Pharmaceutical analysis Unit 1

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PHARMACEUTICAL ANALYSIS:

Pharmaceutical analysis may be defined as the application of analytical procedures used to determine the purity, safety and quality of drugs and chemicals. The term "Pharmaceutical Analysis" is otherwise called Quantitative pharmaceutical chemistry. Pharmaceutical analysis includes both qualitative and quantitative analysis of Drugs and Pharmaceutical substances starts from bulk drugs (starting material) to the finished dosage forms. In modern practice of medicine, the analytical methods are used in the analysis of chemical constituents found in the human body whose altered concentrations during disease states serve as diagnostic aids and used to analyze the medicinal agents and their metabolites found in biological system.

Pharmaceutical analysis is a branch of practical chemistry that involves a series of process for identification, determination, quantification and purification of a substance, separation of the components of a solution or mixture, or determination of structure of chemical compounds.The substance may be a single compound or a mixture of compounds and it may be in any of the dosage form. The substance used as pharmaceuticals are animals, plants, micro organisms, minerals and various synthetic products.

Thesampletobeanalysediscalledas analyte andonthebasisofsizeofsample,theycanbeclassified as macro (0.1 g or more), semi micro (0.01 g to 0.1 g), micro (0.001 g to 0.01 g), sub micro (0.0001 g to 0.001 g), ultramicro (below 10-4 g), trace analysis (100 to 10000 ppm). Among all, the semi micro analysis is widely used.

In modern practice of pharmacy, it is important that Pharmacists have more than an appreciation of quantitative analytical methodology. The term Quality as applied to a drug product has been defined as the sum of all factors which contribute directly or indirectly to the safety, effectiveness, and reliability of the product. These properties are built into drug products through research and during the manufacturing process by procedures collectively referred to as "Quality Control". Quality control guarantees within reasonable limits that a drug product:

1. Is free from impurities

2. Is physically and chemically stable

3. contains the number of active ingredient(s) as stated on the label

4. provides optimal release of active ingredient(s) when the product is administered.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

1 Unit I | PharmaceuticalAnalysis

TYPES OFANALYSIS

The two main types of are:

1. Qualitative (identification)

2. Quantitative (estimation)

1. Qualitative analysis is performed to establish composition of natural/synthetic substances. These tests are performed to indicate whether the substance or compound is present in the sample or not. Various qualitative tests are detection of evolved gas, formation of precipitates, limit tests, colour change reactions, melting point and boiling point test etc.

2. Quantitative analytical techniques are mainly used to quantify any compound or substance in the sample. These techniques are based in (a) the quantitative performance of suitable chemical reaction and either measuring the amount of reagent added to complete the reaction or measuring the amount of reaction product obtained, (b) the characteristic movement of a substance through a defined medium under controlled conditions, (c) electrical measurement, (d) measurement of some spectroscopic properties of the compound.

Qualitative Analysis

In chemistry, qualitative analysis is a branch of the subject that examines the chemical makeup of a material

Qualitative analysis determines the presence or absence of several chemical components in a sample through analytical techniques

Qualitative analysis employs procedures such as distillation, extraction, color change, chromatography, and so on

Quantitative Analysis

In chemistry, quantitative analysis is a section of the subject that deals with the quantities of various components in a sample

Quantitative analysis determines the amount of various chemical components contained in a sample

Titrations, gravimetric analysis, combustion analysis, and other techniques are used in quantitative analysis in chemistry

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

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SCOPE OF PHARMACEUTICAL ANALYSIS

1. In Pharmaceutical Industry: There are different sectors in pharmaceutical industry as research and development (R&D) and Quality control (QC) in which pharmaceutical analysis is utilizes regularly. Pharmaceutical Analysis is one of the most sorts after specializations in Master of Pharmacy. People specialized in pharmaceutical analysis are indispensable to the manufacturing, quality control and analytical manifestations of the industry. They can work in quality control department which oversees the purity, qualitative aspects and the matching of the stringent regulatory limits required by a finished product. Research and development have huge implications on the results of the analysis and detection of new compounds. More and more companies are stressing on a separate analytical R&D department. Pharmaceutical analysis students also finds takers in the medical device’s companies, equipment companies, regulatory agencies etc.

2. In Food Industry: As we all know packed food which consumed by consumer should have all parameters like quality, purity and safety which enhance acceptability by consumer. For this it is require analyzing all these parameters for packed food.

3. In Cosmetic Industry: Preparation of cosmetics, as lipsticks, creams, nail-paints, lotions, shampoo and conditioners etc., play with two things as colour and odour and these coloring agents and fragrances are also built by different chemical ingredients so the quality and quantity of these ingredients should be known which can be analyze by different techniques of analysis.

4. In Environmental study: Pharmaceutical analysis have different techniques which can be applicable in environmental studies as to check pH of rain, river and water resources. Different environmental factors like temperature, humidity etc can be analyze by analytical method.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

3 Unit I | PharmaceuticalAnalysis

DIFFERENT TECHNIQUES OF ANALYSIS

a. The manual method of analysis

The manual method of analysis: Taste, smell, texture, color, and appearance are some of the organoleptic qualities that the senses can perceive.

b. Chemical methods

(i) Titrimetric or volumetric method

It involves reaction of substance to be determined with an appropriate reagent as a standard solution, and volume of solution required to complete the reaction is determined. Volumetric methods require simple and less apparatus and they are susceptible of high accuracy.

Various types of titrimetric methods are:

i)Acid-basetitrations(neutralizationreactions)

ii) Complexometric titrations

iii) Precipitation titrations

iv) Oxidationreductiontitrations

v) Non aqueous titrations

(ii) Gravimetric methods

In gravimetric analysis, a substance to be determined is converted into an insoluble precipitate in the purest form, which is then collected and weighed. It is the time-consuming process.

In electrogravimetry, electrolysis of the sample is carried out on the electrodes is weighed after drying.

Thermogravimetry (TG) records the change in weight, differential thermal analysis (DTA) records the difference in temperature between test substance and an inert reference material, differential scanning calorimetry (DSC) records the energy needed to establish a zero-temperature difference between a test substance and reference material.

