CHE 312-INTRODUCTION TO CHROMATOGRAPHY 11-11-2022 (1).ppt

INTRODUCTION TO
SEPARATION SCIENCE (CHROMATOGRAPHY)

Chromatography
• Chromatography –is a technique for separation of components of a
mixture based on the differences in the rate at which they travel
through a stationary phase (rate of migration) by a gaseous or liquid
mobile phase.
• Stationary phase-is a phase that is fixed in place either in a column or
planar surface.
• Mobile phase-is a phase that moves over or through the stationary
phase carrying with it the analyte mixture
• Separations are based on differences interactions with the stationary
and mobile phase.
• All forms of chromatography works on the same principle.

Forms of chromatography
Thin layer chromatography (TLC)
• Stationary phase is a thin uniform layer of silica or alumina (more
polar) supported on an inert base such as glass, aluminium foil or
insoluble plastic.
• The mobile phase is a suitable liquid solvent or a mixture of solvents.
• A pencil line is drawn near the bottom of the plate to mark where the
sample is applied. The sample (mixture) is ‘spotted’ at the bottom of
the TLC plate and allowed to dry. The plate is placed in a closed
vessel (development chamber) containing solvent (the mobile phase)
so that the solvent level is below the spot.
• The liquid moves up the plate by capillary action. Capillary action-is
the ability of a liquid to flow in narrow spaces without the
assistance of external forces such as gravity or even in
opposition to external forces. It occurs because of
intermolecular forces between the liquid and surrounding solid
surfaces.

TLC
• As the solvent slowly travels up the plate, the different components
of the mixture travel at different rates because of their differences in
intermolecular forces between the analyte and the stationary phase
i.e The component with high affinity towards the stationary phase
travels slower. The component with lesser affinity towards the
stationary phase travels faster.
• As a result the mixture is separated into different coloured spots.
Which are visible if the analyte is coloured.
• When the solvent front gets close to the top, the plate is removed
from the the solvent, the front is marked with a pencil. And retention
factor (Rf) is calculated;
• Rf= distance moved by the solute divided by distance moved by the
solvent

TLC
• If this experiment is repeated under exactly the same conditions,
then the Rf values for each component would always be the same
and can be used to identify the compound.

Thin layer chromatography (TLC)/paper
chromatography
The following are the important components
and steps of a typical TLC system:
–Apparatus (developing chamber)
- mobile phase (solvent, a mixture of solvents)
– Stationary phase layer (TLC plate)- silica or
alumina on a glass support
– Application of sample
– Development of the plate
– Detection of analytes

Visualization of spots
• Visualization of colourless spots can be done by following method,
– Spray of Iodine vapors
– UV exposure for compounds that flouresces– Excites the
compound and as it relaxes it emits some photons within the uv-
visible region in the and a compound must have a chromophore
– Ninhydrin for identification of amino acids- reacts with amino
acids to form coloured compounds.

Column chromatography
• the stationary phase (silica or alumina) is held in a narrow glass
tube (column) and the mobile phase occupies the open spaces
between the particles of the packing.
• A solution containing the mixture of components is introduced at
the top of the column.
• Separation occurs by forcing the components through the column by
continuously adding fresh mobile phase under pressure or gravity.
This carries analytes down the column in a continuous series of
transfers between the two phases.

column chromatography-
Solutes which interact more strongly with
the stationary phase take longer to pass
through the column
Strongly Retained
Weakly Retained
Solutes which only weakly interact with
the stationary phase or have no
interactions with it elute very quickly
• The rate of migration for the different components depend on the extent
of interaction with the stationary phase and is slow for components that
strongly interact with the stat. phase.
• The resulting differences in rates cause the components in a mixture to
separate into bands.
• The mobile phase is added until the individual bands elute at the end of
the column where they can be collected or detected.

Gas chromatography (GC)and Liquid chromatography (LC)
 gas chromatography (GC)- a gas is employed as the mobile phase while the
stationary phase is a solid or a viscous liquid adsorbed on the surface of a
capillary column.
 a sample is injected into the instrument, its vaporized turning the solvent
and analytes into gaseous form, and separating the mixture of compounds
into individual peaks (individual compounds)
 Liquid chromatography(LC)- Mobile phase is a liquid and stationary phase is
a solid uniformly packed in a stainless steel column or a viscous liquid
bonded on a solid support.
• Liquid chromatography completes the same process as GC except
the separations occur in a liquid phase.

