ERROR, PRECISION AND ACCURACY PHARMACEUTICAL ANALYSIS

1 ERROR,
PRECISION
AND ACCURACY
ERROR

 Error is the difference between the true result (accepted true result) and the measured result.
 If the error in an analysis is large, serious consequences may result.

TYPE OF ERROR

A. DETERMINATE ERROR
 Determinate errors are caused by faults in the analytical procedure or the instruments used
in the analysis.
 Determinate errors are systematic errors.
 Determinate error can arise from uncalibrated balances, improperly calibrated volumetric
flasks or pipettes, malfunctioning instruments, impure chemicals, incorrect analytical procedures
or techniques and analyst error.
 Systemic error is under the control of the analyst
TYPE OF DETERMINATE ERRORS
1. Personal errors - They are exclusively caused due to ‘personal equation’ of an analyst
and do not due to either on the prescribed procedure or methodology involved.
2. Instrumental errors - These are invariably caused due to faulty and uncalibrated
instruments, such as: pH meters, uv-spectrophotometers, potentiometers etc.
3. Reagent errors - The errors that are solely introduced by virtue of the individual
reagents, for instance impurities inherently present in reagents ; high temperature
volatalization of platinum (Pt) ; unwanted introduction of ‘foreign substances’ caused by
the action of reagents on either porcelain or glass apparatus.
4. Constant Errors - They are observed to be rather independent of the magnitude of the
measured amount ; and turn out to be relatively less significant as the magnitude enhance
5. Proportional Errors - The absolute value of this kind of error changes with the size of
the sample in such a fashion that the relative error remains constant. It is usually incorporated
by a material that directly interferes in an analytical procedure
6. Errors due to Methodology - Both improper (incorrect) sampling and incompleteness of
a reaction often lead to serious errors. A few typical examples invariably encountered in
titrimetric and gravimetric analysis
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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

B. INDETERMINATE ERROR
 Indeterminate errors are not constant or biased.
 They are random in nature and are the cause of slight variations in results of replicate
samples made by the same analyst under the same conditions
 Sources of random error include the limitations of reading balances, electrical noise in
instruments and vibrations caused to the building by heavy vehicular trafficking , which are
beyond anyone’s control.

GROSS ERROR

 Gross errors differ from indeterminate and determinate errors.

 They usually occur only occasionally, are often large, and may cause a result to be either
high or low. They are often the product of human errors.
ABSOLUTE ERROR

 Observed value – True or most probable value

 Absolute error may be negative or positive.
RELATIVE ERROR

 Absolute error /True value1000 per thousand

ACCURACY AND PRECISION

 Accuracy - is the closeness of experimental value to the true value.

 Accuracy is inversely proportional to the error i.e. the greater the accuracy, smaller is the
error
 Precision - Precision is the closeness of measurement from one another.
 Reproducibility - Reproducibility expresses the precision between laboratories
 Repeatability - Repeatability expresses the precision under the same operating conditions
over a short interval of time.

High Accuracy Low Accuracy High Accuracy Low Accuracy

High Precision High Precision Low Precision Low Precision

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 ERROR, PRECISION AND ACCURACY PHARMACEUTICAL ANALYSIS

PRECISION MEASURE

 Mean or average or relative mean deviation - Mean is obtained by dividing the sum of a
set of measurements by the number of individual results in the set.

mean, m 
 Mn
Where,
n
M is individual measurement
n is the total number of measure
 Median - Median is a about which all the other value are equally distributed.
 Mean deviation - Mean deviation of a single measurement is the mean of the derivations
of all the individual measurements.

d 
  Mn  m
N
d  = mean deviation

 Mn  m = Absolute value of the deviation of the Mnth number

 Relative mean deviation - Relative mean deviation is the mean deviation divided by the
mean.
mean deviation
Relative mean deviation =  100
mean
 Average deviationAverage deviation
d
D
n
d  = Average deviation
n = Total number of measurements

  Mn  m 
2

 Standard deviation - s 
N

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

2 INTRODUCTION
OF TITRATION

INTRODUCTION

Titration: A titration is a technique where a solution of known concentration is used to determine

the concentration of an unknown solution.
KEY TERMS

Qualitative Determine the chemical composition in a sample using chemical

chemical analysis reactions and interactions.
Quantitative Determine the content in a sample of one or more chemical
chemical analysis substances using chemical reactions and interactions.
In this type of titration, the titrate (unknown concentration)
Double Titration
solution contains more than one component.
An assay is a test of a substance to find out what chemicals it
Assay
contains.
Analyte The solution of unknown concentration but known volume.
Titrant The solution of known concentration.
Standard solution A solution of known concentration is called the standard solution.
Euivalence Point Point where the amount of two reactants are just equivalent.
The endpoint is the point where the system changes when the
Endpoint moles of the reacting titrant exceed the moles of the substance
being titrated.
An auxiliary substance which helps in the usual detection of the
Indicator completion of the titration process at the end point. For examples:
- Methyl orange, Phenolphthalein, Cresol red, Thymol blue.
A titration curve is the plot of the pH of the analyte solution versus
Titration curve
the volume of the titrant added as the titration progresses

STANDARDS

 Primary standard
1. Extremely pure
2. Highly stable
3. Can be weighed easily
4. Stable in air

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 INTRODUCTION OF TITRATION PHARMACEUTICAL ANALYSIS

5. High molecular weight

6. Readily soluble
7. Undergoes stoichiometric and rapid reactions
Example: Na2CO3, Potassium hydrogen pthalate
TITRATION METHOD PRIMARY STANDARD
Acid-base reactions Na2CO3, Na2B4O7, KH (C8H4O4), HCl
Complex formation reaction AgNO3, NaCl
Precipitation reactions AgNO3, KCl
Redox reactions K2Cr2O7, Na2C2O4, I2
 Secondary standard: - A substance that can be used for standardisation, and whose
concentration of active substance has been determined by comparison to a primary standard.

METHOD OF EXPRESSING CONCENTRATION

Number of gram equivqents of Weight 1000

Eq.weight Volume  ml 
Normality solute present per liter of N N= ×
solution.
Number of moles of solute
Moles of solute
Molarity dissolved one litre (or one M M=
Volume of solution inL/dm 3
dm3) of solution.
Number of moles of solute per Moles of solute
Molality m m=
1 kg of the solvent. Mass of solvent in kg
Formality Number of formula mass In Weight 1000
GFM Volume  ml 
grams present per liter of F= ×
solution. F where,
GFM = Gram formula mass of
solute
Ratio of number of moles of
Mole one component to the total nA
X xA =
fraction number of moles of all the n A + nB
components.

TYPES OF TITRATIONS

1. Acid base titration

2. Redox titration
3. Precipitation titration
4. Non aqueous titration
5. Complexometric titration

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

3 ACID BASE
TITRATION
INTRODUCTION

An Acid-Base titration involves strong or weak acids or bases. Specifically, an acid-base titration
can be used to figure out the following:
 The concentration of an acid or base
 Whether an unknown acid or base is strong or weak.
 PKa of an unknown acid or pKb of the unknown base.

ACI D
 An Acid is a substance that can release ion (H+) when dissolved in water.
 Acid converts blue litmus paper to red having the pH <7.
Example: HCl  H+ + Cl-

BASE
 A Base is a substance that can release a hydroxyl ion (OH-) when dissolve in water.
 Base converts red litmus paper to blue having the pH >7.
Example: NaOH  Na+ +OH-

ARRHENIUS CONCEPT
 An Acid is a substance that dissociated completely to give hydrogen ions when dissolved in
water.
Example: HCl+ Water  H+-(aq) + Cl- (aq)
 A Base is a substance that dissociates to give hydroxyl ions when dissolved in water.
Example: NaOH + Water    Na+(aq)+ OH- (aq)

H+ H+H+ OH+ OH+

acid Base
Water Water Water Water
H+ OH+
H+ OH+

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 ACID BASE TITRATION PHARMACEUTICAL ANALYSIS

BRONSTED LOWRY CONCEPT

 An Acid is proton donor.

Example: HCl H+ + Cl-
 A Base is proton acceptor.
Example: NH3+ H+  NH4

CONJUGATE ACID AND BASE

 Conjugated Acid: Conjugated acid is the substance which can be formed by the loss of a
proton.
 Conjugated Base: Conjugated base is the substance which can be formed by the gain of a
proton.
Adds proton

NH3(aq) + H 2O (l) NH4 (aq) + OH (aq)

-

Conjugate Conjugate
Base Acid Acid Base

Losses proton
 Example of Conjugate Acid and Base
ACID CONJUGATE BASE BASE CONJUGATE ACID
H2F+ HF NH3 NH4+
H2SO4 HSO4- OH− H2O
HNO3 NO3− CH3NH2 CH3NH3+
H3O+ H2O C6H5NH2 C6H5NH3+

LEWIS CONCEPT

 An Acid is any species that can accept an electron-pair to form a coordinate bond.
Example : Ag+, Fe+, Zn2+, Na+, Al3+
 Base is any species that can donate an electron-pair to form a coordinate bond.
Example : NH3, H2O, OH- Cl-

LAW OF MASS ACTION

The law of mass action states that the rate of a reaction is proportional to the product of the
concentrations of each reactant.
 The Equilibrium Constant (Kc)
The concentration of reactants and products, at equilibrium, are constant at a given temperature.
Consider the following simple reversible reaction where A & B are the reactants whereas C &
D are the products. A + B C + D
 A mixture of products and reactants in a state of chemical equilibrium is known as an
equilibrium mixture. There exists a relation between the concentration of products and the
concentration of reactants for an equilibrium mixture. This relation can be equated as follows

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

C D
 A B
Kc =

Here, Kc is called the equilibrium constant.

BUFFER SOLUTION

Buffer Solution is a water solvent based solution which consists of a mixture containing a weak
acid and the conjugate base of the weak acid, or a weak base and the conjugate acid of the weak
base. They resist a change in pH upon dilution or upon the addition of small amounts of acid/alkali
to them.
 Acidic Buffer: Combination of weak acids and its salt with a strong Base or Weak acid and salt
with strong base (Conjugate base).
Example: Mixture of Acetic acetic and Sodium acetate
 Basic Buffer: Combination of weak base and salt with a strong acid Or Weak base and salt
with strong acid (Conjugate acid).
Example: Mixture of ammonium hydroxide and ammoium chloride
 Neutral Buffer: The solution having a mixture of weak base and its salt.
Example: Mixture of acidic acid and ammonium hydroxide.
Role of solvent
 Water is amphiprotic in nature i.e. it can act either as an acid or as a base depending upon
experimental conditions. So aqueous titration methods are used.
 The solvent selected for acidimetry should be either neutral or acidic in nature
 Neutral solvent – Acetonitrile, Alcohols, Chloroform, Benzene, dioxane ethyl acetate.
 Neutral solvent do not enhance dissociation to a great degree.

COMMON ION EFFECT

 A weak electrolyte AB, when dissolved in water will dissociate to a small extent into A+ & B-
ions
 If, in this solution, we add a strong electrolyte CB, it will dissociate into C+ & B- ions.
 So both the electrolytes will give B- which is called the common ion effect.
AB A+ + B- (POORLY DISSOCIATED)
CB  C+ + B- (STRONGLY DISSOCIATED)
 The effect of addition of CB to solution of AB will thus increase the conc. of B- ions.
 This will push equilibrium to backward direction as per the law of mass action
AB  A+ + B-
 So, that the poor dissociation of AB will further decrease.
 Thus, when to a solution of a weak electrolyte a strong electrolyte with common ion is added,
the dissociation of the weak electrolyte is suppressed, this is known as the common ion effect.

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 ACID BASE TITRATION PHARMACEUTICAL ANALYSIS

PROCEDURE OF ACID-BASE TITRATION

 The required volume of the base is taken whose concentration is known in a pipette ad is
poured into the titrating flask.
 The acid whose concentration is unknown is taken in the Burette and is allowed to react
with the base drop by drop.
 An indicator which is used for detecting the endpoint is also added in the titration flask.
 When the reaction reaches completion the colour of the solution in the titration flask changes
due to the presence of the indicator.
 The indicator used for this purpose can be phenolphthalein which forms pink colour in
basic solution and colourless in acid and neutral solution.
 Therefore the endpoint is detected when the pink coloured solution turns colourless.

ACID BASE INDICATORS

TITRATIONS INDICATORS
Strong acid strong base Methyl orange, Methyl red, Phenolphthalein Bromothymol
Blue, Phenol Red
Weak acid strong base Phenolphthalein, Thymolphthalin, and Thymol Blue
Strong acid weak base Methyl orange, Methyl red, bromophenol, Bromocresol green
Weak acid weak base Mixed Indicators

INDICATOR pH RANGE COLOUR ON ACIDIC SIDE COLOUR ON BASIC SIDE

Methyl Violet 0.0 to 1.6 Yellow Violet
Bromophenol Blue 3.0 to 4.6 Yellow Blue
Methyl orange 3.1 to 4.4 Red Yellow
Methyl red 4.4 to 6.2 Red Yellow
Phenol red 6.8 t0 8.4 Yellow Red
Cresol red 7.2 to 8.8 Yellow Red
Naphtholphthalein 7.3 to 8.7 Yellow Blue
Phenolphthalein 8.3 to 10.0 Colourless Pink
Alizarin yellow 10.1 to 12.0 Yellow Red

TYPES OF ACID-BASE TITRATION

S. NO TYPES EXAMPLES
1. Strong acid-strong base Hydrochloric acid and sodium hydroxide
2. Weak acid-strong base Ethanoic acid and sodium hydroxide
3. Strong acid-weak base Hydrochloric acid and ammonia
4. Weak acid-weak base Ethanoic and ammonia

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

4 PRECIPITATION
TITRATION
INTRODUCTION

 Precipitation is the formation of a solid in a solution and solid formed is called the precipitate.
 A precipitation reaction occur when water solutions of two different ionic compounds are
mixed and an insoluble solid separates out of solution.
KCl + AgNO3  AgCl + KNO3
(Cl- solution) (Precipitating agent) (Precipitate)
 The precipitation is itself ionic; the cation comes from one solution and anion from another.
 The requirement for a reaction to be useful in titrimetric analysis are:
1. The precipitate must be practically insoluble
2. The precipitation reaction should be rapid and quantitative
3. The titration result should not be hampered by adsorption (co-precipitation) effects
4. It must be possible to detect the equivalence point during titration

ARGENTOMETRIC TITRATIONS

 Titration methods based upon utilizing AgNO3 (silver nitrate) as a precipitating agent.
 Silver ions is extremely useful in precipitation reactions including
 Halides (Cl-, Br- , I-)
 Pseudohalides (S2-, HS-, CN-, SCN-)
 Requirements:-
1. The precipitate formation is stoichiometric.
2. Equilibrium should be attained rapidly.
3. The precipitate must be of low solubility in the solution.
4. Method to detect the stoichiometric point of the titration must be available.

