- :Chromatography: -

Contents

- :Introduction: - .................................................................................. 2
- :Thin Layer Chromatography: - ......................................................... 6
- :Paper Chromatography: - .............................................................. 10
-:Column Chromatography:- ............................................................. 12
- :High Pressure Liquid Chromatography:- ........................................ 16
- :Ion-exchange Chromatography: - .................................................. 22
- :Gas Chromatography: - .................................................................. 27
- :High Performance Thin Layer Chromatography: -.......................... 33
- : Supercritical Fluid Chromatography:- ........................................... 37

1
- :Introduction: -

 What is chromatography?
- It is a separating technique which physically separates the components
of the mixture.
 It involves following terms.
- Stationary phase
- Mobile phase
- Retention time or retardation factor
 There are mainly two types of separation techniques available.
- Classical methods
- Modern methods
 Classical method involves
- Precipitation
- Distillation
- Extraction
 Growing demands in last few years have helped in emerging with newer
separation techniques involving chromatography.

 Historical Development of chromatography:-

 Mikhail Semyonowich Tswett, a Russian-Italian botanist invented
chromatography in 1906 separating plant pigments.
 Chromatography word is derived from tow Greek word “Chroma” which
means “colour” and “Graphein” which means “to write”.
 Further, Arther John Portor Martin and Richard Laurence Millington
Syring discovered gas-liquid chromatography. They both were awarded
with Nobel Prize in 1952.
 Further partition chromatography was also introduced.

 Definition according to IUPAC:-

2
  “Chromatography is a special technique of separation in which the
components to be separated are distributed between the two phases,
one of which is stationary (stationary phase) while other moves (mobile
phase) in a definite direction.

 Principle of Chromatography:-
 The proper choice of the two phases constituting a chromatographic
system permits selective adjustment of the relative migration rates of
the mixture components and of the extent of eventual separation.
 Components which are strongly retained on the stationary phase moves
slowly with the flow of mobile phase.
 In contrast, components that are weakly held by the stationary phase
will moves rapidly.
 As a consequence of the differences in mobility, sample components
separate into discrete bands or zones that can be analyzed qualitatively
or quantitatively.

 Stationary Phase:-
 Solid: - ‘Absorbent’ having large surface area and having accessible
pores.
 Liquid coated on solid support: - Avoid shifting of stationary phase due
to mobile phase.
 Immobilized polymers
 Surface bonded shorted chemical species

 Mobile Phase:-
 Liquid
 Gas
 Super critical fluids

 Classification of Chromatography:-
 Chromatography can be classified into following four ways.
1.) According to purpose of experiment:-
 Preparative chromatography:-
3
 - Separates component for further use
- Sample required in larger amount
- Sample eluted is used further.
 Analytical Chromatography:-
-Identify and quantify the analyte in gives sample
- Sample required in small quantity
- Sample eluted is disposed
2.) According to physical state:-
 Planner chromatography:-
- It is two dimensional chromatography.
- It involves paper chromatography and thin layer chromatography.
- It works on the principle of capillary motion (against gravity).
 Column chromatography:-
- It is mathematically three dimensional chromatography.
- It involves gas chromatography and high pressure liquid
chromatography.
- It works on principle of adsorption or partition
3.) According to mobile phase:-
 Liquid chromatography:-
- It can be both planner and column chromatography.
- It involves liquid-solid chromatography and liquid-liquid
chromatography (according to stationary phase).
 Gas chromatography:-
- It can only be column chromatography.
- It involves gas-solid chromatography and gas-liquid chromatography
(according to stationary phase).
4.) According to mechanism of separation:-
 Adsorption chromatography:-
- Stationary phase must be solid.
- It can be both planner and column.
 Partition chromatography:-
- Stationary phase must be coated on solid.
- It can be planner and column both.
 Ion exchange chromatography:-
- It can only be column.
4
 - Here, ion exchange resins containing cationic exchangers and anion
exchangers are used.
 Size exclusion chromatography:- (Gel permeation / Gel filtration /
Molecular exclusion)
- Stationary phase is a porous gel with specific pore size.
- As the mobile phase passes through a porous gel, larger solute
molecules passes through the void spaces while small molecules are
trapped inside the pores of the gel.
 Boned phase chromatography:-
- Separation is based on the distribution of solute between polar/non-
polar stationary phase and polar/non-polar mobile phase.
- Normal phase chromatography: - polar stationary phase and non-polar
mobile phase.
- Reversed phase chromatography: - Non-polar stationary phase and
polar chromatography.
 Affinity chromatography:-
- Separation is based on the specific interaction between the analyte
molecule and another compatible molecule immobilized on stationary
phase.
- Separation of antigen, an antibody may be immobilized on a matrix
that forms a stationary phase.
- It is mainly used in drug industries for separation of bio-molecules and
enzymes.

