CHROMATOGRAPHY A POWERFUL
SEPARATION TECHNIQUE
Why this name ?
First used to separate plant pigments
Such as Chlorophyll and Xanthophylls
By passing solutions of their compounds through glass column packed with
CaCO3
Separated species appeared as colored bands in the column
CHROMA Color
GRAPHEIN Writing
GENERAL DESCRIPTION
Chromatography is
A group of different methods which allows
Separation , Identification and Determination
Of closely related components of complex mixtures
-
Whose separation is otherwise impossible
CHROMATOGRPAHIC SEPARATION
INVOLVES
The sample dissolved in a mobile phase
-
Gas
Liquid
Supercritical fluid
Mobile phase forced through an immiscible stationary phase
Stationary phase fixed in place in a column or on a solid surface
SELECTION OF MOBILE & STATIONARY PHASES
Selected in such a way that
The components of the sample(mixture) distribute themselves between
mobile and stationary to varying degrees
Component Strongly held / retained by the stationary phase
Moves slowly with flowing mobile phase
Component Weakly held / retained by the stationary phase
Moves rapidly with flowing mobile phase
HOW SEPARATION OCCURS
Difference in migration rates help separation of components into
BANDS or ZONES
Which is then analyzed qualitatively and quantitatively
CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUES
Based on physical means by which stationary and mobile phases are
brought into contact
COLUMN CHROMATOGRAPHY
Stationary phase held in a narrow tube through which mobile phase is
forced under pressure
PLANAR CHROMATOGRAPHY
Stationary phase suspended on a flat paper or plate, mobile phase
moves through stationary phase by capillary action or under the
influence of gravity
*** Theory behind both the techniques are the same
II Based on the types of mobile and Stationary phases
Technique
Gas Chromatography(GC)#
Mobile Phase
Gases
(only column)
Liquid Chromatography(LC)
Liquids
(both column and planar)
Supercritical Fluid Chromatography
(only column)
Supercritical Fluids
ELUTION IN COLUMN CHROMATOGRAPHY
Substances A & B separated on a packed column by elution
COLUMN
Narrow bore tubing packed with finely divided inert solid - the stationary
phase
Solution of the sample (Mixture A & B) in mobile phase is poured at time t0
A & B gets distributed between mobile and stationary phases
Elution involves washing a species through a column by addition of Fresh
mobile phase
In MODERN CHROMATOGRAPHY
Continuous addition of mobile phase achieved by
using pump in LC and SFC
application of pressure in GC
With introduction of fresh mobile phase ELUENT
Sample moves down the column and gets partitioned between the mobile
and stationary phase
Rate at which a solute zone migrates down the column depends on the
fraction of time it spends in mobile phase (t1)
For solutes Strongly retained by stationary phase Fraction is small
For solutes Not strongly retained by stationary phase Fraction is large
Separation into Bands
Resulting difference in rates cause components in a mixture to separate into
bands/ zones
Isolation of Separated Species
By passing more mobile phase to cause individual zones to be eluted from
the column further they can be detected/ collected.
Chromatogram
A detector that responds to solute concentration placed at the end of the
column whose signal is plotted as a function of time
-
Series of peaks obtained CHROMATOGRAM
Distribution constant is fundamental to chromatography
-cannot be measured
Retention time is a function of distribution constant
-can be measured
Small peak for species not retained by the column (often mobile phase
contains one or added)
tm time for un retained species to reach detector --------- Dead / Void time
All components spend tm in mobile phase
Large peak corresponds to Analyte time required for this zone to reach
Detector after injection Retention time tR
tR = tS + tM
Average linear rate of solute migration = L / tR
Average linear velocity u of mobile phase, u = L / tM
Retention factor
Important experimental quantity used to compare migration rates of solutes
in column
k independent of column geometry / flow rate
ka =
tRtM
tM
k < 1 solute emerges out of column at a time near void time
k = 20 30 elution time is long
Ideally Separations performed where retention factor for solutes in a
mixture are between
1 & 10
Selectivity factor of a column for two solutes
( t R ) Bt M
( t R ) A t M
Band Broadening & Band Separation
Movement down the column increases distance between bands
Also, broadening of both zones take place, which reduces the efficiency of
column Band broadening is inevitable
Reasons for Band Broadening
Rate theory
Shapes and breadths of elution bands are described quantitatively based on
a random walk mechanism
For migration of molecules through the column
Gaussian shape of chromatographic band attributed to additive combination
of random motions of various molecules as they move down the column
Single solute undergoes many thousands of transfers between stationary
and mobile phase during elution. Residence time in either phase is highly
irregular
Transfer between phases requires energy
It is acquired from surroundings
Thus residence time is very short and after some time long.
