Chromatographic separation

Introduction
 What is Chromatography?
 Chromatography is technique the separation, identification, and purification
of the components of a mixture.
 Chromatography(from Greek chroma“ color and graphe in“ to write") is the
collective term for a set of laboratory techniques for the separation of
mixtures.
 Tswett (1990’s) stated that “chromatography is a method in which the
components of a mixture are separated on adsorbent column in a flowing
system.
 IUPAC definition (International Union of pure and applied Chemistry)
(1993): Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases,
 Phase one of which is stationary (stationary phase) while the other (mobile
1
phase) moves in a definite direction.
 Chromatography is the ability to separate molecules using various
partitioning characteristics of molecule to remain in a stationary
phase versus a mobile phase.
 Stationary phase - Immobilized phase
 Immobilized on the support particles or on the inner wall of the
column tubing.
 The stationary phase in chromatography is the phase that is either a
solid -liquid particle attached to a glass or a metal surface on
which the components of the mixture to be separated is absorbed.
 Most substances used as stationary phases are porous, thus allowing
the attachment of components during chromatography
 Examples : Silica - Thin layer chromatography
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 Mobile phase is moves in a definite direction.
 The term mobile indicates that the phase is moving down the
chromatographic system
 It is moves through the chromatography column (the stationary
phase) where the sample interacts with the stationary phase and is
separated.
 It is the solvent that carries the mixture as it moves down the
stationary phase.
 Substances used as mobile phases are selected for a
chromatographic process depending on the nature of the
components to be separated and the type of chromatography.
 Alcohol, water, acetic acid, acetone, or some gases are the
commonly used mobile phase in different chromatographic
techniques.
 The solutes (analytes) are separated due to differences in how they
interact with the two phases
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 Principles of Chromatography

 Chromatography is based on the principle where molecules in

mixture applied onto the surface or into the solid, and fluid
stationary phase is separating from each other while moving with
the aid of a mobile phase.
 Principle of chromatographic separation: Different distribution of
the analytes between mobile and stationary phase results in
different migration velocities
 Any Chromatography system is composed of three Components:
• Stationary phase
• Mobile phase
• Mixture (analytes) to be separated 4
 Three components form the basis of the chromatography
technique.
1. Stationary phase: This phase is always composed of a
“solid” phase or “a layer of a liquid adsorbed on the surface
solid support”.
2. Mobile phase: This phase is always composed of “liquid” or
a “gaseous component.”
3. Separated molecules
 The type of interaction between the stationary phase, mobile
phase, and substances contained in the mixture is the basic
component effective on the separation of molecules from each
other

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 Principle of Chromatography (how does chromatography
work)

 Eluent - Fluid entering column/ solvent that carries the analyte.

 Eluate - Mobile phase leaving the column.
 Sample (Anylate) :Substance analyzed in chromatography.
 Solvent : Any substance capable of solubilizing another
substance.
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 Types of Chromatography
A. Based on Mobile phase
1. Liquid Chromatography(LC)
2. Gas Chromatography (GC)

B. Based on the stationary phase

1. Planar or Plane Chromatography: -the stationary phase is used in
the form of layer. Planar Chromatography includes:
A. Thin layer Chromatography (TLC) B. Paper Chromatography (PC)
2. Column Chromatography (CC)
The stationary phase is held into a tube made of glass or metal.

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 Column chromatography
 Column chromatography is the separation technique where the
components in a mixture are separated on the basis of their
differential adsorption with the stationary phase,
 Resulting in them moving at different speeds when passed through
a column.
 It is a solid-liquid chromatography technique in which the
stationary phase is a solid & mobile phase is a liquid or gas.
 The stationary phase is held in a narrow tube, and the mobile phase
is forced through the tube under pressure or gravity.
 The column consists of narrow tubing packed with a finely divided
inert solid that holds the stationary phase on its surface.
 The two components distribute themselves between the mobile
phase and stationary phase.
 Elution occurs by forcing the sample components through the
column by continuously adding fresh mobile phase. 9
 Principle of Column chromatography