 Elution: - Elution is a process of allowing mobile phase flow through the

column to carry out the phenomena of washing under the effect of gravity
or pumping.

5
- :Thin Layer Chromatography: -

 Stationary Phase:-
 Silica gel with binder (gypsum/starch)
 Alumina
 Cellulose powder
 Acryl amides
 Charcoal and activated carbon

 Requirements:-
Small particle size
Can be polar, weak polar or non-polar
Should be homogenous
Applied on the thin glass or aluminum plate.
Bonder must be used for binding stationary phase

 Mobile phase:-
 Non-polar:- Ethyl ether, Petroleum ether, Hexane, Acetone
 Polar:- Chloroform, Methanol, Ammonia, Water

 Experiment:-
 Preparation of TLC plate:-
- Prepare slurry (e.g. silica gel-G with water)
- There should be no lumps.
- Clean glass surface
- Pour slurry uniformly
- allow it to dry in oven for 24 to 48 hours.
 Performing the experiment:-
- Take the dry plate

6
 - Place the sample of spot at the bottom with capillary
- Place it in a jar filled with mobile phase
- Allow it to run
 Observation:-
- Suppose, if polar stationary phase is used, polar component of sample
will run slow on thin layer and non-polar component will run fast on thin
layer.

 Retardation factor:- (Rf value)

distance travelled by analyte
 R value = distance travelled by mobile phase
 Note:- distance travelled by mobile phase is always longer than distance
travelled by analyte. Therefore, Rf value must be >0 and <1.
 Ideal value of Rf is 0.25 to 0.75
 If Rf=0 then analyte has no interaction with the stationary phase.
 If Rf=1 then analyte has strong interaction with mobile phase and no
interaction with stationary phase.

 Factors affecting Rf value:-

 Temperature
 Solvent
 Concentration of sample (sample must be diluted with mobile phase and
small spots are mandatory)
 Thickness of layer
 Impurities of sample

 Detection in TLC:-

7
 Detection

Destructive
Non-destructive
method (oxidise
method
the sample)

Spraying of
UV light source
Spraaying of dilute ninhydrine/iodine
(short or long
H2SO4 (for organic
wavelength)
compound)

 Applications:-
 Purity of sample
 Identification of compound qualitatively
 Evaluation of reaction process
 Checking performance of other separation

 Chromatograph:-
 Process:-

8
 Calculations:-

9
- :Paper Chromatography: -

 Stationary phase:-
 Paper (cellulose)=polar

 Mobile phase:-
 Solution or solvent that does not affect
- The paper
- The solubility of paper
- The reactivity of paper

 Classification:-
 Ascending paper chromatography:-
- A paper is hanging from a support into a jar filled with mobile phase
such that the end of paper just touches the mobile phase.
- The mobile phase runs upwards carrying the sample with it up to
particular height.
- All the components of the mixture get separated depending upon their
polarity. (Non-polar travels more, polar travels less)
 Descending paper chromatography:-
- Here, the flow of direction of mobile phase will be reversed from the
ascending chromatography.
- The reservoir placed at the top of the jar and mobile phase will flow
upside down.
- Sample gets separated at the bottom of the paper depending upon the
gravity.
- Note: - Sometimes it gives abrupt results which lead to a variation in Rf
value. Thus, it is less preferred technique.
 Circular paper chromatography:-
- A paper in a square or round shape is kept on a paltry dish filled with
mobile phase which travels through the paper from the centre of the
periphery of a circular paper.

10
 - A wick made of the same paper is used for the displacement of mobile
phase from the reservoir.
- This technique is highly precised.

 Comparison with TLC:-

 TLC gives better separation and resolution because papers have pores
which sometimes adsorb sample.
 Variety of choice of mobile phase and stationary phase is large in TLC.
 Speed of mobile phase is much faster in TLC.
 Therefore, TLC is superior technique the paper chromatography.

