Chromatographic Techniques

BSMRSTU CHE 361

Liquid Chromatography

Dr. Bijan Mohon Chaki

Associate Professor
Department of Chemistry
Begum Rokeya University, Rangpur
 Liquid Column
Chromatography

Adsorption Ion-exchange
Chromatography Chromatography

High Performance Liquid Size-exclusion

Chromatography (HPLC) Chromatography

Gas Chromatography Affinity

(GC) Chromatography
 Other Liquid Chromatography
Types of Liquid Chromatography: Techniques in LC are
classified according to the method of solute separation
 Ion-exchange Chromatography (IEC)
 Ion exchange chromatography that separates ionizable solutes or charged
molecules present in a mixture by their adsorption onto a support containing fixed
charges on its surface. A high concentration of a competing ion is often added to
the mobile phase (salt solutions or buffer solutions) to elute the analytes from the
column
 Mixture of similar charged ions separated by using ion exchange resin
 Reversible exchange of similar charged ions
 Cations or Anions can be separated

PRINCIPLE
 Reversible exchange of ions
 b/w ions present in the solu. & ion exchange resin

Ion chromatography includes all chromatographic methods that separate ionic

substances and substances that dissociate easily. Analytic and mobile phase are
initially always polar and/or ionic.
xRSO3-H+ + Mx+ (RSO3-)xMx+ +xH+
xRN(CH3)3OH- + Ax-  [RH(CH3)3+]xAx- + xOH-
 CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
 According to the Source they can -

Natural : Cation – Zeolytes (Al2O5Si,

aluminosilicate), Clay(chunapathor)
Anion - Dolomite, CaMg(CO₃)₂.
Synthetic : Inorganic & Organic resins

◘Organic resins are polymeric resin matrix

The resin composed of –
Polystyrene (sites for exchangeable functional
groups), Divinyl benzene (Cross linking
agent)-offers stability
Organic resins are polymeric resin matrix, e.g. Phenol-formaldehyde resin
The resin composed of –
Polystyrene (sites for exchangeable functional groups), Divinyl benzene, cation
exchange resin synthesis
Ion exchange resin should have following
requirements
»It must be chemically stable
»It should be insoluble in common solvents
» It should have a sufficient degree of cross
linking
»The swollen resin must be denser than water
»It must contain sufficient no. of ion exchange
groups
Two General Types of Stationary Phases Can be Used in IEC:
- Cation-exchangers: have fixed negatively charged groups, used to separate
positively-charged ions
- Anion-exchangers: have fixed positively-charged groups, used to separate
negatively-charged ions

Chemical Structure Functional Group Chemical Nature Type of Exchange

-SO3¯ H+ Sulfonic acid Strong acid Cation
-COO¯H+ Carboxylic acid Weak acid Cation
-CH2COO¯H+ Carboxymethyl Weak acid Cation
-CH2N+(CH3)3Cl- Quaternary ammonium Strong base Anion

CH3 Quaternary ammonium Strong base Anion

CH2N+ CH2CH2CH(Cl-)

CH3

CH3 Tertiary ammonium Weak base Anion

CH2NH+ OH-

CH3
CH2CH3 Diethylaminoethyl Weak base Anion
CH2CH2NH+ OH- (DEAE)

CH2CH3
The charged groups that make up the stationary
phase can be placed on several different types of
support materials:
Cross-linked polystyrene resins: for use with the
separation of inorganic ions and small organic ions
Carbohydrate-based resins: for low-performance
separations of biological molecules (dextran,
agarose, cellulose)
Silica-based supports: for high-performance
rigid polystyrene/divinyl benzene beads
separations of biological molecules
A strong mobile phase in IEC:
- contains a high concentration of a competing ion for displacement of the
sample ion from the stationary phase
cation exchange resin (Kex):
Tl+ > Ag+ > Cs+ > Rb+ >K+ >NH4+ > Na+ > H+ > Li+
Ba2+ > Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+ > UO22+
anion exchange resin (Kex):
SO42- > C2O42- > I- > NO3- > Br- >Cl- > HCO2- > CH3CO2- > OH- > F-
or - a solvent that has a pH which decreases ionization of the analyte stationary
phase
Factors That Affect Mobile Phase Strength Are:
- Mobile phase pH
 especially for weak acid or base analytes and weak acid or base
stationary phases
- Mobile phase concentration of competing ion
- Type of competing ion