Purpose of Chromatography
• Preparative
Preparative –to purify and collect one or more components of a
sample for further analysis, examples TLC and column
chromatography. (mostly employed in natural products)
• Analytical
Analytical – separate, identify or quantify chemical components of
a sample.
• Chromatographic techniques used for analytical purpose are gas
chromatography or liquid chromatography coupled to a suitable
detector.
• HPLC can also be preparative

Mechanisms of separation in chromatography
• They are divided on the basis of interaction mechanism of the solute
and stationary phase
1. Adsorption chromatography (Used in gas or liquid chromatography
when the stationary phase is solid)
 A solid stationary phase and a liquid or gas mobile phase
 Solute adsorb/adhere on the surface on the solid particles through
hydrophobic or hydrophilic interactions (Molecules that have dipole
charge are attracted to the charges within the stationary phase).
 the stronger the hydrophobic interactions the more strongly the
solute is adsorbed the slower it migrates through the column.
Different solutes will have different extent of interactions with the
stationary phase causing the separation of components of a
mixture.

2. Partition chromatography
• (Used in gas or liquid chromatography when the stationary phase is
a viscous liquid)
• The stationary phase is a thin film of viscous liquid chemically
bonded on the surface of support (SiO2). Solute equilibrates
between the stationary liquid and the mobile phase, which is flowing
gas in gas chromatography or a liquid in liquid chromatography.
• It is based on differences in solubility of a solute in the mobile phase
(liquid or a gas) and a stationary liquid.
Organo chlorosilane
Silica particles

3. Ion exchange chromatography
• The column contains high ,molecular mass polymers that contain
large number of ionic functional group on the stationary phase.
• retention is based on interaction between solutes ions and charged
sites on the stationary phase. Anions such as SO-
3or cations (-
trimethl amine ion -N(CH3)+
3) are covalently bonded to a stationary
solid phase (resin).
• Solute ions of the opposite charge are attracted to the stationary
phase. After the separation the molecules are washed with the
suitable solvent.
• Weak base types contain secondary and primary amines.

Separating ions by ion exchange
Anion exchange:
xRN(CH3)3
+
OH-
+ Ax-
 [RN(CH3)3]xAx-
+ xOH-
solid soln solid soln
where: Ax-
represents an anion and R a part of resin containing
trimethyl ammonium group
After ion exchange of cations or anions on the resin, a
suitable solvent is used to elute them.

CHE 312-INTRODUCTION TO CHROMATOGRAPHY 11-11-2022 (1).ppt

Affinity chromatography
 Is the most selective kind of chromatography
 Affinity chromatography is a method of separating biochemical mixture
based on highly selective interaction between the solute molecule and
another molecule covalently attached to the stationary phase called an
affinity ligand.
 Examples of affinity ligands are antibodies (antigen), enzyme ( substrate),
protein (nucleic acid)
 When a sample passes through the column, only the molecules that
selectively bind to the affinity ligands are retained. Molecules that do not
bind pass through the column with the mobile phase. The retained analyte is
later eluted by changing the mobile phase conditions (solvent that does not
support the binding of the analyte to the affinity ligand).

Terms used in chromatography
• Eluent is the solvent used to carry the components being separated
through the chromatographic system.
• Eluate the fluid emerging from the end of the column containing the
separated components.
• Elution-The process by which the solutes are extracted from the
stationary phase by washing them with a solvent. Compounds with
greatest affinity for mobile phase elute faster. Compounds with high
affinity for the stationary phase are held longer (elute late)

Chromatogram
• a detector that respond to solute concentration is placed at the end of
the column to detect components eluting from the column, resulting
signal is a plot of detector response as a function of elution time.
• This graph showing the detector response as a function of elution time
in chromatography is called chromatogram.
• The mobile phase (unretained solute ) has less interaction with the
stationary phase, therefore it travels through the stationary phase in
the minimum time possible called void time or dead volume
designated tm.
• The void time provides a measure of the average rate of migration of
the mobile phase called the linear velocity of the mobile phase (cm/s)
where L is the length of the column, tm dead
volume
µ = L/tm (cm/s)

Chromatogram
• Each component of a mixture is separated as a peak. The time
required for analyte to reach the detector after sample injection is
called the retention time (tR). It is characteristic of a compound under
the given conditions.
• The average linear velocity of solute migration is given by (cm/s)
R
t
L