(iii)

Gasometric analysis

Gasometry involves measurement of the volume of gas evolved or absorbed in a chemical reaction. Some of the gases which are analysed by Gasometry are CO2 , N2O, cyclopropane, amyl nitrate, ethylene, N2, helium etc.

c) Instrumental analysis method:

Instrumental methods are classified based on the property of the analyte to be tested. Many of the techniques apply neighboring to both qualitative and quantitative research.

(i) Spectroscopy: Nuclear Magnetic Resonance, Atomic Absorption Spectroscopy, UltravioletVisible Spectrophotometry, Fluorescence Spectroscopy, Magnetic Resonance Spectroscopy

(ii) Electrochemical Analysis

(iii) Thermal Analysis

(iv) Separation

(v) Microscopy

(vi) Precipitation Analysis

(vii) Colorimetric Analysis

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter

(B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

4 Unit I | PharmaceuticalAnalysis

(i) Spectroscopy: Spectroscopy is a technique for determining how molecules deal with electromagnetic radiation. Spectroscopy encompasses a wide range of techniques, including atomic absorption spectroscopy, atomic emission spectroscopy, ultravioletvisible spectroscopy, x-ray spectroscopy, fluorescence spectroscopy, infrared spectroscopy, Raman spectroscopy, dual-polarization interferometry, nuclear magnetic resonance spectroscopy, and photoemission spectroscopy. Some of the spectroscopy is discussed below.

➢ Nuclear Magnetic Resonance: Different spectral regions of absorption correspond to different physical processes occurring within the analyte. In the presence of a magnetic field, absorption of energy in the radiofrequency region is sufficient to cause a spinning nucleus in certain atoms to shift to a different spin state. As a result, nuclear magnetic resonance spectrometry may be used to determine the number and types of various nuclei present in the groups attached to the atom containing the nucleus under investigation. It's particularly useful for analyzing organic compounds qualitatively.

➢ Atomic Absorption Spectroscopy: Atomic absorption spectroscopy (AAS) andatomic emission spectroscopy (AES) are Spectro analytical procedures that use the absorption of optical radiation (light) by free atoms in the gaseous state to determine chemical elements quantitatively. The absorption of light by free metallic ions is the basis for atomic absorption spectroscopy. The technique is used in analytical chemistry to determine the concentration ofa certainelement (the analyte) ina sample to beanalyzed.

➢ Ultraviolet-Visible Spectrophotometry: UV–Vis spectroscopy, also known as ultraviolet-visible spectrophotometry (UV–Vis or UV/Vis), is the study of absorption and reflectance spectroscopy in the ultraviolet and visible regions of the electromagnetic spectrum. This means it makes use of visible and neighboring light.

➢ Fluorescence Spectroscopy: Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is an electromagnetic spectroscopy technique for analyzing fluorescence in a sample. It entails the use of a beam of light, generally ultraviolet light, to excite the electrons in certain compounds' molecules and cause them to emit light; normally, though not always, visible light.

➢ Magnetic Resonance Spectroscopy: The spectroscopic technique of nuclear magnetic resonance spectroscopy, also known as NMR spectroscopy or magnetic resonance spectroscopy (MRS), is used to observe local magnetic fields around atomic nuclei. The Nuclear magnetic resonance signal is provided by excitation of the nuclei sample with radio waves into nuclear magnetic resonance, which is detected with sensitive radio receivers, and the sample is placed in a magnetic field. The resonance frequency of an atom in a molecule is changed by the intramolecular magnetic field surrounding it, allowing access to knowledge of the molecule's electronic structure and individual functional groups. The spectroscopic technique of nuclear magnetic resonance spectroscopy, also known as Nuclear magnetic resonance spectroscopy or magnetic resonance spectroscopy (MRS), is used to observe local magnetic fields around atomic nuclei. The NMR signal is provided by excitation of the nuclei sample with radio waves into nuclear magnetic resonance, which is detected with sensitive radio receivers, and the sample is placed in a magnetic field. The resonance frequency of an atom in a molecule is changed by the intramolecular magnetic field surrounding it, allowing access to knowledge of the molecule's electronic structure and individual functional groups.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

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(ii) Electrical methods

Electrical methods of analysis involve the measurement of electric current, voltage or resistance in relation to the concentration of some species in the solution. Electrical methods of analysis include: (a)Potentiometry (b)Conductometry (c)Polarography (d)Voltammetry (e)Amperometry

Potentiometry measures electrical potential of an electrode in equilibrium with an ion to be determined. Conductometry measures electrical conductivity of an electrode with a reference electrode while Polarography, Voltammetry and Amperometry measures electrical current at a micro-electrode.

(iii) Thermal Analysis

The interaction of a substance and heat is measured using calorimetry and thermogravimetric analysis.

(iv) Separation

Separation methods are used to make content mixtures less complicated. This field includes chromatography, electrophoresis, and field flow fractionation.

(v) Microscopy

In analytical science, visualization of single molecules, single cells, biological tissues, and nanomaterials is an effective and appealing approach. Analytical science is now being revolutionized by the hybridization of other conventional analytical methods. Optical microscopy, electron microscopy, and scanning probe microscopy are the three different types of microscopies. Because of the rapid growth of the computer and camera industries, this sector has recently accelerated.

(vi) Precipitation Analysis

Cations are generally classified according to their properties into one of six groups and anions into one of three groups. The details of the classification vary slightly from source to source. Each group has a common reagent that can be used to separate cations or anions in solution. Separations must be performed in a specific order to achieve meaningful results. This is because some ions from the former group may react with reagents from the latter group. This is because cation analysis is based on ionic solubility products. When the cations reach the desired optimal concentration, they are precipitated for detection. Copper in the precipitation analysis wire displaces silver, silver is immersed in silver nitrate solution, and precipitates solid silver. Sediment analysis can be used to determine the chemical composition of a solution. The cationic group includes:

Cation forms insoluble chlorides such as lead, silver, and mercury. Formation of acid-insoluble sulfides such as cadmium, bismuth, copper, antimony, and tin. With the formation of insoluble hydroxide complexes such as iron, aluminum and chromium.