MOHR’S METHOD

 The falls under the category of determining the end point by formation of colored
precipitate and is used, for the determination of chlorides and bromides is a neutral solution.
 Titrant: AgNO 3 (Secondary standard solution) so AgNO 3 is standardized by primary
standard solution of NaCl (measured volume)

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 PRECIPITATION TITRATION PHARMACEUTICAL ANALYSIS

AgNO3 + NaCl  AgCl + NaNO3

 Indicator: 5% Potassium chromate (K2CrO4) which forms reddish brown precipitate.
2AgNO3 + K2CrO4  Ag2CrO4 + 2KNO3
 Determination substance: Chloride, Bromide.
 Medium: pH range 6.5 - 10.3
 Usage: Quantitative definition of NaCl, KCl, NaBr, KBr etc.

VOLHARD METHOD

 In this method formation of a solute color compound at the end point.

 Silver ion is titrated with thiocynate in as acid solution using ferric ion as an indicator.
 Titrant
 AgNO3 (Silver Nitrate),
 NH4SCN (Ammonium thiocyanide),
 KSCN (Potassium thiocyanide)
 AgNO3 is standardized by NaCl (Primary standard solution).
 NH4SCN and KSCN is standardized on standardize solution of AgNO3
AgNO3 + NH4SCN AgSCN + NH4NO3
AgNO3 + KSCN  AgSCN + KNO3
 Medium: In presence of nitric acid.
 Determinate Substance
 Halogenides, Thiocyanide, Cyanides, Sulphides, Carbonates, Chromate, Oxalates, Arsenates
etc.
 The titration is done in acidic pH medium to present precipitation of iron hydroxides, Fe(OH)3

FAJAN’S METHOD

 Indicators are adsorbed on the surface of the precipitation at the equivalence point and
this absorption is accompanied by a color change.
 These indicator are either
 Acid dyes - Fluorescein, Eosin etc. or
 Basic dyes - Rhodamine series.
 Titrant: AgNO3 (Secondary standard solution) so standardized on primary standard solution
of NaCl (by a measured volume).
 Medium: pH :- 6.5-10.3 (for chloride); pH :- 2.0-10.3 (for Bromides & Iodides)
 Indicators: Dichlorofluoroscein (for Chlorides); Eosine (for Bromides & Iodides)
 Mechanism of Indicator action
 The property of a colloidal precipitate to adsorb its own ions which are in excess, is made
use of in the case.
 When a NaCl solution is titrated with AgNO3 the AgCl precipitate will adsorb chloride ions
which are initially in excess.

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

 This the primary adsorbed layer will be formed by chloride ions, which in taken will hold
the secondary adsorbed layer of oppositely charged Na+
 At the equivalence point, Ag+ ions in excess and hence AgCl ions adsorb Ag+ ions as primary
adsorb later and NO3- as secondary adsorb layer.
 Now, if the Na + salt of Fluorescein is also present in the solution then the negatively
charged fluorescein ions would be adsorbed instead of NO3 as secondary adsorbed layers and
this adsorption occur along with a change to pink colored complex of Ag+ and fluorescein
ions.

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 NON- AQUEOUS TITRATIONS PHARMACEUTICAL ANALYSIS

5 NON- AQUEOUS
TITRATIONS
INTRODUCTION

 Non aqueous titrations are the titrations in which weakly acidic or basic substances are carried
out using non – aqueous solvents to get sharp end point.
 The moisture content in non aqueous titrations should not be more than 0.05%.
 Moisture and carbon dioxide are to be avoided in non aqueous methods.
 Reasons for Non aqueous titrations
 The reactant is insoluble in water
 The reactant is reactive with water
 The sample is too weak acid or too weak base.

TYPES OF SOLVENTS IN NON-AQUEOUS TITRATIONS

1. Protogenic solvents
 These are acidic solvents and used to enhance the basicity of weak bases.
Examples: Glacial acetic acid, HCl, H2SO4
2. Protophilic solvents
 These are basic solvent and used to enhance the acidity of weak acids.
Examples: Pyridine, Ethylenediamine and Dimethylformamide (DMF).
3. Amphoteric solvents
 These solvents behave as acid as well as base depending on the substance dissolved in it.
They can accept or donate protons.
Examples: Alcohol, Water, Weak organic acids
4. Aprotic solvents
 These solvents neither accept proton nor donate proton. They are used in dissolving the
drugs especially those are insoluble in water.
Examples: Benzene, Carbon tetrachloride, Chloroform

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

COLOUR CHANE OBSERVED

NAME OF INDICATOR
ACIDIC NEUTRAL BASIC
Crystal violet (0.5% w/v in glacial acetic Yellowish green Bluish Violet
acid) green
Oracet Blue B(0.5% in glacial acetic acid) Pink Purple Blue
α-Naphtholbenzein (0.2% in glacial acetic Dark green Orange Blue
acid)
Quinalidine Red (0.1% in methanol) Colourless Magenta

TYPES OF NON-AQUEOUS TITRATIONS

1. Acidimetry: It involves the quantitative determination of weak bases by non-aqueous titration.
2. Alkalimetry: It involves the quantitative determination of weak acids by non-aqueous titration.
DETAILS ACIDIMETRY ALKALIMETRY
Samples Basic drugs such as: Ephedrine, Acidic drugs such as:
Adrenaline, Caffeine, Acyclovir. Nalidixic acid, Flurouracil.
Solvent Protogenic solvents such as: glacial Protophilic solvents such as:
acetic acid DMF
Titrant Perchloric acid HClO4 Sodium methoxide
Crystal violet (0.5% in glacial Thymol blue (0.5 % in
Indicator
acetic acid) Color changes from methanol) Color change from
violet to yellowish green. pink to blue
NON-AQUEOUS TITRATIONS AND DIFFERENT INDICATORS
NAME OF SUBSTANCES INDICATOR USED
Acidimetric Assays
Amantadine hydrochloride Crystal violet
Chlorpromazine hydrochloride Methyl orange
Clonidine hydrochloride α -Naphthol benzein
Cyproheptadiene.HCl Crystal violet
Dehydroemetine.HCl Crystal violet
Ephedrine hydrochloride Crystal violet
Imipramine hydrochloride Crystal violet
Isoprenaline hydrochloride Crystal violet
Lignocaine hydrochloride Crystal violet
Morphine hydrochloride Crystal violet
Morphine sulphate Crystal violet
Phenylephrine hydrochloride Crystal violet
Phenytoin sodium α -Naphthol benzein
Promethazine hydrochloride Methyl orange
Thiabendazole Crystal violet
Alkalimetric Assays
Acetazolamide Potentiometric determination
Bendrofluazide Azo violet
Allopurinol Thymol blue
Mercaptopurine Thymol blue
Amylobarbitone Quinaldine Red
Nalidixic acid Thymolphthalein

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 REDOX TITRATION PHARMACEUTICAL ANALYSIS

6 REDOX
TITRATION
CONCEPT OF OXIDATION AND REDUCTION

 OXIDATION: It may be defined as a loss of electrons to an oxidizing agent (that undergoes

reduction) to yield more positive or higher oxidation state.
Examples:
1. Fe+2 (ferrous ion) into Fe+3 (ferric ion)
2. Cu (copper) into Cu+2 (cupric ion)
3. Cl-(chloride) into Cl2 (chlorine)
 REDUCTION: It may be defined as gain of electrons from reducing agent (that undergoes
oxidation) to give more snegative or lower oxidation state.
Example:
1. Ce+4 (cerric form) + e-
2. Ce+3 (cerrous form)

REDOX REACTION

 It is defined as the reaction which takes place rapidly between two typically specific entities,
one being a reducing agent and other one is an oxidising agent.
 A good example is the reaction between hydrogen and fluorine in which hydrogen is being
oxidized and fluorine is being reduced:
H2 + F2  2 HF
We can write this overall reaction as two half-reaction
 Oxidation reaction: H2  2H+ + 2e –
 Reduction reaction: F2 + 2e  2F
in the above reaction H2 acts as a reducing agent and F2 acts an oxidising agent.

OXIDISING AGENTS

 An oxidizing agent (oxidant, oxidizer) is a substance that has the ability to oxidize other
substances, in other words to cause them to lose electrons.
 Common oxidizing agents are Oxygen, Hydrogen Peroxide and Halogens.
 Examples include MnO 4, CrO2 4, OsO4, and especially ClO 4 .

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

REDUCING AGENTS

 A reducing agent (also called a reductant or reducer) is an element (such as calcium) or

compound that loses (or “donates”) an electron to another chemical species in a redox chemical
reaction.
 Since the reducing agent is losing electrons, it is said to have been oxidized.
 Examples of reducing agents include the Alkali metals, Alkaline earth metals, Formic acid, and
Sulfite compounds.

REDOX POTENTIAL

 Redox potential (also known as oxidation-reduction potential / ORP) is a measure of the

tendency of a chemical species to acquire electrons and thereby be reduced.
 Reduction potential is measured in volts (V), or millivolts (mV).
 It is determined by using half-cell with standard hydrogen electrode.
 The potential difference between this 2 electrodes is determined which gives the redox potential.

NERNS’T EQUATION
 The Nernst Equation enables the determination of cell potential under non-standard conditions.
 It relates the measured cell potential to the reaction quotient and allows the accurate
determination of equilibrium constants (including solubility constants).
 The Nernst Equation is derived from the Gibbs free energy under standard conditions.
RT
E = E0 - 2.303 log 10Q
nF
Where,
Eo – Standard electrode (or reduction) potential
E – Potential observed at absolute temperature (T)
R – Gas constant = 8.314 joules/deg/mol-1
F – Faraday’s constant = 96500 coulumbs
T – Absolute temperature (T) = 298 °K (25 °C)
n – Number of electrons gained by an oxidant in being converted to reducing agent

TYPES OF INDICATOR

1. SELF INDICATOR: A substance is said to be self indicator if it itself acts as an indicator in

titration.
Example: KMnO4 acts as a self indicator during the titration of Potassium permanganate
(KMnO4) and oxalic acid (COOH)
2. INTERNAL INDICATOR: The indicator which is used within the solution is called internal
indicator. These indicators takes part in the reaction.
Example: N-phenyl anthranilic acid, Starch.
3. EXTERNAL INDICATOR: The indicator which are not added to the solution are called

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 REDOX TITRATION PHARMACEUTICAL ANALYSIS

external indicator. These do not take part in the reaction.

Example: Potassium Ferro cyanide K3[Fe(CN)6] can acts as external indicator during titration
of ammonium or ferrous sulphate (FeSO4) with Potassium dichromate (K2Cr2O7).
INDICATOR COLOUR CHANGE
Diphenylamine Colourless to Blue
Diphenylamine Sulphonate Colourless to Blue
Diphenyl Benzidine Colourless to Reddish Violet
Methylene Blue Colourless to Blue
Starch Colourless to Blue

OXIDATION REDUCTION TITRATION CURVE

1. Initial region: It is the point at which the titration starts (0% titration).
2. Buffering region: It is a region where the reducing agent takes small amount of time to lose
all the electrons and subsequently the electrons are gained by an oxidizing agent to reach the
equivalence point (>0% and < equivalence point).
3. Equivalence point: It is the region where the percent of oxidized and reduced form are
equivalent to each other. At this point there is sharp increase in curve till the end point is
reached.
4. Inflection point: A point on a curve at which tangent crosses the curve itself or a point on a
curve at which the curvature changes sign. The curve changes from being concave upwards
(positive curvature) to concave downwards (negative curvature).
5. Over titration: It is the region showing the constant curve or potential and colour showing
by the indicator should be deep in this significant region

5. Over titration

4. Inflection point

3. Equlvalence
point
Ec ell

2. Buffer region

1. Initial region
Percent titration
(or volume of titrant)

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

7 IODIMETRY
AND IODOMETRY
INTRODUCTION

 Iodine can be very conveniently and accurately titrated with standard thiosulphate solution.
 Two types of iodine titrations are possible:
1. Iodimetry: Standard Iodine solution used.
2. Iodometry: The liberated iodine during chemical reaction is titrated.

IODIMETRY

 I2 is a weak oxidant, and can be reduced by reductants.