• A series of peaks is obtained depending on the number of
components in the mixture. Concentration of each component is
directly proportional to the area below the peak.

dichlorvos (retention time of 3.097 minutes) dimethoate 10.69 mins,
chlorpyrifos at 14.433 minutes and malathion at 15.774 minutes
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 min
0.0
0.5
1.0
1.5
2.0
2.5
3.0
uV(x10,000)
Chromatogram
Dichlorvos
Dichlorvos
Chlorpyrifos
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
uV(x100,000)
Chromatogram
D
ic
hlorv
os
D
im
ethoate
C
hlorpy
rifos
M
alathion

• For two components the relative retention(α)
Relative retention (α)- is independent of the flow rate, and can help identify the
peaks even if the flow rate changes. Relative retention is also known as selectivity
factor.

• Capacity factor k’=
• The longer the component is retained by the column, the greater is its capacity
factor.
• A capacity factor much less than unity means that the solute emerges from the
column at a time near that of the void time.
• Used to monitor the performance of the column, it is good practice to periodically
measure the capacity factor of a standard. Changes in the capacity factor reflect
degradation of the stationary phase.
m
m
r
t
t
t
'
k

 = = t’r/tm

where: Vs = volume of the stationary phase
Vm = volume of the mobile phase
Keq = partition coefficient
Cs – concentration of solute in the stationary phase
Cm –concentration of solute in the mobile phase
Relationship btwn capacity factor and distribution coefficient
• The capacity factor is directly proportional to the partition co-
efficient.
• The larger the equilibrium constant/capacity factor the longer
the material is retained by the column

Example
• A mixture of benzene, toluene and methane was injected into a gas
chromatograph. Methane gave a sharp peak at 42 s, whereas benzene required
251 s and toluene was eluted in 333 s. Find the adjusted retention time and
capacity factor for each solute and the relative retention.
The adjusted retention times
t’r = tr – tm
Benzene t’r = 251- 42 = 209 s toluene t’r = 333-42= 291 s
Relative retention α= tr’toluene/ tr’benzene =6.9/5 =1.39
Capacity factor k’ benzene= t’r /tm = 5 toluene =291/42= 6.9

Efficiency of separation
Resolution
• Resolution (Rs): is the degree of separation between two
chromatographic peaks as recorded by the detector.
• Two parameters/factors determine how well one solute is separated
from another
i. Difference in retention times; the further apart the peaks are, the better
the separation.
ii. How broad the peaks are-the broader the peaks, the poorer the
separation between them. The width of the peak can be measured at
the baseline or at half the peak height.
Measurement of peak width
(i) Width at baseline (w= 4 δ)
(ii) w1/2 –width at 1/2 the peak height (w1/2= 2.35δ)

Efficiency of separation
Resolution
• Solute moving through a column spreads into a Gaussian shape
with a standard deviation δ

Improving resolution
Therefore improved separation can be realized by control of variable that either
increase the rate of band separation or decrease band spreading.
Two methods of improving separation is
(i) Increase band separation
(ii) Decrease peak width

Resolution
2
/
)
(
)
(
1
2
1
2
w
w
t
t
R
r
r
s



where: tr2,tr1 = retention times of solutes 1 and 2 (tr2 > tr1)
wb2,wb1 = baseline widths of solutes 1 and 2

a resolution of 1.5 gives an essentially complete separation of the two components

Plate theory
• Plate theory tries to explain how separation takes place in a column.
• It considers a column as divided into adjacent imaginary segments called
theoretical plates. One theoretical plate (N) can be defined as the part of the
column, where quasi-equilibrium takes place between stationary and mobile
phase.
• 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 height of each plate is called height equivalent to a theoretical plate
(HETP). Plate Height is the constant of proportionality btwn the variance (δ
2
)
of the band and the distance it has travelled. (H=δ
2
/L).