Adding ammonia chloride, ammonium hydroxide, and hydrogen sulfide gases to zinc, nickel, cobalt, and manganese will determine the element. The colour of the sediment indicates the metal. Insoluble carbonate group. (Many initial cations precipitate as carbonate but will be detected up to this point if you follow the steps inacertainorder.)Barium,calcium,andstrontiumprecipitateatthispoint,butnotearlier.Magnesium,lithium, sodium, potassium, and ammonium are difficult to precipitate and are usually identified by the colour of the flame. There are three groups of negative ions, and there are several ways to detect them. However, deposition methods such as those mentioned above are often used.

(viii) Colorimetric Analysis

Usingacolored reagent,colorimetricanalysismeasurestheconcentrationofachemicalinasolution.Inorganic and organic compounds are examined in this way. In medical laboratories and industrial water treatment, this method is widely used to analyze water samples.

d) Biological and microbiological methods

Biological methods are used when potency of a drug or its derivative cannot be properly determined by any physical or chemical methods. They are called bioassays.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

6 Unit I | PharmaceuticalAnalysis

Microbiological methods are used to observe potency of antibiotic or anti- microbial agents. In antimicrobial assay, inhibition of growth of bacteria of the sample is compared with that of the standard antibiotic. These methods include cup plate method and turbidimetric analysis.

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7 Unit I | PharmaceuticalAnalysis

APPLICATIONS

➢ Manufacturing industries require both qualitative and quantitative analysis to ensure that their raw materials meet certain specifications, and to check the quality of final product.

➢ Raw materials are to be checked to ensure that the essential components are present within the predetermined range of composition and there are not any unusual substances present which might upset the manufacturing process or it may appear as a harmful impurity in the final product.

➢ In the development of new products which contains mixtures other then the pure material, it is necessary to ascertain composition of mixture which shows the optimum characteristics for which the material has been developed.

➢ Geographical surveys require analysis to determine the composition of soil sample and numerous rock samples collected from the field.

➢ Most of the industrial processes give rise to pollutants which may cause health related problems. So quantitative analysis of air, water and soil sample should be carried out to determine the level of pollution and to establish the safe limits for pollutants.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

8 Unit I | PharmaceuticalAnalysis

METHODS OF EXPRESSING CONCENTRATION

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

9 Unit I | PharmaceuticalAnalysis

PRIMARY AND SECONDARY STANDARDS STANDARDS:

In Pharmaceutical Analysis, the word standard means a material containing a substance of our interest with a known concentration We can express this with definite numbers with proper units.

FUNCTIONS:

➢ To provide a reference using which we can determine unknown

➢ Concentration of solution

➢ To standardization of volumetric solutions

➢ Preparation of secondary standard

➢ To calibrate an instrument

PRIMARY STANDARDS:

➢ Primary standard is a reagent which is very pure, generally representative of the number of moles the substance contains and easily weighed.

➢ A Primary standard is a reagent that’s stable, it’s not a hydrate /has no water of hydration and has a high molecular weight.

➢ Primary standards are typically used in titration to determine an unknown concentration and in other analytical techniques.

➢ High level of purity, low reactivity (high stability), high equivalent weight (to reduce error from mass measurements)

➢ Not hygroscopic (to reduce changes in mass in humid versus dry environments), non-toxic, inexpensive and readily available

➢ It should have a high relative molecular weight so that weighing errors may be negligible.

SECONDARY STANDARDS:

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

➢ A secondary standard is a substance which may be used for standardization.

➢ A secondary standard is a standard that is prepared in the laboratory for a specific analysis. It is usually standardized against a primary standard.

➢ It follows that a secondary standard solution is a solution in which the concentration of dissolved solute has not been determined from the weight of the compound dissolved but by reaction (titration) of a volume of the solution against a measured volume of a primary standard solution.

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10 Unit I | PharmaceuticalAnalysis

PREPARATION AND STANDARDIZATION OF VARIOUS MOLAR AND NORMAL SOLUTIONS

Oxalic acid:

Oxalic acid Solution Preparation

➢ Weigh accurately 6.3 gm of oxalic acid & dissolve in distilled water & finally make up the ➢ volume to one litre in a volumetric flask.

Oxalic acid Solution Standardization

➢ Take 20 ml of prepared oxalic acid in a conical flask

➢ Add 5 ml of sulphuric acid, and warm at 70 0C

➢ Rinse the burette with distilled water and pre rinse with the portion of potassium permanganate soln.

➢ Start the titration with 0.1N KMnO4 until the end point.

➢ End point is the appearance of pink colour that persists for more than 30 seconds.

➢ Record the reading repeat the titration 3 times to get the precise values

Sodium hydroxide:

Sodium Hydroxide Solution Preparation

➢ Take about 100ml of distilled water in a cleaned and dried 1000 ml volumetric flask.

➢ Add about 4.2 gm of Sodium hydroxide with continues stirring.

➢ Add more about 700ml of distilled water, mix and allow to cool to room temperature.

➢ Make up the volume 1000 ml with distilled water. Mix solution thoroughly.

➢ Keep the solution for at least an hour and then carry out the standardization.

Sodium Hydroxide Solution Standardization

➢ Accurately weigh about 0.5 g of potassium biphthalate, previously crushed lightly and dried at 120° for 2 hours.

➢ Dissolve in 75 ml of carbon dioxide free water.

➢ Add 2 drops of phenolphthalein, and titrate with the sodium hydroxide solution to the production of a permanent pink color.

➢ Each 20.42 mg of potassium biphthalate is equivalent to 1 ml of 0.1N sodium hydroxide.

➢ Calculation: Wt. in gm of potassium biphthalate

M= 0.20423 x ml NaOH solution

Hydrochloric acid: Hydrochloric acid Solution Preparation

➢ Take about 100 ml of water in a cleaned and dried 1000 ml volumetric flask.