 This technique is based on the principle of differential adsorption where

different molecules in a mixture have different affinities with the absorbent
present on the stationary phase.
 The molecules having higher affinity with stationary phase remain
adsorbed for a longer time decreasing their speed of movement through the
column.
 However, the molecules with lower affinity with stationary phase move
with a faster movement, thus allowing the molecules to be separated in
different fractions.
 Here, the stationary phase in the column chromatography also termed the
absorbent is a solid (mostly silica) and the mobile phase is a liquid that
allows the molecules to move through the column smoothly. 10
Figure: Column chromatography

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 A single portion of the sample dissolved in the mobile phase is
introduced at the head of the column (at time t0 ),
 Components A and B distribute themselves between the two
phases as depicted in figure 1(a) below
 Additional of mobile phase (the eluent) forces the dissolved
portion of the sample down the column,
 Where further partition between the mobile phase and fresh
portions of the stationary phase occurs (time t1).
 Partitioning between the fresh solvent and the stationary phase
takes place simultaneously at the original site of the sample

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 The separation of a mixture of components A and B is due to the
differences in rates cause the components in a mixture to separate
into bands, or zones, along the length of the column
 Isolation of the separated species is then accomplished by passing
a sufficient quantity of mobile phase through the column to cause
the individual bands to pass out the end (to be eluted from the
column), where they can be collected (times t3 and t4 in Figure 1
below).
 If a detector that responds to solute concentration is placed at the
end of the column and
 its signal is plotted as a function of time (or of volume of added
mobile phase)
 Such a plot, called a chromatogram, is useful for both qualitative
and quantitative analysis. As shown Figure 1 below.

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The positions of the peaks on the time axis can be used to identify
the components of the sample;
The areas under the peaks provide a quantitative Figure 1 (a)
measure of the amount of each species Diagram showing
the separation of
a mixture of
components A
and B by column
elution
chromatography.

(b) The output of

the signal
detector at the
various stages of
elution shown in
(a).
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 If a detector that responds to solute concentration is placed at the
end of the column and its signal is plotted as function of time, a
series of peaks is obtained.
 Such a plot, called a chromatogram, is useful for both qualitative
and quantitative analysis.
 Uses of Column chromatography
 Column chromatography is routinely used for the separation of
impurities and purification of various sample mixtures.
 Column chromatography is increasingly used for the detection of
drugs in crude extracts.

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 Gas chromatography
 Gas chromatography (GC) is a separation technique used to isolate
volatile components of a mixture depending on differences in the
mode of partitioning between a flowing mobile phase and a stationary
phase.
 It is a separation technique in which the molecules are separated on the
basis of their retention time depending on the affinity of the molecules
to the stationary phase.
 The sample is either liquid or gas that is vaporized in the injection
point.
 Principle of Gas chromatography
 Gas chromatography is based on the principle that components having
a higher affinity to the stationary phase have a higher retention time as
they take a longer time to come out of the column.
 However, the components having a low affinity to the stationary phase
have less retention time as they move along with the mobile phase.
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• The mobile phase is a gas, mostly helium that carries the sample
through the column.
• The sample once injected in converted into the vapor stage is then
passed through a detector to determine the retention time.
• The components are collected separately as they come out of the
stationary phase at different times.

Figure: Gas chromatography

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 Uses of Gas chromatography
 This technique is used to calculate the concentration of different
chemicals in various samples.
 It can also be used in forensic science to identify and quantify
various biological samples found in the crime scene.
 Pharmaceutical residual solvent analysis
 Food and beverages component analysis, food safety
analysis, analysis of alcohol
 Environmental air, water, soil analysis
 Chemicals material, polymer, additive, gas purity,
gas emission in automotives analysis

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 Liquid chromatography
 Liquid chromatography is a separation technique where the
mobile phase used is liquid, and the separation can take place
either in a column.
 Principle of Liquid chromatography
 The process of liquid chromatography is based on the principle for
the affinity of the molecules to the mobile phase.
 If the components to be separated have a higher affinity to the
mobile phase, the molecules move along with the mobile phase
and come out of the column faster.
 However, if the components have a lower degree of interaction
with the mobile phase, the molecules move slowly and thus come
out of the column later.

