Chromatographic

Separation
Techniques

Assoc. Prof. Dr. Wafa Farooq

1
 To Know

1- Definition of Chromatography.
2- Terms used in chromatography.
3- Classification of Chromatography.
4- High-performance liquid chromatography.
5- Gas and flash Chromatography.
6- Thin layer Chromatography.

Dr. Wafa Farooq 2

Pretest:
1-Define the Chromatographic methods?
2-What is the difference between the normal phase HPLC and
reverse phase HPLC?
3- What is the difference between the isocratic and gradient
elution?

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Chromatography : is a physical method in which separation of
components takes place between two phases-a stationary phase and a
mobile phase.

• Stationary phase : The substance on which adsorption or partition

of the analyte (the substance to be separated during
chromatography) takes place . It can be a solid, a gel, or a solid
liquid combination. It is fixed in place either in a column or on a
planar surface.

• Mobile phase : solvent which carries the analyte (a liquid , a gas

or supercritical fluid).

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Elution is a process in which solutes are washed through a stationary phase
by the movement of a mobile phase. The mobile phase that exits the column
is termed the eluate.
An eluent is a solvent used to carry the components of a mixture through a
stationary phase.

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(a) Diagram showing the
separation of a mixture of
components A and B by column
elution chromatography. (b) The
detector signal at the various
stages of elution shown in (a).

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 Dr. Wafa Farooq

Chromatograms
✓ Is a plot of some function of solute concentration versus elution time or
elution volume. It is useful for both qualitative and quantitative analysis.
✓ The positions of peaks on the time axis may serve to identify the
components of the sample; the areas under the peaks provide a quantitative
measure of the amount of each component.

Chromatogram of two substance A, B. 7

tR Total retention time of the compound
t′R Corrected retention time of the compound (tR- tM)
tM "Dead time" (retention time-mobile phase)
W0.5 Peak width at half height
h Height of a signal 8
• The baseline is any part of the chromatogram where only mobile
phase is emerging from the column.

• The peak maximum is the highest point of the peak.

• The injection point is that point in time/position when/where the

sample is placed on the column.

• The dead point is the position of the peak-maximum of an

unretained solute.

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• The corrected retention time (t′R) is the time elapsed between the
dead point and the peak maximum.

• The dead time (tM) is the time elapsed between the injection
point and the dead point.

• The retention time (tR) Total retention time of the compound in

the whole chromatographic system

tR = t′R + tM

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➢ Retention Time
The time it takes after sample injection for the analyte peak to reach the
detector is called the retention time and is given the symbol tR.
The time tM for the unretained species to reach the detector is called the
dead OR void time. The rate of migration of the unretained species is the
same as the average rate of motion of the mobile phase molecules. The
average linear rate of solute migration  is
 = L/tR
where, L is the length of the column packing.
The average linear rate of movement μ of the molecules of the mobile
phase is:

μ = L/tM
Where tM, the dead time.
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The rate of migration of a solute through the stationary phase is
determined by its distribution ratio D, which is determined by its
relative affinity for the two phases.

𝐶𝑠
D=
𝐶𝑀

Cs; total solute in stationary phase

CM; total solute in the mobile phase.

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For a particular compound, the retention time will vary
depending on:
• The pressure of the column (because that affects the flow rate
of the solvent).
• The nature of the stationary phase (not only what material it is
made of, but also particle size, diameter and lenght).
• The exact composition of the solvent.
• The temperature of the column.

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Classification of Chromatographic Methods

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Dr. Wafa Farooq 15
16
High-performance liquid chromatography, HPLC
An analytical separation technique that involves the high-pressure flow of a
liquid through a column that contains the stationary phase.
Mobile phase: Liquid.
Stationary phase: Can be a solid (LSC) or a liquid (LLC)
➢ A mixture of compounds injected at one end of the column separates as the
compounds pass through.
➢ Separated compounds are detected electronically as they elute at the other
end of the column.
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Applications of Chromatography
Qualitative Analysis: retention time in Chromatographic separation is an
essential step in qualitative spectroscopic analyses.
Quantitative Analysis
* Calibration and Standards.
* Analysis Based on Peak height: 5-10% error.
* Analysis Based on Peak area: 2-5% error.