Plate theory
• Within each theoretical plate, complete solute equilibration
of analytes must take place between the mobile phase and
the stationary phase before the solute moves to another
plate.
• The theory explains As HETP decreases, more separation
steps (N) per column length (N) are possible and that
greater separation will take place. Also the smaller the plate
height the narrower the band improving resolution.
Small plate height =narrow peaks
• Efficient column has more plates than an inefficient column.
Therefore the ability of a column to separate is improved by
decreasing H.
• For a column length L, the number of theoretical plates (N)
in the entire column.
N
/
L
H 

Measure of Column Efficiency
 The efficiency of a column is measured by the number of theoretical plates (N)
N
/
L
H 
where: L = length of column
N = number of theoretical plates
H

Band broadening
• Encompasses any process that results in spreading of analyte as it
migrates through the chromatographic column. In simple terms, it
means increase in peak width, which results in loss of efficiency in
separation (loss of resolution) and deterioration of chromatographic
performance of a method.
• J.J Van Deemter described various factors that contributes to loss of
efficiency of separation. Which relates column efficiency measured
as the HETP to the linear velocity of the mobile phase as it flows
through the column.

Van Deemter equation
 Factors include:
- Resistance to mass transfer/Finite equilibration time
- Longitudinal diffusion
- Multiple flow paths/eddy diffusion
• Describes three terms which contributes to band broadening and relates them to the
mobile phase linear velocity
Composite curve

Causes of band broadening in chromatography
A. Multiple Flow Paths/ Eddy diffussion – Solute molecules travelling through the
column many take any of the many different paths through the stationary phase at
random. Different paths have different lengths.
This means that some analyte molecules arrive at the end sooner than others simply due
to the different path traveled around the support particles in the column that result in
different travel distances. Larger difference in travel time is seen with larger
particles and non uniform packing.
Molecules enter the column
at the same time
Molecules exit the column at
different times due to different path
lengths
The A-term may be reduced using smaller particles of the stationary phase with a
narrow particle size distribution and
using narrower columns for open tubular columns

(B) Longitudinal diffusion – band slowly broadens as molecules diffuse
from high concentration band to regions of lower concentration (More dilute
region) on either side (towards and opposed to direction of flow). Therefore
a concentration gradient exist with high analyte conc at the centre of the band
(i) This is the most common source of band broadening in GC where the
rate at which molecules diffuse is high. It can be lowered by lowering
the oven temperature.
Can be lowered by optimizing flow rate- If the velocity of the mobile phase is
high then the analyte spends less time on the column, which decreases the
effects of longitudinal diffusion. Reducing the temperature can also minimize
diffusion.
•LC-insignificant- diffusion rates are smaller for liquids.

• With gaseous mobile phases, the rate of longitudinal diffusion can be
reduced appreciable by lowering the temperature and thus the diffusion
coefficient . The consequence is significantly smaller plate heights at low
temperatures.

C. Finite Equilibration Time Between Phases/ Resistance to Mass transfer – a finite
time is required for the solute to equilibrate between stationary and mobile phase at
each plate
- Analyte in the mobile phase will travel ahead of those in the stationary
phase
- For analyte that is strongly adsorbed to stationary phase- will be stuck in
the stationary phase, leading to slower rate of mass transfer.
Distribution of solute between
mobile and stationary phase
Solute in mobile phase moves
down column  broader peaks

How to lower band spreading
• Two important controllable variables that affect column efficiency ;
1) the diameter of the particles making up the packing for (packed
columns)
2) the diameter of the column (for open tubular columns).
To take advantage of the effect of column diameter, narrower and
narrower columns have been used in recent years.

Columns
• Columns/stationary phases are considered the “heart” or “brain” of
the chromatography and are responsible for the separation process.
• We have two types of columns used in chromatography;
• Packed column: its a metal column tubing made of stainless steel
filled with small spherical particles of the stationary phase (Silica gel
or porous polymer).
• Packed columns are used in liquid chromatography and they require
high pressure to force the material through the stationary phase
hence the name (High pressure liquid chromatography/ high
performance liquid chromatography)-HPLC.

Gas chromatographic column evolution
• Open tubular column/ Capillary columns-it is a narrow hollow
capillary made of fused silica. Internal diameter of capillary columns
is 0.1-0.75 mm and the length is 5-100 m.
• Polyimide coating is made on the surface of the capillary columns to
protect it from mechanical damage (the column is made of fused
silica (highly purity glass).
• The gas flow faces less resistance.
– We have different types of capillary columns.

Types of capillary columns
• Porous layer open tubular (PLOT) columns contain a porous layer of
a solid adsorbent such as alumina and Porapak (polystyrene
beads ) coated on the wall of the capillary column.
• Wall-coated open tubular (WCOT) columns, the wall of a capillary
column is directly coated with a thin film a liquid stationary
phase( film thickness of 0.05–3 μm).
• Support coated open tubular column (SCOT)- The inner surface of a
capillary column is coated with a layer of porous solid support
material. The liquid stationary phase is coated on this support
material. SCOT have more sample capacity than WCOT.

