Titration

Titration is an analytical technique which allows the quantitative determination of a specific

substance (analyte) dissolved in a sample. It is based on a complete chemical reaction between
the analyte and a reagent (titrant) of known concentration which is added to the sample. A well-
known example is the titration of acetic acid (CH3COOH) in vinegar with sodium hydroxide,
NaOH:

The titrant is added until the reaction is complete. In order to be suitable for a determination the
end of the titration reaction has to be easily observable. This means that the reaction has to be
monitored (indicated) by appropriate techniques, e.g. potentiometry (potential measurement with
a sensor) or with colour indicators.
The measurement of the dispensed titrant volume allows the calculation of the analyte content
based on the stoichiometry of the chemical reaction. The reaction involved in a titration must be
fast, complete, unambiguous and observable.

BASIC TERMINOLOGY
Titration: A titration is a technique where a solution of known concentration is used to
determine the concentration of an unknown solution. Typically, the titrant (the known solution)
is added from a burette to a known quantity of the analyte/titrate (the unknown solution) until the
reaction is complete. Knowing the volume of titrant added allows the determination of the
concentration of the unknown. Often, an indicator is used to usually signal the end of the
reaction, the endpoint
Titrate: Substance to be analyzed or to be determined. Titrant is a substance added from the
burette. Titrant used and the reaction principle that proceeds usually defines name of the titration
- like acid-base (or alkalimetric) titration if we use strong acid (or strong base) as a titrant, or
redox when the reaction that proceeds is of a redox type.

Titrant/analyte: Reagent of know concentration. Titrate is (analytical chemistry) to ascertain the

amount of a constituent in a solution (or other mixture) by measuring the volume of a known
concentration (the "standard solution") needed to complete a reaction. Analyte is a substance
whose chemical constituents are being identified and measured

Indicator: Indicator is a substance that changes color in response to a chemical change. An acid-
base indicator (e.g., phenolphthalein) changes color depending on the pH. Redox indicators are
also used. A drop of indicator solution is added to the titration at the beginning; the endpoint has
been reached when the color changes.

Equivalence point: The point at which complete chemical reaction takes place and equivalent
quantity of reagent is used. Or the equivalence point is the exact point in a titration when moles
of one titrant equal the moles of the substance being titrated.

End Point: The point at which the indicator changes its color. Or The endpoint is the point
where the system changes when the moles of the reacting titrant exceed the moles of the
substance being titrated.
 
Difference between Equivalence Point and End Point:

The equivalence point in a titration is the point at which the added titrant is chemically
equivalent completely to the analyte in the sample. End point is the point where the indicator
changes its color.

Equivalence point comes before the end point.

Concentration: A defined quantity of substance in a defined volume of solution.

STANDARDIZATION: Standardization is the process of determining the exact concentration

(molarity) of a solution. Basically standardardization is carried to know the strength of the
secondary standard prepared for carring out the assay. It is done to ensure the strength of
secondary standard prepared. The strength calculated after standardization is used to calculate
the content or percentage purity.

Primary Standard: A primary standard is a reagent which is very pure, representative of the
number of moles the substance contains and easily weighed. Primary standards are typically used
in titration to determine an unknown concentration and in other analytical chemistry techniques.
Primary standards are often used to make standard solutions. 

Secondary Standard: A related term is "secondary standard". A secondary standard is a

chemical that has been standardized against a primary standard for use in a specific analysis.
Secondary standards are commonly used to calibrate analytical methods

METHODS OF EXPRESSING CONCENTRATION:

Molarity: Molarity(M) is the number of moles of solute dissolved in one liter of a solution and

the unit for molarity is moles/L.
M = moles of solute / liters of solution 
Let’s say you dissolve 1.00mol of a solute into 0.500L of solution. The molarity(M) would be
1.00mol/0.500L = 2.00mol/L = 2.00M

Molality: Molality (m) is the number of moles per kilogram of solvent. It is determined by

dividing the number of moles (n) of the solute by the mass of the solvent in kg.
m = moles of solute / mass of solvent in kg
Lets say you dissolve 0.75mol of a solute into 2.50L of water. Since the density of water is
1.00g/mL and one liter of water is 1000mL, the mass of a liter of water is 1.00kg. So 2.50L of
water has a mass of 2.50kg.
The molality, m , of this solution would by 0.75mol/2.50kg = 0.3mol/kg = 0.3 m .

Normality: Normality (N) is defined as the number of mole equivalents per liter of solution:

N = number of mole equivalents / 1 L of solution

Like molarity, normality relates the amount of solute to the total volume of solution; however,
normality is specifically used for acids and bases.

Percent Solution: The concentration is expressed in terms of per cent (parts per hundred) also.
Per cent Composition of a solution can be expressed as:
1. Per cent W/W = Weight of solute/ Weight of solution X100
2. Per cent V/V =Volume of solute/ Volume of solution X 100
3. Per cent W/V= Weight of solute/ Volume of solution X100

Parts Per Million: Parts per million is frequently employed to express the concentration of very
dilute solutions and is express as PPM. it also can be expressed as milligrams per liter (mg/L).
This measurement is the mass of a chemical or contaminate per unit volume of water.