 Example: Stannous Chloride (SnCl2); Sodium thiosulphate (Na2S2O3)
 They reduces rapidly and completely with I2
Sn2+ + I2 Sn4+ + 2I-
2S2O2-3 + I2 S4O2-6 + 2I-
 In an iodimetric method, a known volume of solution of the reductant is taken in a glass
stopper conical flask (Iodine flask).
 Add 1ml starch solution or sodium starch glycollate (better than starch) as an indicator.
 Standard solution of iodine is gradually added from burette, swirling the flask frequently.
 Titration solution becomes blue and stops the solution (from colorless)
 But color change detection is more clearly visible when color changes from blue to colorless.
 So, Iodine standard solution is taken in Iodine flask and starch indicator is added to the flask
which gives blue color.
 Then titrated against the thiosulphate test solution taken in the burette.
 Finally Normality of the thiosulphate is calculated taking iodine as a standard.
 End point is detected when blue color changes to colorless.
 Iodine is reduces to iodine.
I2+e-  2I- (Blue color)

IODOMETRY
 If we have a solution of strong oxidant, such as
 CuSO4
 F2Cr2O7

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 TITRATION PHARMACEUTICAL ANALYSIS

 KMnO4
 Add large excess of KI solution (in the presence of acid)
 Iodide ions oxidized to iodine
2I- - 2e-  I2 (Eq. a)
 Electrons are given out by I- ions (Reductants)
 The oxidant accepts these electrons and thereby gets reduced.
 If oxidant is CuSO4 then
2Cu2+ + 2e-  2Cu+ (Eq. b)
 Cu2+ ions (oxidant) accepts electron supplied by iodide ions (reductant) and themselves get
reduced to Cuprous state (CuII) is reduced to Cu(I)
 On adding partial equation (Equation a) and (Equation b)
2Cu2+ + 2I-  2Cu+ + I2
2Cu2+ + 4I-  2Cu2I2 + I2
 Because I- ions present in large excess.
 The quantity of 2 liberated will be equivalent to conc. of Cu2+ (or amount of CuSO4)
 Thus, 2CuSO4 = 2Cu2+ = I2
 Two molecules of CuSO4 will produce a molecule of iodine.
 Hence, if we find out the quantity of the liberated Iodine, the amount of Cu2+ ions or that of
CuSO4 can be calculated.
 The quantity of liberated iodine is found out by titration with standard thiosulphate solution.
I2 + 2Na2S2O3  Na2S4O6 + 2NaI
 Thus, 2CuSO4 = I2 = 2Na2S2O3

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

8 COMPLEXOMETRIC
TITRATION

INTRODUCTION
 Complexometric titration is a form of volumetric titration in which the formation of a
colored complex is used to indicate the end point of a titration.
 The complexes are formed by the reaction of a metal ion (an acceptor, a central atom or a
cation) with an anion, a neutral molecule or very rarely a positive ion.
 Complexometric titrations are particularly useful for the determination of a mixture of
different metal ions in solution.
 An indicator capable of producing a distinct color change is usually used to detect the end
point of the titration.
 Complex: Metal ion + Ligand it may be electrically positive, negative or neutral. Metal ions
are Lewis acids and ligands are Lewis bases

COMPLEX TITRATION WITH EDTA

 EDTA, ethylene diamine tetra acetic acid, has four carboxyl groups and two amine groups
that can act as electron pair donors, or Lewis bases.
 The ability of EDTA to potentially donate its six lone pairs of electrons for the formation of
coordinate covalent bonds to metal cations makes EDTA a hexadentate ligand.
 However, in practice EDTA is usually only partially ionized, and thus forms fewer than six
coordinate covalent bonds with metal cations.
 Masking Agent: Masking agent is a reagent that protects some component of the analyte
from reaction with EDTA.
 Demasking Agent: Demasking Agent is a reagent that release of a metal ion from a
masking agent.

TYPES OF COMPLEXOMETRIC TITRATIONS

1. Direct Titration
 It is the simplest and the most convenient method used in chelometry.
 In this method, the standard chelon solution is added to the metal ion solution until the
end point is detected.
 This method is analogous to simple acid-base titrations. Example -calcium gluconate
injection, calcium lactate tablets and compound sodium lactate injection for the assay of
calcium chloride (CaCl2.6H2O).
 Limitations: Slow complexation reaction; interference due to presence of other ions.
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 COMPLEXOMETRIC TITRATION PHARMACEUTICAL ANALYSIS

2. Back Titration
 In this method, excess of a standard EDTA solution is added to the metal solution, which is
to be analyzed, and the excess is back titrated with a standard solution of a second
metal ion.
Example: Determination of Mn
 This metal cannot be directly titrated with EDTA because of precipitation of Mn (OH)2.
 An excess of known volume of EDTA is added to an acidic solution of Mn salt and then
ammonia buffer is used to adjust the pH to 10 and the excess EDTA remaining after
chelation, is back titrated with a standard Zn solution kept in burette using Eriochrome
blackT as indicator.
 This method is analogous to back titration method in acidimetry.Example - Zn
3. Replacement Titration
 In this method the metal, which is to be analyzed, displaces quantitatively the metal from
the complex.
 When direct or back titrations do not give sharp end points, the metal may be determined
by the displacement of an equivalent amount of Mg or Zn from a less stable EDTA
complex.
Mn+2 + Mg EDTA-2  Mg+2+ Mn EDTA-2
 Mn displaces Mg from Mn EDTA solution.
 The free Mg metal is then directly titrated with a standard EDTA solution.
 In this method, excess quantity of Mg EDTA chelate is added to Mn solution. Mn
quantitatively displaces Mg from Mg EDTA chelate.
 This displacement takes place because Mn forms a more stable complex with EDTA.
 By this method Ca, Pb, Hg may be determined using Eriochrome black T indicator.
4. Indirect Titration
 This method also known as Alkalimetric titration.
 It is used for the determination of ions such as anions, which do not react with EDTA
chelate.
 Protons from disodium EDTA are displaced by a heavy metal and titrated with sodium
alkali.
INDICATORS
 To carry out metal cation titrations using EDTA, it is almost always necessary to use a
complexometric indicator to determine when the end point has been reached.
INDICATOR pH Range COLOUR CHANGE
Sodium alizarie sulphate 4 Blue to red
Alizarine 4.3 Red to yellow
Xylenol 1 to 6 Lemon to yellow
Methyl blue 4 to 5 Blue to yellow
Murexide 12 Violet to blue
Mordant Black II 6 to 7 Red to Blue
Enoehrome Black T 6 to 7 Red to Blue
Solochrome black T 6 to 7 Red to Blue
Catechol Violet 8 to 10 Violet to red
Thymol blue 10 to 12 Blue to gray

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

9 GRAVIMETRIC
ANALYSIS

INTRODUCTION

 Gravimetric analysis is a method in analytical chemistry to determine the quantity of analyte

based on the mass of a solid. Example: Measuring the solids suspended in the water sample –
Once a known volume of water is filtered, the collected solids are weighed.
 Gravimetric analysis is a process of precipitation, isolation and weighing of the isolated
product. It is mainly based on the measurement of the analyte.
 Gravimetric analysis is based on conversion of ions/elements/radicals into a stable and
pure compound through precipitation, which can be directly weighed and quantified.

PRINCIPLE
 The principle involved in this method is that the sample is dissolved in a solvent and then
the precipitating agent is added. The resulting precipitate is filtered dried and weighed. Types
of Gravimetric Analysis
1. Volatilization gravimetry: Volatilization Gravimetry involves separating components of
our mixture by heating or chemically decomposing the sample.
2. Precipitation gravimetry: Precipitation Gravimetry uses a precipitation reaction to
separate one or more parts of a solution by incorporating it into a solid.
3. Electrogravimetry: Electrogravimetry is a method used to separate and quantify ions of
a substance, usually a metal.
4. Thermogravimetric: Thermogravimetric is a method of thermal analysis in which
changes in physical and chemical properties of materials are measured as a function of
increasing temperature or as a function of time.

STEPS INVOLVED IN GRAVEMETRIC ANALYSIS

1. Preparation of the solution: Sample solution has to be first prepared for the analysis. The
precipitation is carried out using dilute solution. Adjustment of volume and pH is important to
get the desired properties of precipitate.
2. Precipitation: This step requires the addition of the precipitating agent in the form of
solution to the sample solution. After the addition of the first drop of the precipitating agent,
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 GRAVIMETRIC ANALYSIS PHARMACEUTICAL ANALYSIS

supersaturation occurs, and nucleation starts to occur, where the molecules of precipitate
aggregate together and forms a nucleus. At this point, addition of extra precipitating agent
will either form new nuclei or will build up on existing nuclei to give a precipitate.
3. Digestion of the precipitate: The precipitate formed in the solution is left hot for 30min to
1 hour. The primary precipitate formed may be crystals of imperfect structure, such particles
have to be subjected to digestion. Digestion involves the dissolution of small particles and re-
precipitation of larger ones resulting in particle growth and better precipitate characteristics.
This process is called Ostwald ripening. The most important advantage of digestion is the
conversion of the colloidal particles to coagulated particles. We get precipitate with good crystal
characteristics.
4. Filtering and washing: In this operation, the precipitate is separated from the mother
liquor. Various types of filter media is used for this purpose like filter paper, gooch crucible,
filter pulp etc. After filtering the precipitate, the impurities on the surface of the precipitate can
be removed by washing. Large amount of water must not be used for washing because some
of the precipitate may be lost. In the case of colloidal particles, we can use dilute solution of
nitric acid, ammonium nitrate or dilute acetic acid.
5. Drying and Ignition: The main objective of drying to remove the water trapped in
between the precipitate particles. The drying and ignition process will depend upon the weight
of the precipitate. Drying can be using an oven at temperature 120-150 °C. Ignition can be
done using a muffle furnace at temperature 600-1200 °C. A constant weight must be obtained.
This can be achieved only when the process of heating, cooling and weighing is repeated.
6. Weighing and Calculation: In the usual gravimetric analysis, the precipitate is weighed and
from this value the weight of the analyte in the sample is calculated with the help of a gravimetric
factor.

APPLICATIONS OF GRAVIMETRIC ANALYSIS

1. Extensive number of inorganic ions can be easily determined.

2. It is the most widely applicable analytical procedure.
3. A variety of organic substances can be easily determined like lactose in milk products,
cholesterol in cereals etc.
4. To determine atomic mass of many elements.
5. Elemental analysis of the organic compound can be done. The composition of the elements
of the compound can be done.
6. The analysis of rocks, ores, soils etc and other inorganic samples for their major
components can be carried out.

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

10 KARL FISCHER
TITRATION

INTRODUCTION

 Karl Fischer titration is a titration method that uses volumetric or coulometric titration to determine the
quantity of water present in a given analyte

PRI NCIP LE

 The principle of Karl Fischer titration is based on the oxidation reaction between iodine
and sulphur dioxide.
 Water reacts with iodine and sulphur dioxide to form sulphur trioxide and hydrogen iodide.
An endpoint is reached when all the water is consumed.
 Equation I2 + SO2 + H2O  2HI + SO3
 Karl fisher reagent - Iodine, Buffer (Imidazole), sulphur dioxide, solvent (methanol)

KARL FISCHER TITRATION PROCEDURE

 Volumetric determination: This technique is suitable to determine water content down to

1% of water. The sample is dissolved in KF methanol and the iodine is added to KF Reagent.
The endpoint is detected potentiometrically.
 Coulometric determination: The endpoint is detected in this experiment electrochemically.
Iodine required for KF reaction is obtained by anodic oxidation of iodide from solution.

APPLICATIONS

1. It is used in technical products such as plastics, oils, gases.

2. It is used in pharmaceutical products.
3. It is used in cosmetic products.
4. It is used in the industry.
5. Sodium tartarate dihydrate is used in standardization of Karl-Fisher reagent.

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 INTRODUCTION OF CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

11 INTRODUCTION OF
CHROMATOGRAPHY

INTRODUCTION

Chromatography is a physical method of separation in which the components to be

separated are distributed between two phases, one of which is stationary (stationary phase)
while the other (the mobile phase) moves in a definite direction.
 Analytical chromatography: Analytical chromatography is to separate the components of
a sample.
 Preparative chromatography: Preparative chromatography is to isolate and purify a
reasonable quantity of a specific substance from a sample.

KEY TERMS

Stationary phase or Substance that stays fixed inside the column

Adsorbent
Mobile phase or Solvent moving through the column
Carrier
Bonded phase Stationary phase that is covalently bonded to the support particles
or to the inside wall of the column tubing
Eluate Fluid exiting the column (that is collected in flasks)
Eluent Fluid entering the column
Elution The process of washing out a compound through a column using a
suitable solvent
Eluotropic Series A series of solvents with an increasing degree of polarity, generally
used to explain solvent strength
Analyte Mixture whose individual components have to be separated and
analyze
Solvent Any substance capable of solubilizing another substance.
Isoelectric Point The pH point at which a molecule no longer has a net charge
Retention time Time takes for a particular analyte to pass through the system (from
the column inlet to the detector) under set conditions
Resolution A measure of the separation of two adjacent peaks. The higher the
resolution value the greater the separation
Chromatogram Visual output of the chromatograph
Separation Different peaks or patterns on the chromatogram correspond to
different components of the separated mixture
Isocratic Analysis The procedure in which the composition of the mobile phase
remains constant during the elution process.
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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

Gradient Elution The procedure in which the composition of the mobile phase is
changed continuously or stepwise during the elution process.
Stepwise Elution The elution process in which the composition of the mobile phase is
changed in steps during a single chromatographic run.

CLASSIFICATION OF CHROMATOGRAPHY

According to Adsorption
the principle Partition
Ion exchange
Size/molecular exclusion
Affinity
According to Normal Phase An elution procedure in which the stationary
the Polarity of phase is more polar than the mobile phase.
the Phases Reversed Phase In which the mobile phase is significantly
more polar then the stationary phase.
According to Liquid Liquid Liquid Chromatography
the Mobile Chromatography Example: Paper Chromatography
Phases Ion Exchange Chromatography
Droplet Counter Current Chromatography
Liquid Solid Chromatography
Example: Column, TLC, HPLC, HPTLC
Gas Gas Solid Chromatography
Chromatography Gas Liquid Chromatography
Supercritical Fluid Chromatography
According to Planar Paper Chromatography
the Geometry chromatography Thin Layer Chromatography
HPLC
Columnar Column Chromatography
chromatography Gas Chromatography
Size Exclusion Chromatography
HPLC
Flash Chromatography
Ion Exchange Chromatography
Droplet Counter Current
Affinity chromatography

TYPES OF CHROMATOGRAPHY

TECHNIQUE STATIONARY PHASE MOBILE PHASE

Column/Adsorption Chromatography Solid Liquid
Partition Chromatography Liquid Liquid
Paper Chromatography Liquid Liquid
Thin Layer Chromatography Liquid/Solid Liquid
Gas – Liquid chromatography Liquid Gas
Gas – Solid Chromatography Solid Gas
Ion Exchange Chromatography Solid Liquid

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 INTRODUCTION OF CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

CHROMATOGRAM DEVELOPMENT TECHNIQUE

FRONTAL A procedure in which the sample (liquid or gas) is fed continuously

ANALYSIS into the chromatographic bed. In frontal chromatography no
additional mobile phase is used.
DISPLACEMENT Sample mixture is dissolved in large volume of solvent and applied to
ANALYSIS the top of the column. Mobile phase containing displacement agent is
passed through the column.
ELUTION Isocratic Solvent composition or strength is not changed during
ANALYSIS elution column development
Gradient Solvent composition or strength is changed during
elution column development.
It is also known as solvent programming.

