Chromatography

Introduction to Chromatographic •chroma – “color”; graphein -“to write”

Methods •1st use was to separate pigment components of plant
extracts
•Now: a group of techniques used to separate the
components of a mixture Identify
- What are the
components present?
Components of a Mixture Separate
Jonyl L. Garcia, M.Sc., RCh.
Quantify
Assistant Professor 3
-How much of the
Chemistry Department components are present?

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Origin of Chromatography Components of Chromatography

•Stationary phase
–The component that
does not move
Plant extract
+
CaCO3 column
+
•Mobile phase
Pet. ether –The component that
moves
Separation of plant pigments into Mikhail Tswett (1872-1919)
colored bands Whether an analyte goes toward the mobile phase
or stationary phase will depend on its polarity, and in
turn its affinity with either phases.

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Types of Chromatography Planar Chromatography

Based on the configuration of the Stationary phase Key features of planar

Thin layer Paper
•Planar chromatography chromatography (TLC) chromatography chromatography:

–Stationary phase is supported on a flat plate or in pores • Mobile phase (solvent system) moves
of paper up or along the plate by capillary
action
–Mobile phase moves by capillary action • Use of a developing chamber
• To maintain equilibrium
•Column chromatography • Prevent solvent evaporation
• Color development or UV irradition of
–Stationary phase packed inside a narrow tube / column Stationary phase is a
silica-coated glass film the plate is usually employed after
–Mobile phase moves under the influence of pressure or solvent has reached the solvent front
• Used for detection of the presence
gravity. Stationary phase is a
paper of known porosity & relative migration of the analytes

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 Column chromatography

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Types of Chromatography

Based on the State of the Mobile Phase

•Liquid Chromatography
•Gas Chromatography
•Supercritical Fluid Chromatography

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The Separation Process
•Elution → the process in which solutes are washed
through a stationary phase by movement of mobile
phase
•Eluent → solvent used to elute components through
the stationary phase
The Separation Process
• Addition of solvent carry solute
molecules through the stationary
phase in a continuous series of
transfers between the two phases
• Different solutes have relatively
different degrees of “attraction”
towards the stationary phase and
the mobile phase
• More retained more attracted to
stationary phase
• Less retained less attracted to
stationary phase

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Chromatogram
• A plot of some function of solute
concentration versus elution time
or elution volume
• Useful for both qualitative and
quantitative analysis
• Retention time/volume → identity
• Peak height/area → quantity

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Recall: the chromatogram

• Goal of chromatography
–Complete separation of components

– In a chromatogram, each peak /

band represents a component

– Therefore, we want our

chromatograms to be well-
resolved:
• Peaks / bands do not overlap
CHROMATOGRAPHIC • Peaks / bands are far apart from
each other as much as possible
PARAMETERS • Peaks / bands are less broad and
trailing

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Effect of variables on band broadening and
separation
Migration Rates of Solutes
• Improved separation of • Separation of solutes → depends upon relative rates of solute elution
components, as exhibited by • Rate of elution → determined by partition ratios of solutes between two phases
chromatographic bands / peaks,
can be achieved by:
–Increasing the rate of band separation
–Decreasing the rate of band spreading
Partition coefficient

• Different partition rates → lead to different rates of elution → lead to separation

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Rate of Solute Migration → Average linear

Migration Rates of Solutes rate & Average linear velocity
• Retention time (tR)
– time it takes after sample injection for the analyte peak to reach the detector
• Dead time / void time (tM)
•Average linear rate of solute migration
– time for unretained species to reach the detector / average rate of motion of mobile phase ~ rate of
migration of unretained species
• (tS)
– time spent by the analyte on the stationary phase
• L – column length

•Average linear velocity of mobile phase

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Rate of Solute Migration → Capacity Factor Sample Problem - 1

• Capacity factor (or retention factor) → an experimental
parameter used to describe the migration rates of solutes in a
column

• If k’<1 – elution occurs rapidly

• If k’ is large (20-30) – elution time is very long
• Ideal k’ value – 1 to 5
• In GC → k’ varied by changing temperature and column
packing
• In LC → k’ varied by changing mobile phase and stationary
phase composition

