CHEMICAL AND BIOPROCESS

INSTRUMENTATION

JCB 40104
Chromatography (Principles and Application)
 is Chromatography?

“Chromatography is the term used to

describe a separation technique…”
 Chromatography is a
technique employed for
separation of the
components of mixture
by continuous
distribution of the
components between two
phases
WHY use Chromatography?
Analytical Preparative (Measured)

• Determine Chemical composition • Used to purify sufficient quantities

of a sample of a substance
 Chromatography Terms
 Chromatogram - Data obtained from chromatography process.
 Stationary phase - Immobilized phase, on the support particles or on the
inner wall of the column tubing.
 Mobile phase - Moves in a definite direction, The mobile phase moves
through the chromatography column (the stationary phase) where the
sample interacts with the stationary phase and is separated.
 Retention time - Time takes for a particular analyte to pass through the
system (from the column inlet to the detector) under set conditions.
 Sample (Analyte) - Substance analyzed in chromatography.
 Solvent - Any substance capable of solubilizing another substance.
 Introduction to the Chromatography

 Chromatography is the laboratory techniques used for the separation of

mixtures which is a mobile phase carrying a mixture is caused to move
in contact with a selectively absorbent stationary phase.
 The mixture is dissolved in a fluid called the mobile phase, which
carries it through a structure holding another material called the
stationary phase.
 The various constituents of the mixture travel at different speeds, various
nature of interaction, and etc. causing them to separate.
 The separation is based on differential partitioning between the mobile
and stationary phases.
 Types of
Chromatography
Techniques
based on

PHYSICAL STATE SEPARATION

OF MOBILE PHASE MECHANISM

CHROMATOGRAPHIC
BED SHAPE
Techniques by physical state of
mobile phase
1. Gas chromatography - mobile phase is inert gas
2. Liquid chromatography - mobile phase is inert liquid (as
solvent)
3. Affinity chromatography - utilizes a biomolecule's affinity for a
metal (Zn, Cu, Fe, etc.)
4. Supercritical fluid chromatography - mobile phase is a fluid
above and relatively close to its critical temperature and pressure
Techniques by separation
mechanism
1. Ion exchange chromatography – ion exchange mechanism to
separate analytes (samples) based on their respective charges
2. Size-exclusion chromatography - separates molecules according
to their size (or more accurately according to their hydrodynamic
diameter or hydrodynamic volume)
3. Expanded bed adsorption chromatographic separation - for a
biochemical separation process, capture of proteins directly from
unclarified crude sample
Techniques by chromatographic
bed shape (stationery phase)
1. Column chromatography – the stationary bed is within a tube
2. Planar chromatography - the stationary phase is present as or
on a plane
 Paper chromatography
 Thin layer chromatography (TLC)
3. Displacement chromatography - A molecule with a high
affinity for the chromatography matrix (the displacer) competes
effectively for binding sites, and thus displace all molecules with
lesser affinities
12
 GAS
CHROMATOGRAP
HY (GC)
 Introduction to Gas Chromatography

 Gas chromatography is a type of chromatography used in

chemistry for analyzing and separating compounds that can be
convert to gas without decomposition.
 Uses of this technique include testing the purity of a gaseous
substance and separating the different components of a mixture.
 It may help in identifying the compound.
 Gas chromatography is similar to fractional distillation, as both
processes separate the mixture components primarily based on
their vapor pressure differences.
 Gas Chromatography
There are five basic components of GC

1. Pneumatic system – gas supply (flow control and measurement).

2. Injection system – a heated injector port, where the sample is vaporized if
necessary.
3. Column – where the separation occurs.
4. Oven –The coiled column is wholly contained in a thermostatically controlled
oven.
5. Detector – integral detector or link to a mass spectrometer.
Schematic of Gas Chromatography
Detection Systems of Gas
Chromatography
The Stationary Phase for Gas Chromatography
Working Principles of Gas Chromatography
1. A carrier gas, (e.g. Helium or Neon) flows through the system. A valve
controls the flow rate.

2. A sample of the volatile mixture is injected into the carrier gas. The sample is
vaporised in the heated injector port .