Isoelectric point
Range of
Stability
Net Charge On Protein

Attached to anion
exchangers

Attached to
cation
exchangers
 Structural types of ion exchange resins

a) Pellicular type with ion exchange resins:

»30 - 40µ with 1-2µ film thickness
»Very low exchange capacity
b) Porous resin coated with exchanger beads
» Size 5 - 10µ
» Porous & highly efficient
c) Macroreticular resin bead
» Not highly efficient & low exchange capacity
d) Surface sulfonated & bonded electrostatically
with anion exchanger
» less efficient & low exchange capacity
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contracGon
Swelling also affected electrolyte conc.
Particle size & Porosity
↑surface area & ↓parGcle size will ↑rate of ion
exchange
 Particle size 50-100 mesh / 100-200 mesh

Regeneration
 Cation exchange resin are regenerated by treatment
with acid, then washing with water
 Anion exchange resin are regenerated by treatment
with NaOH, then washing with water until neutral
 PRACTICAL REQUIREMENTS

1. Column
» glass, stainless steel or polymers
Length: diameter ratio 20:100 to 100:1
2. Packing the column
» Wet packing method
3. Application of the sample
After packing, sample is added to the top of the
column, use syringe or pipette
4.Mobile phase
Acids, alkalis, buffers…
5.Elution
Components of mixture separate & move down the
column at different rates depending upon the affinity
of the ion for ion exchanger.
» the eluates are collected at different stages
6. Analysis of the eluate
> spectrophotometric, flame photometry
polarographic, conductometric…
Factors affecting ion exchange separations

a. Nature & properties of ion exchange resins

Cross linking & swelling is important
If more cross linking , they are more rigid, but
swelling is less
swells less → separaGon of ions of different sizes is
difficult
b. Nature of exchanging ions
1. valency of ions
2.Size of ions
3.Polarizability
4.Concentration of solution
5. Concentration & charge of ions
Common applications of IEC:
- Removal or replacement of ionic compounds in samples
(sample pretreatment)
- Separation of inorganic ions and organic ions
- Analysis/purification of charged biological compounds
e.g. amino acids, proteins, peptides, nucleic acids

Purposes of IEC
 softening of water
 demineralisation of water
 purification of solutions free from ionic impurities
 separation of inorganic ions
 separation of sugars, amino acids
 ion exchange column in HPLC
Figure: Ion Exchangers for the Production of Deionized Water (DI
water)
What is Deionized Water?
Deionized water is the water from which ions are removed. In other words, deionized water
does not contain ionic species. We can get deionized water by passing the water through
an ion exchange process, from which the ions are removed from the water. The ion
exchange process results in high-quality, ion-free water that is suitable for research
purposes. Deionized water is also abbreviated as DI water in common.

Usually, tap water contains a number of ions that come from the soil, including sodium
cations and calcium cations as the major cations. Moreover, tap water may also contain ions
coming from the pipes through which water passes, e.g. ferrous ions and cuprous ions.
When we remove these ions, we get deionized water.

What is Demineralized Water?

Demineralized water is the purest form of water that does not contain any charged or
uncharged mineral particles that come from the soil. We can also name it purified water.
This form of water can be produced from mechanical filtration to remove impurities, which
makes it suitable for use. Although people often use the terms demineralized water
and distilled water interchangeably, they are different from each other because distilled
water may contain some particles due to the less efficient process from which it is
produced, i.e., distillation process.
When producing demineralized water, we can use a range of processes, including capacitive
deionization, reverse osmosis, carbon filtering, microfiltration, ultrafiltration, ultraviolet
oxidation, and electro-deionization. Moreover, we can use some of these techniques in
combination to produce “ultrapure water”.

There are many different uses of demineralized water, which include the production of
pharmaceutical products, food industry, laboratory uses for research studies, beverage
industry, lead-acid battery production, semiconductor productions, automotive cooling
systems, etc.

What is the Difference Between Deionized Water and Demineralized Water?