TITRATION PROCESS

Equipments used:
 Burette, (is a cylindrical tube with a stopcock at one end).
 Pipette: Delivers an accurate volume of a solution. Often this is 25 cm3.
 Volumetric flask: Used to make up an accurate volume of a solution, for example,
250 cm3. This could be a standard solution (of exactly known concentration and
known solute).
 pH meter.
 Indicators: provide some visual clue that the reaction is complete.
 Some commomly used indicators are:
 Figure: Titration.

STANDARD METHOD OF TITRATION:

1. A known volume VA of the analyte is placed in a titration flask.

2. The burette is filled by a standard solution (titrant,) of known concentration cT (M).
3. Before the titration is started, 1-3 drops of indicator (phenolphthalein) is placed in the
titration flask with the analyte. The chosen indicator must be one color when the solution
is acidic.
4. A base solution is then slowly added from the burette, drop by drop.
5. The titration continues, drop by drop, until the indicator suddenly achieves the
intermediate color (weak pink) between that of the acid and the color of the base
(fuchsia). At that point the titration ceases.
 6. The point at which the system is neither acidic or basic is referred to as the endpoint. The
endpoint will correspond to a perfect stoichiometric relationship between the acid and the
base.
7. Once the endpoint has been reached, the burette must be read. The bottom of the
meniscus line determines the quantity of the base VT that was required to reach the
endpoint.
8. Once the titration is completed, the final calculations can be done.

Calculations:
Calculating the concentration cA (M) of the analyte (acid):
i. From the balanced chemical equation
moles analyte = moles titrant
nA = nT……………….. (2)
ii. moles titrant: nT= VT . cT…………. (3)
iii. because nT = nA , the concentration of the analyte cA=nA/VA …………(4)

Titrating with a pH meter:

Titration with a pH meter follows the same procedure as a titration with an indicator, except that
the endpoint is detected by a rapid change in pH, rather than the color change of an indicator.
Arrange the sample, stirrer, burette, and pH meter electrode so that you can read the pH and
operate the burette with ease.

TYPES OF TITRATION:
Titration Reaction types
1) Acid-Base Titrations
2) Redox titrations
3) Complexometric Titrations
4) Zeta- potential Tittrations
5) Miscellaneous titration
6) Iodimetry titration
 7) Precipitation titration
8) KjeldahlTitration
9) Argentometric Titrations
10)Back titrations

Classification of titration by end-point techniques

1) Conductometric titration
2) Potentiometric titration
3) Spectrophotometric titration
4) Amperometric titration
5) Thermometric or enthalpimetric titration
6) Nonaqueous titration
7) Automatic titration
8) electrochemical titration

Acid-Base Titrations:
Titration is a process of neutralization whereby a titrant (a solution of known concentration) is
delivered into an analyte (unknown solution) until the unknown solution is completely
neutralized. This will allow information about the unknown solution to be determined. An
indicator is (often) a weak acid that is placed into the unknown solution to determine the
endpoint of the titration (the pH at which the indicator changes color). The equivalence point of
the titration is the point when the moles of H+ are equal to the moles of OH- in a titration. The
progress of an acid-base titration is often monitored by plotting the pH of the solution being
analyzed as a function of the amount of titrant added. The graph produced is called a titration
curve.

Types of acid-base Titrations:

(i) Strong Acid / Strong Base: pH at equivalence point = 7
(ii) Weak Acid / Strong Base: pH at equivalence point >7
(iii) Strong Acid / Weak Base: pH at equivalence point <7
*Note: weak acid / weak base titrations are too complicated and are almost never carried
Out
Applications of acid-base titration:
 In the determination of iron in pharmaceutical preparations.
 First of all, acid-base titration, to control acidity or alkilinity of solutions, to
do neutralisation tests and analyze mixtures of acids.
 Wide use is in titration processes.

Red-ox reaction:
A redox reaction (reduction and oxidation reaction) is a reaction in which there is a transfer of
electrons. When an element is reduced, it gains electrons and its oxidation number is reduced.
When an element is oxidized, it loses electrons and its oxidation number increases. Reduction
and oxidation always happen at the same time.

Application of Red-ox titrartion:

 In the determination of iron in pharmaceutical preparations
 Wide use is in titration processes
 We also perform redox titration for quantitative determination of metals such as Ca, Mg,
Zn, Co, Ni, Fe(II) and (III), Mn(II), (IV) and (VII), Cr(III) and (VI), U (IV) and (VI),
V(II), (IV) and (V) and PGMs;
 for determination of dissolved O2, H2O2 and anions such as NO2-, S2-, SO32-, Cl-and
Br-
 to determine the oxidation state of elements.

Complexometric Titrations:
The technique involves titrating metal ions with a complexing agent or chelating agent (Ligand)
and is commonly referred to as complexometric titration. A technique of volumetric analysis in
which the formation of a colored complex is used to indicate the end point of a titration. Also
known as chelatometry. Also spelled compleximetric titration. These titrations are based on the
formation of a complex between the analyte and the titrant.