3. The carrier gas carries the vaporised sample into the column. The columns are
stainless steel or glass tubes and the column is often wound into a coil. The
sample mixture undergoes a series of interactions between the stationary and
mobile phases as it is carried through the system by the carrier gas. Due to the
wide choice of materials available for the stationary and mobile phases, it is
possible to separate molecules that differ only slightly in their physical and
chemical properties.
Working Principles of Gas Chromatography
4. The coiled column is contained in the thermostatically controlled oven.

5. Separated components emerge in the order of increasing interaction

with the stationary phase. The least retarded component comes through
first. Separation is obtained when one compound is sufficiently retarded
to prevent overlap with another component of the sample, as it emerges
from the column.

6. Two types of detector can be used either thermal conductivity detectors

or flame ionisation detection (FID).
Retention Time
• Retention time is defined as the time taken for a component to go from
injection to detection
tR = Distance travelled by a component
Distance travelled by the solvent
Retention Time
Factors that influence retention time;
1. The nature of the interactions between the solute and the phases.
2. The flow rate of the carrier gas.
3. The temperature of the column.
4. The length and diameter of the column.
5. The molecular mass of solute.
Retention Time
•A interacts more strongly with the stationary liquid phase and is
retained relative to B, which interacts weakly with the stationary
phase. Thus B spends less time in the gas phase and advances more
rapidly through the column and has a shorter retention time than A.
• B soluble, low retention time.
Retention Time
• Once GC has separated a mixture, the components can be identified
using known retention times.

• For unknown compounds the solutes are collected individually and

analyzed using another method, e.g. mass spectrometry.
Combining Gas Chromatography
with Spectroscopic Methods
 Applications of GC/MS
 Environmental monitoring : Organic Pollutants (Measuring toxic substances in
soil, air or water)
 Criminal forensics : Analyze the particles (Fibre) from a human body in order to
help link a criminal to a crime.
 Law enforcement : Detection of illegal narcotics,
 Forensic toxicology : Find drugs and/or poisons in biological specimens of
suspects, victims, or the deceased.
 Sports anti-doping analysis : Test athletes urine samples
 Security : Explosive detection.
 Food, beverage and perfume : from spoilage or Adultration - aromatic
compounds, esters, fatty acids, alcohols, aldehydes, terpenes.
 Advantages of Gas Chromatography

 Requires only very small samples with little preparation.

 Good at separating complex mixtures into components.

 Results are rapidly obtained (1 to 100 minutes).

 Very high precision, resolution, speed, accuracy and quantitative.

 The only instrument with the sensitivity to detect volatile organic

mixtures of low concentrations

 Equipment is not very complex (sophisticated oven)

 LIQUID
CHROMATOGRAP
HY (LC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
• HPLC is a form of liquid chromatography used to separate compounds that
are dissolved in solution.
• HPLC instruments consist of a reservoir of mobile phase, a pump, an
injector, a separation column, and a detector.
• Compounds are separated by injecting a sample mixture onto the column.
• The different component in the mixture pass through the column at
differentiates due to differences in their partition behavior between the
mobile phase and the stationary phase.
• The mobile phase must be degassed to eliminate the formation of air
bubbles.
Principle:
• The process involves the interaction of the compounds in the analyte
(which travels along with a mobile phase) across an immobile surface
(stationary phase).
• The compounds bind at specific regions of stationary phase based
on certain physical and chemical properties. These bound
molecules are then eluted with a suitable buffer and the same are
collected with time.
These are –
• Polarity
• Charge
• Molecular weight
• Present of functional group
Introduction
• HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution.
• HPLC instruments consist of a reservoir of mobile phase, a
pump, an injector, a separation column, and a detector.
• Compounds are separated by injecting a sample mixture
onto the column.
• The different component in the mixture pass through the
column at differentiates due to differences in their partition
behavior between the mobile phase and the stationary phase.
• The mobile phase must be degassed to eliminate the
formation of air bubbles.
Principles of Liquid Chromatography
Terminologies for HPLC
• HPLC : High Performance Liquid Chromatography : High Pressure
LC
• Mobile phase: The solvent system which carries the mixture to be
separated.
• Stationary phase: Immobile surface which is particulate in nature. This is
the region over which the compound gets separated.
HPLC system

Flow chart of HPLC mechanism

 HPLC
instrument
COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM

Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors
Waste Collector
Recorder (Data Collection)
 Instrumentation of HPLC
(Describing the 5 major components and their functions….)