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 Paper Chromatography
 The stationary phase is a piece of porous paper with water adsorbed
on it.
 Paper essentially consists of cellulose fibers which are polymers
having -OH functional groups sticking out of the polymer chains.
 These groups lead to retention and separation of surface absorbed
molecules.
 The sample is placed on the paper as a spot and then irrigated by
the solvent system that percolates within the porous structure of the
paper.
 Usually development of the chromatogram is stopped before the
mobile phase reaches the farther edge of the paper, so the solute
zones are distributed in space instead of time.

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 Development system

 The mobile phase rises up the stationary phase by capillary action,

moving the components of the sample at various rates because of their
different degrees of interaction with the matrix and solubility in the
solvent.

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 The ratio b/n distance traveled by solute and distance traveled by
mobile phase(solvent) called Retention Factor (Rf).

Example:

1. What is the Rf value of an ion that moved 5.54 cm up a piece of

chromatography paper, if the solvent moved a distance of 7.91 cm?
2. If a spot travels 2.3 cm during paper chromatography while the
mobile phase travels 6.6 m, what is the Rf value of the substance in
the spot? 23
 Applications of Paper Chromatography
 To check the control of purity of pharmaceuticals,
 For detection of pollutants,
 Detect the contaminants in foods and drinks,
 For the detection of drugs
 In analysis of cosmetics
 Analysis of the reaction mixtures in biochemical labs

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 Thin-Layer Chromatography

 Separation is conducted on a thin layer (100–200µm) of stationary

phase, usually based upon silica gel and deposited on a rectangular
plate made out of glass, plastic or aluminum of a few centimeters
in dimensions.

 To maintain the stationary phase on the support and to assure the

cohesion of the particles, an inert binder like gypsum (or organic
linker) is mixed into the stationary phase during the manufacture
of the plate.

 The constituents can be identified by simultaneously running

standards with the unknown. 25
Figure: Developing chamber and TLC plate.
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 Chromatography nomenclature (Common terms in
Chromatography)
 Baseline: is any part of the chromatogram where only mobile phase is
emerging from the column.
 Peak maximum: is the highest point of the peak.
 Injection point: is the point in time/position when/where the sample is
placed on the column. Dead point: is the position of the peak-maximum
of an unretained solute.
 Dead time (to): is the time elapsed between the injection point and the
dead point.
• tM is the time for the unretained species to reach the detector,
dead time
 Dead volume (Vo): is the volume of mobile phase passed through the
column between the injection point and the dead point. Thus, Vo = Qto
where (Q) is the flow rate in ml/min.

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 Chromatograph: Instrument employed for a chromatography
 Stationary phase: Phase that stays in place inside the column.
 Can be a particular solid or gel-based packing (LC) or a highly
viscous liquid coated on the inside of the column (GC).
 Mobile phase: Solvent moving through the column, either a liquid
in LC or gas in GC.
 Eluent: Fluid entering a column (the solvent that carries the
analyte)
 Eluate: Fluid exiting the column (the mobile phase leaving the
column)
 Elution: The process of passing the mobile phase through the
column.

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 Chromatogram: Graph showing detector response as a
function of a time.
 Flow rate: How much mobile phase passed / minute
(mL/min).
 Linear velocity: Distance passed by mobile phase per
1min
 Detector: refers to the instrument used for qualitative and
quantitative detection of analytes after separation.

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 APPLICATIONS OF CHROMATOGRAPHY

 Chromatography has grown to be the premiere method

for separating closely related chemical species.
 In addition, it can be employed for qualitative
identification and quantitative determination of
separated species.