Applications of Complexometric Titrations:

 Complexometric titrations have been employed with success for determination of various
metals like Ca, Mg, Pb, Zn, Al, Fe, Mn, Cr etc. in different formulations that are official
in I.P., and also for the determination of Hardness of water.
 Determination of total hardness of water by Complexometric method

Zeta potential titration:

It is a titration of heterogeneous systems, such as colloids, emulsions, etc. Solids in such systems
have very high surface area. This type of titration is used to study the zeta potential of these
surfaces under different conditions.
Miscellaneous titration:
A form of titration can also be used to determine the concentration of a virus or bacterium. The
original sample is diluted (in some fixed ratio, such as 1:1, 1:2, 1:4, 1:8, etc.) until the last
dilution does not give a positive test for the presence of the virus. This value, the titre, may be
based on TCID50, EID50, ELD50, LD50 or pfu. This procedure is more commonly known as an
assay.

Iodimetry titration:
Iodometry is a method of volumetric chemical analysis, a titration where the appearance or
disappearance of elementary iodine indicates the end point. Usual reagents are sodium
thiosulfate as titrant, starch as an indicator (it forms blue complex with iodine molecules - though
polyvinyl alcohol has started to be used recently as well), and an iodine compound (iodide or
iodate, depending on the desired reaction with the sample).
The principal reaction is the reduction of iodine to iodide by thiosulfate:
I2 + 2 S2O32− → S4O62− + 2 I−

Application of iodiometry titration:

  A common and illustrative use of iodometry is the measurement of concentration of
chlorine in water. Chlorine in pH under 8 oxidizes iodide to iodine. An overabundance
of potassium iodide is added to the known amount of sample in acidic environment (pH
< 4, the reaction is not complete in more alkaline pH). Starch is added, forming a blue
clathrate complex with the liberated iodine. The blue solution is then titrated with
thiosulfate until the blue color vanishes.
 Determination of Vit-C (ascorbic acid) in fruit juices by Iodimetric method
 Analysis of commercial Hypochlorite solution lodometrically.
 Analysis of hydrogen peroxide Iodometrically.

Precipitation titration:
Amperometric titration in which the potential of a suitable indicator electrode is measured during
the titration. In a precipitation titration, one of the products is a precipitate.

Indicators used:
As this reaction is not of the acid/base type, the end point cannot be detected with indicators such
as the methyl orange or phenolphthalein used in E13. Instead, a small amount of chromate ions,
CrO4 2–, is added as potassium chromate solution. Potassium chromate solution is yellow, whilst
silver chromate is a red.

Applications of precipitation titration:

 To determine the equilibrium constant or the solubility product of a compound.
 We also perform precipitation titration, for example, argentometric determination of
chlorides, cyanides and thiosulphites
 To determine electrode potential
 Precipitation Titrations are used for the analysis of halides and pseudo-halides for
quantitative determination, as well as for some metal ions
.
Kjeldahl Titration:
Developed in 1883-still widely used to determine N content in substances (e.g. protein, milk,
cereal, and flour) In a long-necked flask (Kjeldahl) digestion of solid (decomposition and
dissolution) inboiling H2SO4 is done.

Argentometric Titrations:
Titrimetric methods based on silver nitrate are sometimes called argentometric methods.