Solvent
1 reservoirs
and degassing
2

Not shown 3
here 5
4
5

2 – Pump
3 – Injector
4 – Column
5 – Detector
6 – Computer
1. Solvent Reservoir
2. The Pump System
 controls the flow and measures the volume of solvent (the mobile
phase). The flow rates of HPLC columns are slow – often in 3 -1
the range of 0.5 - 5 cm min
 The role of the pump is to force a liquid (called the mobile
phase) through the liquid chromatograph at a specific flow rate,
expressed in milliliters per min (mL /min).
 Normal flow rates in HPLC are in the 1-to 2-mL/min range.
 Typical pumps can reach pressures in the range of 6000-9000psi
(400-to 600-bar).
 During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient)
3. The Injector System
 The sample to be separated is injected into the liquid phase at this
point
 The injector serves to introduce the liquid sample into the flow
stream of the mobile phase.
 Typical sample volumes are 5-to 20-microliters (μL).
 The injector must also be able to withstand the high pressures of
the liquid system.
 An auto sampler is the automatic version for when the user has
many samples to analyze or when manual injection is not
practical
HPLC system

Flow chart of HPLC mechanism

 Sample Injection……how is a sample
actually put into an LC system
Manual Injector:
1. User manually loads sample into the injector using a syringe
2. and then turns the handle to inject sample into the flowing mobile
phase… which transports the sample into the beginning (head) of the
column, which is at high pressure

Auto sampler:
1.User loads vials filled with sample solution into the auto sampler tray
(100 samples)
2.and the auto sampler automatically
a. measures the appropriate sample volume,
b. injects the sample,
c. then flushes the injector to be ready for the next sample,
etc., until all sample vials are processed ……
…….for unattended automatic operation
Manual Injectors
Sample Loop

Load - Inject

Front View Rear View

Inject

44
 Automatic Injectors

Step 1 Step 2

45 Step 3
 4. The Column

Considered the “heart of the chromatograph” the column’s stationary phase

separates the sample components of interest using various physical and
chemical parameters.
The small particles inside the column are what cause the high back pressure
at normal flow rates.
The pump must push hard to move the mobile phase through the column
and this resistance causes a high pressure within the chromatograph.
Several Column Types
(based on Stationery Phase)

• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
Normal phase
• In this column type, the retention is governed by the
interaction of the polar parts of the stationary phase
and solute.
• For retention to occur in normal phase, the packing
must be more polar than the mobile phase with respect
to the sample
STATIONARY PHASES
(NORMAL POLARITY)

Silica or alumina possess polar sites that interact with polar molecules.

silica
O
Polar Group HO Si
O

Components
Componentselute
eluteininincreasing
increasing
order
orderof
ofpolarity.
polarity.

Most polar…….Least polar

49
Reverse phase
• In this column the packing material is relatively nonpolar and the solvent is polar with
respect to the sample. Retention is the result of the interaction of the nonpolar
components of the solutes and the nonpolar stationary phase.
• Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic
mixtures such as methanol-water or acetonitrile-water.

Common Reverse Phase Solvents –

Methanol CH3OH

Acetonitrile CH3CN

Tetrahydrofuran

Water H2O
 STATIONARY PHASES
(REVERSE POLARITY)

If the polar sites on silica or alumina are capped with non-polar groups,
they interact strongly with non-polar molecules.

silica
C18 phase Me O
Si O Si
Me O

Components
Componentselute
eluteinindecreasing
decreasing
order
orderof
ofpolarity.
polarity.

Most non-polar…….Least non-polar

51
Size exclusion
• In size exclusion the HPLC column is consisted of
substances which have controlled pore sizes and is
able to be filtered in an ordinarily phase according to
its molecular size.
• Small molecules penetrate into the pores within the
packing while larger molecules only partially penetrate
the pores. The large molecules elute (dissolved and
removed) before the smaller molecules.
 STATIONARY PHASES
(SIZE EXCLUSION)

Size exclusion gels separate on the basis of molecular size. Individual gel
beads have pores of set size, that restrict entry to molecules of a
minimum size.