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 Qualitative Analysis

 A chromatogram provides only a single piece of qualitative

information about each species in a sample, namely, its
retention time or its position on the stationary phase after a
certain elution period.
 It is a widely used tool for recognizing the presence or absence
of components of mixtures containing a limited number of
possible species whose identities are known.
 Positive spectroscopic identification would be impossible
without a preliminary chromatographic separation on a complex
sample. 36
 Quantitative Analysis

 Chromatography can provide useful quantitative information

about the separated species.
 Quantitative column chromatography is based upon a comparison
of either the height or the area of the analyte peak with that of one
or more standards.
 For planar chromatography, the area covered by the separated
species serves as the analytical parameter.
 If conditions are properly controlled, these parameters vary
linearly with concentration.

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 THE BASIC EQUATIONS DESCRIBING CHROMATOGRAPHIC
SEPARATIONS
Partition Ratios in Chromatography
All chromatographic separations are based on differences in the
extent to which solutes are partitioned between the mobile and
the stationary phase.
For the solute species A, the equilibrium involved is described by
the equation
Amobile Astationary
The equilibrium constant K for this reaction is called a partition
ratio, or partition coefficient, and is defined as:
K = CS/CM ------------------------------------------------------------------------------------ 1
where : Cs is the molar analytical concentration of a solute in the
stationary phase and CM is its analytical concentration in the
mobile phase.
December 30, 2023 38
 Ideally, the partition ratio is constant over a wide range of solute
concentrations; that is, Cs is directly proportional to CM.
 Retention Time
 The retention time tR is the time between injection of a sample and the
appearance of a solute peak at the detector of a chromatographic column.
 Dead time (tM) is the time for the unretained species to reach the detector,
 The rate of migration of the unretained species is the same as the average rate
of motion of the mobile phase molecules.
Figure 4: A typical
chromatogram for a two
component mixture. The
small peak on the left
represents a solute that is
not retained on the column
and so reaches the detector
almost immediately after
elution analyte is started.

December 30, 2023 39

The average linear rate of solute migration, , is
2
where L is the length of the column packing. Similarly, the average
linear velocity, U, of the molecules of the mobile phase is
3

December 30, 2023 40

The Relationship Between Migration Rate and Partition
Ratio

𝒗= 𝐮 × 𝐟𝐫𝐚𝐜𝐭𝐢𝐨𝐧 𝐨𝐟 𝐭𝐢𝐦𝐞 𝐬𝐨𝐥𝐮𝐭𝐞𝐬𝐩𝐞𝐧𝐝𝐬𝐢𝐧 𝐦𝐨𝐛𝐢𝐥𝐞 𝐩𝐡𝐚𝐬𝐞

𝐦𝐨𝐥𝐞𝐬 𝐨𝐟 𝐬𝐨𝐥𝐮𝐭𝐞 𝐢𝐧 𝐦𝐨𝐛𝐢𝐥𝐞 𝒑𝒉𝒂𝒔𝒆
𝒗= 𝐮 ×
𝐭𝐨𝐭𝐚𝐥 𝐦𝐨𝐥𝐞𝐬 𝐨𝐟 𝒔𝒐𝒍𝒖𝒕𝒆

 The number of moles of solute in the mobile phase is equal to the molar

concentration, CM, of the solute in that phase multiplied by its volume,

VM.

 Similarly, the number of moles of solute in the stationary phase is given

by the product of the concentration, Cs, of the solute in the stationary

phase and its volume, Vs.

December 30, 2023 41
Therefore,
𝑪𝑴𝑽𝑴 𝟏
𝒗 =𝐮× =𝐔 ×
𝑪𝑴 𝑽 𝑴+ 𝑪𝑺 𝑽 𝑺 𝟏 + 𝑪𝑺 𝑽 𝑺 / 𝑪 𝑴 𝑽 𝑴

 Substitution of Eq. 1 into this equation gives an expression

for the rate of solute migration as a function of its partition
ratio as well as a function of the volumes of the stationary
and mobile phases:
K = CS/CM ……1
4

The two volumes can be estimated from the method by which

the column is prepared.
December 30, 2023 42
 The Capacity Factor
 The capacity factor is an important experimental parameter that is
widely used to describe the migration rates of solutes on columns.
 For a solute A, the capacity factor is defined as

Where KA is partition ration of solute, VR is the retention volume,

VM is the mobile phase volume (also called void volume)
 tR and tM can be readily
obtained from the
chromatogram.