Often, the terms deionized water and demineralized water are used interchangeably
though there is a slight difference between them. The key difference between deionized
water and demineralized water is that deionized water is formed from the removal of all
the ionic species from water whereas demineralized water is formed from the removal of
all the mineral particles from water. Moreover, we can easily produce deionized water from
ion exchange processes while forming demineralized water requires more complicated
techniques such as capacitive deionization, reverse osmosis, carbon filtering,
microfiltration, ultrafiltration, ultraviolet oxidation or electro-deionization.
Highly charged ions elutes later than less
charged ions or molecules, e.g. Rf of
Na+<Ca2+<Al3+
 Size Exclusion Chromatography (SEC)
 SEC is a chromatographic techniques in which separation of component is
based on the difference in molecular weight or size.
 The molecules pass through a gel filtration medium packed in a column
that’s why it is also known as gel chromatography.
 Molecules move through a stationary phase containing bed of porous
beads, diffusing into the beads to greater or lesser degrees.
 It is one of the effective method used to isolate and analyze the bio-
macromolecules.
 Protein Isolation and Purification

• Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture,
usually cells, tissues or whole organisms.
Cell debris and
other proteins
are removed by
centrifugation
and other protein
could be
removed by You will have a
Whole Tissue salting out mixture of proteins
that have different
Tissue homogenization charges, Sizes , and
other properties
SEC is based on the use of a support material that has a certain range of pore sizes
- as solute travels through the support, small molecules can enter the pores
while large molecules can not
- since the larger molecules sample a smaller volume of the column, they
elute before the smaller molecules.
- separation based on size or molecular weight

SEC is based on the different interactions of solutes with the flowing mobile phase
and the stagnant mobile phase.
- no true stationary phase is present in this system (carbohydrate cross
linking agents are used as stationary phase, e.g. Dextran (sephadex), Agarose
(Sepharose), polyacrylamide etc.
- stagnant mobile phase acts as the “stationary phase”
 SEC does not have a “weak” or “strong” mobile phase since retention is
based only on size/shape of the analyte and the pore distribution of the
support.
- gel filtration chromatography: if an aqueous solvent or buffer solution is
used as mobile phase
- gel permeation chromatography: if an organic mobile phase is used
Common applications of SEC: (usually tetrahydrofuran)
- Separation of Biological Molecules (e.g., proteins from peptides)
- Separation/analysis of organic polymers
- molecular-weight determination
Stationary phase

It is composed of semi-permeable, porous polymer gel beads with

a well-defined range of pore sizes.

It has the following properties:

Chemically inert
Mechanically stable
With ideal and homogeneous porous structure (wide pore size
give low resolution).
A uniform particle and pore size.
 Gel Filtration Chromatography

Mobile phase: Stationary phase:

NaCl, buffers,.. (column matrix)
In this lab the mobile phase is NaCl. "beads" of hydrated, porous
polymer.

Examples of gel:
1.Dextran (Sephadex) gel: An α 1-6-polymer of
glucose natural gel
2.Agarose gel: A 1,3 linked β-D-galactose and
1,4 linked 3,6-anhydro-α, L-galactose natural gel
3.Acrylamide gel: A polymerized acrylamide, a
synthetic gel
 Stationary phase
• The stationary phase in gel filtration
chromatography is made of a gel consisting of
beads containing pores of a defined size range.
 Example
 A (red) size larger than the pores
 B (blue) size that can enters the pores

 Which one will eluted first?

 A will move only in the space between the
beads and are not retarded by the beads ,
So it will eluted first

 B diffuse in and out of the so they are

slowed down in their movement through
the column
 Principle
If an aqueous solution containing molecules of various sizes is passed
through a column containing such molecular sieve "beads", the molecules that
are larger than the pores move only in the space between the beads and are not
retarded by the beads.

However, molecules smaller than the pores diffuse in and out of the beads
with a probability that increase with decreasing molecular size; by this way,
they are slowed down in their movement through the column.

Therefore, these large molecules cross the column more rapidly in a smaller
elution volume , than the molecules that pass through the pores. Molecules in
the sample can be separated in order of their size by collecting fractions as the
mobile phase is eluted through the column, with the largest molecules eluting
first and the smallest last.