➢ Add about 8.5 ml of Conc. Hydrochloric acid with continuous stirring.

➢ Add more about 700 ml of water, mix and allow to cool to room temperature.

➢ Make up the volume 1000 ml with water. Mix solution thoroughly.

➢ Keep the solution for at least one hour and then carry out the standardization.

Hydrochloric acid Solution Standardization

➢ Accurately weigh about 0.5 g of THAM (Tris (hydroxymethyl)-amino methane (tromethamine), previously dried at 105° for 3 hours and cooled in a desiccator, transfer to a conical flask.

➢ Dissolve in 50 ml of distilled water.

➢ Add 2 drops of Bromocresol Green indicator.

➢ Titrate with 0.1M Hydrochloric acid to a pale yellow endpoint.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

11 Unit I | PharmaceuticalAnalysis

➢ Each 121.14 mg of tromethamine is equivalent to 1 ml of 0.1M hydrochloric acid.

➢ Calculate the molarity by the following formula: THAM in mg M = 121.14 x HCl in ml

Sodium thiosulphate: Sodium Thiosulphate Solution Preparation

➢ Take about 100 ml of water in a cleaned and dried 1000 ml volumetric flask.

➢ Add about 25 gm of Sodium Thiosulphate with continues stirring.

➢ Add about 0.2 gm of Sodium Carbonate with continues stirring.

➢ Add more about 700 ml of water, mix.

➢ Make up the volume 1000 ml with water. Mix solution thoroughly.

➢ Keep the solution for at least one hour and then carry out the standardization.

Sodium Thiosulphate Solution Standardization

➢ Accurately weigh about 210 mg of primary standard Potassium Dichromate, previously pulverized and dried at 120°C for 4 hours.

➢ Dissolve in 100 ml of distilled water in a glass-stoppered, 500 ml conical flask.

➢ Swirl to dissolve the solid, remove the stopper, and quickly add 3 g of Potassium Iodide, 2 g of Sodium Bicarbonate, and 5 ml of hydrochloric acid.

➢ Insert the stopper gently in the flask, swirl to mix, and allow to stand in the dark for exactly 10 minutes.

➢ Rinse the stopper and the inner walls of the flask with distilled water, and titrate the liberated iodine with the Sodium Thiosulfate solution until the solution is yellowish green in color.

➢ Add 3 ml of starch indicator solution, and continue the titration until the blue color is discharged.

➢ Perform a blank determination.

➢ Calculate the molarity by the following formula:

K2Cr2O7 in mg M = 49.04 x Na2S2O3 in ml

Sulphuric acid:

Sulphuric Acid Solution Preparation

➢ Add slowly, with stirring, 6 ml of Sulphuric acid to about 800 ml of purified water.

➢ Makeup to 1000 ml with purified water.

➢ Allow cooling at 25°C.

Sulphuric Acid Solution Standardization

➢ Weigh accurately about 0.2 g of anhydrous Sodium Carbonate, previously heated at about 270°C for 1 hour.

➢ Dissolve it in 100 ml of water and add 0.1 ml of methyl red solution.

➢ Add the acid slowly from a burette, with constant stirring, until the solution becomes faintly pink.

➢ Heat the solution to boiling, cool and continue the titration.

➢ Heat again to boiling and titrate further as necessary until the faint pink color is no longer affected by continued boiling.

➢ 1 ml of 0.1 M sulphuric acid is equivalent to 0.0098 g of Na2CO3.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

12 Unit I | PharmaceuticalAnalysis

➢ Calculate the molarity of solution by the following formula: Na2CO3 in mg M = H2SO4 in ml x 9.8

Potassium permanganate: Potassium Permanganate Solution Preparation

➢ Dissolve 3.2 g of potassium permanganate in 1000 ml of water.

➢ Heat on a water-bath for 1 hour.

➢ Allow to stand for 2 days and filter through glass wool.

➢ Standardize the solution in the following manner.

Potassium Permanganate Solution Standardization

➢ To 25.0 ml of the solution in a glass-stoppered flask add 2 g of potassium iodide, followed by 10 ml of 1 M sulphuric acid.

➢ Titrate the liberated iodine with 0.1 M sodium thiosulphate, using 3 ml of starch solution, added towards the end of the titration, as an indicator.

➢ Perform a blank determination and make the necessary correction.

➢ Store protected from light.

➢ 1 ml of 0.1 M sodium thiosulphate is equivalent to 0.003161 g of KMnO4.

Ceric ammonium sulphate:

Ceric Ammonium Sulphate Solution Preparation

➢ Dissolve 65 g of ceric ammonium sulfate with the aid of gentle heat, in a mixture of 30 ml of sulphuric acid and 500 ml of water.

➢ Cool, filter the solution, if turbid, and dilute to 1000 ml with water.

➢ Standardize the solution in the following manner.

Ceric Ammonium Sulphate Solution Standardization

➢ Weigh accurately about 0.2 g of arsenic trioxide, previously dried at 105° for 1 hour, and transferred to a 500 ml conical flask.

➢ Wash down the inner walls of the flask with 25 ml of a 8.0 % w/v solution of sodium hydroxide, swirl to dissolve, add 100 ml of water and mix.

➢ Add 30 ml of dilute sulphuric acid, 0.15 ml of osmic acid solution, 0.1 ml of ferroin sulfate solution.

➢ Titrate with the ceric ammonium sulphate solution until the pink color is changed to a very pale blue, adding the titrant slowly towards the endpoint.

➢ 1 ml of 0.1 M ceric ammonium sulphate is equivalent to 0.004946 g of As2O3.

➢ Calculate the molarity of the solution by the following formula: As2O3 in mg

M = Ceric Ammonium Sulphate in ml x 4.946

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

13 Unit I | PharmaceuticalAnalysis

ERRORS

Error is the deviation from the absolute value or from the true average of a large number of results. The skill, knowledge, expertise and above all the degree of confidence involved in the ultimate result of an analyst is solely governed by the extent of accuracy and precision achieved by the analytical procedure vis-a-vis the possible sources of error that may be incorporated inadvertently. In fact, the quantitative pharmaceutical analysis is not merely confined to just taking a random sample, performing a single assay quickly, and finally making a loud claim that the result so obtained cannot be challenged. Truly speaking an ideal analyst must have a total in-depth knowledge of the chemistry involved along with the pros and cons of interferences that may be caused due to the host of compounds, elements and ions besides adequate exposure and hands-on experience of the statistical distribution of values.

