78. UV/Visible III
 Advantages
– high sensitivity
– small sample volume required
– linearity over wide concentration ranges
– can be used with gradient elution

79. UV/Visible IV
 Disadvantage
– does not work with compounds that do not absorb light
at this wavelength region

80. Fluorescence I
 For compounds having natural
fluorescing capability
 Fluorescence observed by
photoelectric detector
 Mercury or Xenon source with
grating monochromator to isolate
fluorescent radiation

81. Fluorescence II
 Advantages
– extremely high sensitivity
– high selectivity
 Disadvantage
– may not yield linear response over wide range of
concentrations

82. Conductivity
 Measure conductivity of column
effluent
 Sample indicated by change in
conductivity
 Best in ion-exchange
chromatography
 Cell instability

83. Evaporative Light Scattering I
 Nebulizer converts eluent into mist
 Evaporation of mobile phase leads to formation of fine
analyte particles
 Particles passed through laser beam; scattered
radiation detected at right angles by silicon
photodiode
 Similar response for all nonvolatile solutes
 Good sensitivity

84. Evaporative Light Scattering II

85. Electrochemical I
 Based on reduction or
oxidation of the eluting
compound at a suitable
electrode and measurement of
resulting current

86. Electrochemical II
 Advantages
– high sensitivity
– ease of use
 Disadvantages
– mobile phase must be made conductive
– mobile phase must be purified from oxygen, metal
contamination, halides

87. Data System
 For better accuracy and precision
 Routine analysis
– pre-programmed computing integrator
 Data station/computer needed for higher control levels
– add automation options
– complex data becomes more feasible
– software safeguard prevents misuse of data system

88. Electrophoresis…charged species
migrate in electric field
Separation based on charge or
mobility

89. Capillary electrophoresis
higher voltages can be used as
the heat can be dissipated

90. Capillary electrophoresis