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Relative Migration Rates → Selectivity
Factor
Sample Problem - 2

•Selectivity factor of a column for 2 species →

provides a measure of how well the column separates
the two analytes
Sample Problem - 1

•Where:
–B = more strongly retained species (species with higher tR)
–A = less strongly retained species (species with lower tR)

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Rate Theory of Chromatography

• Elution peaks / bands resemble Gaussian error curves;
–Attributed to additive combination of the random motions of the many molecules in the
band as it moves down the column
• Random individual processes lead to symmetric spread of velocities around
the mean value;
• A peak / band widens as the band moves down the column because more time
is allowed for spreading to occur.

RATE THEORY OF
CHROMATOGRAPHY

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Rate Theory of Chromatography

• Fronting / Tailing → nonideal
elution peaks
• Arises from the variation of the
distribution constant with
concentration
• Fronting also occurs when too much
of the sample is introduced into the
column (overloading)

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The Theoretical Plate Model of • The theoretical plates in this
chromatography model is analogous to
Chromatography the separations in a fractional
distillation column used in petroleum
refinery
• Martin & Synge, 1954
• A chromatographic column is imagined to contain a large number of More fractions better separations higher
separate layers, referred to as theoretical plates efficiency

• Accounts for most experimental

variables that affect solute migration in
both mobile and stationary phases, eg.
solute adsorption & desorption

• For every theoretical plate, separate equilibrations of the sample

between the stationary and mobile phase occur
• The analyte moves down the column by transfer of equilibrated
mobile phase from one plate to the next.

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The Theoretical Plate Model of The Theoretical Plate Model of

Chromatography Chromatography

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Quantitative Definition of Column Efficiency Quantitative Definition of Column Efficiency

According to the Theoretical Plate Model According to the Theoretical Plate Model
•Plate height (H) or height equivalent of theoretical
plate (HETP)
•Number of theoretical plates (N)

•Column efficiency increases as the number of plates

(N) increases, and/or as plate height (H or HETP)
becomes smaller

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Experimental determination of N and H Sample Problem - 3

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Effect of Mobile Phase Flow Rate

• Extent of band broadening depends
on length of time mobile phase is in
contact with stationary phase
• Efficiency studies done by plotting
H as a function of MP flow rate

KINETIC VARIABLES AFFECTING

BAND BROADENING

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Multiple Paths: Eddy Diffusion (A or CMµ) Multiple Paths: Eddy Diffusion (A or CMµ)

• Solute molecules from band passes through different paths along the
column packing during elution

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Multiple Paths: Eddy Diffusion (A or CMµ)
Longitudinal Diffusion B/µ
•Results in migration of a solute from a
concentrated center of a band to the more
dilute regions on either side (i.e. toward and
opposed to the direction of flow)
•Magnitude of diffusion largely determined by
diffusion coefficient, DM, of the analyte in MP

•Contribution of longitudinal diffusion to plate

height is inversely proportional to linear
velocity of the eluent
•As flow rate increases, the analyte is in
column for briefer period hence longitudinal
diffusion has less time to occur hence less
broadening

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Longitudinal Diffusion B/µ Longitudinal Diffusion B/µ

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Finite Equilibration Time Between Phases Finite Equilibration Time Between Phases
C Sµ C Sµ
• Solutes continue to transfer
from the mobile phase into the
stationary phase and vice versa
as they travel down the column;
• The rate of transfer is slower
with thicker film on the support
or when diffusion coefficient in
the film, DS, is low;
• When transfer across SP is slow,
equilibrium between MP and SP
is not established;

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Finite Equilibration Time Between Phases
C Sµ

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How can we achieve the lowest band

broadening?
Van Deemter Equation

H = A (or Cmu) + B/u + Csu

Optimum flow rate!