TYPES OF COLUMNS USED IN CHROMATOGRAPHY

Gas Chromatography stationary phase

Open tubular columns vs packed columns
• Higher resolution- Length 15-100 m, more theoretical plates
• Shorter analysis- higher flow rates are possible because of low resistance
to mass transfer.

Gas Chromatography
1.) Open Tubular Columns
 Increasing Resolution
Decrease tube diameter- lowers eddy diffusion
Increase resolution
Increase Column Length
Increase resolution

Gas chromatography
• It is a process of separating component(s) from the given components of
a mixture by using a gaseous mobile phase.” It involves a sample being
vaporized and injected onto the head of the chromatographic column.
The sample is transported through the column by the flow of inert,
gaseous mobile phase. The column itself contains a liquid stationary
phase which is adsorbed bonded on the surface of an inert solid (Gas-
liquid chromatography) or a solid (Gas-solid chromatography)
• Two major types: Gas-solid chromatography: Here, the mobile phase is
a gas while the stationary phase is a solid. Used for separation of low
molecular gases, e.g., air components, H2 S, CS2 ,CO2 ,rare gases, CO
and oxides of nitrogen .
• Gas-liquid chromatography: The mobile phase is a gas while the
stationary phase is a liquid retained on the surface as an inert solid by
chemical bonding

Principle of separation
• The principle of separation in GLC is “partition.” The
mixture of component to be separated is converted to
vapour and mixed with gaseous mobile phase. The
component which is more soluble in stationary phase
travel slower and eluted later. The component which is
less soluble in stationary phase travels faster and eluted
out first. No two components has same partition
coefficient conditions. So the components are separated
according to their partition coefficient. Partition
coefficient is “the ratio of solubility of a substance
distributed between two immiscible liquids at a constant
temperature

Gas chromatography (GC)
• Used for separation and analysis of volatile (non-polar) and
thermally stable compounds.
• In gas chromatography the mobile phase is a gas while the
stationary phase is a liquid or a solid.
• In GC a Volatile liquid or gas injected through septum into heated
port in the injector through a septum.
• Sample vaporized (high temp of the injector) and is pulled through
the column with carrier gas
• When a sample traverses the column by the flow of an inert gas
employed as the mobile phase, its components are separated owing
to differences in their interactions with the stationary phase.
Column is heated to maintain the analyte in gaseous form

Block diagram of components of a GC
• Upon elution from the column, the separated
compounds pass over a detector that generates a
signal corresponding to the concentration of the
compound. The species present can be
qualitatively identified based on their retention
times.

• Separation in the column is based on the concept of likes dissolves
likes
 Nonpolar columns - components are separated based on
their polarity. The order of elution is the most non-polar
compound will be retained more.
• Separation is also based on volatility- most volatile (small
molecules) will travel through the column faster least volatile = most
retained.

1. Carrier gas
The Mobile phases in GC is a gas and its called carrier gas. All carrier gases are available
in pressurized tanks and pressure regulators, gauges and flow meters are used to
meticulously control the flow rate of the gas; The carrier gas must
Must be highly pure- Most gas supplies fall between 99.995% - 99.9995% purity range .
Must be chemically inert
N2, He and H2 are typical carrier gases
 He:
- Most common ly used and compatible with most detectors
- Solutes diffuse rapidly  smaller mass transfer term
- Better resolution (smaller plate heights)
 N2:
- Lower resolution and solute diffusion rates
 H2:
- Fastest separations
- Can catalytically react with unsaturated compounds on metal surfaces
- Can not be used with mass spectrometers Forms explosive mixtures with air
- Better resolution (smaller plate heights)
- Solutes diffuse rapidly  smaller mass transfer term
Flow rate increases N2 < He < H2
Diffusion coefficients follow: H2 > He > N2

Sample injector
• Calibrated syringes are used to inject liquid samples (either
manually or through and autosampler) through a septum into a
heated liner at the head of the column.
• Injector is kept at about 50oC greater than the bp of the least
volatile component of the sample.