December 30, 2023 43

 When the retention factor for a solute is less than 1.0, elution
occurs so rapidly that accurate determination of the retention
times is difficult.
 When the retention factor is larger than perhaps 20 to 30, elution
times become inordinately long.
 Ideally, separations are performed under conditions in which the
retention factors (capacity factors) K, A for the solutes in a mixture
lie in the range between 2 and 10

44
 Example. Calculate the retention factor (capacitor factors) ( 𝑘’) for
the peaks 1, 2 and 3 in the chromatogram shown below sol

• Solution

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 Conclusion. Since all of 𝑘’ values for 2 and 3 lie in the preferred
range of 2-10, the peaks are suitable for quantitation. However,
peak 1 is not
 Example 2 Calculate the retention factor for the peaks 1 and 11
in the chromatogram as shown below in figure. Comment on the
quality of those peaks for quantitation

46
 Solution

Conclusion
Since 𝑘1′ is equal to 2.00, peak 1 is suitable for quantitation.
However, since 𝑘11′ 𝑖𝑠 𝑙𝑎𝑟𝑔𝑒𝑟 𝑡ℎ𝑎𝑛 10, peak 11 is not.

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 Selectivity Factor
 The selectivity factor for two analytes in a column provides a
measure of how well the column will separate the two.
 The selectivity factor (α) of a column is defined as the degree of
separation between successive peaks (generally called as critical
pair).
 The selectivity factor )of a column for the two species A and B is
separated defined as:
-------------9

 where KBis the partition ratio for the more strongly retained species
B and KAis the constant for the less strongly held or more rapidly
eluted species A.
 According to this definition, is always greater than unity.
December 30, 2023 48
 Substitution of Eq. 5 and the analogous equation for solute B into
Eq. 9 provides after rearrangement a relationship between the
selectivity factor for two solutes and their capacity factors:
10

 where and are the capacity factors for B and A, respectively.

 Substitution of Eq. 8 for the two solutes into Equation. 10 gives an
expression that permits the determination of from an experimental
chromatogram:
11

 In generally, The selectivity factor of a column for the two species

A and B is defined as:

December 30, 2023 49

 The selectivity factor (α) is one of the most critical factors in
chromatography.
 α should be large enough so that each peak is sufficiently
resolved.
 Since A in the above equation is for the substance that is retained
less and B is for the one that is retained more, α is always larger
than 1.0
• Example Calculate the selectivity factor (α) for the peak pairs of
1,2 and 3,4 and 5,6 in the chromatogram shown below in figure.
• Retention times (in min) from left to right are: 0.20, 0.25, 0.53,
0.83, 1.52 and 2.25. (The retention time for the unretained peak is
0.08 min)

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• Chromatogram

Solution
1)For the peak pair 1,2:

2)For the peak pair 3,4

3)For the peak pair 5,6:

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 Example 2 . In a chromatographic analysis of low molecular weight
acids, butyric acid elutes with a retention time of 7.63 min. The
column’s void time is 0.31 min. Calculate the retention factor( capacity
factor) for butyric acid.
Solution

 Example 3.In the chromatographic analysis for low molecular weight

acids described in Example 2., the retention time for isobutyric acid is
5.98 min. What is the selectivity factor for isobutyric acid and butyric
acid? Solution
 First we must calculate the retention factor ( capacityfactor)for
isobutyric acid. Using the void time (unretainedpeak) from Example 2.
we have

 The selectivity factor, therefore, is

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 The Efficiency of Chromatographic Columns
The efficiency of a chromatographic column refers to the amount of
band broadening that occurs when a compound passes through the
column.
 A Quantitative Description of Column Efficiency
 Two related terms are widely used as quantitative measures of the
efficiency of chromatographic columns:
(1) plate height H and
(2) number of theoretical plates N.
The two are related by the equation
𝑵 =𝑳 / 𝑯
where L is the length (usually in cm) of the column packing
 The plate height H is also known as the height equivalent of a
theoretical plate (HETP).
 The efficiency of a column is great when H is small and N is large.
December 30, 2023 53
 Theoretical plates

 A chromatographic column is made up of numerous discrete but

continuous narrow layers called theoretical plates.
 At each plate, equilibration of the solute between the mobile and
stationary phase is assumed to take place.