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Types of HPLC are often classified by :

I. separation mechanism or by the type of stationary phase. These include

(1) Partition, or liquid-liquid, chromatography;
(2) Adsorption, or liquid-solid, chromatography;
(3) Ion-exchange, or ion, chromatography;
(4) Size-exclusion chromatography;
(5) Affinity chromatography; and
(6) Chiral chromatography.
Please read about each type Dr. Wafa Farooq 19
(1) Partition chromatography, or liquid-liquid chromatography is a
Chromatographic technique in which solute are separated based on their
partition between a liquid mobile phase and a liquid stationary phase
coated on a solid support.

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(2) Adsorption, or liquid-solid, chromatography:
adsorption chromatography is a type of LC in which chemicals are retained
based on their adsorption and desorption at the surface of the support,
which also acts as the stationary phase.

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(3) Ion-exchange, or ion, chromatography
Ion exchange chromatography (usually referred to as
ion chromatography) uses an ion exchange
mechanism to separate molecules on the basis of their
electrical charges. Ion exchange chromatography uses
a charged stationary phase to separate charged
compounds including anions, cations, amino acids,
peptides, and proteins.
• Cation exchangers & anion exchangers are used
as ion exchange resins. In conventional methods
the stationary phase is an ion exchange resin that
carries charged functional groups that interact with
oppositely charged groups of the compound to
retain.
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(4) Size-exclusion chromatography
Size-exclusion chromatography (SEC), also known as molecular sieve
chromatography, is a chromatographic method in which molecules in
solution are separated by their size, and in some cases molecular weight. It
is usually applied to large molecules or macromolecular complexes such as
proteins and industrial polymers.
Typically, when an aqueous solution is used to transport the sample
through the column, the technique is known as gel-filtration
chromatography, versus the name gel permeation chromatography,
which is used when an organic solvent is used as a mobile phase. The
chromatography column is packed with fine, porous beads which are
commonly composed of dextran, agarose, or polyacrylamide polymers.

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(5) Affinity Chromatography is essentially a sample purification technique,
used primarily for biological molecules such as proteins.
• It is a method of separating a mixture of (molecules) by specific
interactions with a component known as a ligand, which is immobilized on
a support.

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(6) Chiral chromatography: is employed for the separation of enantiomers, e.
g. in racemates. Chiral HPLC columns are made by immobilizing single
enantiomers onto the stationary phasa, resolution relies on the formation of
transient diastereo isomers on the surface of the column packing.
The compound which forms the most stable diastereo isomer will be most
retained. Whereas the opposite enantiomer will form a less stable diastereo
isomer and will elute first

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Applications of liquid chromatography 27
Types Of HPLC ( …cont)
II. Based On Mode of Separation
Normal-Phase HPLC
• In his separations of plant extracts, Tswett was successful using a polar
stationary phase [chalk in a glass column] with a much less polar [non-
polar] mobile phase. This classical mode of chromatography became
known as normal phase.

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• The stationary phase is polar and retains the polar yellow dye most
strongly.

• The relatively non-polar blue dye is won in the retention competition

by the mobile phase, a non-polar solvent, and elutes quickly.
• Since the blue dye is most like the mobile phase [both are non-polar], it
moves faster.

• It is typical for normal-phase chromatography on silica that the mobile

phase is 100% organic; no water is used.
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Reversed-Phase HPLC
• The term reversed-phase describes the chromatography mode that is
just the opposite of normal phase, namely the use of a polar mobile
phase and a non-polar [hydrophobic] stationary phase.

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• Now the most strongly retained compound is the more non-
polar blue dye, as its attraction to the non-polar stationary phase
is greatest.
• The polar yellow dye, being weakly retained, is won in
competition by the polar, aqueous mobile phase, moves the
fastest through the bed, and elutes earliest like attracts like.

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• Today, because it is more reproducible and has broad
applicability, reversed-phase chromatography is used for
approximately 75% of all HPLC methods.
• Most of these protocols use as the mobile phase an aqueous
blend of water with a miscible, polar organic solvent, such as
acetonitrile or methanol. This typically ensures the proper
interaction of analytes with the non-polar, hydrophobic particle
surface.

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There are several reasons why reverse phase HPLC has become more of a
standard means of HPLC separation than normal phase.
1- Reverse phase columns have a hydrophobic stationary phase which works
well for retention of most organic analytes.
2- Water can be used as a mobile phase in conjunction with less polar
solvents such as acetonitrile and MeOH which can be adjusted at highly
controlled rates to improve chromatographic performance.