The elongated molecules are less likely to penetrate a given gel pore than
spherical molecules of the same molecular weight
The samples are moving by the addition of buffer (mobile phase)
After few mins. At the beginning
 For every column, three volumes
should be distinguished:
1. The void volume, Vo:
The volume of the mobile phase in the space between the beads . [which is the volume
external to the beads].
-Can be determined by the elution of High MW molecules which can not enter the
pores .

2. The total volume, Vt (bed volume):

The total volume of material in the column (both solid and liquid).
[can be calculated from the dimension of the column.]

3. The elution volume, Ve, of molecules:

The amount of liquid (mobile phase) that must be added to produce a peak of
a particular solute in the effluent.

Or the volume required for completely eluting the solute from column.
Gel Filtration Chromatography.
 Stationary phase
• Gel beads come in various sizes large, medium, fine, and superfine.
• All consist of semi-permeable, porous gels of cross linked polymers
with a range of pore sizes.
• The degree of cross linking is controlled to yield a series of gels having
different pore sizes.

• The pore size determines the range of molecular weight in which

fractionation occurs.
• The beads must be inert, uncharged .
• Suitable materials for gel are: agarose, dextran or polyacrylamide
 Mobile phase
• Molecules in the sample can be separated in order of their size by
collecting fractions as the mobile phase is eluted through the column, with
the largest molecules eluting first and the smallest last.
Advantages of gel filtration:
It is the best method for separation of molecules differing in
molecular weight because:

1. It doesn’t depend on temperature, pH, ionic strength and

buffer composition, so, separation can be carried out under
any conditions.

2. There is less zonal spreading than in other techniques.

3. The elution volume is related to the molecular weight.

4. Important method in protein purification.

5.This separation method is unique in fractionating without

requiring protein binding, thus significantly reducing the risk
of protein loss.
The most common application of gel filtration in
biochemistry are:

-Molecular weight determination.

-Fractionation of macromolecules.

-purification.
 elution volume, Ve is proportional to log of molecular weight.

For high molecular weight less elution volume is needed, and for small
molecular weight the large elution volume is needed to elute the
sample.
Cautions must be taken:
It is important that the gel should be:

-Homogenous.

-Free from bubbles.

-Free from cracks.

-Free from spaces between the walls.

- And it should be covered by the liquid "mobile phase" all the

time.
 Gel Permeation Chromatography(GPC)

 Gel permeation chromatography is also called gel filtration or size

exclusion chromatography.

 In size exclusion chromatography, the stationary phase is a porous

matrix made up of compounds like cross-linked polystyrene,
cross-like dextrans, polyacrylamide gels, agarose gels, etc.

 The separation is based on the analyte molecular sizes since the

gel behaves like a molecular sieve.

 This technique is used for the separation of proteins,

polysaccharides, enzymes, and synthetic polymers.
 Principle of Gel Permeation Chromatography

 It is a technique in which the separation of components is based on the difference

in molecular weight or size.
 The stationary phase used is a porous polymer matrix whose pores are completely
filled with the solvent to be used as the mobile phase.
 The molecules in the sample are pumped through specialized columns containing
such microporous packing material (gel).
 The basis of the separation is that molecules above a certain size are totally
excluded from the pores, while smaller molecules access the interior of the pores
partly or wholly.
 The flow of the mobile phase hence will cause larger molecules to pass through
the column unhindered, without penetrating the gel matrix, whereas smaller
molecules will be retarded according to their penetration of the gel.
 Components/ Instrumentation of Gel Permeation Chromatography
1.Stationary Phase
2.The Mobile Phase
3.The Columns
4.The Pump
5.Detectors
A. Stationary phase
It is composed of semi-permeable, porous polymer gel beads with a well-defined
range of pore sizes.
It has the following properties:
 Chemically inert
 Mechanically stable
 With ideal and homogeneous porous structure (wide pore size give low
resolution).
 A uniform particle and pore size.
Examples of gel:
1.Dextran (Sephadex) gel: An α 1-6-polymer of glucose natural gel
2.Agarose gel: A 1,3 linked β-D-galactose and 1,4 linked 3,6-anhydro-α, L-
galactose natural gel
3.Acrylamide gel: A polymerized acrylamide, a synthetic gel
B. The Mobile Phase
It is composed of a liquid used to dissolve the bio-molecules to
make the mobile phase permit high detection response and wet the
packing surface.
C. Columns
Any of the following kinds may be used:
 Analytical column- 7.5–8mm diameters.
 Preparative columns-22–25mm
 Usual column lengths-25, 30, 50, and 60 cm.
 Narrow-bore columns- 2–3mm diameter have been introduced
D. Pumps
They are either syringe pumps or reciprocating pumps with a high
constant flow rate.
E. Detectors
The detectors may be concentration-sensitive detectors, bulk
property detectors, refractive index (RI) detectors, etc.
Steps in Gel Permeation Chromatography
It involves three major steps:
A. Preparation of column for gel filtration
It involves:
1.Swelling of the gel
2.Packing the column semi-permeable, porous polymer gel beads
with a well-defined range of pore sizes.
3.Washing: After packing, several column volumes of buffer
solution is passed through the column to remove any air bubbles
and to test the column homogeneity.