 Applications:-
 UG and PG level experiments for students understanding the concepts of
- Separation of group in organic ions
- Separation of dye and color samples
- Separation of plant pigments

11
-:Column Chromatography:-

 It involves –
- High pressure liquid chromatography
- Gas chromatography
- Ion exchange chromatography
- Size exclusion chromatography

 Definition:-
 Column chromatography is a technique which is used to separate a
single chemical compound from a mixture dissolved in the solvent. It
separates substances based on their adsorption by the adsorbent, as the
compound move through the column at the different rates which allows
them to get separated in fractions.

 Principle:-
 When the mobile phase along with the mixture that needs to be
separated is introduced from the top of the column, the movement of
the individual of the mixture at the different rates.
 The components with the lower adsorption and affinity to stationary
phase travel faster when compared to the greater adsorption and
affinity with the stationary phase.
 The components that move faster are eluted first whereas the
components that move slow are eluted last.

 Stationary phase:-
 Always solid (adsorbent) undergoing the process of adsorption
 Stationary phase should have following properties—
- Uniform and spherical size
-Chemically and mechanically stable
-Inexpensive and easily available

12
  Examples:- silica, alumina, calcium carbonate, calcium phosphate,
magnesia, starch
 Silica attracts polar analytes
 Alumina attracts non-polar analytes

 Mobile phase:-
 Serves several role-
- Introduce sample into column
- To develop bands into column
- To elute components from the column
 Choice of mobile phase on the basis of-
- Components of mixture must be soluble in mobile phase
- Low boiling points so components can be recovered easily
- Depending on polarity
 Examples:- petroleum ether, cyclohexane, acetone, carbon tetrachloride,
toluene, benzene, water

 Column:-
 Inert material like glass
 Long column is used for good resolution of separation
 At the bottom of column collector and at the top of column mobile phase
reservoir
 Preparation of column:-
 Before preparing the column, adsorbent is pretreated to enhance its
adsorption activity by heating
 There are mainly two types of filling
 Dry filling:-
- Dry adsorbent is filled in the dry column from top. After filling the
column with adsorbent, the mobile phase is added up to appropriate
mark.
- This mixes the adsorbent with solvent, sometimes, air bubbles trapped
after filling to remove the air bubbles.
 Wet filling:-

13
 - In this method, slurry of adsorbent is formed in the mobile phase by
stirring. The slurry is added to the column from the top in small portions.
- A careful addition should be done to avoid the cracks and to get uniform
setting of material.
- This solvent is removed by knob at the bottom and material is covered
with cotton plug.

 Sample introduction:-
 Sample is prepared by dissolving it in required amount of mobile phase.
 Solution is added at once and allowed to stand so that it come down and
get adsorbed in the stationary phase.

 Detection:-
 If color bands fractions, they are directly collected and identified.
 If colorless components, they are collected in fractions and identified
using TLC.

 Factors affecting column chromatography:-

 Selection of solvent
 Decreasing the size of particles
 Length and width of column
 Increasing the temperature during elution

 Application:-
 In separation of inorganic ions, organic molecules, cis-trans isomers, plant
extracts, drugs and final products.
 In purification of compounds
 In estimation of drugs in tablets and injections

 Advantages:-
 Separation of almost all compounds
 Wide range of amount of samples

 Disadvantages:-

14
 Time consuming
 Loss of components
 Wastage of solvents
 Lack of accuracy and precision

 Separation:-

15
- :High Pressure Liquid Chromatography:-

 To overcome drawbacks, HPLC used which have following characteristics

- Pressurized pumps (Lesser time consumption)
- Small column size
- Flow rate is 0.5 to 0.2 ml/min (less mobile phase)
- Automated system
- Error free (Effective separation)
- Small sample size (high sensitivity)
 The letter ‘P’ in HPLC is also stands for performance which attributes high
accuracy and high sensitivity.

 Mechanism:-
 Separation of compounds takes places they move at different rates in the
column which is recorded in chromatogram.
 Due to different interactions between sample and the stationary phase,
molecules moves at the different rates.
 Molecules having weaker interactions with stationary phase will be eluted
at less time and will recorded first on chromatograph.
 Molecules having stronger interactions with stationary phase will be
eluted in more time and will be recorded later on chromatograph.