Large
Largemolecules
moleculeselute
elutefast
fast(restricted
(restrictedpath),
path),
while
whilesmall
smallmolecules
moleculeselute
eluteslowly
slowly(long
(longpath
pathlength)
length)

Larger molecules…….Smaller molecules

53
Ion exchange
• In this column type the sample components are
separated based upon attractive ionic forces between
molecules carrying charged groups of opposite charge
to those charges on the stationary phase.
• Separations are made between a polar mobile liquid,
usually water containing salts or small amounts of
alcohols, and a stationary phase containing either
acidic or basic fixed sites.
 STATIONARY PHASES
(CATION EXCHANGE)

Silica is substituted with anionic residues that interact strongly with

cationic species (+ve charged)

Cations exchange Na+ silica

O
Na O S
O

+ve
+vecharged
chargedspecies
speciesadhere
adheretotothe
thesupport
support
and
andare
arelater
latereluted
elutedwith
withacid
acid(H
(H+))
+

Most +ve…….Least +ve

55
 STATIONARY PHASES
(ANION EXCHANGE)

Silica is substituted with cationic residues that interact strongly with

anionic species (-ve charged)

Anions exchange Cl- silica

Me
Cl Me N CH2
Me

-ve
-vecharged
chargedspecies
speciesadhere
adheretotothe
thesupport
support
and
andare
arelater
latereluted
elutedwith
withacid
acid(H
(H+))
+

Most -ve…….Least -ve

56
 HPLC Columns
Within the Column is where separation occurs.
Key Point –Proper choice of column is critical for success in HPLC

Materials of construction for the tubing

• Stainless steel (the most popular; gives high pressure capabilities)
• Glass (mostly for biomolecules)
• PEEK polymer (biocompatible and chemically inert to most solvents

Packing material:
The packing material is prepared from SILICA particle, ALUMINA particle and ion
exchange RESIN.
Porous plug of stainless steel or Teflon are used in the end of the columns to retain the
packing material.
According to the mode of HPLC , they are available in different size , diameters, pore
size or they can have special materials attached ( such as antigen or antibody ) for
immuno affinity chromatography.
 Modes of High Performance Liquid
Chromatography

Types of Compounds Mode Stationary Mobile Phase

Phase
Neutrals Reversed C18, C8, C4 Water/Organic
Weak Acids Phase cyano, amino Modifiers
Weak Bases

Ionics, Bases, Acids Ion C-18, C-8 Water/Organic

Pair Ion-Pair Reagent

Compounds not Normal Silica, Amino, Organics

soluble in water Phase Cyano, Diol

Ionics Inorganic Ions Ion Anion or Cation Aqueous/Buffer

Exchange Exchange Counter Ion
Resin
High Molecular Weight Size Polystyrene Gel Filtration-
Compounds Exclusion Silica Aqueous
Polymers Gel Permeation-
Organic

58
Columns in HPLC
1. Guard Column
2. Fast Column
3. Preparative
4. Capillary
Guard Column
 These are placed anterior to the separating column. This serves as
protective factor.
 They are dependable columns designed to filter or remove :
 Particles that clog the separation column
 Compounds and ions that could ultimately cause “ Baseline drift ”,
 Decreased resolution, decreased sensitivity and create false peaks.
 These columns must be changed on a regular basis in order to
optimize their protective function.
 Fast Column
• One of the primary reasons for using these column is to obtain
improved sample output ( amount of compound per unit time).
• Fast column are designed to decrease the time of chromatographic
analysis
• Here internal diameter is same but length is short and packed with
smaller particles , that are 3 μm diameter.
• Advantages-
Increased sensitivity
Decreased analysis time
Decreased mobile phase usage
Increase reproducibility
Capillary Column
• It is also known as micro columns

• It has a diameter much less than a millimeter and there 3 types:

Open tubular
Partially packed
Tightly packed

They allow the user to work with nanoliter sample volume , decreased flow
rate and decreased solvent usage volume , led to cost effectiveness
Preparatory Column
• Used when objective is to prepare bulk ( milligrams) of sample for
laboratory preparatory application.