Movement can occur only if the solute is in the mobile phase
Certain particles travel rapidly by their accidental inclusion in the mobile
phase for most of the time
Whereas after lag as they are incorporated in stationary phase for > average
length of time
Result of these random individual processes is a symmetric spread of
velocities around the mean value which represents behavior of analyte
molecules
Breadth of a zone increases as it moves down the column because more time
is allowed for spreading
Zone breadth residence time ( length of the time the mobile phase is in
contact with stationary phase)
& 1 / velocity of mobile phase flow
Qualitative aspects of column efficiency
Plat height, H
Plate count, No. of theoretical plates N
N= L/H
L length of column packing in cm
Efficiency increases as the plate height becomes smaller and plate count
increases
Theoretical plate = distillation column made of numerous discrete but
continuous narrow layer
Each plate equilibration of the solute between mobile and stationary phase
is assumed to take place
Movement of solute down the column treated as step wise transfer of
equilibrated mobile phase from one to next
Plate theory doesnt account for peak broadening
Plate Height:
H=
2
L
W= width of the peak
Band broadening Reflects column efficiency
No. of plates more column efficiency more
Made mere by increasing column ( height) length
Diameter of the plate column
Decreasing plate height narrower & narrower
Reducing linear velocity leading to reducing multipath
Increasing velocity leading to less longitudinal diffusion
Reduction in G
Optimal flow rate make plate height minimum
OPTIMIZATION
Reduce zone broadening
Altering relative migrations of components
Column Resolution
How apart two bands are relative to their widths
R=
Effect of retention and velocity factor
Increases N but takes more time to complete.
Reduce H by increasing the length of the column
Variation in H
H reduced
Decrease in packed particle size
Reducing solvent viscosity
Variation in Retention factor
Selecting factor
Increases by 2
increases with increase in KB
with increase in selective resolution
Changing composition of mobile phase
Column Temperature
Composition of stationary phase
Using special effects
PARTITION CHROMATOGRAPHY
Stationary phase is a second liquid
Immiscible with the liquid mobile phase
In the past chromatography was applicable to
Non ionic
Polar compounds of low molecular mass
Recently
To partition separation of ionic compounds too
TYPES OF COLUMNS
i.
ii.
Liquid-Liquid columns where liquid is held by physical adsorption
Liquid Bonded Phase columns held by chemical bonding Highly stable
Bonded Phase is more versatile
Support for bonded phase packings from
Rigid silica
Silica based compositions
Uniform, porous, mechanically sturdy particles
Fully hydrolysed silica with reactive silanol
Siloxanes most useful bonded phase coatings
Normal and Reversed Phase packings
Based on the relative polarities of mobile and stationary phases
Normal Phase
Highly polar stationary phase eg. Triethylene glycol, water
Non- polar solvent as mobile phase eg. Hexane
Least polar compounds eluted first
Increasing the polarity of the mobile phase increases elution time
Reversed Phase
Non polar stationary eg hydrocarbon
Polar mobile phase eg,. Water , methanol
Elution with highly polar mobile phase
Bonded phase packings
are reversed phase when bonded coating is non polar
normal when coating is polar
Mechanism of retention of solutes not clear