PRINCIPLE

ADSORPTION
 Stationary phase – Solid
 Mobile phase – Gaseous and Liquid
 Principle of separation - Utilizes a mobile liquid or gaseous phase that is adsorbed onto
the surface of a stationary solid phase.

Solute adsorbed
on surface of
stationary phase

Fig: ADSORPTION CHROMATOGRAPHY

PARTITION
 Stationary phase – Nonvolatile liquid
 Mobile phase - Gas and liquid
 Principle - Partition of component of sample between sample and liquid/gas stationary
phase retard some components of sample more as compare to others. This gives the basis of
separation.

Solute dissolved
in liquid phase
bonded to the
surface of
column

Fig: PARTITION CHROMATOGRAPHY

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

ION EXCHANGE
 Stationary phase – Coated solid (resin)
 Mobile phase – Liquid
 Principle - Ion exchange mechanism separates analytes based on their respective charges.
Mobile anions
- -
held near cations
+ -
- that are covalently
- +
- - attached to
+ - - stationary phone
+ - + - +
+
- + -
+
- Small molecules
- - -
+
+ - + penetrate pores
- + -
- - of particles
+ - + -
+ - - +
- + + - Anion-exchange
-
+ -
-
+
- -
- resin; only
- attracted to it

Fig: ION-EXCHANGE CHROMATOGRAPHY

MOLECULAR EXCLUSION
 Stationary phase - Dextran (Sephadex), Polyacrylamide gels
 Mobile phase – Toluene, Tetrahydrofuran
 Principle - Uses porous particles to separate molecules of different sizes.
Large molecules
are excluded

Small molecules
penetrate pores
of particles

Fig: MOLECULAR EXCLUSION CHROMATOGRAPHY

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 PAPER CHROMATOGRPHY PHARMACEUTICAL ANALYSIS

12 PAPER
CHROMATOGRPHY

INTRODUCTION
 Paper chromatography is considered to be the simplest and most widely used of the
chromatographic techniques because of its applicability to isolation, identification and
quantitative determination of organic and inorganic compounds.
 Two types
 Paper Adsorption Chromatography: Paper impregnated with silica or alumina acts as
adsorbent (stationary phase) and solvent as mobile phase.
 Paper Partition Chromatography: Moisture / water present in the pores of cellulose
fibers present in filter paper acts as stationary phase & another mobile phase is used as
solvent.

PRI NCIP LE
 The principle involved can be Partition chromatography or Adsorption chromatography.
 When the mobile phase moves, the separation of the mixture takes place.
 The compounds in the mixture separate themselves based on the differences in their
affinity towards stationary and mobile phase solvents under the capillary action of pores in
the paper.

STATIONARY PHASE AND WHATMAN FILTER PAPERS

 These types of papers are prepared from cotton linters selected to be especially low in
lorganic and inorganic impurities and uniform in physical characteristics
 Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc
are used.
 Genrally whatman paper 31 ET are used for separation.
COMPONENTS PERCENTAGE
α Cellulose 98-99
β Cellulose 0.3-1.0
Pentosans 0.4-0.8
Ether soluble matter 0.015-0.03
Ammonia 0.001-0.06
Organic nitrogen <0.01
Inorganic material 0.008-0.06
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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

CHARACTERISTICS OF WHATMANN CHROMATOGRAPHIC PAPERS

RATE OF FLOW
PAPER
Fast Medium Slow
No. 4 No. 7 No. 2
Thin paper No. 54 No. 1 No. 20
No. 540 - -
No. 31 No. 3 -
Thick paper
No. 17 No. 3 MM -

TYPE OF FILTER PAPER

 Modified Papers – Acid or base washed filter paper, glass fiber type paper.
 Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc.
 Hydrophobic papers – Acetylation of group’s leads to hydrophobic nature, hence can be used
for reverse phase chromatography.

FACTORS THAT GOVERNS THE CHOICE OF PAPER

 Nature of sample and solvents used.
 Based on quantitative or qualitative analysis.
 Based on thickness of the paper.

PREPARATION OF PAPER
 Cut the paper into desired shape and size.
 The starting line is marked on the paper with an ordinary pencil 5cm from the bottom edge.
 On the starting line marks are made 2 cm apart from each other.

PREPARATION OF SOLUTION

 Pure solutions can be applied direct on the paper but solids are always dissolved in small
quantity of a suitable solvent.
 Biological tissues are treated with suitable solvents and their extracts obtained.
 Proteins can be precipitated with alcohol and salts can be removed by treatment with ion
exchange resin.

APPLICATION OF SAMPLES
 The sample to be applied is dissolved in the mobile phase and applied as a small spot on
the origin line, using capillary tube or micropipette.
 Very low concentration is used to avoid larger zone.
 The spot is dried on the filter paper and is placedin developingchamber.

CHOICE OF SOLVENT
 The commonly employed solvents are the polar solvents, but the choice depends on the
nature of the substance to be separated.
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 PAPER CHROMATOGRPHY PHARMACEUTICAL ANALYSIS

 If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity
may be applied.

MOBILE PHASE

HYDROPHILIC MOBILE PHASE HYDROPHOBIC MOBILE PHASE

N-butanol : Glacial acetic acid : Water (4:1:5) Dimethyl ether : cyclohexane
Methanol : water (4:1) Kerosene : 70% isopropanol
Isopropanol : ammonia : water (9:1:2) Dimethyl formamide : cyclohexane
 Eutropic series: N-Hexane > Cyclohexane > Carbontetrachloride > Benzene > Toluene >
Trichloromethylene > Diethyl Ether > Chloroform > Ethyl Acetate > N-Butanol > N-Propanol >
Acetone > Ethanol > Methanol > Water.

DEVELOPMENT TECHNIQUES

Ascending  The solvent flows against gravity.

development  The spots are kept at the bottom
(Go up) portion of paper and kept in a
chamber with mobile phase solvent
at the bottom

Decending  This is carried out in a special

development chamber where the solvent holder is
(Downward at the top.
slope)  The spot is kept at the top and the
solvent flows down the paper.
 In this method solvent moves from
top to bottom so it is called
descending chromatography
Ascending  A hybrid of above two techniques is
Decending called ascending-descending
development chromatography.
 Only length of separation increased,
first ascending takes place followed
by descending
Circular  Spot is kept at the centre of a circular
Radial paper.
development  The solvent flows through a wick at
the centre & spreads in all directions
uniformly.

Two  In this method the paper is

dimentional developed in one direction and after
development development, the paper is developed
in the second direction allowing
more compounds to be separated
into individual spots.
 In the second direction, either same
solvent/different solvent system can
be used for development.

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

CHROMATOGRAPHIC CHAMBER

 The chromatographic chambers are made up of many materials like glass, plastic or
stainless steel.
 Glass tanks are preferred most.

DETECTING AND VISUALIZING AGENTS

 Physical method: The fluorescent compounds can be detected by using UV chamber at 254
nm.
 Chemical method: In this metod the spots, spray reagents or visilizing reagents are used.
Lid

Solvent
front Paper

Solvent

Solvent front
New position
of compound 2.9 cm
2.4 cm

Origin

2.4
Rf = = 0.82
2.9

RETENTION FACTOR (R f )
 Retention factor (Rf): Distance travelled by solute/ Distance travelled by solvent. Rf Value
cannot be greater than 1.
Distance travelled by solute
Rf =
Distance travelled by solvent
 If Rf value of a solution is zero, the solute remains in the stationary phase and thus it is
immobile.
 If Rf value = 1 then the solute has no affinity for the stationary phase and travels with the
solvent front.
 Factors affecting Rf value

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32
 PAPER CHROMATOGRPHY PHARMACEUTICAL ANALYSIS

1. Quality and dimension of the paper.

2. Solvent system, its flow rate and direction of flow.
3. Temperature of the environment
4. Size of the vessel in which chromatogram is developed.

APPLICATION

1. Separation of mixtures of drugs

2. Separation of carbohydrates, vitamins, antibiotics, proteins, etc.
3. Identification of drugs
4. Identification of impurities
5. Analysis of metabolites of drugs in blood, urine.

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

13 THIN LAYER
CHROMATOGRAPHY

INTRODUCTION

 Thin Layer Chromatography can be defined as a method of separation or identification of a

mixture of components into individual components by using finely divided adsorbent solid (liquid)
spread over a glass plate and liquid as a mobile phase.
 Synonyms: Drop, strip, spread layer, surface chromatography and open column
chromatography.

PRI NCIP LE
 It is based on the principle of adsorption chromatography or partition chromatography or
combination of both, depending on adsorbent, its treatment and nature of solvents employed
 The components with more affinity towards stationary phase travels slower.
 Components with less affinity towards stationary phase travel faster.

STATIONARY PHASE
 Finely divided powder is used as a stationary phase.
 Spread over supporting plate. (Aluminum or glass)
 Thickness of the stationary phase will be ~250µm/2.5mm
 Particle size range for TLC is 1-40µm.
 Available Size of prepared plates: 20x20cm/ 20x10cm/ 20x5cm/ 10x10cm
STATIONARY PHASE USE’S
Silica gel (SiO2) Most widely used adsorbent
Surface of the silica gel is acidic in nature.
Alumina (Al2O3) The active groups on the surface of chromatography alumina are
hydroxyl groups, oxide (O2–) ions and an aluminum cation.
Diatomaceous earth Chemical composition of oven-dried diatomaceous earth is 80-
(Kieselguhr) 90% silica, with 2-4% alumina and 0.5-2% iron oxide.
Magnesium Silicate Used for separation of sugars.
Cellulose Separating hydrophilic substances like amino acids, sugars.
Charcoal Specific property of adsorbing strongly aromatic substances.
Starch and talc To analyze food components
Cerium Separation of cations
Titanium silicate Separation of metal ions

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34
 PAPER CHROMATOGRPHY PHARMACEUTICAL ANALYSIS

MOBILE PHASE
 A mixture of an organic solvent and water with the addition of acid, base or complexing
agent to optimize the solubility of the components of a mixture can be used.

PREPARATION OF TLC PLATE

TLC PLATE IS PREPARED BY USING FOUR METHODS

1. Pouring: In these method slurry is put on to the plate which is kept on a labeled surface,
then the slurry is spared on the surface of the plate.
2. Dipping: in these method two plates of standard dimensions are dipped into the slurry at a
time, back to back which is made up of from chloroform-methanol as a solvent.
3. Spraying: Slurry is spared onto glass plate by using a spray gun with an air pressure.
4. Spreading: Most preffered method. Spreader used to spred the slurry on the glass plate.
 Suspend 100 g of silica gel G (G stand for Gypsum) in 200-250 ml of water, mix with a
stirrer to get homogeneous slurry.
 Take the air dried TLC glass plates or dried in oven at 110 °C and pour the silica gel G
slurry into the glass plate. (Thickness should be around 250 m) Slurry should be used
within 2 minute of preparation otherwise slurry will dry and needs more water to maintain
the fluidity.

TLC Chamber

Spotting line
TLC Plate

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

APPLICATION OF SAMPLE
 Sample solution in a non-polar solvent is applied.
 The concentration of a sample or standard solution has to be minimum of a 1% solution of
either standard or test sample is spotted using a capillary tube or micropipette.
 The area of application should be kept as small as possible for sharper and greater resolution.
 Sample should be applied 2 cm above from bottom.

TLC COLUMN DEVELOPMENT

 Place the prepared TLC plate in the developing beaker, cover the beaker with the watch
glass, and leave it undisturbed on your bench top.
 The solvent will rise up the TLC plate by capillary action. Make sure the solvent does not
cover the spot.
 Allow the plate to develop until the solvent is about half a centimeter below the top of the
plate.
 Remove the plate from the beaker and immediately mark the solvent front with a pencil.

VISUALIZING METHOD
 Nonspecific method: Number of spot can be detected.
1. Iodine chamber method
2. UV chamber for fluorescent compound
 Specific method: specific spray or detecting or visualizing agents are used to find out the
nature of compound or for identification purposes.

DETECTING REAGENT

REAGENT COLOUR COMPOUND

Day light Various colours Coloured compound
UV light Fluorescent spot Organic compound
Iodine vapour Brown Most organic substance
Bromo cresol green Yellow Acids
2, 4-Di nitro phenyl hydrazine Yellowish red Aldehyde and Ketone
Ninhydrin Purple Amino acid
Mercuric nitrate Yellowish brown Alkaloids
Aniline phthalate Gray black Carbohydrate
Bromothymol blue Light green Lipids
Antimony trichloride Purple Steroids
FeCl3 Purple Phenolic compound

QUALITATIVE ANALYSIS
 Rf VALUE
 A parameter often used for qualitative evaluation is the RF value (Retention Factor).
 Rf values are between 0 and 1
Distance travel by solute (spot)
 Rf =
Distance travel by solvent front
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36
 PAPER CHROMATOGRPHY PHARMACEUTICAL ANALYSIS

 Rx value: Distance travelled by sample/distance travelled by standard. It is always closer to 1.

 Rm value: To find whether compounds belong to a homologous series. It is a combined
value.