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To Reduce Band Broadening… Column Resolution, RS

• Use smaller packing diameter • Provides a quantitative measure of
column’s ability to separate 2
• Use smaller column diameter analytes
(narrower column)
• Use lower temperature (for
GC)
• Use thinner layer of stationary
phase (for open tubular • RS > 1.5: complete resolution w/
columns) 0.3% overlap
• Optimize flow rate • RS < 0.75: incomplete separation
• RS = 1.0: ~4% overlap
• RS can be improved by increasing
column length at the expense of
analysis time

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Relationship between RS and properties of Relationship between RS and properties of
column and solute column and solute
For solutes A and B, where B is the slower moving species

Number of plates needed to achieve a given resolution

Time required to elute two species with a resolution RS

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Relationship between RS and properties of

Factors that affect the retention time (tR)
column and solute

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Sample Problem - 4 Optimization of Column Performance

• Substances A and B have retention times of 16.40 and 17.63 min, respectively, on a
30.0-cm column. An unretained species passes through the column in 1.30 min. The
•Objectives
peak widths (at the base) for A and B are 1.11 and 1.21 min, respectively. –Reduce band broadening
Calculate:
–Alter relative migration
–Column resolution rates of components
–Average number of plates in column
•Choose conditions that
–Plate height
lead to a desired
–Length of column required to reach a resolution of 1.5
degree of resolution
–The time required to elute substance B on the column that
with a minimum
gives a resolution of 1.5
expenditure of time

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Optimization of Column Performance Factors that affect the retention time (tR)
• Variation of Plate Height
• L x2 → tR is doubled
• Variation of Capacity (retention)
factor • µ x 2 → tR is halved
–Change temperature (in GC)
–Change MP composition (in LC)
• Increase K (partition coefficient) → increased tR
• Variation in Selectivity factor • Increase VS → increased tR
–Changing MP composition
–Changing column T
• Inc VM → decreased tR
–Changing SP composition
–Using special chemical effects

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General Elution Problem

• Hypothetical chromatogram for a six-component mixture

APPLICATIONS
• (a) parameters optimized for 1 and 2
• (b) parameters optimized for 5 and 6
• (c) parameters optimized for 3 and 4
–*gradient elution (solvent programming)
–*temperature
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Qualitative Analysis Qualitative Analysis Example

Given this chromatogram of
•“Fingerprinting” cyclooctene derivatives…
•Basis of identification → Retention time • … you can identify the presence of a
cyclooctene derivative in an unknown
–Comparison of retention times of standard and unknown
mixture, provided that the unknown’s
using exactly the same chromatographic parameters (i.e. FR, chromatogram gives off a signal at the
column specs, solvent system, detector, etc) retention time corresponding to any of
the known cyclooctene derivatives.
•Limitation → retention times are only good for
species whose structures are known
•Confirmation of identity would require spectral or
chemical investigation of isolated compounds.

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 Quantitative Analysis:
Quantitative Analysis Area Normalization or Area % calculation
–Requires complete elution of
•Peak height or peak area vary linearly with all components of the sample
concentration –Limited to GLC
•Analysis is based upon a comparison of either the –Peak areas of all components
are summed up, ratio of peak
peak height or area of the chromatographic peak of area of a particular peak
the analyte with that of one or more standards divided over total peak area
–Analysis of peak heights → used when provisions are made –Used to estimate the %
composition the sample
as to not alter peak widths during recording; peaks should
not be distorted or column overloaded
–Analysis of peak area → less sensitive to small changes in
operating conditions; independent of broadening effects

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Quantitative Analysis:
Internal standard addition
Sample Problem

•Internal Standard •In a chromatography experiment, a solution containing

–A known amount of a compound different to the analyte that
0.0837M X and 0.0666M internal standard gave peak areas
of AX = 423 and AIS = 347. Determine the response factor
is added to an unknown sample.
F.
–Elutes close to the analyte
–Similar in concentration to peaks of interest
•An unknown mixture containing X was analyzed using the
–Chemically inert and absent in the sample same chromatograph. Ten milliliters (10.0 mL) of 0.146 M
•Particularly useful for chromatographic analysis. internal standard were added to 10.00 mL of the
Ax = peak area of unknown X
unknown, and the mixture was diluted to 25.00 mL in a
As = peak area of internal standard volumetric flask. The mixture gave the chromatogram
[X] = concentration of unknown X
[S] = concentration of internal standard
with peak areas AX = 553 and AIS = 585. Find the
F = response factor concentration of X in the unknown.

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Assignment

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 QUESTIONS?

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