Commonly used modes of injection
1.) Split Injection
 Delivers only a fraction of sample to the column
- Split ratio is applied in the method; example a split ratio of 1:10
deliver 1/10 th of the sample and the the rest is discarded
 Good for highly concentrated sample
- Best resolution is obtained with smaller amount of sample
- ≤ 1 L with ≤ 1 ng of each compound (0.5 mL of gas volume)
 A good indicator for highly concentrated (overloaded column)
sample is unsymmetrical peaks (peak tailing and peak fronting).
 the split ratio is must be constant for the standards and the method
otherwise the method will not be accurate.
2.) Splitless Injection
All the sample is delivered into the column
Suitable for trace analysis, where analytes of interest
are in low concentrations.

oven
• The column is housed in a thermostatic oven to control temp.
• Optimum temp, depends on the b.p of the analyte and the required
resolution
• For compounds with broad boiling range, a temperature program is used-
temp is increased continuously or in steps

Oven
The oven can be operated in two modes;
•Isothermal elution- temperature of the oven is maintained constant.
•Temperature programming – temp is increased with time during separation
to simulate gradient elution since a solute retention in the GC is related to its
volatility..

• the sample is completely vaporized before the separation column and
then transferred to the column in gaseous form. This is where the
interaction with the stationary phase starts.
• The analyte has to form an equilibribrium between the stationary phase
and the mobile phase, this interaction is a gas phase equilibrium between
the mobile and stationary phases. This equilibrium is different for each
compound and is expressed in terms of separation into the individual
components.
• The equilibrium is also temperature dependent. This means that the
higher the temperature, the more the equilibrium is shifted towards the
mobile phase. An isothermal temperature of 300°C would shift the
equilibrium maximum towards the mobile phase, so that all sample
components migrate through the column at the same time and no
separation takes place.

• The temperature program is needed to shift the equilibrium for these
substances to the mobile phase so that they are transported through
the column sufficiently fast. At low column temperature high-boiling
substances, will move through the column slowly. This brings about
separation of different components.

Advantages of Temperature programming
• Decrease retention times of late eluting components
increase in temp Increase flow of the carrier gas decrease
retention time. A constant temp e.g 150o
C compounds elute
emerge close. If the temp is increased from 50-250 o
C at 8o
C/
min all compounds are eluted at their boiling points, thus
separated and the separation of peaks is fairly uniform.
NB; Every column has a maximum operating temperature and
the temp should not be raised above the optimum operation
temp of the column to avoid degradation of stationary phase
(Column bleeding)

oven
The oven is maintained at high temp to maintain the components
being separated in gaseous form
1.) Improving Column Efficiency
 Temperature programming:
- Temperature is raised
during the separation
(gradient)
- Increase flow of the carrier
gas decrease retention
time
Temperature gradient improves
resolution while also decreasing
retention time

Qualitative analysis
• A chromatogram provides only a single piece of qualitative
information about each species in a sample, its retention time
• 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 using standard reference
materials.
• thirty or more amino acids in a protein hydrolysate can be identified
with a relatively high degree of certainty by using chromatography.
• Thus, if the sample does not produce a peak at the same retention
time as a standard run under identical conditions, it can be assumed
that the compound in question is either absent or is present at a
concentration level below the detection limit of the procedure.

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.
• the area covered by the separated species serves as the analytical
parameter.
• If conditions are properly controlled, these parameters vary linearly
with concentration.

Calibration and standards
• The most straightforward method for quantitative chromatographic
analyses involves the preparation of a series of external standard
solutions that approximate the composition of the unknown.
• Chromatograms for the standards are then obtained and peak
heights or areas are plotted as a function of concentration.
• A plot of the data should yield a straight line passing through the
origin.

Detectors:
Common detectors
1.) Flame Ionization
Detector (FID)
– Measures the concentration
of organic species in a
gaseous stream eluting
from the column
– Sample is mixed with H2
(fuel) and air (Oxidant) and
burned in a flame
– Carbon atoms produce CH
radicals/ions which react
with O atoms to produce
CHO+
.