QUATITATIVE ANALYSIS
 Direct method
1. Visual assessment of chromatogram
2. Detemination of measurement of spot area
 Indirect metod
1. Gravimetric
2. Colorimetry
3. Polarography
4. Radiometry
5. Fluorimetry

APPLICATION

1. Purity of any sample

2. Identification of compounds
3. Examination of reactions
4. Biochemical analysis
5. Separation of multicomponent pharmaceutical formulations.

DIGESTER
6. In food and cosmetic industry

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

14 HIGH PERFORMANCE
THIN LAYER
CHROMATOGRAPHY
INTRODUCTION

 HPTLC is a sophisticated and automated form of TLC.

 Efficient separation in short time.
 HPTLC is a form of thin-layer chromatography (TLC) that provides superior separation power
using optimized coating material, novel procedures for mobile-phase feeding, layer
conditioning, and improved sample application.
 The basic difference between conventional TLC and HPTLC is only in particle and pore size
of the sorbents.
 The principle of separation is similar that of TLC (adsorption).
 It is very useful in quantitative and qualitative analysis of pharmaceuticals.

STEPS INVOLVED IN HPTLC

1. Sample preparation
 Normal phase chromatography - Non polar solvents
 Reverse phase chromatography - Polar solvents
2. Selection of chromatographic layer - Depends on the nature of material to be separated
commonly used materials - Silica gel 60F, Alumina, Cellulose etc.
3. Plates
 HPTLC plates 10 × 10 cm and 20 × 10 cm
 TLC and HPTLC sheets up to 20 × 20 cm
4. Pre-washing
 To remove water vapors and volatile impurities this might get trapped in the plates.
 To avoid this, plates are cleaned by using methanol as solvent by ascending or
descending etc.
5. Conditioning - Plates activated by placing them in an oven at 120 °C for 15 to 20 minutes.
6. Sample application
 Application of 1 - 5l for HPTLC
 Application carried out by - LinomatIV applicator on the plates which give uniform, safe
& standard results.
7. Pre-conditioning
 Also called chamber saturation
 Unsaturated chamber - cause high Rf values
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38
 HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

8. Mobile phase - Selection of appropriate mobile phase is based on the trial and error.
9. Chromatographic development
 Ascending, descending, horizontal, continuous, gradient, and multidimensional.
 HPTLC – migration distance of 5-6 mm is sufficient, after development, plates removed &
dried.
10. Detection of spots
 Detection can be done by iodine vapor in iodine chamber.
 Visual inspection at 254 nm of UV region in UV cabinet.
11. Scanning & documentation
 HPTLC plates are scanned at selected UV regions wavelength by the instrument & the
detected spots are seen on computer in the form of peaks.
 The scanner converts band into peaks & peak height or area is related to the
concentration of the substance on the spot.

COMPARISON BETWEEN TLC AND HPTLC

PARAMETER TLC HPTLC

Technique Manual Instrumental
Efficiency Less High
Layer Lab made/ pre-coated Pre-coated
Mean particle size 10-12µm 5-6 µm
Layer thickness 250 µm 100 µm
Plate height 30 µm 12 µm
Solid support Silica gel, Alumina, Silical gel – normal phase
Kieselghur C8 and C18 – reverse phase
Sample spotting Manual spotting (capillary) Auto sampler (syringe)

APPLICATION OF HPTLC

1. Multidimensional and multimodal seperation by HPTLC in photochemistry

2. Stability-indicating HPTLC determination of imatinib mesylate in bulk drug and pharmaceutical
dosage
3. A Quality control for authentication of herbal photochemicals
4. Herbal drug quantification
5. Determination of artemisinin and its derivatives in bulk pharmaceutical dosages.
6. Biomedical application.

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

15 HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY (HPLC)

INTRODUCTION

 It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through
a column filled with a solid adsorbent material
 Each component in the sample interacts slightly differently with the adsorbent material,
causing different flow rates for the different components and leading to the separation of the
components as they flow out the column.

PRI NCIP LE

 The basic principle of HPLC in normal phase and reverse phase mode is adsorption. The
sample is introduced into HPLC column, different components of the sample move according
to their affinities towards the stationary phase.
 Components having high affinity gets adsorb on stationary phase while components
having less affinity towards stationary phase travels faster down the column.
 As no two components have the same adsorption and affinity towards stationary phase,
the components get separated by this method.

TYPES OF HPLC TECHNIQUES

CHARACTERISTIC NORMAL PHASE REVERSE PHASE

Stationary phase Polar (silica gel) Non polar (octa decyl silane C18)
Mobile phase Non polar Polar
Mechanism Adsorption Partition
Compound eluted first Non polar Polar

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40
 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) PHARMACEUTICAL ANALYSIS

INSTRUMENTATION

Solvent Sample Injector

Reservoir

Analytical
PreColumn Column
or
Recorder
Guard
HPLC Column
Pump
Vacuum Pump

Degasser

Solvent Mixing Detector

Valve

SOLVENT RESERVOIR AND DE-GASSER

 Mobile phase reservoir will carry the mobile phase solution and through the pump it will
be pumped into the column.
 Glass or stainless steel (1 liter)
 Stainless steel should be avoided for use with solvents containing halide ions.
 Degassing of mobile phase is done to prevent the formation of gas bubbles in the pump or
detector by sonication, sparing with helium.

PUMPS
 To pass the mobile phase through the column at a high pressure, and at a constant a
controlled flow rate.
 3000 - 5000 psi force is needed.
 Flow rate: 0.1 to 10mL/min.
 Types
 Mechanical pump: constant flow rate (by reciprocating piston)
 Syringe (Displacement pump)
 Reciprocating Pump (RP)
i. Single piston reciprocating pump
ii. Double piston reciprocating pump
iii. Reciprocating diaphragm pump
 Pneumatic pump: Constant pressure
i. Direct pressure pump
ii. Amplifier pump
 Pulse damping device is used to correct the variation in base line.

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41
PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

SAMPLE INJECTION SYSTEM

 Septum Injectors: They usually permit the introduction of the sample by a high pressure
syringe through a self-sealing elastometer septum
 Stop-flow Septum less Injection: The flow of the mobile-phase through the column is
stopped for a while, and when the column reaches an ambient pressure the top of the column
is opened and the sample introduced at the top of the packing.
 Micro volume Sampling Valves: Highly sophisticated modern HPLC frequently make use
of micro volume sampling valves for injection which not only give fairly good precision, but
also are adaptable for automatic injection.

HPLC COLUMS

Precoloumn • Optional, stationary phase coated with liquid.

(25 – 100 cm length, • Mainly used to remove the impurities from the
6 mm diameter) solvent.
• Prevents contamination of analytical column.
• Also called Guard column or protective column.
• Having large particle size.
Analytical column • Particles are > 10μm, pressure 6000 psi.
(25 – 100 cm length, • Actual separation is carried out here.
2 - 4.6 mm diameter) • Stainless steel tube
• The solid support can be silica gel, alumina.

DETECTORS

Uv-visible spectroscopic Measures the ability of solutes to absorb light at a particular

detectors wavelength in the ultraviolet or visible wavelength range.
Refractive index detector measures the molecule’s ability to deflect light in a flowing
mobile phase in a flow cell relative to a static mobile phase
contained in a reference cell.
Photo diode array monitoring absorbance of solutes at several different
detector wavelengths
Fluorescence detectors sensitivity depends on the fluorescence properties of the
components in the elute
Electrical conductivity conductivity detectors measures electronic resistance and
detector measured value is directly proportional to the concentration
of ions present in the solution.
 Retention time (Rt): The time taken for a particular compound to travel through the column
to the detector is known as its retention time. This time is measured from the time at which
the sample is injected to the point at which the display shows a maximum peak height for that
compound.
 Recorder and integrator: Recorder are used to record the response obtained from
detector after amplification.

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42
 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) PHARMACEUTICAL ANALYSIS

COMPARISON ON COLUMN, HPLC

PARAMETER COLUMN HPLC

Stationary phase 70 – 150 µm 3 – 20 µm
Column size Large Small
Pressure 50 – 150 psi 500 – 3000 psi

COMPARISON ON COLUMN, HPLC

COLUMN INTERNAL DIAMETER HPLC GAS CHROMATOGRAPHY

Preparative 100 mm -
Normal bore 3.9 to 5.0 mm 0.53 mm
Mini bore 2.1 to 3.9 mm 0.18 mm
Micro bore 1.0 to 2.1 mm 0.1 mm
Capillary 50 µm to 1 mm 0.2, 0.25

APPLICATION

1. Therapeutic drug monitoring

2. Qualitative and quantitative analysis
3. Structural determination
4. Biochemical genetics
5. Clinical application
6. Diagnostic studies
7. Drug discovery
8. Proteomics

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43
PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

16 COLUMN
CHROMATOGRAPHY

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 components of the mixture is at
different rates.
 The components with 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 fast are removed first whereas the components that move
slowly are eluted out last.
 Column chromatography are two types
1. Column adsorption chromatography
2. Column partition chromatography

COLUMN EFFICIENCY

HETP (High It is defined as the length or height of stationary phase within

equivalent to which there exist equilibrium of solute between stationary phase
theoretical plate) and mobile phase or it is a height equivalent to one theoretical
plate.
N = L/H
Where, N = Number of theoretical plate
L = Total length of stationary phase
H = Height equivalent to theoretical plate
Peak Resolution
1.18(t B - t A )
R=
WhB + WhA
Where, ChB and WhA = Width of height of components B and A
respectively
When R = 1 There is 98% separation
When R = 1.5 There is 100 % separation
When R > 1.5 There is highest efficiency

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44
 COLUMN CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

Factors affecting Column dimension

column efficiency Nature of solvent
Particle size of adsorbent
Temperature of the column
Column dimension
Nature of solvent
Particle size of adsorbent
Temperature of the column
Pressure

COLUMN CHARACTERISTICS

 The main function of all the columns is to support the stationary phase.
 The material of the column is mostly good quality neutral glass since it shouldn’t be affected
by solvents.
 Column dimensions - length & diameter ratio (10:1, 30:1 or 100:1)
 The length of the column depends upon
1. Number of compounds to be separated
2. Type of adsorbent used
3. Quality of the sample
4. Affinity of compounds towards the adsorbent used

Mobile
Loaded
Phase
sample
Sample
separation Stronger
interactions

Stationary Weaker
phase interactions

Eluted
Molecules

PACKING OF COLUMNS

SLURRY PACKING (WET METHOD) DRY PACKING

The adsorbent is suspended in the In this method the dry adsorbent is poured to
mobile phase and stirred very well to the column directly. Vibration is the applied
drive off all air bubbles. The resulted to get rid of air bubbles then the mobile phase
slurry is then poured into the column. as passed through the adsorbent.
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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

COMPONENTS OF COLUMN CHROMATOGRAPHY

STATIONARY PHASE

 The usual adsorbents employed in column chromatography are silica, alumina, calcium
carbonate, calcium phosphate, magnesia, starch.
 Particle size range: 60-200 µ.
 Alumina is generally suitable for chromatography of less polar compounds.
 Silica gel gives good results with compounds containing polar functional groups.
 Weak adsorbent: Sucrose, Starch, Talc
 Intermediate adsorbent: CaCO3, Calcium Phosphate, Silica Gel
 Strong adsorbent: Alumina, Activated Charcoal, Fuller’s Earth

MOBILE PHASE

 They act as Solvent, Developer and Eluent

 Solvent: To introduce the mixture into the column
 Developer: To develop the zone for separation
 Eluent: To remove pure component out the column
INTRODUCTION OF SAMPLE

 The sample which is usually a mixture of components is dissolved in minimum quantity of

the mobile phase.
 The entire sample is introduced into the column at once and gets adsorbed on the top
portion of the column.
 From this zone, individual sample can be separated by a process of elution.
DEVELOPMENT TECHNIQUE (ELUTION)

In this elution technique, same solvent composition or

ISOCRATIC ELUTION solvent of same polarity is used throughout the process of
TECHNIQUE separation.
Example: Chloroform only
Solvents of gradually increasing polarity or increasing
GRADIENT ELUTION
elution strength are used during the process of
TECHNIQUES
separation.
(GRADIENT
Example: Initially benzene, then chloroform, then ethyl
GRADUALLY)
acetate then chloroform

DETECTION OF COMPONENTS

 The detection of coloured components can be done visually.

 Different coloured bands are seen moving down the column which can be collected separately.
 For colourless compounds, the technique depends upon the properties of the components,
different properties which can be used are
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46
 COLUMN CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

1. Absorption of light - Using Uv/Visible Detector

2. By monitoring the fractions by thin layer chromatography
3. Evaporation of the solvent and weighing the residue
4. Fluorescence or light emission characteristics - Using Fluorescence Detector
5. By using flame ionization detector
6. Refractive index detector - Based on the refractive index difference between the mobile
phase and mobile phase component

APPLICATIONS

1. Separation of mixture of compounds

2. Purification process
3. Isolation of active constituents
4. Estimation of drugs in formulation
5. Isolation of active constituents
6. Determination of primary and secondary glycosides in digitalis leaf.
7. Separation of diastereomers

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

17 GAS
CHROMATOGRAPHY

PRINCIPLE

 Gas chromatography is a term used to describe the group of analytical separation

techniques used to analyse volatile substances in the gas phase.
 In gas chromatography, the components of a sample are dissolved in a solvent and vaporized
in order to separate the analytes by distributing the sample between two phases: (A) Stationary
phase (B) Mobile phase.
 The mobile phase consists of an inert gas loaded with the vaporized mixture of solutes
flowing through the stationary phase at a suitable temperature.

GAS - SOLID • Mobile phase is a gas while the stationary phase is a

CHROMATOGRAPHY solid.
• Used for separation of low molecular gases
• Example: Air components, H2S, CS2, CO2 rare gases,
CO and oxides of nitrogen.
GAS - LIQUID • The mobile phase is a gas while the stationary phase is
CHROMATOGRAPHY a liquid retained on the surface as an inert solid by
adsorption or chemical bonding.