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3- Gradient separations in normal phase are much more complicated
because of UV cutoff variation as well as differences in compressibility of
common hydrophobic solvents, which would have an effect on flow rate.
4- Reverse phase chromatography also has the advantage of being able to
use pH selectivity to improve separations.
5- There are also many more choices in stationary phases for reverse phase
vs. normal phase.

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When is normal phase HPLC used?
1- It can be used for compounds that are too hydrophobic or
hydrophilic for separation using the reverse phase.
2- Compounds that are not soluble in water or that may
decompose in water.
3- For separation of isomers.

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III. Based On Elution Technique

1. Isocratic elution
A separation in which the mobile phase composition remains constant
throughout the procedure is termed isocratic elution.

2. Gradient elution
A separation in which the mobile phase composition is changed during the
separation process is described as a gradient elution.

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1. Isocratic mode

CH3OH / H2O = 6 / 4
Long analysis time!!

Poor CH3OH / H2O = 8 / 2

separation!!

(Column: ODS type) Dr. Wafa Farooq 38

2. Gradient elution

• If the eluent composition is changed gradually during analysis...

Concentration of methanol in eluent

95%

30%

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Asst. Prof. Dr. Wafa Farooq 40
✓ This mode is useful for samples that contain compounds that
span a wide range of chromatographic polarity.

✓ As the separation proceeds, the elution strength of the mobile

phase is increased to elute the more strongly retained sample
components.

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IV. Based On Scale Of Operation:
1. Analytical HPLC
No recovery of individual components of substance.
2. Preparative HPLC
Individual components of substance can be recovered.

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V. Based On Type of Analysis

1.Qualitative analysis
Analysis of a substance in order to ascertain the nature of its chemical
constituents .We can separate individual components but cannot assess the
quantity in this analysis.
2.Quantitaive analysis
Determining the amounts and proportions of its chemical constituents .
Quantity of the impurity and individual components can be assessed.

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Qualitative Analysis
• Identification based on retention time.(sample/standard)
• Acquisition of spectra with detector.
• UV spectra
• MS spectra
• Transfer to other analytical instruments after preparative separation.

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Quantitative Analysis

• Quantitation performed with peak area or height.

• Calibration curve created before by using a standard.

• Absolute calibration curve method.
• Internal standard method.
• Standard addition method.

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Calibration Curve for Absolute Calibration Curve Method

Area
Concentration
A1 Calibration curve
C1
A4

A2

Peak area
A3
C2

A2
A3
C3
A1

A4
C4 C1 C2 C3 C4
46 Concentration
Calibration Curve for Internal Standard Method

Concentration Area

Area for target substance / Area for internal standard

Target Internal
substance standard A1 AIS Calibration curve
C1 CIS A4 /AIS

A2 AIS A3 /AIS
C2 CIS

A2 /AIS
A3 AIS
C3 CIS
A1/AIS

A4 AIS
C4 CIS C1/CIS C2 /CIS C3 /CIS C4 /CIS
Concentration of target substance /
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Concentration of internal standard
HPLC involves following functional instruments:

1. Mobile phase reservoir.

2. Pump.
3. Injector.
4. Stationary phase ( Column).
5. Detector.
6. Data processing unit (Computer).

HPLC Instrumentation.
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Instrumentation
1. Mobile phase reservoir:
• Commonly glass bottles with caps are used.
• Vaccum for 5-10 min is also used for degassing.

Mobile phase considerations

Must be filtered (to prevent tiny solids from depositing at the column head)
and degassed.
Degassing is done by helium sparging. Because bubbles could interfere
with detection.
⚫ Problems caused by dissolved air in the eluent
❖ Unstable delivery by pump.
❖ More noise and large baseline drift in detector cell.
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Major components
Solvent or mobile phase
✓ Usually a mixture of an organic solvent (Ex. methanol, ACN) and
water or nonpolar solvent (Hexane).
✓ Sometimes buffered - keeps solutes in electrically neutral form.
✓ Solvent polarity affects the separation process.

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2. Pump:
• It forces the mobile phase to pass through the column.
• Flow rate is 1-2 ml/ min.
Manual Injection
Three common types of pumps are constant pressure
pumps, syringe-type pumps and reciprocating piston
pumps
3. Injector:
• Manually (syringe) or automated.
• Sample volume 5-20 μl. Automated Injection
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4. Stationary phase (Column):
• Heart of HPLC.
• Separate sample components on basis of physical and chemical
parameters.
• Length 10-30 cm.
• Diameter 4-10 mm.
• Made of stainless steel,
PEEK (poly-ether ether ketone) and glass.