Back titrations:
Back titrations are like normal titrations, except that a known excess of a standard reagent is
added to the solution being titrated. The solution is then titrated back, taking into account the
addition of the excess. Back titrations are useful if the end point of the reverse titration is easier
to identify than the end point of the normal titration.
Classification by end-point techniques:
The precision and accuracy with which the end point can be detected is a vital factor in all
titrations. Because of its simplicity and versatility, chemical indication is quite common,
especially in acid-base titrimetry.
1) Potentiometric titration
If a pH meter is used, its associated electrodes are first standardized by use of a buffer solution of
known pH. By suitable choice of electrodes, potentiometric methods can also be applied to
combination titrations and to oxidation-reduction titrations. The advent of modern ion-selective
electrodes has greatly extended the scope of potentiometric titration and of other branches of
titrimetry.
2) Conductometric titration
uses the change of conductivity of the solution. Conductometric titration is sometimes successful
when chemical indication fails. The underlying principles of conductometric titration are that the
solvent and any molecular species in solution exhibit only negligible conductance; that the
conductance of a dilute solution rises as the concentration of ions is increased; and that at a given
concentration the hydrogen ion and the hydroxyl ion are much better conductors than any of the
other ions.
3) Spectrophotometric titration
We can use absorption of light to monitor the progress of the reaction.The spectrophotometer is
an optical device that responds only to radiation within a selected very narrow band of
wavelengths in the visual, ultraviolet, or infrared regions of the spectrum. The response can be
made both quantitative and linearly related to the concentration of a species that absorbs
radiation within this band.
Titrations at wavelengths within the visual region are by far the most common.
4) Amperometric titration
By use of a dropping-mercury or other suitable microelectrode, it is possible to find a region of
applied electromotive force (emf) in which the current is proportional to the concentration of one
or both of the reactants in a titration.
Biamperometric titration is a closely related technique. An emf that is usually small is applied
across two identical micro-electrodes that dip into the titrand solution. This arrangement, which
involves no liquid-liquid junctions, is valuable in nonaqueous titrations, but also finds much use
in aqueous titrimetry.
5) Thermometric or enthalpimetric titration
Many chemical reactions proceed with the evolution of heat. If one of these is used as the basis
of a titration, the temperature first rises progressively and then remains unchanged as the titration
is continued past the end point. If the reaction is endothermic, the temperature falls instead of
rising. Thermometric titration is applicable to all classes of reactions.
6) Nonaqueous titration
This technique is used to perform titrations that give poor or no end points in water. Although
applicable in principle to all classes of reactions, acid-base applications have greatly exceeded all
others. Nonaqueous titrations in which the solvent is a molten salt or salt mixture are also
possible.
7) Automatic titration
Automation is particularly valuable in routine titrations, which are usually performed repeatedly.
One approach is to record the titration curve and to interpret it later. Another method is to stop
titrant addition or generation automatically at, or very near to, the end point. Although a
constant-delivery device is desirable, an ordinary buret with an electromagnetically controlled
valve is often used. Microcomputer control permits such refinements as the continuous
adjustment of the titrant flow rate during the titration. In some cases, it is possible to automate an
entire analysis, from the measurement of the sample to the final washout of the titration vessel
and the printout of the result of the analysis.
8) Electrochemical titration
In this we Detect a voltage changes between electrodes.

Different Chromatography Techniques

Chromatography is an important biophysical technique that enables the separation, identification,

and purification of the components of a mixture for qualitative and quantitative analysis.

Chromatography is a very useful technique as it allows the separation of components of a

mixture on the basis of their nature, structure, size, and other properties.
Chromatography, in general, is based on the principle that components of a mixture are separated
when the mixture added to a mobile phase is moved through a stationary phase (which mostly is
a solid surface), resulting in some components of the mixture being attached to the stationary
phase. At the same time, the rest is passed along with the mobile phase.
Thus, there are two essential components of all chromatography techniques.

Stationary Phase: The stationary phase in chromatography is the phase that is either a solid or
liquid particle attached to a glass or a metal surface on which the components of the mixture to
be separated is absorbed selectively.
 The term stationary refers to the fact that this phase remains stationary while the other
phase moves.
  Most substances used as stationary phases are porous, thus allowing the attachment of
components during chromatography.
 The stationary phase to be selected for a chromatographic process depends on the nature
of the components to be separated and the type of chromatography.
 Depending on the type of chromatography gel beads, thin uniform paper, silica, glass,
some gases, or even liquid components are used as a stationary phase.

Figure: Stationary & Mobile Phase

Mobile Phase: The mobile phase in chromatography is the phase that is either liquid or gas that
is passed through a chromatographic system where the components of the mixture are separated
at different rates by adsorbing them to the stationary phase.
 The mobile phase is the solvent that carries the mixture as it moves down the stationary
phase.
 The term mobile indicates that the phase is moving down the chromatographic system,
whereas the other phase remains stationary.
 Substances used as mobile phases are selected for a chromatographic process depending
on the nature of the components to be separated and the type of chromatography.
 Alcohol, water, acetic acid, acetone, or some gases are the commonly used mobile phase
in different chromatographic techniques.

Basic Principle:
In all chromatography there is a mobile phase and a stationary phase. The stationary phase is the
phase that doesn't move and the mobile phase is the phase that does move. The mobile phase
moves through the stationary phase picking up the compounds to be tested. As the mobile phase
continues to travel through the stationary phase it takes the compounds with it. At different
points in the stationary phase the different components of the compound are going to be
absorbed and are going to stop moving with the mobile phase. This is how the results of any
chromatography are gotten, from the point at which the different components of the compound
stop moving and separate from the other components. In paper and thin-layer chromatography
the mobile phase is the solvent. The stationary phase in paper chromatography is the strip or
piece of paper that is placed in the solvent. In thin-layer chromatography the stationary phase is
the thin-layer cell. Both these kinds of chromatography use capillary action to move the solvent
through the stationary phase.
The Retention Factor, Rf:

The retention factor, Rf, is a quantitative indication of how far a particular compound travels in a
particular solvent. The Rf value is a good indicator of whether an unknown compound and a
known compound are similar, if not identical. If the Rf value for the unknown compound is close
or the same as the Rf value for the known compound then the two compounds are most likely
similar or identical.

The retention factor, Rf, is defined as Rf = distance the solute (D1) moves divided by the
distance traveled by the solvent front (D2)

Rf = D1 / D2

Where,

D1 = distance that color traveled, measured from center of the band of color to the point where
the food color was applied
D2 = total distance that solvent traveled.
Chromatogram
A graphical or other presentation of detector response, concentration of analyte in the effluent or
other quantity used as a measure of effluent concentration versus effluent volume or time. In
planar chromatography "chromatogram" may refer to the paper or layer with the separated zones.