The terminology ‘error’ invariably refers to the difference in the numerical values between a measured value and the true value. It has become universally accepted in methods of comparison that the percentage composition of a ‘standard sample’ provided and certified by the National Institute of Standards and Technology (NIST) or the British Pharmacopoea Chemical Reference Substance (BPCRS) or the European Pharmacopoea Chemical Reference Substance (EPCRS) must be regarded and treated as absolutely correct, pure and authentic while evaluating a new analytical method. Consequently, the differences thus obtained between the standard values and those by the new analytical methods are then treated as ‘errors’ in the latest procedure.

Sources of errors:

Sample preparations

Error by analyst

Equipment problem

Calibration

Reporting error

Calculation error

Error in method selection

Sampling error

Laboratory environment

Error during transport

Types of error:

Determinate (systematic) Errors Indeterminate (random) Errors

Personal Errors

Instrumental Errors

Reagent Errors

Constant Errors

Proportional Errors

Errors due to Methodology

Additive Errors

Indeterminate (random) Errors

(i) Determinate (systematic) Errors

Systematic error is predictable and either constant or else proportional to the measurement. Systematic errors primarily influence a measurement's accuracy. Typical causes of systematic error include observational error, imperfect instrument calibration, and environmental interference. For example: • Forgetting to tare or zero a balance produces mass measurements that are always "off" by the same amount. An error caused by not setting an instrument to zero prior to its use is called an offset error.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

14 Unit I | PharmaceuticalAnalysis
➢ Observational ➢ Environmental

• Not reading the meniscus at eye level for a volume measurement will always result in an inaccurate reading. The value will be consistently low or high, depending on whether the reading is taken from above or below the mark.

• Forgetting to tare or zero a balance produces mass measurements that are always "off" by the same amount. An error caused by not setting an instrument to zero prior to its use is called an offset error.

• Not reading the meniscus at eye level for a volume measurement will always result in an inaccurate reading. The value will be consistently low or high, depending on whether the reading is taken from above or below the mark.

• Measuring length with a metal ruler will give a different result at a cold temperature than at a hot temperature, due to thermal expansion of the material.

• An improperly calibrated thermometer may give accurate readings within a certain temperature range but become inaccurate at higher or lower temperatures.

• Measured distance is different using a new cloth measuring tape versus an older, stretched one. Proportional errors of this type are called scale factor errors.

• Drift occurs when successive readings become consistently lower or higher over time. Electronic equipment tends to be susceptible to drift. Many other instruments are affected by (usually positive) drift, as the device warms up.

Various types of Determinates (systematic) Errors has been discussed below:

a. Personal Errors: They are exclusively caused due to ‘personal equation’ of an analyst and have no bearing whatsoever either on the prescribed procedure or methodology involved.

b. Instrumental Errors: These are invariably caused due to faulty and uncalibrated instruments, such as : pH meters, single pan electric balances, uv-spectrophotometers, potentiometers etc.

c. Reagent Errors: The errors that are solely introduced by virtue of the individual reagents, for instance: impurities inherently present in reagents; high temperature volatilization of platinum (Pt) ; unwanted introduction of ‘foreign substances’ caused by the action of reagents on either porcelain or glass apparatus.

d. 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 enhances.

Example: Assuming a constant equivalence point error of 0.10 ml is introduced in a series of titrations, hence for a specific titration needing only 10.0 ml of titrant shall represent a relative error of 1% and only 0.2% for a corresponding 50 ml of titrant consumed.

e. 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.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

15 Unit I | PharmaceuticalAnalysis

Example: Estimation of ‘chlorate’ an oxidant by iodometric determination. In this instance two things may happen, namely: (i) Presence of ‘Bromate’ another oxidizing agent would give rise to positively higher results, and hence, it must be duly corrected for, and (ii) Absolute error might increase while dealing with large samples, whereas the relative error would remain more or less constant if the sample is perfectly homogenous,

f. 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 are cited below :

g. Additive Errors: It has been observed that the additive errors are independent of the quantum of the substances actually present in the assay.

Examples: (i) Errors caused due to weights, and (ii) Loss in weight of a crucible in which a precipitate is incenerated. Detection of this error is ascertained by taking samples of different weights.

(ii) Indeterminate (random) Errors

The main reasons for random error are limitations of instruments, environmental factors, and slight variations in procedure. For example:

➢ When weighing yourself on a scale, you position yourself slightly differently each time.

➢ When taking a volume reading in a flask, you may read the value from a different angle each time.

➢ Measuring the mass of a sample on an analytical balance may produce different values as air currents affect the balance or as water enters and leaves the specimen.

➢ Measuring your height is affected by minor posture changes.

➢ Measuring wind velocity depends on the height and time at which a measurement is taken. Multiple readings must be taken and averaged because gusts and changes in direction affect the value.

➢ Readings must be estimated when they fall between marks on a scale or when the thickness of a measurement marking is considered.

Various types of Determinates (systematic) Errors has been discussed below:

a. Observational: When the observer makes consistent observational mistakes (such not reading the scale correctly and writing down values that are constantly too low or too high)

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b. Environmental: When unpredictable changes occur in the environment of the experiment (such as students repeatedly opening and closing the door when the pressure is being measured, causing fluctuations in the reading).

Methods of Minimizing Errors

(i) Calibration of Instruments, Apparatus and Applying Necessary Corrections: Most of the instruments, commonly used in an analytical laboratory, such as: UV-Spectrophotometer, IR-Spectrophotometer, single pan electric balance, pH-meter, turbidimeter and nephelometer, polarimeter, refractometer and the like must be calibrated duly before use so as to eliminate any possible errors. In the same manner all apparatus, namely: pipettes, burettes, volumetric flasks, thermometers, weights etc., must be calibrated duly, and the necessary corrections incorporated to the original measurements In some specific instances where an error just cannot be avoided it may be convenient to enforce an appropriate correction for the effect that it ultimately causes; for instance : the inherent impurity present in a weighed precipitate can be estimated first and then deducted duly from its weight.