B. Loading the sample onto the column using a syringe

C. Eluting the sample and detection of components

Applications of Gel Permeation Chromatography
 Proteins fractionation
 Purification
 Molecular weight determination.
 Separation of sugar, proteins, peptides, rubbers, and others on the
basis of their size.
 Can be used to determine the quaternary structure of purified
proteins.
Advantages of Gel Permeation Chromatography
 Short analysis time.
 Well defined separation.
 Narrow bands and good sensitivity.
 There is no sample loss.
 The small amount of mobile phase required.
 The flow rate can be set.
Limitations of Gel Permeation Chromatography
 The limited number of peaks that can be resolved within the short
time scale of the GPC run.
 Filtrations must be performed before using the instrument to
prevent dust and other particulates from ruining the columns and
interfering with the detectors.
 The molecular masses of most of the chains will be too close for
the GPC separation to show anything more than broad peaks.
 Application of Chromatography with Spectroscopy

Different Types of MS
• GC-MS - Gas Chromatography MS
– separates volatile compounds in gas column and ID’s by mass
• LC-MS - Liquid Chromatography MS
– separates delicate compounds in HPLC column and ID’s by mass
• GC-MS/MS - Tandem Mass Spectrometry
– separates compound fragments by magnetic field and ID’s by mass
• LC/LC-MS/MS-Tandem LC and Tandem MS
– Separates by HPLC, ID’s by mass and AA sequence
 Gas chromatography-Mass spectrometry (GC-MS)

It’s a Hyphenated Technique. Invented By James & Martin in 1952. Gas

chromatography-mass spectrometry (GC-MS) is a method that combines the
features of gas-liquid chromatography and mass spectrometry to identify different
substances within a test sample.
 Gas chromatography-Mass spectrometry (GC-MS)

 The GC-MS instrument consists of two main components.

 GC can separate volatile and semi-volatile compounds with great resolution,
but it cannot identify them.
 MS can provide detailed structural information on most compounds such that
they can be exactly identified, but it cannot readily separate them.
 The Fig. 1 is the schematic diagram of GC-MS.
 Gas chromatography-Mass spectrometry (GC-MS)
 Gas chromatography: Gas chromatography leads to separation of volatile
organic compounds.
 The gas chromatography portion separates different compounds in the sample
into pulses of pure chemicals based on their volatility by flowing an inert gas
(mobile phase), which carries the sample, through a stationary phase fixed in
the column (Skoog et al., 2007). Spectra of compounds are collected as they
exit a chromatographic column by the mass spectrometer, which identifies and
quantifies the chemicals according their mass-to-charge ratio (m/z).
 The helium gas is most commonly used, however, argon, nitrogen, and
hydrogen are also used.
 Gas chromatography-Mass spectrometry (GC-MS)
•Gas Chromatography uses a gaseous mobile phase to transport sample
components through columns either packed with coated silica particles or hollow
capillary columns containing, the stationary phase coated onto the inner wall.
Capillary GC columns are usually several meters long (10-120 m is typical) with
an internal diameter of 0.10-0.50 mm, whilst packed GC columns tend be 1-5
meters in length with either 2 or 4 mm internal diameter.
 Gas chromatography-Mass spectrometry (GC-MS)
•There are several very popular types of mass analyzer associated with routine
GC-MS analysis and all differ in the fundamental way in which they separate
species on a mass-to-charge basis.
 Gas chromatography-Mass spectrometry (GC-MS)
 The interface b/w the GC&MS is an important role to play in the overall
efficiency of the instrument.
 Both system are heated (200 -300 ∘C) both deal with compounds in the vapour
state.
 Only one problem is that the atmospheric pressure output of the GC must be
reduced to vacuum of 10-5 – 10-6 torr for the MS inlet