THEORY

1. PLATE THEORY: Each single equilibration between the phases is termed a theoretical plate
and the length of the column required for one equilibration is called the height equivalent a
theoretical plate (HETP).
HETP= L/N
Where,
N is number of theoretical plates in a column
L= Length of the column
 If HETP is less – Column is more efficient
 If HETP is more – Column is less efficient
2. VAN DEEMETER PLOT
 The term ‘A’ is independent of flow of the mobile phase
 The term B/u decrease drastically in the beginning with increase in the flow rate of mobile

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48
 GAS CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

phase. Increase in the flow rate beyond particulars value , leads to slow decrease value in the
value of B/u
 The term Cu increase with increase in the rate
12

10

08 C
/+
B
A+ C
06 T P=
HE
HETP (mm)

04
B/
02
A
00
0 10 20 30 40 50 60 70 80
Fig: VAN DEEMETR PLOT

3. RATE THEORY: It describes the effect of an elution band as well as its time of elution. Van
Demeter equation describes the relation of the height of a theoretical plate H and the average
linear velocity of the mobile phase.
B
Van Demeter Equation H = A + + Cμ
μ
Where, H = Height of theoretical plate,
 = Average liner velocity of mobile phase
A = Eddy diffusion
B = Longitudinal or ordinary diffusion term
C = Non equilibrium or resistance to mass transfer
EDDY DIFFUSION
 The mobile phase moves through the column which is packed with stationary phase.
 Solute molecules will take different paths through the stationary phase at random.
 This will cause broadening of the solute band, because different paths are of different
lengths.
 Eddy diffusion (A) is negligible in the case of GC.
LONGITUDINAL DIFFUSION
 The concentration of analyte is less at the edges of the band than at the center.
 Analyte diffuses out from the center to the edges. This causes band broadening.
 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.
RESISTANCE TO MASS TRANSFER
 The analyte takes a certain amount of time to equilibrate between the stationary and mobile
phase.

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

 If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary
phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary
phase.
 The band of analyte is broadened. The higher the velocity of mobile phase, the worse the
broadening becomes.
 RETENTION TIME: Difference between point of injection and appearance of peak maxima.
Target Compound Peak

Injection
Unretained Compound Peak

to Time
Adjustedt Retention Time
Gas hold-up time or
tor
Retention Time
 RETENTION VOLUME: Volume of carrier gas required to elute 50 % of component from
the column
 RESOLUTION
 To obtain high resolution, the three terms must be maximized.
 An increase in N, the number of theoretical plates,
 By lengthening the column leads to an increase in retention time
 By increasing band broadening, which may not be desirable.
 Instead, to increase the number of plates, the height equivalent to a theoretical plate can be
reduced by reducing the size of the stationary phase particles.

INSTRUMENTATION

Soap-bubble meter
Recorder

Syringe
Detector Electrometer
or
Two-stage bridge
pressure Rotometer Flow Injector
regulator splitter

ADC
Flow
controller
Carrier Data
gas Column system
supply

Column oven
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 GAS CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

STATIONARY PHASES

 Particles of the stationary phase should be passed through 80-120 mesh size.
 Average particle size is 125-180µm.
 Three types of stationary phases are available.
1. Uncoated Solid Material: Activated Charcoal, Alumina, Styrene, Silica Gel, Glass beads, Divinyl
Benzene.
2. Inert Solid coated with thin layer of liquid: Diatomaceous Earth and it is coated with1-
5% silicone polymers (Phenyl/ Cyano/ Thrifluoropropyl) or 1-5% Ethylene Glycol.
3. Direct coating of liquid: Silicone polymers and Ethylene Glycol are chemically bonded
with inner wall of the column.

MOBILE PHASE / CARRIER GAS

 Highly pure (> 99.9%)
 Inert, higher density
 compatible with the detector
 cheap and available.
 The carrier gas pressure ranges from 10-50 psi.
 Conventional analytical columns usually use flow rates in the range from 20-50 mL/min
while capillary columns use flow rates from 1-5 mL/min.
Helium Good thermal conductivity, good flow rate, low density, inert
but expensive.
Nitrogen Less expensive, easily available, reduces peak broadening.
Hydrogen Easily available, low density but reactive with unsaturated
compounds.
Argon Inert but occasionally used.
Carbon dioxide Occasionally used.
Air When O2 is beneficial for detection in detector then only used.

SAMPLE INJECTION SYSTEM

 The most common injection method is where a micro syringe is used to inject sample
through a rubber septum into a flash vaporizer port at the head of the column.
 The temperature of the sample port is usually about 50°C higher than the boiling point of
the least volatile component of the sample.
 For packed columns, sample size ranges from tenths of a microliter up to 20 microliter.
 Capillary columns, on the other hand, need much less sample, typically around 10–3
microliter.
 For capillary GC, split/split less injection is used.

FLOW REGULATOR
 Rotameter
 Soap bubble flow meter

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

COLUMNS

WCOT SCOT
MICRO
PACKED (WALL-COATED (SUPPORT - COATED
PACKED
OPEN TUBULAR) OPEN TUBULAR)
Diameter 2 mm 1 mm 0.25 mm 0.50 mm
Length 1-4 meter 1-6 meter 10-100 meter 10-100 meter
Smple 10 ng - 1 mg 10ng -10 µg 10 -100 ng 10 ng -1 µg
size
Efficacy 500 1000 1000-3000 600-1200
plate/meter plate/meter plate/meter plate/meter
Pressure High Very high Low Low
required
Figure

DETECTORS

SUPPORT
DETECTORS TYPE REMARKS DETECTABILITY
GASES
Thermal Concentration He Hot wire detector/ 10-9g
conductivity Katharometer
(TCD) Tungsten Rhenium alloy
wire
Universal detector
Flame Mass flow H2 and Air Sensitive to most 10-11g
ionization organic compounds
(FID) Insensitive to water &
inorganic subs.
Electron Concentration Make up 63Ni emits beta particles 10-9 - 10-11g
capture gas Halides, nitrates,
(ECD) nitriles, peroxides,
anhydrides,
organometallics,
pesticides.
Thermionic Mass flow H2 and Air Electrically heated bead 10-11g (N)
Specific of solid alkali metal 10-12g (P)
(TSD) or N-P compound (Rubidium
Detector silicate) Nitrogen,
phosphorus
Flame Mass flow H2 and Air Sulphur and 105 greater
photometric phosphorus, tin, boron, sensitive for S & P.
(FPD) arsenic, germanium,
selenium, chromium

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 GAS CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

Photo Concentration Make up Aliphatics, aromatics, 20-9g

Ionization gas ketones, esters,
(PID) aldehydes, amines,
heterocyclic, organ
sulphurs, some
organometallics
Mass Mass Flow He All kind of materials 10-12 - 10-15g
Spectral
(MSD)

APPLICATIONS OF GAS CHROMATOGRAPHY

1. QUALITATIVE ANALYSIS
 When 2 substance gives coincident peak (one known and one unknown), it is evidence
that the compounds may same.
 Retention characteristics of unknown compound determined by:
A. Specific Retention volume (Vg)
 Flow rate of carrier gas adjusted Retention time)
 But in this, reproducibility is very low due to varying packing density, liquid loading,
and activity of support, age etc.
B. Relative retention (rA / B)
 Adjusted retention volume of substance A related to that of reference standard B.
 Here reproducibility is good.
2. QUANTITATIVE ANALYSIS
 Size of the chromatographic peak is proportional to amount of the compound.
 By measuring accurately the peak area or height, quantitative analysis can be done.
A. Peak Area Determination
1. Mechanical or Electronic integration
2. Triangulation
3. Planimetry
4. Cut and Weigh method
B. Peak Height determination
1. Recorders and integrators
2. Measuring with ruler
C. Data Interpretation
1. External standardization
2. Internal standardization
3. Presence of impurities
4. Used in the quality control of :
 Antibiotics - Penicillin, Gentamycin
 Anti T.B drugs - Isoniazid, Ethambutol
 Antivirals - Amantadine, Idoxuridine
 Anti neoplastic - Fluorouracil, Doxorubicin

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

18 SIZE-EXCULSION
CHROMATOGRAPHY

NTRODUCTION

 Size-exculsion chromatography (SEC), also called gelfiltration or gel-permeation

chromatography (GPC), uses porous particles to separate molecules of different sizes.
 It is generally used to separate biological molecules, and to determine molecular weights and
molecular weight distributions of polymers
 It is usually applied to large molecules or macromolecular complexes such as proteins and
industrial polymers.
 When an aqueous solution is used to transport the sample through the column, the technique
is known as Gel-filtration chromatography.
 When an organic solvent is used as a mobile phase, the technique is known as Gel-permeation
chromatography.
 The separation of molecules is called fractionation.
 Size of pores in beads determines the exclusion limit (what goes through the beads and what
goes around the beads)

PRI NCIP LE

 A mixture of molecules dissolved in liquid (the mobile phase) is applied to a

chromatography column which contains a solid support in the form of microscopic spheres,
or “beads” (the stationary phase).
 The mass of beads within the column is often referred to as the column bed.
 The beads act as “traps” or “sieves” and function to filter small molecules which become
temporarily trapped within the pores.
 Larger molecules pass around or are “excluded” from the beads.
 Large sample molecules cannot or can only partially penetrate the pores, whereas smaller
molecules can access most or all pores.
 Thus, large molecules elute first, smaller molecules elute later, while molecules that can access
all the pores elute last from the column.
 Particles of different sizes will elute (filter) through a stationary phase at different rates.

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 SIZE-EXCULSION CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

STATIONARY PHASE

Soft gel Dextran (sephadex), polyacrylamide gels : Separation of proteins.

Semi-rigid Bio beads : Separation of non-polar polymers in non-polar solvents
Highly rigid Gels and glasses : Separation of polar systems

MOBILE PHASE

MATERIAL SOLVENT
Synthetic elastomers ( polybutadiene , polyisoprene ) Toluene
Poly styrene, PVC, Styrene-Butadiene Tetrahydrofuran
Rubber , Epoxy resins
Polyolefins Tri- chloro -benzene
Polyurethane Di- methylformamide
Proteins, polysaccharides Water / Buffers

STEP INVOLVED IN SIZE EXCLUSIO CHROMATOGRAPHY

1. Gel preparation: For gel preparation dry powder is placed in a particular solvent and is
allowed to swell in It generally required amount of dry powder is mixed with excess of liquid
and is left as such until equilibrium is reached and this process takes a very long time.
2. Packing of the column: Gel is allowed to swell, deaerated and then allowed to settle, poured
into the column Packing should be done in single step as it gives uniform packing. Dry packing
the column is avoided as it will results in cracking of the column due to swelling of the gel. So,
wet packing is generally preferred.
3. Sample preparation: Sample is dissolved in a suitable solvent. The sample volume should be
1-3% of total bed volume. Smaller the sample volume smaller will be concentration of the
component in elute.
4. Sample application: The sample is applied at the top of the gel bed and flow is started. The
sample solution rum down the column Sample is applied with the help of pipette. Viscous
samples are introduce with the help of valve loop.

PUMP
 A highly constant flow rate has to be maintained during the entire chromatogram.
 A change of the flow rate of only 0.1% can cause an error in molar mass of up to 10%.
Most pumps can only reproduce the flow rate to 0.2–0.3%.
 Types - Syringe pumps, Reciprocating pumps

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

DETECTOR

Concentration • Bulk Property Detectors- Refractive index detector

sensitive • Solute Property Detectors- Ultraviolet absorption detector
detectors • Evaporative Detectors- Evaporative Light Scattering Detector
Molar mass • Light Scattering Detectors
sensitive A. Low Angle Light Scattering Detectors
detectors B. Multiangle Light Scattering detectors
• Viscosity Detectors- Differential Vscometers
Other • Flame Ionization Detector
• Mass Spectrometer
• Fourier Transform Infrared Spectrometer

APPLICATION

1. Proteins fractionation, Purification

2. Molecular weight determination.
3. Separation of sugar, proteins, peptides, rubbers and others on the basis of their size.
4. This technique can be for determining the quaternary structure of purified proteins.
5. SEC is a widely used technique for the purification and analysis of synthetic and biological
polymers, such as protein, polysaccharides and nucleic acid.
6. Various species of RNA and viruses have been purified using agarose gels.

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 ION EXCHANGE CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

19 ION EXCHANGE
CHROMATOGRAPHY

INTRODUCTION

 Ion exchange chromatography is a process by which a mixture of similar charged ions can be
separated by using an ion exchange resin which exchanges ions according to their relative
affinities.
 Anion exchangers contain bound positive groups, where as cation exchangers contain
bound negative groups.
 Columns used for ion exchange are characterized by the presence of charged groups
covalently attached to the stationary phase.
 The most common properties of all ion exchangers are:-
1. They are almost insoluble in water and organic solvents such as benzene, carbon
tetrachloride, ether etc.
2. They are complex in nature.
3. They have active or counter ions that will exchange reversible with other ions in a
surrounding solution without any substantial change in the material.

PRI NCIP LE

ANION EXCHANGERS: The anion exchangers have positively charged exchanger with negatively
charged mobile counter ion available for exchange.
 If the Basic functional groups are introduced, the resin becomes anion exchanger.
 X+ + R-K+ X+R- + K+
 Tertiary amines  Strong anion exchangers
 Secondary amines  Weak anion exchangers
CATION EXCHANGERS: The cation exchangers have negatively charged exchanger with
positively charged mobile counter ion available for exchange.
 If Acidic functional group are introduced, then the resin becomes cation exchangers.
 X- + R+Cl-  X-R+ + Cl-
 Sulphonic acid  Strong cation exchangers

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

 Carboxylic acid ’! Weak cation exchangers

Positively Charged
Negatively Charged Analyte [Cation]
Analyte [Anion] Attracted to

- +
Attracted to Negative Surface
Positive Surface

+ ++ +
+ + +
+ + Anion + + Cation
+ Exchanger +
+ Stationary-phase+
Exchanger
+ Stationary-phase
+ Particles + Particles
+ + +
+ ++ + + +

X- + R K+ X +R + K (cation exchange)
- - +

X- + R Cl+ X+R + Cl (anion exchange)

+ - -

 ION EXCHANGE GELS: Cellulose and dextran ion exchangers, which are polymers of the
sugar glucose, posses larger pore sizes and lower charge densities.Because they are much
softer than polystyrene resins, dextran and its relatives are called gels.