(ii) Performing a Parallel Control Determination: It essentially comprises of performing an altogether separate estimation under almost identical experimental parameters with a quantity of a standard substance that consists of the same weight of the component as is present in the unknown sample. Thus, the weight of the component present in the unknown sample may be calculated with the help of the following expression:

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17 Unit I | PharmaceuticalAnalysis

where, X = Weight of the component present in the Unknown Sample.

(iii) Blank Determination: In order to ascertain the effect of the impurities present in the reagents employed and reaction vessels used ; besides establishing exactly the extent to which an excess of standard solution required to locate the exact end-point under the prevailing experimental parameters of the unknown sample a blank determination is an absolute necessity. It may be accomplished by performing a separate parallel estimation, without using the sample at all, and under identical experimental parmeters as employed in the actual analysis of the given sample.

(iv) Cross-checking Results by Different Methods of Analysis: In certain specific cases tha accuracy of a result may be cross-checked by performing another analysis of the same substance by an altogether radically different method. Examples : (a) HCl-Solution : It may be assayed either by titration with a standard solution of a strong alkali (NaOH), or by precipitation and weighing as AgCl ; and (b) Fe3+ : It may be assayed either by gravimetric method as Fe(III) hydroxide after getting rid of the interfering elements and igniting the precipitate to Fe(III) oxide, or by titrimetric method i.e., first reducing to the Fe(II) state and then titrating with a suitable oxidizing agent, for instance Ce(IV) sulphate, K2Cr2O7. In short, the results thus obtained by the two fundamen-tally different techniques must be concordant thereby justifying and ascertaining the fact that the values obtained are fairly small limits of error.

(v) Method of Standard Addition: Here, a small known quantity of the component under estimation is added to the sample, which is subsequently subjected to analysis for the total amount of component present. The actual differ-ence in the quantity of components present in samples with or without the added component ultimately gives the recovery of the quantum added component. A good satisfactory recovery builds up the confidence in the accuracy of the method of analysis.

(vi) Method of Internal Standards: The specific method is of immense value both in chromatographic as well as spectroscopic determinations. Here, a fixed quantity of a reference substance (i.e., the ‘internal standard’) is added to a series of known concentrations of the material to be assayed. A graph is plotted between the concentration values and the ratios obtained from the physical value (i.e., peak area of absorption) of the ‘internal standard’ and the series of known concentra-tions, thereby producing a straight line. Any unknown concentration may be determined effec-tively by adding the same amount of ‘internal standard’ and locating exactly where the ratio obtained falls on the concentration scale.

Accuracy:

➢ The concordance between the data and the true value.

➢ It is an agreement between the data and true value.

➢ If true value is not known the mean calculated from results obtained from several different analytical methods which are very precise and in close agreement with one another may be considered the true value in practical sense.

➢ The difference between the mean and the true value is known as absolute error.

➢ The relative error is found by dividing the absolute error by the true value.

➢ Accuracy is the agreement between the data the true value.

Precision:

➢ The concordance of a series of measurements of the same quantity.

➢ The mean deviation or the relative mean deviation is a measure of precision.

➢ It is a measure of reproducibility of data within a series of result.

➢ Results within a series which agree closely with one another are said to be precise. Precise results are not necessarily accurate for a determined error may be responsible for the inaccuracy of each result in a series of measurement.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

18 Unit I | PharmaceuticalAnalysis

➢ It is usually reported as the average deviation,standard deviation or range.

➢ Precision is a measure of the agreement among the values in a group of data.

Significant figures:

➢ Each of the digits of a number that are used to express it to the required degree of accuracy, starting from the first non-zero digit.

➢ All non-zero digits are considered significant. For example, 91 has two significant figures (9 and 1), while 123.45 has five significant figures (1, 2, 3, 4 and 5). Zeros appearing anywhere between two non-zero digits are significant: 101.1203 has seven significant figures: 1, 0, 1, 1, 2, 0 and 3.

➢ Example:

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

19 Unit I | PharmaceuticalAnalysis

PHARMACOPOEIA

➢ Pharmacopoeia Derived from Greek word ‘Pharmakon’ means drug and ‘Poiea’ means to make.

➢ It is a legal and official book issued by recognized authorities usually appointed by Government of each country.

➢ It comprises list of pharmaceutical substances, formulae along with their description and standards.

➢ A drug included in pharmacopoeia is termed official and sections dealing with official drugs preparation and substances are known as monograph.

➢ List of pharmacopoeias and some standard reference books common use in india:

• Indian Pharmacopoeia (I.P.)

• British Pharmacopoeia (B.P.)

• United State Pharmacopoeia (U.S.P.)

• European Pharmacopoeia (E.P.)

• International Pharmacopoeia

• Japanese Pharmacopoeia

• Martindale Extra Pharmacopoeia

• British Pharmaceutical Codex

• Pharmaceutical Codex

• Merck Index

• British National Formulary

• United State National Formulary

• National Formulary (N.F.)

• United State Dispensatory

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

20 Unit I | PharmaceuticalAnalysis

• Indian Pharmaceutical Codex (I.P.C.)

• National Formulary if India (N.F.I.)

➢ INDIAN PHARMACOPIA:

• First official Pharmacopeia of India appeared in 1868 which was edited by Edward John Waring.

• In preindependence days, British Pharmacopeia was used in India.

• In 1946 Government of India issued one list known as The Indian Pharmacopeial list‟

• Committee under chairmanship of Sir R. N. Chopra alongwith other nine members prepared „The Indian Pharmacopeial list‟

• It was prepared by Dept. of Health, Govt. of India, Delhi in 1946.