 Configuration:-
 HPLC analysis can be carried out in the following two manners.
1.) Isocratic elution
2.) Gradient elution
 Isocratic elution:-
- One reservoir and one pup
- When a mixture is required as mobile phase, pre- mixing is required.
- It doesn’t give good results as the proportion of mobile phase can’t be
changed between the analysis. Thus, the extent of separation won’t be
good.
 Gradient elution:-
16
 - Gradient elution is also divided in two categories as
1.) Low pressure gradient (one pump controls 4 or more reservoir) (here
pressure drop is also observed)
2.) High pressure gradient (each pump for each reservoir is used)
(excellent gradient and accuracy in pressure)

 Instrumentation:-
 Instrument contains following parts:-
1.) Solvent delivering system
2.) High pressure pump
3.) Sample injection
4.) Column and oven
5.) Detectors
 The sketch diagram of HPLC instrument is given as follows.

1.) Solvent delivering system:-

 Solvent reservoir:-
- Glass bottle with valve
- Anti-corrosive and transparent
17
 Degassers:-
- Generally placed before reservoir to remove air bubbles and
undesirable gas.
- Some of the following methods are used for the degasification.
i.) Sonification: - Vibrating mobile phase
ii.) Vacuum filtration: - Mobile phase passed through vacuum
iii.) Helium sparge: - Helium passed through mobile phase
iv.) Online membrane degassing: - passed through membranes
 High pressure pumps:-
- Reciprocating pump (most preferred)
- Syringe type pump
- Constant pressure pump
- Pneumatic pump
 Mixer:-
- Binary, ternary or quaternary mixtures can be prepared using mixing
valve
- It is computerized system
 Pulse dampener:-
- Controls pulse dampening in HPLC

2.) Injectors:-
 Auto-sampler (more preferred)
 Manual sampling
 Sampling valve is mainly used having sample loop of 10 µL, 20 µL or 30 µL.
 The simple diagram of sampling valve is shown as follows.

18
3.) Column:-
 HPLC instrument have guard column and analyte column
 Guard column used to saturate mobile phase and absorb impurities of
mobile phase.
 The following type of column is used in HPLC
- Packed column
- Wall coated open tubular column
- Support coated open tubular column
- Porous layer open tubular column
- Fused silica open tubular column

4.) Detectors:-
 UV-visible detector
 PDA detector
 Fluorescence detector
 Mass spectrophotometer
 Evaporating light scattering detectors
 Conductive detectors
 Optical detectors
 Chiral detectors
 Electrochemical detector

19
 Chromatogram:-

- The following type of chromatogram is

obtained which is used for qualitative or
quantitative analysis.

 Working:-
 Size exclusion
 Ion exchange
 Affinity
 Bonded phase support

 Bonded phase support chromatography:-

 Normal phase:-
- Stationary phase = polar or hydrophilic
- Mobile phase = non-polar or hydrophobic
 Reversed phase:-
- Stationary phase = non-polar and hydrophobic
- Mobile phase = polar of hydrophilic

 Applications:-
 In pharmaceuticals
 In food, flavor and fragrances
 In environmental analysis
 Forensic science
 In clinical analysis
 In petrochemical industries
 Bioactive natural products
20
 In cosmetic industries
 In scientific research

21
- :Ion-exchange Chromatography: -

 Separation of analytes is mainly based on net surface charge of

molecules (electrostatic interaction)
 Stationary phase = anionic or cationic resins
 Mobile phase = liquid

 Principle:-
 Molecules possessing the opposite charge (to resin) will bind tightly to
the resin, whereas molecules having the same charge (to resin) will flow
through the column and eluted first.
 Positively charged resin = anionic exchanger
 Negatively charged resin = cationic exchanger

 Approach in operating IEC :-

 Classical approach by using only column
 Derivatized approach by HPLC

 Separating mechanism:-

 An anionic exchanger
resin carries a net positive
charge and thus, ions carrying
net negative charge will bind to
resin and the ions carrying net
positive charge will be eluted
fast.
 Thus, duel mechanism is
observed here as electrostatic
attraction and repulsion at the
same time.