Bonded Phase
A stationary phase which is covalently bonded to the support particles or to the inside wall of the
column tubing.

Immobilized Phase
A stationary phase which is immobilized on the support particles, or on the inner wall of
the column tubing, e.g., by in situ polymerization (cross-linking) after coating.

Elute (verb)
To chromatograph by elution chromatography. The process of elution may be stopped
while all the sample components are still on the chromatographic bed or continued until
the components have left the chromatographic bed.
Effluent
The mobile phase leaving the column.
Sample
The mixture consisting of a number of components the separation of which is attempted
on the chromatographic bed as they are carried or eluted by the mobile phase.
Sample Components
The chemically pure constituents of the sample. They may be unretained (i.e., not
delayed) by the stationary phase, partially retained (i.e., eluted at different times) or
retained permanently. The terms Eluite or Analyte are also acceptable for a sample
component.
Solute
A term referring to the sample components in partition chromatography.
Solvent
A term sometimes referring to the liquid stationary phase in partition chromatography.
Note: In liquid chromatography the term "solvent" has been often used for the mobile
phase. This usage is not recommended.
Zone
A region in the chromatographic bed where one or more components of the sample are
located. The term Band may also be used for it.

DIFFERENT TYPES OF CHROMATOGRAPHY TECHNIQUES

Some commonly used chromatographic techniques are:

1. Column chromatography
2. Gas chromatography
3. Liquid chromatography
4. Paper chromatography
5. Thin-layer chromatography (TLC)
6. High-performance liquid chromatography (HPLC)
7. Ion exchange chromatography
8. Affinity chromatography
 9. Reverse-phase chromatography
10. Gel filtration chromatography

COLUMN CHROMATOGRAPHY

Column chromatography is the separation technique where the components in a mixture are
separated on the basis of their differential adsorption with the stationary phase, resulting in them
moving at different speeds when passed through a column.

It is a solid-liquid chromatography technique in which the stationary phase is a solid & mobile
phase is a liquid or gas.

Principle of Column chromatography

 This technique is based on the principle of differential adsorption where different
molecules in a mixture have different affinities with the absorbent present on the stationary
phase.
 The molecules having higher affinity remain adsorbed for a longer time decreasing their
speed of movement through the column.
 However, the molecules with lower affinity move with a faster movement, thus allowing
the molecules to be separated in different fractions.
 Here, the stationary phase in the column chromatography also termed the absorbent, is a
solid (mostly silica) and the mobile phase is a liquid that allows the molecules to move
through the column smoothly.

Figure: Column Chromatography

Steps of Column chromatography
 The column is prepared by taking a glass tube that is dried and coated with a thin,
uniform layer of stationary phase (cellulose, silica).
 Then the sample is prepared by adding the mixture to the mobile phase. The sample is
introduced into the column from the top and is allowed to pass the sample under the
influence of gravity.
 The molecules bound to the column are separated by elution technique where either
solution of the same polarity is used (isocratic technique), or different samples with
different polarities are used (gradient technique).
 The separated molecules can further be analyzed for various purposes.
Uses of Column chromatography
 Column chromatography is routinely used for the separation of impurities and
purification of various biological mixtures.
 This technique can also be used for the isolation of active molecules and metabolites
from various samples.
 Column chromatography is increasingly used for the detection of drugs in crude extracts.
Examples of Column chromatography
 Extraction of pesticides from solid food samples of animal origin containing lipids,
waxes, and pigments.
 Synthesis of Pramlintide which is an analog of Amylin, a peptide hormone, for
treating type 1 and type 2 Diabetics.
 Purification of bioactive glycolipids, showing antiviral activity towards HSV-1 (Herpes
Virus).

GAS CHROMATOGRAPHY

Gas chromatography is a separation technique in which the molecules are separated on the basis
of their retention time depending on the affinity of the molecules to the stationary phase.

The sample is either liquid or gas that is vaporized in the injection point.
Principle of Gas chromatography
 Gas chromatography is based on the principle that components having a higher affinity to
the stationary phase have a higher retention time as they take a longer time to come out of
the column.
 However, the components having a higher affinity to the stationary phase have less
retention time as they move along with the mobile phase.
 The mobile phase is a gas, mostly helium, that carries the sample through the column.
 The sample once injected in converted into the vapor stage is then passed through a
detector to determine the retention time.
 The components are collected separately as they come out of the stationary phase at
different times.

Figure: Gas chromatography.

