Pharmaceutical Analysis: General & Chemical Tests

• Assay refers to the determination of the content of a specific component without evaluating other components

General Methods in Pharmaceutical Analysis

• Principles of Volumetric analysis include neutralization methods, direct and residual titrations, acidimetry and alkalimetry, and nonaqueous titrations and alkalimetry.
• Precipitation methods include the Mohr and Volhard methods
• Complexation methods include EDTA titration
• Oxidation-Reduction methods include permanganate method, direct, indirect, and residual methods and use oxidizing and reducing agents
• Iodimetry and iodometry
• Cerimetry
• Gravimetric Methods
• Special Methods include ash and water determination, extractive and crude fiber determination.
• Special Methods include determining the constants of fats, assaying volatile oils, and assaying alkaloids and amine drugs.

Chemical Tests

• Quantitative analysis determines the amounts of various substances in a sample.
• Volumetric or titrimetric methods determine the volume of a solution with known concentration required to react with a given amount of substance to be analyzed.
• Gravimetric methods separate the constituent to be determined by extraction, precipitation, or other means, and then weighing the resulting product
• Special methods require distinct techniques such as analysis of crude drugs, assay of fats, fixed oils, volatile oils and alkaloids
• Physico-Chemical methods test the physical and chemical properties of a substance using instrumental tests like spectrometric, chromatographic, electrometric and polarographic.

Principles of Titrimetric (Volumetric) Analysis

• Four requirements must be met for a reaction to be used for titrimetric analysis: the reaction must proceed to completion, it must proceed in a stoichiometric manner, require a suitable endpoint detecting device (indicators) and the reaction must be rapid for a sharp endpoint.
• Volumetric Analysis determines the volume of a solution of known concentration required to react with a given amount of a substance.
• Titrimetric methods uses the volume of a solution of known concentration consumed during analysis to measure the amount of active constituent in a sample analyzed.
• Analyte is the active constituent in the sample
• Titrant is the standard solution of known concentration
• Titration is the process by which a standard solution is brought into reaction until the desired reaction is accomplished
• Indicator is a chemical which changes color at a point when equivalent quantities of analyte and titrant have reacted
• Stoichiometric or Equivalence Point is the theoretical point at which equivalent amounts of reactants have reacted
• Endpoint is the practical, sudden change apparent when using indicators.

Solutions

• Standard Solution is a solution of known normality or molarity
• Normality is one gram equivalent weight of solute in one liter of solution, expressed as eq/L or one gram milliequivalent weight in one mL of solution, expressed as meq/mL.
• Molarity is the number of moles of solute in a liter of solution, expressed as moles/L or mmoles/mL.
• Molality is the number of moles per 1000g of solvent
• Titer is strength in mg of analyte per mL of titrant, or the weight of a substance chemically equivalent to 1 mL of a standard solution, e.g., 84.01mg MaHCO3 mg of analyte/mL of titrant
• Equivalent weight equals molecular weight in grams divided by n
• Milliequivalent weight = equivalent weight divided by 1000
• n is number of reacting entities.
• Normality can be calculated = weight / (Volume x meq. wt.) = g / (mL x g/mole)
• Molarity can be calculated = moles /L = mmoles/mL
• In acids the # of replaceable Hydrogen: for example, HCI = 1, H2 SO4 = 2, CH3 COOH = 1
• In bases the # of replaceable OH: for example, NaOH = 1, Mg(OH)2 = 2, Al(OH)3 = 3
• Salts total positive or negative charges/cation or anion for example: NaCl = 1, MgO = 2, Ca3 (PO4) = 6

Standardization

• Standardization determines the exact concentration of a solution
• Solutions are standardized using primary and secondary standards
• Primary standard: pure solid substance used for direct standardization
• Secondary standard: standard solution of known concentration also known as volumetric solutions (VS)
• A Primary Standard needs to have a high purity (99.9%), definite and known composition, not be affected by drying and soluble in water
• Examples of primary standards are potassium acid phthalate (KHP) / C6H4COOK.COOH, sulfamic acid (HSO3), benzoic acid and potassium acid iodate, used in the standardization of NaOH
• HCL is standardized titrimetrically using sodium carbonate of known purity as a primary standard or using standard NaOH as a secondary standard
• Secondary standards are solutions of known concentration used to standardize another solution

Reagents

• NaOH uses KHP/sulfamic acid
• HCL uses Na2CO3
• AgNO3 uses NaCl
• EDTA uses CaCO3
• KMnO4 uses Na2C2O4
• Iodine uses As2O3
• Sodium Methoxide uses Benzoic acid
• Ceric sulfate uses As2O3
• Karl Fischer reagent uses Sodium tartrate
• Na2S2O3 uses K2Cr2O7

Secondary Standard Reagents

• HCL reagents use NaOH
• NH4SCN reagent uses AgNO3
• Na2S2O3 reagent uses Iodine

Apparatus

• Volumetric apparatus can be "to deliver" such as burets and pipets, or "to contain" such as volumetric flasks and graduated cylinders.
• Burets are graduated glass tubes with a uniform bore, closed at the bottom by glass or stopcock, and volumes are read at the bottom of the meniscus except highly colored liquid
• Burets are calibrated using an Ostwald pipet or Kiehl buret
• Mohr burets are economical, and have a rubber tubing at the tip
• Geissler burets are more expensive, and are for acids and bases
• Volumetric flasks must be calibrated to contain the indicated volume when filled to the mark. They are used to make up standard solutions
• Graduated cylinders are used to make approximate measures of volume
• Pipets must be graduated
• Class A and B pipets