 Movement of the solute down the column is then treated as a stepwise

transfer of equilibrated mobile phase from one plate to the next.
 The number of theoretical plates can be estimated based on peak
retention times and widths
 The smaller the plate height or the greater the number of theoretical
plates (N), the more efficient the analyte exchange is between two
phases and the greater is the efficiency of the column, which mean the
better the separation.
 That is why column efficiency is measured by N. 54
 The movement of a solute along the column is viewed as a stepwise
transfer from one theoretical plate to the next .
 These terms are related as follows:

 Where, L is the length of the column.

 The number of the theoretical plates (N) in the column is usually
calculated using the following equation:

 Where, σ is the standard deviation as a measure for peak width w =

4σ, w1/2 = 2.354σ, w is the peak width measured in time units as
the distance between the intersections of the tangents to the peak
inflection points with the baseline and w1/2 is the peak width at
half height.

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 In general, the H value is smaller for small stationary phase
particle sizes (thin), low mobile phase flow rates, less viscous
mobile phases, higher separation temperatures and smaller solute
molecule sizes.
 The width of the chromatographic peak reflects the system band
broadening and thus efficiency.
 The efficiency can be varied by changing physical column
parameters such as the length, diameter and construction material
of the container of the column.
 It can also be varied by changing chemical parameters such as the
size of the particles constituting the packing material or the mobile
phase velocity.

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 The Experimental Evaluation 𝑯 and 𝑵

 𝑁 can be calculated from two time measurements tR and peak

width (𝑊); to obtain 𝐻, the length of the column packing ( 𝐿)
must also be known.
 Another method for approximating 𝑁, is to determine 𝑊1/2, the
width of peak at half its maximum height.
 The theoretical plate number (𝑁) is then given by:

 𝑁 and 𝐻 are widely used in the literature

and by instrument manufactures
as measures of column performance.

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 Column Resolution
 The resolution Rs of a column provides a quantitative measure of
its ability to separate two analytes.
 It is s measure of how completely two neighboring peaks are
separated from one another
 Figure below, consists of chromatograms for species A and B on
three columns with different resolving powers.
 The resolution of each column is defined as

December 30, 2023 58

 The resolution is the ability of the column to resolve two analytes
into separate peaks (or chromatographic zones).
 It is affected by the selectivity, efficiency and capacity of the
column.
 The resolution equation describes those factors and indicates how
they can be manipulated in order to improve the resolution
between two peaks.
 Typically, an Rs value greater than 1 is required for accurate
quantification of two peaks. A value of 1, for two equally sized
peaks, indicates an overlap of about 2% for one band over the
other. A complete separation requires at least Rs > 1.5 units.

59
 It is evident from the figure below that a resolution of 1.5 gives
an essentially complete separation of the two peaks, whereas a
resolution of 0.75 does not.
 At a resolution of 1.0, zone A contains about 4% B and zone B
contains a similar amount of A.
 At a resolution for 1.5, the overlap is about 0.3% . The
resolution for a given stationary phase can be improved by
lengthening the column, thus increasing the number of
theoretical plates.

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December 30, 2023 61
 Exercise. Substances A and B have retention times of 16.40
and 17.63 min respectively, on 30 cm column. unretained
species passes through the column in 1.30 min. The peak widths
at bases A and B are 1.11 and 1.21 min. respectively.
 A)Calculate the column resolution
 B) Calculate number of theotical plates in for substace A
 c) Calculate plate height of substance B.
 d) Calculate capacity factor and selectivity factor

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