• In 1948 Government of India appointed an Indian Pharmacopeia committee for preparing Pharmacopeia of India‟

• Tenure of this committee was five years. Indian Pharmacopeia committee under chairmanship of Dr. B. N. Ghosh Published first edition of IP in 1955.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

21 Unit I | PharmaceuticalAnalysis

SOURCES OF IMPURITIES IN MEDICINAL AGENTS

Impurities are defined as any component of a pharmaceutical that is different from the entity defined as the active ingredient. Additionally, any ingredient that is not an essential component of a drug product is considered an impurity.

Manufacturers of APIs must watch out for three different kinds of impurities in their products.

Organic components

➢ Organic pharmaceutical impurities are often associated with a processing step or a drug.

➢ As the process of synthesis, purification, and storage of drugs takes place, these contaminants will likely occur.

➢ Chemical impurities are residual solvents leftover after drug compounds have been synthesized or when excipients are used to make drug formulations.

Inorganic impurities

➢ Impurities in organic materials are often the result of manufacturing processes.

➢ You may also find reagents, ligands, catalysts, heavy or residual metals, organic salts, filter aids, and charcoal in the sample.

➢ Analyzing and quantifying inorganic contaminants is possible using pharmacopeial standards.

Residual solvents

➢ As a third type of impurity, residual solvents appear in pharmaceuticals.

➢ A solvent used in pharmaceutical production is divided into three classes according to its toxicity.

➢ Solvents in the class one category should never be used since they are human carcinogens or environmentally hazardous.

➢ Solvents classified as class two should only be used in limited quantities since they could pose a harmful level of toxicity.

➢ Humans are not at risk from class three solvents.

Organic impurities aren't the only impurities found in pharmaceuticals, as it is also possible to find inorganic impurities and residual solvents.

There are many causes of impurities, including manufacturing processes, degradation, storage conditions, and excipients and contaminants. Pharmaceutical products would be unsafe, ineffective, and of poor quality without the identification and elimination of impurities.

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

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LIMIT TESTS

Limit Test: Limit tests are quantitative or semi-quantitative tests designed to identify and control small quantities of impurities, which are likely to be present in the pharmaceutical substance. Impurity: The impurities in pharmaceuticals are the unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during formulation, or upon aging of both API and formulated APIs to medicines. The presence of these unwanted chemicals even in small amounts may influence the efficacy and safety of the pharmaceutical products.

Types of Inorganic Impurities:

1. Toxic impurities: This kind of impurities are very harmful and can even cause death when taken once or short period of time. Example: Arsenic

2. Cumulative impurities: This kind of impurities shows their toxicity when it taken over a period. Example: Heavy Metals

3. Harmless impurities: Some impurities may not cause harm to body but reduce the therapeutic activity of active ingredient when it is present in large quantities. Example: Chlorides, Sulphates On considerations of above classification, the pharmacopoeia has fixed the permissible limits of each impurity. For toxic impurities the permissible limit is as less as 5-10 ppm, whereas for cumulative impurities the permissible limit is 20 ppm. For harmless impurities the limits are still high. Impurity profiling (i.e., the identity as well as the quantity of impurity in the pharmaceuticals), is now getting to receive important critical attention from regulatory authorities because practically it is impossible to remove all the impurities from any pharmaceuticals. Some remain in trace even after purification, so it is only desirable that the substance should be sufficiently pure and can be used safely. The different pharmacopoeias (IP, BP, USP etc.) specify the limits up to which various impurities can be tolerated in pharmaceuticals. The limit tests help to check and indicate the presence of various inorganic impurities in pharmaceuticals. Obviously the quantity of any impurity present in official pharmaceuticals is often small, and therefore, the normal visible-reaction-response to any test for that impurity is also quite small. Hence, it is essential to design the individual test in such a manner so as to avoid possible errors in the hands of various analysts. This is accomplished by taking into consideration the following three cardinal factors:

1. Specificity of the tests: Any test used as a limit test must, of necessity, give some form of selective reaction with the trace impurity. Many tests used for the detection of inorganic impurities in official inorganic chemicals are based upon the separations involved in inorganic qualitative analysis. A test may be demanded which will exclude one specific impurity, but highly specific tests are not always the best; a less specific test, which limits several likely impurities, at once, is obviously advantageous, and in fact can often be accomplished. An example of such a test is the heavy metals test applied to alum, which not only limits contamination by lead, but also other heavy metal contaminants precipitated by thioacetamide as sulphide at pH 3.5.

2. Sensitivity: The degree of sensitivity required in a limit test varies enormously according to the standard of purity demanded by the monograph. The sensitivity of most tests is dependent upon a number of variable factors all capable of strict definition, and all favorable towards the production reproducible results. Thus the precipitation of an insoluble substance from solution is governed by such factors as concentration of the solute and of the precipitating reagent, duration of the reaction, reaction temperature, and the nature and concentration of other substances unavoidably present in solution. As a general rule, cold dilute solutions give light precipitates, whereas more granular ones are obtained from hot concentrated solutions. Many of the limit tests, however, are concerned with very dilute solutions, which are often slow to react, and here sensitivity of the reaction can often be increased by extending the duration of the reaction or by raising the reaction temperature. Similar considerations apply in the design of colour and other tests employed as limit tests.

3. Control of personal errors: It is essential to exclude all possible sources of ambiguity in the description of a test. Vague terms such as 'slight precipitate,' should be avoided as far as possible. The extent of the visible reaction to be expected under the specified test conditions should be clearly and

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23 Unit I | PharmaceuticalAnalysis

precisely defined. This is usually accomplished in one of three ways.

(a) Tests in which there is no visible reaction: A definite statement is incorporated in the wording of the test, which states that there shall be no colour, opalescence or precipitate, whichever is appropriate to the particular test. One example of this type of requirement is the test for barium, and calcium in dilute hypophosphorus acid, where the additions of dilute sulfuric acid under precisely controlled condition shall produce 'no turbidity, or precipitate' within one hour. The time factor is used here as a means of increasing the sensitivity of the test. Tests such as these which give negative results do not necessarily imply the complete absence of the impurity, the test as laid down merely indicating the absence of an undesirably large amount of the impurity.