22
 Factors affecting IEC:-
𝐼𝑜𝑛𝑖𝑐 𝑐𝑕𝑎𝑟𝑔𝑒 (𝑧)
 𝐼𝑜𝑛𝑖𝑐 𝑝𝑜𝑡𝑒𝑛𝑡𝑖𝑎𝑙 = 𝐼𝑜𝑛𝑖𝑐 𝑟𝑎𝑑𝑖𝑢𝑠 (𝑟)

 Thus, as charge increases binding increases and as radius decreases

binding increases.

 Distribution coefficients:-
 The difference in binding affinity of analytes. Thus, ions to be separated
from the mixture can be used to calculate the theoretical effectiveness
of the separations in IEC.
 This can be done by comparing the distribution coefficient (D) of analyte
ions adsorbed on the resins.
 D is the ratio of the concentration, at steady state, of ion on the
stationary phase to the concentration of ions in mobile phase.
𝑐𝑜𝑛𝑐 .𝑜𝑓 𝑖𝑜𝑛 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝𝑕𝑎𝑠𝑒 (𝐶𝑠)
 𝐷𝑖𝑠𝑡𝑟𝑖𝑏𝑢𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 = 𝑐𝑜𝑛𝑐 .𝑜𝑓 𝑖𝑜𝑛 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝𝑕𝑎𝑠𝑒 (𝐶𝑚 )

 Stationary phase:-
 Resins are spherical beads typically made up of inert polymeric material
providing the facility to coat negatively or positively charged surfaces.
 If surface area of resin is large, the exchange will be more
 If cross-linking of functional group is large the exchange will be more.
 Group present on stationary phase =
- Strong anionic exchanger = Amino ethyl > Diethyl amino ethyl >
Trimethyl amino ethyl > Diethyl-2-hydroxy propylamine ethyl
- Strong cationic exchanger = Carboxy methyl > Sulphopropyl

 Mobile phase:-
 Aqueous acid or base solution
 Organic acid and non-aqueous solution
 Factors affecting in IEC due to mobile phase-
- Flow rate of mobile phase
23
 - Chemistry of mobile phase with analyte ions
- Ionic strength and concentration of mobile phase

 Instrumentation:-

Mobile phase Ion exchange

Pump Injector port
Reservoir column

Suppresor Detector Data system

1.) Mobile phase Reservoir:-

 Glass or PEEK bottle (poly ether ethyl ketone)
 Two or three solvent system
 Degassing is required

2.) Pump:-
 Role of pumps is to deliver the mobile phase from the reservoir to the
ion exchange column with a constant and desired flow rate.

3.) Injector port:-

 Sampling valve or auto-sampler as same as HPLC

4.) Column:-
 Net surface charge of the resin = cation / anion
 Separation depends on pH, buffer salt, organic solvents, temperature
 Guard column:-
- Guards the separating column and increase its life
- Filters the particle responsible for clogging the separation
 Separating column:-
- Ion exchange occurs depending on the surface charge of the resin

24
5.) Detectors:-
 Electrical conducting detector
 Amperomatic detector
 Mass spectrophotometer

 Applications:-
 In chemical analysis:-
- Investigation of cations and anions
- Separation of Nucleotides, amino-acids and alternative polar molecules
- In evaluation of raw materials, conflicting impurities and cleaning
solutions
- In qualitative analysis and quantitative analysis of chemicals
 In water analysis:-
- In trace or ultra-trace analysis of heavy metal in water
- Softening of hard water
- Demineralization of water
- Removal of interfering radicals
 In industries:-
- Determination of heavy metals in cement
- Determination of petrochemicals
- Analysis of metallurgical compounds
- Analysis of alkaline earth metals
 In Environment:-
- Analysis of acid rain and rain water
- Analysis f terrestrial water ice cores.
- Determination of inorganic ions in soil
 In food/flavor industries:-
- Determination of heavy metal in dairy products
- Evaluation of sulphates, chloride, nitrates and phosphates in
sweeteners and sugar syrups
- Acid contents viz. lactic acid, malic acid, citric acid and acetic acid in
coffee, foods and other beverages

25
 - Fluoride content in teas
- Detection of glucose, lactose and galactose in dairy products in lactose-
free category
- Arsenic in rice and phosphoric acid in cold drinks
- Amino acid content in specialized nutritional products
- Presence of carbonates in drinking water
 In pharmaceuticals:-
- Separates the cations in water solution for injectables
- Analyzing final drug formulation
- Simple manufacturing of process of drugs
- Easiest approach for segregation of anions in decongestant and
analgesic drug

26
- :Gas Chromatography: -

 Conditions required:-
 Sample should be volatile and thermally stable
 Mobile phase must be gaseous
 Stationary phase must have high boiling point or melting point

 Definition:-
 Gas chromatography is technique employed to separate and analyze the
compounds that can be vaporized without decomposition via interaction
with gaseous mobile phase or stationary phase. It is popularly known as
vapor phase chromatography.