Errors

• Sources of error when using volumetric apparatus includes rinse water adhering to walls of apparatus, grease films, dirty apparatus, parallax, variations in temperature, air bubbles trapped beneath liquid surface and hot solutions.
• Good cleaning solutions include sodium dichromate in sulfuric acid, chromic acid, solutions of trisodium phosphate and synthetic detergent

Titration Types

• Direct titration involves introducing a standard solution gradually from a buret into the assayed substance, and uses one titrant and one volumetric solution.
• Residual is adding an accurately measured quantity of standard solution in excess and titrating the excess with another standard solution, which use two volumetric solutions, where the first is added used in excess and the second to titrate the excess.
• Residual Titration is used whenever the direct titration is not practicable because the compounds react too slowly, have poor solubility or involve volatile substances.
• Blank Determination means repeating the procedure but omitting the sample.
• Direct Titration percent purity = (N x V X meq.wt / wt of sample) x100
• Residual Titration percent purity = ((N1V1 - N2V2) X meq. Wt.) / wt of sample) x100
• Results in drug assays are usually expressed in terms of %w/w (g/g, mg/mg, mg/g), %w/v (g/mL, mg/mL) and %v/v (mL/mL, ML/L)

Neutralization Method

• Neutralization reactions are chemical processes in which an acid reacts with a base to produce water and salt.
• Indicators change color at a point equivalent quantities of analyte and titrant have reacted
• Indicators consist of complex organic compounds used to determine endpoint and pH and to indicate a desired change in pH
• Indicators may be weakly acidic or weakly basic, and are capable of existing in 2 forms of different color that are mutually convertible
• The usual concentration of indicator solution is 0.05% and 0.1 to 0.2 mL of the indicator solution is used for 10 mL of liquid examined
• Solutions of basic indicators and the phthaleins are prepared by dissolving in alcohol
• Acidic indicator solutions must be neutralized with NaOH
• Commonly used pH indicators are Malachite green, Methyl Yellow, Bromophenol blue, Methyl Orange and Bromocresol green

Indicators

• Methyl red has a pH change ranging from 4.2 – 6.2 (red to yellow)
• Bromocresol purple has a pH change ranging from 5.2 – 6.8 (yellow to purple)
• Bromothymol blue has a pH change ranging from 6.0 – 7.6 (yellow to blue)
• Phenol red has a pH change ranging from 6.8 – 8.2 (yellow to red)
• Cresol red has a pH change ranging from 7.2 – 8.8 (yellow to red)
• Thymol blue has a pH change ranging from 8.0 – 9.2 (yellow to blue)
• Phenolphthalein has a pH change ranging from 8.0 – 10.0 (colorless to red)
• Thymolphthalein has a pH change ranging from 9.3 – 10.5 (colorless to blue)
• Phenolphthalein has a pH range of 8.0-9.8, an acid form color of clear and a base form color of red-violet
• Methyl range has a pH range of 4.4-6.2 with an acid form color of red and base form color of yellow
• Use 3 drops of indicator TS unless otherwise directed
• When using a Strong Acid and a Strong Base, use methyl orange, methyl red or phenolphthalein
• SA + WB use methyl red
• WA + SB uses phenolphthalein
• WA + WB = no indicator
• SA: HCI, HNO3, H2SO4, HI
• SB: NaOH, KOH
• WA: acetic acid, oxalic acid
• WB: NH3

Indicator Papers

• Indicator papers include lead acetate test paper, litmus paper, mercuric bromide test paper, phenolphthalein paper, potassium iodate-starch paper, starch iodate paper, starch iodide paper and turmeric paper.
• Two types of Neutralization Method: Acidimetric and Alkalimetric
• Acidimetric Analysis and Acidimetry uses direct or residual titrimetric analysis of BASES using an acid as titrant
• Analyte is a Base
• Titrant is an Acid
• Direct titration uses standard acid from buret into a solution of base being assayed
• Residual Titration uses 1st vol. of acid in excess and titrates the excess acid with standard base
• Organic substances can be analyzed this way such as urea, sodium salicylate, diphenhydramine, emetine hydrochloride, meprobamate, paramethadione, and pyrazinamide
• Inorganic substances can be analyzed this way such as sodium bicarbonate, milk of magnesia, ammonium chloride, calcium hydroxide, lithium carbonate, and zinc oxide
• Alkalimetric Analysis and Alkalimetry are direct and residual titrimetric analyses of acids.
• Direct uses a sample that is acid as a titrant as base
• Residual uses a sample that is acid treated with excess standard base then titrated with acid
• Direct Titration: % Purity = (N X V X meq. wt / wt. of sample) X 100

Nonaqueous Titrimetric Analysis

• For substances that are poorly soluble and weakly reactive in water
• Uses organic solvents which lack moisture which needs to be under 0.05%
• Advantages include overcoming poor solubility and weak reactivity with water combined with simplicity, peed, precision and accuracy
• Disadvantages include moisture competing with the weak nitrogen bases for the HClO4, causing a loss in the endpoint sharpness and requiring greater care in the control of temperature during standardization
• Solvents that can be uses are acetonitrile, alcohol, dioxane, benzene, and chloroform
• Titrants used are perchloric acid in glacial acetic acid or dioxane and hydrogen bromide, for example assay of methacholine chloride
• Indicators are Crystal violet, Quinaldine red, a- naphtholbenzein and Malachite green
• Alkalimetry uses Sodium methoxide and Lithium methoxide as titrants. Example: assay of phenytoin Indicators include azo violet, thymol blue, nitroaniline, thymolphthalein and hydroxyazobenzene.

Nitrogen Determination

• Kjeldahl method is for organic compounds containing nitrogen
• Digest the sample with conc. H2SO4 until decomposition is complete
• NH3 is treated with excess H2SO4 with NaOH
• Potassium sulfate, selenium and copper sulfate hasten the digestion of ammonia with sulfuric acid