• It has usually a large column diameter , which is designed to facilitate

large volume injections into the HPLC system
5. The Detector
The detector can see (detect) the individual molecules that come out
(elute) from the column.
 A detector serves to measure the amount of those molecules so that
the chemist can quantitatively analyze the sample components.
 The detector provides an output to a recorder or computer that
results in the liquid chromatogram (i.e., the graph of the detector
response).
HPLC Detectors
 Common HPLC Detectors
 UV-VIS
 Diode Array
 Multiple Wavelength
 Variable Wavelength
 Mass Spectrometers
 Refractive Index
 Fluorescence
 Light Scattering
 Electrochemical
 Radioactivity
 Conductivity

66
 UV-Vis Detectors
Principles: The fraction of light transmitted through the detector cell is
related to the solute concentration according to Beer’s Law.

Detector Flow Cell

I0 c I

Log I0 = A = abc
I

Characteristics: Specific, Concentration Sensitive, good stability,

gradient capability.
Special: UV-Vis Spectral capability (Diode Array Technology ).

67
Fluorescence Detection
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68
Electrochemical Detectors

 Gold for carbohydrates.

 Platinum for chlorite, sulfate, hydrazine, etc.
 Carbon for phenols, amines.
 Silver for chloride, bromide, cyanide.

69
THE COMPUTER

• Frequently called the data system,

The computer not only controls all the modules of the HPLC
instrument but it takes the signal from the detector and uses it to:
1. Determine the time of elution (retention time) of the sample
components (qualitative analysis) and
2. The amount of sample (quantitative analysis).
 What is HPLC used for ?
Separation and analysis of non-volatile or thermally
unstable compounds
HPLC is optimum for the separation of chemical and biological compounds that are
non-volatile .

NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline, etc.), gas
chromatography is a better separation technique.

Typical non-volatile compounds are:

 Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)

 Salts like sodium chloride and potassium phosphate
 Proteins like egg white or blood protein
 Organic chemicals like polymers (e.g. polystyrene, polyethylene)
 Heavy hydrocarbons like asphalt or motor oil
 Many natural products such as ginseng, herbal medicines, plant extracts
 Thermally unstable compounds such as trinitrotoluene (TNT), enzymes
 How can We Analyze the Sample?

For example:
Carbohydrates
1. fructose
2. Glucose 5

3. Saccharose
4. Palatinose
5. Trehalulose 2
3
mAU
6. isomaltose
4
1
6

time

72
Separations
Injector
Separation in based upon differential
migration between the stationary and
mobile phases.
Mixer Stationary Phase - the phase
which remains fixed in the
column, e.g. C18, Silica
Pumps
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Column

Detector

Waste
Solvents

High Performance Liquid Chromatograph

73
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph

74
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

75
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

76
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

77
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

78
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

79
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

80
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

81
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

82
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

83
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

84
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

85
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

86
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

87
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

88
 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

89
 The Chromatogram
to - elution time of unretained peak
tR- retention time - determines sample identity
tR

tR
mAU Area or height is proportional
to the quantity of analyte.

to

Injection time
90
HPLC used for Qualitative Analysis
HPLC used for Quantitative Analysis
 HPLC uses
This technique is used for -

• chemistry and biochemistry research analyzing complex mixtures

• purifying chemical compounds
• developing processes for synthesizing chemical compounds
• isolating natural products, or predicting physical properties.

It is also used in quality control to ensure the purity of raw

materials, to control and improve process yields, to quantify assays
of final products, or to evaluate product stability and monitor
degradation.
HPLC Applications
Bioscience
Chemical proteins
peptides
polystyrenes nucleotides
dyes
phthalates

tetracyclines
Pharmaceuticals corticosteroids
antidepressants
barbiturates Consumer Products
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine
liquid chromatography
 Uses of LC;

- HPLC has many uses such as drug testing, testing for vitamins in
food and growth promoters in meat. In each case components of the
mixture are separated and detected.

 Comparison between HPLC over GC

- Less volatile and larger samples can be used with HPLC.

95
Conclusion
• Gasand Liquid Chromatography are currently the most frequently
used technique for determining trace organic compounds in
environmental samples.
• Chromatography coupled with mass spectrometry will have high
selectivity and compounds can be authenticated by analysing
retention times and unique mass spectra.