 Mechanism:-
 It involves two mechanisms: -
- Adsorption (Gas-Solid chromatography)
- Partition (Gas-Liquid chromatography)

 Principle:-
 In the case of GLC, principle of GC is that it separation takes place on the
basis of differences in partition coefficients of volatilized sample
between mobile phase and liquid stationary phase as sample is passed
through column.
 In the case of GSC, Solid stationary phase is used and hence separation
of components occurs via adsorption.
 Generally GLC is more preferred. Thus, GLC is referred as GC.

 History:-
 GC was the first analytical instrument associated with computer for
analysis, data processing and result generation.
 First GC instrument involved-
- Separation of fatty acids

27
 - N2 gas as mobile phase
- Silicon oil as stationary phase
- Thermostatic column
 Comparison with HPLC:-
 LLC was grounded with the invention of GLC as the gaseous mobile
phase enhanced the diffusion of solutes.
 GLC gives faster and better separation compared to LLC.

 Stationary phase:-
 In GSC:- (Solid)
- Activated carbon
- Silica
- Aluminum
 In GLC:- (Liquid)
- Solid supports are Glass powder, Diatomaceous earth, powdered
Teflon, Crushed fire bricks, Carbon black
- Liquid layers (non-volatile) are Polyethylene glycol, Dimethyl silicon,
Diethylene glycol succinate

 Advantages of GLC over GSC:-

 Shorter time analysis
 Better resolution between peaks
 Used for qualitative and quantitative analysis with better results
 Large range of concentration of samples can be evaluated

 Partition coefficient:- (Kc)

𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝𝑕𝑎𝑠𝑒 [𝐶𝑠]
 𝑃𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 = 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝𝑕𝑎𝑠𝑒 [𝐶𝑚]

 It is also known as distribution coefficient

 Larger value of Kc results in more retention of analyte.
 The values of partition coefficient might get affected by following
factor:-
- Column temperature

28
 - Chemical nature of stationary phase
 If sample is more volatile, it will be eluted fast with the flow of mobile
phase as volatility is inversely proportional with partition coefficient.

 Retention factor:- (K)

𝑇𝑟−𝑇𝑚
 𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 = 𝑇𝑚
Where Tr is retention time of sample
Tm is time taken by mobile phase to pass through column

 Instrumentation:-

1.) Mobile phase cylinder (carrier gas source):-

 There are mainly five carrier gases used which are
- Helium
- Hydrogen
- Nitrogen
- Argon
- Methane
 Cylinder is mainly made of steel with two stage regulators

2.) Injectors:-
29
  On column injectors
 Split injectors = high concentration of sample is required and only
fraction of sample is injected while remaining is rejected
 Split less injectors = low concentrations is required and sample in
completely injected into the column
 Cryogenic focusing injectors (in case of highly volatile sample)
 Auto-sampler injectors = are most preferred because-
- Electronic pneumatic control + robotic sampler
- Automatic introduction
- Better reproducibility and time optimization
- Extreme small quantity and précised handling

3.) Column oven:-

 Temperature must be maintained throughout the separation process
 Injector connections, column connections and detector connections all
are inside the column oven.
 Must have rapid heating and cooling controls
 Isothermal operation = steady temperature throughout the separation
 Temperature controlled operation = Temperature programmed
chromatograph as per requirement of sample

4.) Column:-
 Packed column:-
- 1 - 5 meters long and 1- 5 mm diameter
 Capillary column:-
- 50 - 70 meters long and 0.1 – 0.5 mm diameter
 Column are made of Ni, Cu or stainless steel
 But mainly fused silica is used as column material as it have following
characteristics
- Inert
- Larger surface area
- Good conductor of heat
- Wall coated support
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5.) Detectors:-
 Flame ionization detector:-
- Formation of ions from organic molecules combusted in hydrogen-air
flame.
 Thermal conductivity detector:-
- Change in thermal conductivity of column effluents is measured due to
alteration of sample in mobile phase
 Electron capture detector:-
- Electrons generated by imparting column effluents on photosensitive
material are measured.