HPLC Columns Materials.

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▪ HPLC column hardware comprising PEEK (poly ether ether ketone)
polymer is used when analytes are sensitive to commonly used stainless
steel. This includes some peptides and proteins.
• Glass is used in chromatography situations where the sample might
interact with the walls of the tube. It is standard to use glass columns
when working with pesticides and biochemicals such as steroids and
hormones. Glass is more inert than the metals and rarely causes tailing or
decomposition of the samples.

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Octadecyl-silica Dr. Wafa Farooq 56
Column packing is usually silica gel because of its particle shape surface
properties , and pore structure give us a good separation.
Other material used include alumina, a polystyrene-divinyl benzene synthetic
or an ion-exchange resin.
✓ Pellicular particle: original, Spherical, nonporous beads, use for proteins
and large biomolecules separation (dp: 5 μm).
✓ Porous particle: common used, dp: 3 ~ 10 μm. Narrow size distribution,
porous microparticle coated with thin organic film.

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 Dr. Wafa Farooq

✓ Core-Shell particle: dp: 2.6 μm . packed with special core-shell

particles.
The dimensions of the analytical column are usually
-straight, Length (5 ~ 25 cm), diameter of column (3 ~ 5 mm).
- eg: Column-C18 ,12 x 4.0 mm, 5μm

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• Silica has an active, hydrophilic [water-loving] surface containing acidic
silanol [silicon-containing analog of alcohol] functional groups. Si–O–H

• The activity or polarity of the silica surface may be modified selectively

by chemically bonding to it less polar functional groups [bonded phase].
In order to decrease polarity,
• cyanopropylsilyl-[CN],
• n-octylsilyl-[C8],
• n-octadecylsilyl-[C18, ODS]
moieties is added on silica.
The latter is a hydrophobic [water-hating], very non-polar packing.
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Separation Column for Reversed Phase
Chromatography
• C18 (ODS) type • Phenyl type
• C8 (octyl) type • TMS type
• C4 (butyl) type (trimethylmonochlorosilane)
• Cyano type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

Si -O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)
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 Effect of Chain Length of Stationary Phase

C8

Medium
C18 (ODS)

Strong C4

Weak
in general, longer chains have a
greater retention strength 61 Dr. Wafa Farooq
 Phenyl Bihenyl Alkyl Amide

Cyano Propyl Pentafluorophenyl Propyl Amino Propyl 62

Relationship Between Retention Time and
Polarity

C18 (ODS) OH

Weak
Strong
CH3

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Column Chemistry Choices Based on Sample Solubility

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The decision about the dimensions of the column should be based on
the goals for the chromatography.
- Resolution of a critical pair of constituents? – longer column bed
- Fast analysis? – shorter column bed
- Maximum resolution for all? – smaller particles
- Minimize solvent consumption? – narrower inner diameter

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➢ To summarize, the best combination of a mobile phase and
particle stationary phase with appropriately opposite polarities
must be chosen.

➢ Then, as the sample analytes move through the column, the

rule like attracts like will determine which analytes slow down
and which proceed at a faster speed.

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Column Storage
• Storage Solution
• It is generally safe to use the same storage solution as used at shipment.
• In order to prevent putrefaction, alcohol or some other preservative
substance may be added.

• Storage Conditions
• Insert an airtight stopper in the column end. Never allow the packing
material to dry.
• Make a record of the storage solution and final usage conditions and
store it together with the column.
• Store the column in a location not subject to shocks or sudden
temperature changes.
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Guard column is used to remove particular matter and contamination in
sample, it protect the analytical column and contains similar packing.

HPLC Guard Columns.

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 Guard Column and Pre-column

Guard column

Pre-column

To remove or temporarily trap impurities contained in the mobile phase

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5. Detector:
• Detection of elutes from column.
• Quantitative analysis of sample components.
• Output transferred to recorder/ computer.
Commonly used detectors in HPLC
✓ Ultraviolet (UV).
✓ Fluorescence.
✓ Mass Spectrometry.
✓ Electrochemical.
✓ Refractive Index (RI) Detection and others.
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6. Data processing unit (Computer)
• Data system that controls modules of HPLC.
• Signals from detector are interpreted to determine elution time, quantitative
and qualitative analysis of sample.

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References:
1- Skoog, D.A., West, D.M., Holler, F.J., Crouch, S.R., Fundamentals of
Analytical Chemistry.

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