Steps of Gas chromatography
 The sample is injected into the column where it is vaporized into a gaseous state. The
vapourised component than mixes with the mobile phase to be carried through the rest of
the column.
 The column is set with the stationary phase where the molecules are separated on the
basis of their affinity to the stationary phase.
 The components of the mixture reach the detector at different times due to differences in
the time they are retained in the column.
Uses of Gas chromatography
 This technique is used to calculate the concentration of different chemicals in various
samples.
  This is used in the analysis of air pollutants, oil spills, and other samples.
 Gas chromatography can also be used in forensic science to identify and quantify various
biological samples found in the crime scene.
Examples of Gas chromatography
 The identification of performance-inducing drug in the athlete’s urine.
 The separation and quantification of a solid drug in soil and water samples.

LIQUID CHROMATOGRAPHY

Liquid chromatography is a separation technique where the mobile phase used is liquid, and the
separation can take place either in a column or a plain surface.

Principle of Liquid chromatography

 The process of liquid chromatography is based on the principle for the affinity of the
molecules to the mobile phase.
 If the components to be separated have a higher affinity to the mobile phase, the
molecules move along with the mobile phase and come out of the column faster.
 However, if the components have a lower degree of interaction with the mobile phase, the
molecules move slowly and thus come out of the column later.
 Thus, if two molecules in a mixture have different polarities and the mobile phase is of a
distinct polarity, the two molecules will move at different speeds through the stationary
phase.

Figure: Liquid chromatography.

Steps of Liquid chromatography
  The column or paper is prepared where the stationary phase (cellulose or silica) is applied
on the solid support.
 The sample is added to the liquid mobile phase, which is then injected into the
chromatographic system.
 The mobile phase moves through the stationary phase before coming out of the column or
the edge of the paper.
 An elution solution is applied to the system to separate the molecules from the stationary
phase.
Uses of Liquid chromatography
 Liquid chromatography is an effective method for the separation of a colored solution as
they form two separate bands after separation.
 This method can also be used over other techniques as it is quite simple and less
expensive.
 It can be used for the separation of solid molecules that are insoluble in water.
Examples of Liquid chromatography
 High-performance liquid chromatography is a modified form of liquid chromatography
that is used in the research regarding biological molecules.

PAPER CHROMATOGRAPHY

Paper chromatography is a separation technique where the separation is performed on a

specialized paper.

Principle of Paper chromatography

 Paper chromatography is of two types based on two different principles.
 The first is the paper adsorption chromatography that is based on the varying degree of
interaction between the molecules and the stationary phase.
 The molecules having higher affinity remain adsorbed for a longer time decreasing their
speed of movement through the column.
  However, the molecules with lower affinity move with a faster movement, thus allowing
the molecules to be separated in different fractions.
 The second type of paper chromatography is the paper partition chromatography. It is
based on the principle that the moisture on the cellulose paper acts as a stationary phase for
the molecules moving with the mobile phase.
 The separation of the molecules is thus based on how strongly they adsorb onto the
stationary phase.
 An additional concept of ‘retention factor’ is applied during the separation of molecules
in the paper chromatography.
 The retention value for a molecule is determined as a ratio of distance traveled by the
molecule to the distance traveled by the mobile phase.
 The retention value of different molecules can be used to differentiate those molecules.

Figure: Paper chromatography.

Steps of Paper chromatography
 The stationary phase is selected as a fine quality cellulosic paper.
 Different combinations of organic and inorganic solvents are taken as the mobile phase.
 About 2-200 µl of the sample solution is injected at the baseline of the paper, and it is
allowed to air dry.
 The sample loaded paper is then carefully dipped into the mobile phase not more than the
height of 1 cm.
 After the mobile phase reaches near the edge of the paper, the paper is taken out.
 The retention factor is calculated, and the separated components are detected by different
techniques.
Uses of Paper chromatography
 Paper chromatography is performed to detect the purity of various pharmaceutical
products.
 It can also be employed to detect contamination in various samples, like food and
beverages.
 This method can also be used for the separation of impurities from various industrial
products.
 The analysis of the reaction mixtures in chemical labs is also conducted via paper
chromatography.
Examples of Paper chromatography
 Paper chromatography is used in the separation of mixtures of inks or other colored
drinks.

THIN-LAYER CHROMATOGRAPHY

Thin-layer chromatography is a separation technique where the stationary phase is applied as a

thin layer on a solid support plate with a liquid mobile phase.

Principle of Thin-layer chromatography (TLC)

 This chromatography technique is based on the principle that components of a mixture
are separated when the component having an affinity towards the stationary phase binds to
the stationary phase. In contrast, other components are eluted with the mobile phase.
 The substrate/ ligand is bound to the stationary phase so that the reactive sites for the
binding of components are exposed.
 Now, the mixture is passed through the mobile phase where the components with binding
sites for the substrate bind to the substrate on the stationary phase while the rest of the
components are eluted out with the mobile phase.
 After separation, the molecules are seen as spots at a different location throughout the
stationary phase.
 The detection of molecules is performed by various techniques.
 Figure: Thin-layer chromatography (TLC).
Steps of Thin-layer chromatography (TLC)
 The stationary phase is uniformly applied on the solid support (glass, thin plate or
aluminum foil) and dried.
 The sample is injected as spots on the stationary phase about 1 cm above the edge of the
plate.
 The sample loaded plate is then carefully dipped into the mobile phase not more than the
height of 1 cm.
 After the mobile phase reaches near the edge of the plate, the plate is taken out.
 The retention factor is calculated as in paper chromatography, and the separated
components are detected by different techniques.
Uses of Thin-layer chromatography (TLC)
 Thin-layer chromatography is routinely performed in laboratories to identify different
substances present in a mixture.
 This technique helps in the analysis of fibers in forensics.
 TLC also allows the assay of various pharmaceutical products.
 It aids in the identification of medicinal plants and their composition.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