 Applications:-
 Qualitative analysis:-
- Comparison of retention time or volume to the standard
 Quantitative analysis:-
- Measure the area and height of peak gives quantitative idea of sample
- Comparing purity with standard
 In food / flavor / fragrance industry:-
- Analysis of volatile compound in food
- Monitoring legislation of food products
- Controlling diet formulation
- Recognizing change in food during storage
 In environmental analysis:-
- Detection of pollutants
- Water emission analysis
- Water discharging plant analysis
- Air quality analysis
- Remaining pesticides in agriculture products, oils, fruits, vegetables and
drinking water
- Analysis of environmental toxins like dibenzofurans, dioxanes,
polychlorinated biphenyls, polyatomic aromatic hydrocarbon
- Identification of unknown compounds in hazardous waste dump

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 - Estimation of industrial products as well identification of reaction
products
 In chemical industries:-
- Assuring the quality of products and identification of harmful gases
- Determination of toxic substance in soil, water, air
- Detection of harmful gases like SO2, CO2, benzene and 1-3 dibutadiene
 In forensic science:-
- Detection of fluids and compounds present in human body responsible
for death
- In blood and fiber sample therapy
- Detection of flammable liquid on the site of fire accident
- Alcohol analysis in human body
 In petrochemicals:-
- Natural gas analysis, gasoline characterization and fraction quantitation
- Mapping of oil reserves and tracing of reserves
- Monitoring of petroleum refining and finished product analysis
 In drug industry:-
- Analysis of residual solvent used
- Drug assays
- Quality control and analysis of drug product
- Purity and impurity profiling

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- :High Performance Thin Layer Chromatography: -

 HPTLC is a most ultimate modification of Thin Layer Chromatography.

 Emergence of HPTLC:-
 TLC plates are commercialized by replacement of glass with aluminum
 Effect of small sized silica was observed on separation and resolution.
So, the plates were called nano-plates.

 Characteristics:-
 Small sized and high quality plates
 Automated sample application
 Minimum sample preparation
 Full optimization and increased resolution of components
 Densitometric scanning

 Principle:-
 Principle of HPTLC is same as TLC

 Key features:-
 Complex information about the sample-visible chromatogram
 Simultaneously both sample and standard analysis
 Automatically converts visible chromatogram into peak data
 Cost effective and low cost of maintenance
 No filtration or degassing required

 Instrumentation:-
 HPTLC instrument is consist of following parts
- Sample applicator machine
- Chromatogram development
- TLC visualizer

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 - Detection and data system
 Flow-chart:-

Sample Application of
Selection of layer
preparation sample

Chromatogram Quantitative
Scanning
Development evaluation

1.) Selection of layer:-

 In HPTLC plates are coated with small sized particles and narrow size
distribution is used. THUs, the surface area of plates is smooth.
 The size of plate is comparatively large as compared to TLC.
 The selection of HPTLC is stationary phase is based upon the type of
analyte.
 Normal phase stationary phase = Silica gel (polar)
 Reversed phase stationary phase = C18, C8, C4, C, phenyl or chemically
modified silica gel
 Chiral stationary phase = C18 modified silica gel, optically active
adsorbent
 Others = Al2O3, MgO, Silicates, Cellulose, Polyamide, Polar modified silica
gel

2.) Sample preparation:-

 The samples may be prepared using
- Methanol
- Chloroform + Methanol (1:1)

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 - Ethyl acetate + Methanol (1:1)
- Chloroform + Methanol + Ammonia (90:10:1)
- Methylene chloride + Methanol (1:1)

3.) Applications of sample:-

 Samples are applied with the help of some applicators such as
- Capillary tube
- Micro-tube pipettes
- Micro syringe
- Automated sample applicator
 The sample is applied in the form of spots.
 The concentration range of sample is 0.1 – 1 µgm/litre

4.) Development of chromatogram:-

 The chromatogram development is the most important step in the
HPTLC procedure.
 The HPTLC chamber is pre-coordinated with solvent to get uniform
vapors of the solvents in the chamber.
 Chromatogram can be developed in the following four ways.
- Automatic multiple development
- Vertical methods
- Horizontal methods
- Vario methods

5.) Scanning:-
 The developed plates can be detected by using UV cabinet or chamber
which provides a non destructive analysis.
 These days, designs of UV cabinets are improved, which allows fixing of
digital camera for recording images of the plates.