TYPES OF RESIN

ACCORDING TO THE SOURCE

 Cation - Zeolytes, Clay
Natural resins
 Anion - Dolomite
Synthetic resins Inorganic & Organic resins
Organic resins polymeric resin matrix

ACCORDING TO THE CHEMICAL NATURE

TYPE STATIONARY PHASE MOBILE PHASE
Strongly acidic cation Sulphonated polystyrene Cations, inorganic separations,
exchange resin resins vitamins, amino acids
Weakly acidic cation Carboxylic Cations, transition elements,
exchange resin polymethacrylate resins amino acid, antibiotic
Strongly basic anion Quarternary ammonium Anion, halogen, alkaloids,
exchange resin polystyrene resin vitamin B complex
Weakly basic anion Phenol formaldehyde and Anioic complexes of metals,
exchange resin polyamide polystyrene vitamins and amino acid

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 ION EXCHANGE CHROMATOGRAPHY PHARMACEUTICAL ANALYSIS

COMPONENTS OF ION EXCHANGE CHROMATOGRAPHY

 Columns are generally made up of glass.

COLUMN  Length: diameter ratio of 20: 1 to 100:1 is used for
higher efficiency.
 Type of ion exchange resin depends upon the type and
nature of the ions which are to be separated.
ION EXCHANGE RESIN  Efficiency of a particular ion exchange resin depends
upon the particle size, structural type and amount of
cross linking agent present in the resin.
 Wet packing (commonly used)
PACKING OF THE COLUMN
 Dry packing
Different types of acids, bases and buffers are used as a
MOBILE PHASE
mobile phase.
DEVELOPMENT OF  Isocratic elution
CHROMATOGRAM  Gradient elution
 Spectrophotometric method
 Polarographic method
ANALYSIS OF THE ELUTE
 Conductometric method
 Radiochemical method
 Regeneration of cation exchange resin is done by
charging the column with strong acid like hydrochloric
REGENERATION OF THE acid.
ION EXCHANGE RESIN  Regeneration of anion exchange resin is done by using
strong alkali like sodium hydroxide or potassium
hydroxide

APPLICATIONS

1. Softening and demineralisation of water.

2. For extraction of enzymes from tissues.
3. Purification of solutions free from ionic impurities.
4. Separation of inorganic ions.
5. Separation of sugars, amino acids and proteins

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

20 DIFFERENTIAL
THERMAL ANALYSIS

INTRODUCTION

 Thermal analysis: The measurement of some physical parameter of a system as a function

of temperature.
 Types Of Thermal Methods

Diffrential thermal analysis DTA Temperature ∆T vs temp.

difference
Diffrential scanning calorimetry DSC Enthalpy dH/dt v/s temp.
Dynamic reflactance DRS Reflactance %Reflactance v/s
spectroscopy tempreture
Thermogravimetric analysis TGA Mass Mass v/s temp.
Dynamic meachincal analysis DM Deformation -
A
Dielectric thermal analysis DEA Deformation -
Evolved gas analysis EGA Gaseous Thermal
decomposition conductivity v/s
temp.
Thermoptical analysis TOA Optical properties -

DIFFRENTIAL SCANNING CALORIMETRY

INTRODUCTION
 Differential scanning calorimetry (DSC) is a technique for measuring the energy necessary
to establish a nearly zero temperature difference between a substance and an inert reference
material, as the two specimens are subjected to identical temperature regimes in an
environment heated or cooled at a controlled rate.
 It is the most widely used method of thermal analysis in pharmaceutical field.

PRI NCIP LE

The difference in heat supplied to the sample, and the reference material per unit time is
recorded and plotted as dH/dt vs the average temperature to which the sample and reference to
be raised.
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 DIFFERENTIAL THERMAL ANALYSIS PHARMACEUTICAL ANALYSIS

APPLICATION

1. Purity determination of sample directly

2. Detection of polymorphism
3. Quantification of polymorph
4. Detection of metastable polymorph
5. Detection of isomerism
6. Stability/ compatibility studies
7. Percentage crystallinity determination
8. Lyophilization studies
9. Lipid/ Protein determination

DIFFERENTIAL THERMAL ANALYSIS (DTA)

PRINCIPLE

 The basic principle involved in DTA is the temperature difference (“T) between the test
sample and an inert reference sample under controlled and identical conditions of heating
or cooling is recorded continuously as a function of temperature or time, thus the heat
absorbed or emitted by a chemical system is determined.
 If any reaction takes place in the sample, then the temperature difference will occur
between the sample and the reference material.
 In an endothermic change (such as melting or dehydration of the sample) the temperature
of the sample is lower than that of the reference material i.e. “T = ve (for endothermic process)
 In an exothermic change or process the sample temperature is higher than that of the
reference material i.e. “T = + ve (exothermic process).

APPLICATION
1. DTA curves for two substances are not identical. Hence they serve as finger prints for
various substances.
2. Used to study the characteristic of polymeric material.
3. This technique is used for testing the purity of the drug sample and also to test the quality
control of number of substances like cement, soil, glass, etc.
4. Used for the determination of heat of reaction, specific heat and energy change occurring
during melting etc.
5. Trend in ligand stability (thermal stability of the ligands) gives the information about the
ligands in the coordination sphere.

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

THERMO-GRAVIMETRIC ANALYSIS

PRINCIPLE

 Thermo-gravimetric analysis, the sample is heated in a given environment (Air, N2, CO2, He,
Ar, etc.) at controlled rate. The change in the weight of the substance is recorded as a
function of temperature or time.
 The temperature is increased at a constant rate for a known initial weight of the substance
and the changes in weights are recorded as a function of temperature at different time interval.
 This plot of weight change against temperature is called thermo-gravimetric curve or
thermo-gram, this is the basic principle of TGA.
 TGA curve
 The instrument used for themo-gravimetry is a programmed precision balance for rise in
temperature known as Thermo-balance.
 Results are displayed by a plot of mass change versus temperature or time and are known
as Thermogravimetric curves or TG curves.

APPLICATION

1. From TGA, we can determine the purity and thermal stability of both primary and
secondary standard.
2. Determination of the composition of complex mixture and decomposition of complex OR
composition of complex systems.
3. For studying the sublimation behavior of various substances.
4. TGA is used to study the kinetics of the reaction rate constant.
5. Used in the study of catalyst: The change in the chemical states of the catalyst may be
studied by TGA techniques. (Zn- ZnCrO4) Zinc-Zinc chromate is used as the catalyst in the
synthesis of methanol.
6. Analysis of the dosage form.
7. Oxidative stability of materials.
8. Estimated lifetime of a product.

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 KJELDAHL METHOD OF NITROGEN ESTIMATION PHARMACEUTICAL ANALYSIS

21 KJELDAHL
METHOD OF NITROGEN
ESTIMATION

INTRODUCTION

 This method was specifically developed for determining the nitrogen contents in organic and
inorganic substances.
 Kjeldahl nitrogen determinations are used on several samples like waste water, soil,
fertilizers, meat, feed, grain, and many other substances. The method is also used for estimating
the protein content in foods.

PRI NCIP LE
Kjeldahl method is divided into three main steps:
1. Digestion: In this method, a certain substance or sample is heated in the presence of sulphuric
acid
2. Distillation: The distillation of the solution now takes place and a small quantity of sodium
hydroxide is added to convert the ammonium salt to ammonia
3. Titration: The amount of ammonia or the amount of nitrogen present in the sample is then
determined by back titration.

Kjeldahl’s
Trap

Contents of
Kjeldahl’s flask
after digestion Condenser
+
NaOH

Known volume
of standard
acid

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

FORMULA FOR CALCULATION

 The percentage of nitrogen can be determined using the given formula:

1.4 V * N
Percentage of nitrogen in the sample =
W
Where,
V = acid used in titration (ml)
N = normality of standard acid
W = weight of sample (g)

LIMITATIONS
 While the Kjeldahl method of nitrogen analysis has become the worldwide standard, this
method is not suitable for compounds containing nitrogen in azo and nitro groups or in rings
(quinoline, pyridine, etc.).

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64
 OXYGEN FLASK COMBUSTION METHOD PHARMACEUTICAL ANALYSIS

22 OXYGEN
FLASK COMBUSTION
METHOD
INTRODUCTION

 The oxygen flask method for the determination of halogens and sulfur in organic compounds
consists of a combustion procedure followed by appropriate titrimetric determination.
 Combustion of the organic material in oxygen yields water-soluble inorganic products,
which are determined as directed for the individual element
 Determination of F, Cl, I, sulphur, mercury, phosphorus, arsenic, carbon, and boron in the
organic compounds.
 The apparatus is known as Schoniger apparatus.

IGNITION
SAMPLE POINT
SAMPLE IN
PAPER
WARPPER
PLATINUM
SAMPLE CARRIER
ABSORPTION
LIQUID
STOPPER WITH
SAMPLE GROUND JOINT
FOLDER IN
PAPER WRAPPER

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

23 PHARMACOPOEIAL
ASSAYS

NON AQUEOUS TITRATIONS

S. NO. NAME OF THE DRUG TITRANT

1 Acetazolamide 0.1 MN (C4H9)4 OH [tetrabutyl
ammonium hydroxide]
2 Adrenaline 0.1 MHClO4
3 Albendazole 0.1 MHClO4
4 Allopurinol 0.1 MN(C4H9)4 OH
5 Amiloride HCl 0.1 MHClO4
6 Aminocaproic acid 0.1 MHClO4
7 Amitripty line HCl 0.1 MHClO4
8 Astemizole 0.1 MHClO4
9 Atenolol 0.1 MHClO4
10 Atropine metuonitrate 0.1 MHClO4
11 Benzhexol HCl 0.1 MHClO4
12 Bisacodyl 0.1 MHClO4
13 Bromocriptinemesylate 0.1 MHClO4
14 Bupremorphine HCl 0.1 MHClO4
15 Caffeine 0.1 MHClO4
16 Calcium Pantothenate 0.1 MHClO4
17 Carbenoxolone sodium 0.1 MN(C4H9)4 OH
18 Carbidopa 0.1 MHClO4
19 Chlordiazepoxide 0.1 MHClO4
20 Chlorhexidine gluconate 0.1 MHClO4
21 Chloroquine PO43- 0.1 MHClO4
22 Chlorpheniramine maleate 0.1 MHClO4
23 Chlopromazine HCl 0.1 MHClO4
24 Chlorthalidone 0.1 MN(C4H9)4 OH
25 Cimetidine 0.1 MHClO4
26 Clofazimine 0.1 MHClO4
27 Clotrimazole 0.1 MHClO4
28 Codeine PO43- 0.1 MHClO4
29 Colchicine 0.1 MHClO4
30 Cyclizine HCl 0.1 MHClO4
31 Cyproheptadine HCl 0.1 MHClO4

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66
 PHARMACOPOEIAL ASSAYS PHARMACEUTICAL ANALYSIS

32 Cytarabine 0.1 MHClO4

33 DehydroemetineHCl 0.1 MHClO4
34 Dequalinium Cl 0.1 MHClO4
35 Diazepam 0.1 MHClO4
36 Diclofenaec sodium 0.1 MHClO4
37 Dicyclomine HCl 0.1 MHClO4
38 Diethyl carbamazine 0.1 MHClO4
39 Di- iodohydroxyquinoline 0.1 MN(C4H9)4 OH
40 Diloxanide furoate 0.1 MN(C4H9)4 OH
41 Diphenhydramine HCl 0.1 MHClO4
42 Dothiepin HCl 0.1 MHClO4
43 Doxepin HCl 0.1 MHClO4
44 Econazole NO3 0.1 MHClO4
45 Emetine HCl 0.1 MHClO4
46 Ergometrine maleate 0.1 MHClO4
47 Ergotaminetartarate 0.1 MHClO4
48 Ethambutol HCl 0.1 MHClO4
49 Ethionamide 0.1 MHClO4
50 Ethopropazine HCl 0.1 MHClO4
51 Ethosuximide 0.1 MN(C4H9)4 OH
52 Fenfluramine 0.1 MHClO4
53 Fluorouracil 0.1 MN(C4H9)4 OH
54 Fluphenazine decanoate 0.1 MHClO4
55 Gallamine triethiodide 0.1 MHClO4
56 Glycine 0.1 MHClO4
57 Haloperidol 0.1 MHClO4
58 Histamine PO43- 0.1 MHClO4
59 Homatropine HBr 0.1 MHClO4
60 Hydro chlorothiazide 0.1 MN(C4H9)4 OH
61 Hyoscine butyl bromide 0.1 MHClO4
62 Idoxuridene 0.1 MN(C4H9)4 OH
63 Imipramine HCl 0.1 MHClO4
64 Isoprenaline 0.1 MHClO4
65 Isoxsuprine 0.1 MHClO4
66 Ketamine HCl 0.1 MHClO4
67 Ketaconazole 0.1 MHClO4
68 Labetalol HCl 0.1 MHClO4
69 Levodopa 0.1 MHClO4
70 Lignocaine HCl 0.1 MHClO4
71 Mebendazole 0.1 MHClO4
72 Mebeverine HCl 0.1 MHClO4
73 Meclizine HCl 0.1 MHClO4
74 Mepyraminemaleate 0.1 MHClO4
75 Mercaptopurine 0.1 MN(C4H9)4 OH
76 Metformin HCl 0.1 MHClO4
77 Methadone HCl 0.1 MHClO4
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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