 Ionization Techniques: Electron ionization (EI): By far the most common

and perhaps standard form of ionization is electron ionization (EI). The
molecules enter into the MS (the source is a quadrupole or the ion trap itself
in an ion trap MS) where they are bombarded with free electrons.
 Free electrons are emitted from a heated filament (usually made of tungsten
or rhenium) and are accelerated across the source by using an appropriate
potential (5-100V) to achieve the required electron energy (sufficient to
ionize the molecule). The electrons bombard the molecules causing a hard
ionization that fragments the molecule.
 Gas chromatography-Mass spectrometry (GC-MS)
 Chemical Ionization (CI) Chemical ionisation involves the ionisation of a
reagent gas, such as ammonia or methane at relatively high pressure (~1 mbar)
in a simple electron impact source. Once produced, the reagent gas ions collide
with the analyte molecules producing ions through gas phase reaction
processes such as proton transfer. Chemical Ionization (CI)

Electron ionization (EI)

 Gas chromatography-Mass spectrometry (GC-MS)

 All the mass spectrometers now employ computer control of same functions
and also use a computerised display and output. The amount of data generated
even by a fairly modest mass spectrometer is very large indeed, a single run
may store data for up to 100 fragments from each type of molecule and if,
GCMS analyses is being performed, a complete mass spectrum is generated
and stored every sec for up to 90 min
 Gas chromatography-Mass spectrometry (GC-MS)
 Pharmaceutical applications: GC-MS analysis of urine sample know to
contain cocaine.
 Criminal forensics: GC-MS can analyze the particles from a human body in
order to help link a criminal to a crime.
 GC-MS especially useful here as samples often contain very complex matrices
& results used in court.

GC of Cocaine MS of Cocaine
 Gas chromatography-Mass spectrometry (GC-MS)

 Sports antidoping analysis: GC-MS is main tool used in sports anti doping
laboratories to test athletes urine samples for prohibited performance
enhancing drugs. e.g. : anabolic steroids.

 New-born screening (NBS): In

born errors of metabolism are now
detectable by new born screening
tests, especially the testing using
GC-MS. It can determine
compounds in urine even in minor
concentration.
 Disorders related to newborns:
Amino Acid Disorders. eg. PKU.
 Fatty Acid Oxidation Disorders
 Organic Acid Disorders
 Gas chromatography-Mass spectrometry (GC-MS)

 Law Enforcement: GC-MS is increasingly used for detection of illegal

narcotics, and may eventually supplant drug-sniffing dogs. It is also commonly
used in forensic toxicology to find drugs and/or poisons in biological
specimens of suspects, victims, or the deceased.

 Security: A post-September 11 development, explosive detection systems

have become a part of all US airports. These systems run on a host of
technologies, many of them based on GC-MS. There are only three
manufacturers certified by the FAA to provide these systems, one of which is
Thermo Detection (formerly Thermedics), which produces the EGIS, a GC-
MS-based line of explosives detectors.

 Food, Beverage and Perfume Analysis: Foods and beverages contain

numerous aromatic compounds, some naturally present in the raw materials
and some forming during processing. GC-MS is extensively used for the
analysis of these compounds which include esters, fatty acids, alcohols,
aldehydes, terpenes etc. It is also used to detect and measure contaminants
from spoilage or adulteration which may be harmful and which is often
controlled by governmental agencies, for example pesticides.
Example 14: (a) In preparing a hexane-acetone gradient for an alumina HPLC column, is
it desirable to increase or decrease the proportion of hexane as the
column eluted?
(b) Describe the fundamental difference between ion-exchange and size
exclusion chromatography?