High-performance liquid chromatography is a modified form of column chromatography where

the components of a mixture are separated on the basis of their affinity with the stationary phase.
Principle of HPLC
 This technique is based on the principle of differential adsorption where different
molecules in a mixture have a varying degree of interactions with the absorbent present on
the stationary phase.
 The molecules having higher affinity remain adsorbed for a longer time decreasing their
speed of movement through the column.
 However, the molecules with lower affinity move with a faster movement, thus allowing
the molecules to be separated in different fractions.
 This process is slightly different from the column chromatography as in this case; the
solvent is forced under high pressures of up to 400 atmospheres instead of allowing it to
drip down under gravity.

Figure: High-performance liquid chromatography (HPLC).

Steps of HPLC
 The column is prepared by taking a glass tube that is dried and coated with a thin,
uniform layer of stationary phase (cellulose, silica).
 Then the sample is prepared by adding the mixture to the mobile phase. The sample is
introduced into the column from the top, and a high-pressure pump is used to pass the
sample at a constant rate.
 The mobile phase then moves down to a detector that detects molecules at a certain
absorbance wavelength.
 The separated molecules can further be analyzed for various purposes.
Uses of HPLC
 High-performance liquid chromatography is used in the analysis of pollutants present in
environmental samples.
 It is performed to maintain product purity and quality control of various industrial
productions.
 This technique can also be used to separate different biological molecules like proteins
and nucleic acids.
 The increased speed of this technique makes the process faster and more effective.
Example of HPLC
 High-performance liquid chromatography has been performed to test the efficiency of
different antibodies against diseases like Ebola.

ION EXCHANGE CHROMATOGRAPHY

Ion exchange chromatography is the separation technique for charged molecules by their
interaction with the oppositely charged stationary phase in the form of ion-exchange resin.

Principle of Ion exchange chromatography

 This technique is based on the principle of attraction of charged resin and the oppositely
charged analyte. Here the exchange of negatively/ positively charged ions takes place to
remove the charged molecules.
 The stationary phase is first coated with particular charges where the components of the
mixture with opposite charges will bind.
 A cation or anion exchange resin with a higher affinity to the charged components then
binds the components, displacing the oppositely charged resin.
 The cation or anion exchange resin-component complex then is removed by using
different buffers.
 Figure: Ion exchange chromatography.

Steps of Ion exchange chromatography

 A column packed with charged resin that can either be positively charged or negatively
charged is taken as the stationary phase.
 The mixture with the charged particles is then passed down the column where the
charged molecules bind to the oppositely charged resins.
 If a cation exchange resin is used, the positively charged molecules now bind to the
cation exchange resin displacing the negatively charged resin.
 Similarly, if an anion exchange resin is used, the negatively charged molecules bind to
the anion exchange resin displacing the positively charged resin.
 Now an appropriate buffer is applied to the column to separate the complex of charged
exchange resins and the charged molecules.
Uses of Ion exchange chromatography
 Ion exchange chromatography is used in the purification of water where the positively
charged ions are replaced by hydrogen ions, and the negatively charged ions are replaced by
hydroxyl ions.
 This method also works as an effective method for the analysis of the products formed
after hydrolysis of nucleic acids.
 The separation of metals and other inorganic compounds is also facilitated by the ion-
exchange chromatography.
Examples of Ion exchange chromatography
 The separation of positively charged lanthanoid ions obtained from the earth’s crust.
 The separation of proteins from the crude mixture obtained from the blood serum.

AFFINITY CHROMATOGRAPHY

Affinity chromatography is a separation technique where the components of a mixture are

separated based on their affinity towards the stationary phase of the system.

Principle of Affinity chromatography

 This chromatography technique is based on the principle that components of a mixture
are separated when the element having an affinity towards the stationary phase binds to the
stationary phase. In contrast, other components are eluted with the mobile phase.
 The substrate/ ligand is bound to the stationary phase so that the reactive sites for the
binding of components are exposed.
 Now, the mixture is passed through the mobile phase where the components with binding
sites for the substrate bind to the substrate on the stationary phase while the rest of the
components are eluted out with the mobile phase.
 The components attached to the stationary phase are then eluted by changing the pH,
ionic strength, or other conditions.

Figure: Affinity chromatography.