(b) Comparison methods: Tests of this type require a standard containing a definite amount of impurity, to be set up at the same time and under the same conditions as the test experiment. In this way the extent of the reaction is readily determined by direct comparison of the test solution with a standard of known concentration. The official limit tests for chlorides, sulfates, iron and heavy metals are based on this principle. The limit tests for lead and arsenic are, in practice, also comparison methods. They are, however, so designed that they can be readily applied as quantitative determinations.

(c) Quantitative determinations: Quantitative determination of impurities is only applied in special circumstances, usually in those cases where the limit is not readily susceptible to simple and more direct chemical determination. The method is used in the following different types of tests:

i. Limits of insoluble matter ii. Limits of soluble matter iii. Limits of moisture, volatile matter, and residual solvents iv. Limits of non-volatile matter v. Limits of residue on ignition vi. Loss on ignition vii. Ash values viii. Precipitation methods.

Differences between assay and limit test

1. The assay is quantitative whereas the limit test is semi-quantitative or qualitative test.

2. The assay result provides the exact amount of substances whereas the limit tests for range of impurities.

3. The assay will be done for substances as well as impurities whereas the limit tests particularly for impurities.

Limit Test for Chlorides

Principle: It is based upon the chemical reaction between silver nitrate and soluble chlorides to obtain silver chloride in presence of dilute nitric acid. The silver chloride produced in the presence of dilute nitric acid makes the test solution turbid, the extent of turbidity/opalescence depending upon the amount of chloride present in the substance is compared with a standard turbidity/opalescence produced by addition of silver nitrate to a standard solution having a known amount of chloride and the same volume of dilute nitric acid as used in the test solution. If the turbidity/opalescence from the sample has been less than the standard solution, the sample will pass the limit test and vice versa. Dilute nitric acid is used in the limit test of chloride to make solution acidic and which helps silver chloride precipitate to make solution turbid at the end of process.

Chemical Reaction:

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24 Unit I | PharmaceuticalAnalysis

Limit Test for Sulphates

Principle: The limit test for sulphate is carried out on the basis of the reaction between barium chloride and soluble sulphates in the presence of Acetic acid. Then, the comparison of the turbidity produced by a given amount of the substance is done with a standard turbidity obtained from a known amount of sulphates. The barium chloride has been replaced by barium sulphate reagent which is having barium chloride, sulphate-free alcohol, and a solution of potassium sulphate. Potassium sulphate has been added to increase the sensitivity of the test. The ionic concentrations in the reagent has been so adjusted that the solubility product of barium sulphate gets exceeded, and the very small amount of barium sulphate present in the reagent acts as a seeding agent for precipitation of barium sulphate, if sulphate be present in the substance under test. Alcohol helps to prevent supersaturation and thus produces a more uniform opalescence/turbidity. Acetic acid helps to make solution acidic and barium sulphate precipitate formed is insoluble which gives turbidity/opalescence.

Reactions:

Limit Test for Iron

Principle: It depends on the reaction of iron in an ammonical solution with thioglycollic acid in the presence of citric acid when a pale pink to deep reddish purple colour is produced. The colour is due to the formation co-ordination compound, ferrous thioglycollate which is stable in the absence of air bud fades in air due to oxidation. Therefore, the colour should be compared immediately after the time allowed for full development of colour is over. Ferrous thioglycollate is colourless in neutral or acid solutions. The colour develops only in the presence of alkali. The original state of iron is immaterial, as thioglycollic acid reduces ferric (Fe3+) to ferrous (Fe2+) form. Citric acid forms a soluble complex with iron and prevents its precipitation by ammonia as ferrous hydroxide. Interference of other metal cations is eliminated by making use of citric acid, which forms complex with other metal cations.

Reactions:

Limit Test for Heavy Metals

Principle: The limit test for heavy metals is based on the reaction of metallic impurities with hydrogen sulfide in acidic medium; the reaction product will be the sulphides of the respective metals. The sulphides so formed are distributed in colloidal state and produce brownish or black color solution. Metals that response to this test are lead, mercury, bismuth, arsenic, antimony, tin,

From the desk of Abhijit Sir, Asst. Professor, NIET (Pharmacy Institute), Greater Noida Innovation Ambassador, IIC – Ministry of Education, Govt. of India| Innovation Cell SPOC, NPTEL Local Chapter (B. Pharm, M.Pharm, O-level, HMX Fundamentals (Harvard Medical School, Harvard University, Drug Discovery and Drug Development (University of California San Diego, USA), Drug Discovery and Medical Chemistry (Davidson, USA) Website: theabhi.in Facebook Page: https://www.facebook.com/TheAbhijitDN/ Twitter: https://twitter.com/TheAbhijitDN

25 Unit I | PharmaceuticalAnalysis

cadmium, silver, copper, and molybdenum. The metallic impurities in substances are expressed as parts of lead per million parts of the substance. The usual limit as per Indian Pharmacopoeia is 20 ppm.

Reactions:

Limit Test for Arsenic

Principle: Arsenic is an undesirable and harmful type of impurity in pharmaceutical substances because it is toxic and cumulative in nature. The IP prescribes the limits for the presence of arsenic (NMT 2 ppm) as an impurity in various pharmaceutical substances [for example, NaCl should not contain more than 1 ppm]. When the sample is dissolved in acidic medium, the arsenic present in the sample is converted into arsenic acid. The arsenic acid is reduced by reducing agents (like Zinc and Hydrochloric acid, potassium iodide, stannous chloride) to arsenious acid. The nascent hydrogen (Zinc and Hydrochloric acid) produced in the reaction further reduces arsenious acid to arsine gas, which reacts with mercuric chloride paper producing yellow stain. The depth of yellow stain on mercuric chloride paper will depend upon the quantity of arsenic present in the sample. The intensity of stain produced varies according to the quantity of arsenic present in the sample. The stain is compared with that produced from a known amount of arsenic.

Reactions:

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26 Unit I | PharmaceuticalAnalysis

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