FID
 ions produced are collected at an cathode and measured
 Number of electrons is proportional to number of C atoms in
the sample
Advantages;
• Very sensitive to wide range of organic compounds
• It works over wide range of concentrations

GC: Detectors
2.) Electron Capture Detector (ECD)
It is a selective detector;
 Sensitive to halogen-containing cpds
and other compounds containing
electronegative groups (S, P, halogen
containing compounds)
 Based on the capture of electrons by
electronegative atoms
 Gaseous sample entering detector
capture e- produced by from
radioactive 63
Ni causing a current drop
 Number of electrons captured is directly
proportional to conc.
 Extremely sensitive
Steady current (flow of electrons)
disrupted by compounds with high
electron affinity

Gas Chromatography
3.) Mass Spectrometer (MS)
 Is a powerful detector, it’s a universal detector
 Combination of gas chromatography and mass
spectrometry is known as GC-MS
 Gaseous sample entering the mass spectrometer is
ionized by high energy e from tungsten filament
 The ions are accelerated through a flight tube, small
ions moves fast and reach the detector (separated
based on m/z ratio)
 A mass spectrum is produced which is characteristic of
the analyte (finger print)

 Mass spectrometer can identify peaks by
comparing spectrum with a library of spectra (in-
built library)- ie matches the unknown with a
known in the library
 Standards may not be used in qualitative GC-MS
work.

HIGH PERFOMANCE LIQUID CHROMATOGRAPHY (HPLC)

• Pumps-Pumping pressures are required to achieve reasonable flow
rates with column packing
• Sample Injector- graduated syringe- injection can be manual or
automated injection
• Column(s)- packed columns are used, guard columns are used to
increase the lifetime of the analytical columns.
• Detector
• Data System

Components of HPLC
1. Solvent Reservoirs/ Mobile phase reservoirs-
HPLC uses solvents as mobile phase. Solvents such as water,
methanol acetonitrile, cyclohexane, propanol, ethanol etc
buffers may also be employed if separation is to be done at
relatively constant pH.
•Highly pure grade solvents (HPLC or GC grade are employed).
•Dissolved gases may affect the operation of detectors
provisions are made to remove dissolved gases. (vacuum
pumping system) - Degassers

Isocratic elution or gradient elution
• An elution with a single solvent or solvent mixture of constant
proportion is called isocratic elution.
• Gradient elution involve use of two or more solvents that differ
significantly in polarity and varied in composition during separation
process. Gradient elution improves separation efficiency just like
temperature programming helps in gas chromatography.

4. Columns
 LC columns are constructed from stainless steel. Most column range
from 5-25 cm and have internal diameter of 3-5 mm (Straight columns
are used) packed with 5-10 um particles.
 Guard column- A pre-column may be used called a positioned between
the injector and the analytical column. A guard column is a short column
packed with a similar phase as the analytical column. The purpose of the
guard column is to prevent impurities such as highly retained compounds
and particulate matter that may contaminate the analytical column.
Column temperature control.
 Most HPLC are operated at room temperature. But better and more
reproducible chromatograms are obtained by maintaining constant
column temperature

Packing
• Porous particles of silica, alumina, polystyrene divinyl benzene or
ion exchange resin may be used. Silica is the most commonly used
in LC.
• Silica particles are coated with thin organic films which are
chemically bonded (Bonded phase). Formed by reaction of
organochlorosilane with the OH group formed by hydrolysis in hot
dilute HCl on the surface of silica particles.
• The product is organosiloxanes
• R is a straight chain or octyldecyl group, aliphatic amines, ethers,
nitriles.
• Thus many different polarities of the stationary phase are available.

Types of chromatographic separation
Two types of chromatographic separation are distinguishable
in HPLC based on polarities of the mobile and the stationary
phases.
1.Normal phase chromaography- highly polar stationary
phase (triethylene glycol) with a relatively non-polar mobile
phase such as hexane.
In normal-phase chromatography, the least polar component
(non polar)is eluted first because it is the most soluble in the
mobile phase; increasing the polarity of the mobile phase
has the effect of decreasing the elution time.

2. Reverse phase chromatography- non polar
stationary phase (C18, C8) and a polar mobile phase
(water ,methanol, acetonitrile)
– the most polar component appears first, and increasing the
mobile phase polarity increases the elution time increasing the
polarity of the mobile phase decreases the polarity of the elution
time of the polar compounds.

HPLC Advantages vs GC
• Not limited by sample volatility or thermal stability
• Two interacting phases .
• Room temperature analysis .
• Ease of sample recovery.

Common detectors
• UV (diode array detector (DAD))- uses absorbance for
measurement of conc.
• Electrochemical- amperometry, polarography, courometry,
conductometry
• Refractive index- based on bending of light through a medium
• Mass spectrometer -

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