6.) Quantitative evaluation:-

 In desitometry, separation tracks are evaluated with the help of a
reflected light is measured by the photo-sensor and the difference
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 between optical response of blank and sample zone is correlated with
various sample zones to calculate the quantitative idea of each analyte.

 Applications:-
 HPTLC is utilizes for the recognition of constituents, identification and
detections of impurities and quantitative evaluation of active
substances.
 HPTLC can be used in following fields:-
- Botanical and herbal drugs
- Fingerprinting of pharmaceutical components and plant extracts.
- Forensic analysis
- Biochemistry
- Toxicology
- Cosmetics

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- : Supercritical Fluid Chromatography:-

 Each substance has its characteristics critical point. When such

substances are subjected to temperature and pressure above critical
point, they attain ‘supercritical state’ where distinct liquid and gas state
don’t exist.
 Two ways to attain a ‘supercritical state’
- By increasing pressure above critical pressure (Pc)
- By increasing temperature above critical temperature (Tc)
 Tc is temperature above which gas can’t be liquefied.
 Pc is pressure required for liquefying a gas at its critical temperature.
 The change in pressure and temperature can bring about higher change
in density, viscosity and diffusivity of the supercritical fluid leading to its
higher use as an eluent.

 Principle:-
 Supercritical fluids chromatography is based on the partitioning of the
sample components between the mobile phase and the stationary
phase.
 Here, mobile phase is supercritical fluid and stationary phase is similar to
that used in HPLC or GC.

 Mobile phase:-
 The following properties are required to serve as mobile phase in
supercritical fluid chromatography.
- Critical parameters should be easily achievable.
- It should be inert
- Compatible with detector
 Some examples of mobile phases are Carbon dioxide, Ammonia, n-
Butane, Diethyl ether, Tetrahydrofuran, Ethane

 Modifiers:-

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  There several issues with carbon dioxide are-
- Very high retention time
- Non-elution of analyte
- Separation of highly polar analytes is not possible
 Thus, organic modifiers are added for-
- Increasing solubility of polar analytes
- Reduce the interaction between sample and stationary phase to
facilities the reduction in retention time.
 Usually, Methanol is use as modifier because-
- Easily available
- Cost-effective
- Low toxic

 Instrumentation:-
 Instrument of supercritical fluid involves following components
- Solvent delivering system
- Injector
- Thermo-stated column
- Resistor or back pressure device
- Detector
- Data handling system
 Flow chart:-

1.) Solvent delivering system:-

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  It is responsible for transport of solvent through injector and to column,
also conducting controlled flow of mobile phase rate and pressure of the
system.
 It is consist of-
- Supercritical mobile phase
- Liquid modifier pump
- Back pressure regulator

2.) Pumps:-
 Control density and solubility of mobile phase
 Must be capable of pulse-less flow
 Examples:-
- Syringe pump = open tubular columns
- Reciprocating pump = packed column
 Two separate pumps for CO2 and modifier
 CO2 and pump head are pre-cooled to about 4-5◦c in order to prevent
evaporation.

3.) Injector:-
 In-line injector
 Loop injectors
 In-line injectors is more preferred because-
- Flexibility in volume of injection as well as non-diluted sample can be
injected
- A high pressure pump directly helps the sample to enter into flow of
eluent.
4.) Guard column:-
 Activated carbon or alumina based adsorption filters are used.
5.) Oven and column:-
 Packed column (stainless steel)
 Capillary column (purified silica, internal diameter small)
6.) Back pressure regulator:-

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  Back pressure regulator is installed before the detector to maintain the
pressure throughout supercritical fluid.
7.) Detectors:-
 UV-visible detector
 Flame ionization detector
 Mass spectrophotometer detector
 Refractive index detector
 Evaporating light scattering detector
 Electrochemical detector
 Diode array detector
 Thermal conductivity detector
 Fluorescence detector
 Electrical conductivity detector
 Electron capture detector
 IR detector

 Advantages:-
 High speed analysis
 Low pressure drop
 Broad analysis spectrum
 Analysis of thermo-mobile compounds
 Variety of detectors
 Use of eco-friendly mobile phase

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