78 Methoxamine HCl 0.1 MHClO4

79 Methyldopa 0.1 MHClO4
80 Metoprololtartarate 0.1 MHClO4
81 Metronidazole
0.1 MHClO4
82 Miconazole NO3-
83 Morphine SO42- 0.1 MHClO4
84 Neostigmine Br- - do-
85 Niclosamide 0.1 MN(C4H9)4 OH
86 Nicolinamide 0.1 MHClO4
87 Nikethamide 0.1 MHClO4
88 Nitrazepam - do-
89 Noradrenalinebirartarate - do-
90 Norfloxacin - do-
91 Nortriptyline HCl - do-
92 Noscapine 0.1 MHClO4
93 Oxprenolol HCl - do-
94 Penicillamine - do-
95 Pentamidineissothionate 0.1 MN(C4H9)4 OH
96 Pentazocine 0.1 MHClO4
97 Pethidine HCl - do-
98 Phenformin HCl - do-
99 Phenindamine maleate 0.1 MHClO4
100 Pheniramine maleate 0.1 MHClO4
101 Phentolamine mesylate 0.1 MN(C4H9)4 OH
102 Pholcodine 0.1 MHClO4
103 Physostigminesalicylate 0.1 MHClO4
104 Pilocarpine NO3 0.1 MHClO4
105 Piperazine adipate 0.1 MHClO4
106 Potassium citrate - do-
107 Prazosin 0.1 MHClO4
108 Primaquin PO43- - do-
109 ProchlorperazineMaleate - do-
110 Proguanine HCl - do-
111 Propantheline Br- - do-
112 Propylphenazone - do-
113 Psuedo ephedrine HCl - do-
114 PYridenine HCl - do-
115 Pyrimethamine - do-
116 Quinidene SO42- - do-
117 Quinine bisulphate - do-
118 Quiniodochlor 0.1 MN(C4H9)4 OH
119 Salbutamol 0.1 MHClO4
120 CH3COONa - do-
121 Sodium benzoate - do-
122 Sodium CMC - do-
123 Sodium citrate - do-
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 PHARMACOPOEIAL ASSAYS PHARMACEUTICAL ANALYSIS

124 Sodium cromoglycate - do-

125 NaF - do-
126 Sodium salicylate - do-
127 Sodium valproate - do-
128 Succiyl choline chloride - do-
129 Sulfafurazole 0.1 MN(C4H9)4 OH
130 Tamoxifen citrate 0.1 MHClO4
131 Terbutaline SO 2-4 - do-
132 Thiobendazole - do-
133 Thiamine HCl - do-
134 Thiopentone NA 0.1 M Limethoxide
135 Timolol maleate 0.1 MHClO4
136 Tinidazole - do-
137 Trimterene - do-
138 Trifluoperazine HCl - do-
139 Triflupromazine HCl - do-
140 Trimethoprim 0.1 MHClO4
141 Tripolidine HCl - do-
142 Tropicamide - do-
143 Verapamil HCl - do-
144 Xylometazole - do-

COMPLEXOMETRIC TIRATIONS

S.NO CHEMICAL TITRANT

1 Al2(SO4)3 0.5 M disod. edetate
2 Bismuth subcarbonete 0.1 M disod. edetate
3 CaCO3 0.05 M disod. edetate
4 CaCl2 0.05 M disod. edetate
5 Calcium gluconate 0.05 M disod. edetate
6 Calcium Lactate 0.05 M disod. edetate
7 Calcium levulinate 0.05 M disod. edetate
8 Dibasic Ca phosphate 0.1 M disod. edetate
9 Heavy MgCO3 0.05 M disod. edetate
10 MgCl2 0.05 M disod. edetate
11 Mg(OH)2 0.05 M disod. edetate
12 Heavy MgO 0.05 M disod. edetate
13 Magnesium stearate 0.1 M disod. edetate
14 MgSO4 0.05 M disod. edetate
15 Magnesium trisilicate 0.05 M disod. edetate
16 ZnCl2 0.1 M disod. edetate
17 ZnO 0.1 M disod. edetate
18 Zinc stearate 0.1 M disod. edetate
19 ZnSO4 7H2O 0.1 M disod. edetate

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

ACID BASE TITRATIONS

S.NO NAME OF DRUGS TITRANT

1. Amantadine 0.1 M NaOH
2. NH4Cl 0.1 M NAOH
3. Aspirin 0.5 M NaOH (back titration)
4. Benzoic acid 0.5 M NaOH
5. Boric acid 1 M NaOH
6. Bromohexine HCl 0.1 M NaOH
7. Bupivocaine 0.01 M ethanolic NaOH
8. Busulphan 0.1 M NaOH
9. Calamine 0.5 M H2SO4
10. Chlorambucil 0.1 M NaOH
11. Chlorpropamide 0.1 M NaOH
12. Citric acid 1 M NaOH
13. Clonidine 0.1 M ethanolic NaOH
14. Cycloserine 0.1 M NaOH
15. Dextromethorphan 0.1 M NaOH
16. Ephedrine 0.1 M NaOH
17. Ethacrynic acid 0.1 M HCl
18. Ethinyl oestradiol 0.1 M NaOH
19. Flurbiprofen 0.1 M NaOH
20. Frusemide 0.1 M NaOH
21. Fusidic acid 0.1 M NaOH
22. Glibenclamide 0.1 M NaOH
23. Glycerime 0.1 M NaOH
24. HCl 0.1 M NaOH
25. Ibuprofen 0.1 M NaOH
26. Indomethacin 0.1 M NaOH
27. Ketoprofen 0.1 M NaOH
28. Lactic acid 1 M NaOH
29. Levamisole HCl 1 M NaOH
30. Lithium Carbonate 1 M HCl
31. Mefenamic acid 0.1 M NaOH
32. MephentermineSO42- 0.05 M H2SO4
33. Methylsalicylate 0.1 M NaOH (back titration)
34. MetoclopromideHCl 0.1 M NaOH
35. Mianserin HCl 0.1 M NaOH
36. Nalidixic acid 0.1 M ethanolic NaOH
37. Nicolinic acid 0.1 M NaOH
38. Norethisterone 0.1 M NaOH
39. Oxypnenbutazone 0.1 M NaOH
40. Pentobarbitontesodium 0.1 M ethanolic NaOH
41. Phenobarbitone 0.1 M ethanolic NaOH
42. Phosphoric acid 1 M NaOH
43. Phthalyl sulphathiazole 0.1 M NaOH
44. Probenecid 0.1 M NaOH
45. Promethazine 0.1 M NaOH

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 PHARMACOPOEIAL ASSAYS PHARMACEUTICAL ANALYSIS

46. Propranolol HCl 0.1 M NaOH

47. Propyl thiouracil 0.1 M NaOH
48. Pyrazinamide 0.05 M H2SO4 (back titration)
49. Quinal barbitone sodium 0.1 M ethanolic NaOH
50. Saccharin 0.1 M NaOH
51. Salicylic acid 0.1 M NaOH
52. NaHCO3 1 M HCl
53. NaH2PO4.2H2O 1 M NaOH
54. Sodium fusidate 0.1 M HCl
55. NaOH 1 M HCl
56. Sod. Lactate injection 0.05 M H2SO4 (back titration)
57. Urea 0.1 M HCl
58. Sorbic acid 0.1 M NaOH
59. Tartaric acid 1 M NaOH
60. Theophylline 0.1 M NaOH
61. Thiotepa 0.1 M HCl
62. Tolbutamide 0.1 M NaCOH
63. Undeconic acid 0.5 M NaCOH
64. Warfarin sodium 0.01 M NaOH
65. Vanillin 0.1 M NaOH

REDOX TITRATIONS

1 Ascorbic acid Tablets 0.1 M cerric ammonium sulphate

2 Ferrous fumarate -do-
3 Ferrous gluconate 0.1 M cerric ammonium nitrate
4 Ferrous sulphate - do –
5 H2O2 soln. (100vol) 0.02 M KMnO4
6 Menadione 0.1 M cerric ammonium sulphate
7 Nifedipine - do –
8 Paracetamol - do –
9 Tocopheryl acetate - do –

NITRITE TITRATIONS

1 Benzocaine 0.1 M NaNO2

2 Dapsone 0.1 M NaNO2
3 Procainamide HCl 0.1 M NaNO2
4 Procaine HCl 0.1 M NaNO2
5 Sod PAS 0.1 M NaNO2
6 Succinylsulphathiazole 0.1 M NaNO2
7 Sulphacetamidesodium 0.1 M NaNO2
8 Sulphadiazine 0.1 M NaNO2
9 Sulpha dimethoxine 0.1 M NaNO2
10 Sulphadimidine 0.1 M NaNO2
11 Sulphadoxine 0.1 M NaNO2

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

12 Sulphamethizole 0.1 M NaNO2

13 Sulphamethoxazole 0.1 M NaNO2
14 Sulfaphenazole 0.1 M NaNO2

POTENTIOMETRIC TITRATIONS

1 Acebutolol HCl 0.1M NaOH

2 Dipenoxylate HCl 0.1M rhanolic NaOH
3 Hydralazine HCl 0.05M HIO3
4 Phenytoin Sodium 0.1M NaOH

ARGENTOMETRIC TITRATIONS

1 NaCl 0.1M AgNO3

2 AgNO3 0.1M Ammonium thiocyanate
3 KCl 0.1M AgNO3
4 Melphalan 0.1M AgNO3
5 Disulfiram 0.1M AgNO3
6 Lomustine 0.05M AgNO3

IODOMETRY & IODIMETY TITRATIONS

1 Analgin Iodimetry 0.05M I2

2 Ascorbic acid Iodimetry 0.05M I2
3 Cephalexin Iodimetry 0.02M Na2S2O3
4 Chlorocresol Iodimetry 0.1M Na2S2O3
5 Chloroxylenol Iodimetry 0.1M Na2S2O3
6 Dimercaprol Iodimetry 0.1M Na2S2O3
7 Guaiphenesin Iodimetry 0.05M I2
8 I2 Iodimetry 0.1M Na2S2O3
9 Mannitol Iodimetry 0.05M I2
10 Methyl paraben Iodimetry 0.1M Na2S2O3
11 Monothioglycerol Iodimetry 0.05M I2
12 Phenol Iodimetry 0.1M Na2S2O3
13 KI Iodimetry 0.05M KIO3
14 KMnO4 Iodimetry 0.1M Na2S2O3
15 Sod. ascorbate Iodimetry 0.05M I2
16 Sod. diatrizoate Iodimetry 0.05M KIO3
17 Sorbitol Iodimetry 0.05M I2
18 Na2S2O35H2O Iodimetry 0.05M I2
19 Sodium metabisulphite Iodimetry 0.1M I2
20 Thyroxine sodium Iodimetry 0.1M Na2S2O3

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 PHARMACOPOEIAL ASSAYS PHARMACEUTICAL ANALYSIS

DRUGS ANALYZED BY GRAVIMETRIC ANALYSIS

1. Thioacetazone sodium. 2. Fluorescein 3. BaSO4

DRUGS ANALYZED BY HPLC

1. Alprazolam 2. Cefadroxil 3. Cefazolin sodium 4. Cefotaxime sodium

5. Ceftazidime 6. Cefuroxim 7. Ca Folinate 8. Cholecalciferol
9. Ciprofloxacin 10. Cisplatin 11. Diltiazem 12. Doxorubicin HCl
13. Enalapril 14. Ergocalciferol 15. Guaggul resin 16. Insulin
maleate
17. Methotrexate 18. Opium 19. Omeprazole 20. Ormeloxifene HCl
21. Piroxica 22. Ranitidine 23. Vinblastin SO 2- 4 24. Vincristine SO 2- 4

MICROBIOLOGICAL ASSAYS

NAME OF THE DRUG MICRORGANISM USED

Amikacin Staphylococcus aureus
Amphotericin B Saccharomyces cerevisiae
Erythromycin Micrococcus luteus
Doxycycline HCl Staphylococcus aureus
Gentamicin SO42- eye drops Staphylococcus epidermidis
Kanamycin SO42- B. pumilus, S. aureus
Kanamycin B B. subtilis
Novobiocin Na S. epidermidis
Nystatin Saccharomyces cervisiae
Neomycin Sraphylococus epidermides
Rifampicin B. subtilis
Streptomycin SO42- Klebsiella pneumonia & B. subtilis
Tetracycline Staphylococcus aureus, B. cereus

SPECTROMETRY

NAME OF THE DRUG ABSORBANCE

1. Amodiaquine HCl 343nm
2. Calciferol Capsules 500 & 550nm
3. Carbamezapine 285nm
4. Carbimazole 291nm
5. Chloramphenicol 278nm
6. Cyanocobalamn 361nm
7. Danazol 285nm
8. Deslanoside 420nm
9. Dienoestrol 245nm
10. Digitoxin 495nm

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PHARMACEUTICAL ANALYSIS GPAT DISCUSSION CENTER : MAKES STUDY EASY

11. Dydrogesterone 286nm

12. Folic acid 550nm
13. Furazolidine 367nm
14. Gresiofulvin 291nm
15. Hydroxocobalamin 351nm
16. Hydroxyprogesterone hexanoate 240nm
17. Lanatoside C 484nm
18. Levonorgestrol 241nm
19. Megesterol acetate 287nm
20. Methylergometrine maleate 550nm
21. Nalorphine HCl 285nm
22. Nandrolone decanoate 239nm
23. Nitrofurantoin 367nm
24. Nitrofurazone 375nm
25. Norgesterol 241nm
26. Oestradiol benzoate 231nm
27. Phenolphthalein 555nm
28. Reserpine 388nm
29. Riboflavin 444nm
30. Rifampicin 475nm
31. Spironolactone 238nm

MISCELLANEOUS

NAME OF THE DRUG TITRANT

Thiomersal 0.1 M ammonium thiocyanate
Sodium stilbogluconate 0.05 M ferric ammonium SO42-
Sodium lauryl sulphate 0.04 Benzethomium Cl-
Phenyl mercuric acetale 0.1 M amm thiocyanate
Isoniazid 0.0167 M KBrO3
Desferrioxamine mesylate 0.1 M ferric ammonium sulphate
Cyclophosphamide 1.1 M AgNO3
Cloxacillin sodium 1.2 M mercuric NO -3
Cetrimide 0.05 M KIO3
Captopril 0.025 M KIO3

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