Steps of Affinity chromatography
 The column is prepared by loading it with solid support like agarose or cellulose, onto
which the substrate/ ligand with the spacer arm, is attached.
 The mobile phase containing the mixture is poured into the column at a constant rate.
 Once the process is complete, the ligand-molecule complex is eluted from the stationary
phase by changing the conditions that favor the separation of ligand and components of the
mixture.
Uses of Affinity chromatography
 Affinity chromatography is used as a staple separation technique from enzymes and other
proteins.
 This principle is also applied in the in vitro antigen-antibody reactions.
 This technique is used for the separation of components as well as the removal of
impurities from a mixture.
 Affinity chromatography can be used in the detection of mutation and nucleotide
polymorphisms in nucleic acids.
Examples of Affinity chromatography
 The purification of coli β-galactosidase from a mixture of proteins using the p-
aminophenyl-1-thio-β-D-galactopyranosyl agarose as the affinity matrix.
 The removal of excess albumin and α2-macroglobulin from the serum albumin.

REVERSE-PHASE CHROMATOGRAPHY

Reverse-phase chromatography is a liquid chromatography technique where the separation of

molecules is achieved through hydrophobic interaction between the liquid mobile phase and the
stationary phase.

Principle of Reverse-phase chromatography

 The principle of reverse phase chromatography is based on the interaction between two
molecules with hydrophobic groups.
 Here, the stationary phase is solid support applied with both hydrophobic and hydrophilic
groups.
  The solvent molecules containing hydrophobic regions interact with the hydrophobic
groups, thus separating them from the molecules with hydrophilic groups.
 The interaction is then reversed by applying an elution solution with decreasing salt
gradient, which causes the molecules with hydrophobic groups to be separated from the
stationary phase.

Figure: Steps of a reversed-phase chromatography separation.

Steps of Reverse-phase chromatography
 The column is prepared with a glass tube applied with solid support like silica gel, upon
which hydrophobic groups like phenyl, octyl butyl, are attached.
 The sample is prepared by adding the mixture to the mobile phase of organic and
inorganic solvents.
 The sample is then injected into the column from the top of the column.
 The molecules with hydrophobic groups form an interaction with the hydrophobic groups
of the stationary phase. In contrast, the molecules without such groups move out of the
column with the mobile phase.
 Then a particular elution solution with decreasing salt gradient is then passed into the
column that removes the bound molecules from the stationary phase.
Uses of Reverse-phase chromatography
 Reverse chromatography, in combination with high-performance liquid chromatography,
is increasingly used for the separation of biomolecules.
 This is also used in the study of the analysis of drugs, metabolites, and active molecules.
 It can also be used to remove impurities from various environmental samples.
Examples of Reverse-phase chromatography
 Hydrophobic interaction chromatography is an example of reverse phase chromatography
where this technique is used to separate proteins from their mixtures.

GEL-FILTRATION CHROMATOGRAPHY

Gel-filtration chromatography is a form of partition chromatography used to separate molecules

of different molecular sizes.

This technique has also frequently been referred to by various other names, including gel-
permeation, gel-exclusion, size- exclusion, and molecular- sieve chromatography.

Principle
 Molecules are partitioned between a mobile phase and a stationary phase as a function of
their relative sizes.
 The stationary phase is a matrix of porous polymer which have pores of specific sizes.
 When the sample is injected with the mobile phase, the mobile phase occupies the pores
of the stationary phase.
 If the size of the molecules is appropriate enough to enter the pores, they remain in the
pores partly or wholly.
 However, molecules with a larger size are retained from entering the pores, causing them
to be moved with the mobile phase, out of the column.
 If the mobile phase used in an aqueous solution, the process is termed gel filtration
chromatography.
 If the mobile phase used is an organic solvent, it is termed as gel permeation
chromatography.
 Figure: Gel-filtration chromatography.
Steps
 The column is filled with semi-permeable, porous polymer gel beads with a well-defined
range of pore sizes.
 The sample, mixed with the mobile phase, is then injected into the column from the top
of the column.
 The molecules bound to the column are separated by elution solution where either
solution of the same polarity is used (isocratic technique), or different samples with
different polarities are used (gradient technique).
 Elution conditions (pH, essential ions, cofactors, protease inhibitors, etc.) can be selected,
which will complement the requirements of the molecule of interest.
Uses
 One of the principal advantages of gel-filtration chromatography is that separation can be
performed under conditions specifically designed to maintain the stability and activity of
the molecule of interest without compromising resolution.
 The absence of a molecule-matrix binding step also prevents unnecessary damage to
fragile molecules, ensuring that gel-filtration separations generally give high recoveries of
activity.
 Because of its unique mode of separation, gel-filtration chromatography has been used
successfully in the purification of proteins and peptides from various sources.
 Gel-filtration chromatography has been used to separate various nucleic acid species such
as DNA, RNA, and tRNA as well as their constituent bases, adenine, guanine, thymine,
cytosine, and uracil.
Examples
 The separation of recombinant human granulocyte colony-stimulating factor (rhG-CSF)
from inclusion bodies in high yield by urea-gradient size-exclusion chromatography.
 The separation of hen egg lysozyme using both acrylamide- and dextran-based gel
columns.
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