6.4 QUALITATIVE QUANTITATIVE ANALYSIS.pdf

MODULE 6│PHARMCHEM 3 QUALITATIVE & QUANTITATIVE ANALYSIS QUALITATIVE AND QUANTITATIVE ANALYSIS C. ERRORS IN ANALYSIS I. INTRODUCTION...

MODULE 6│PHARMCHEM 3 QUALITATIVE & QUANTITATIVE ANALYSIS QUALITATIVE AND QUANTITATIVE ANALYSIS C. ERRORS IN ANALYSIS I. INTRODUCTION Types of Errors: A. DEFINITION a. Random (Intermediate) Errors Due to uncontrollable variables Qualitative Analysis Quantitative Analysis Variations in a series of observations (by the same Reveals the identity of the Indicates the amount of observer under identical conditions) sample elements and each substance in the Affect measure precision compounds in a sample sample Presence or absence of a Exact amount or proportion component of component (expressed in E.g., USP ID Tests 1% purity and compared to official compendia) E.g., Gravimetric, Volumetric, Physicochemical and Special methods of analysis B. TYPES OF ANALYSIS 1. Based on the amount of sample b. Systematic (Determinate) Errors With definite value and identifiable cause a. Ultra-micro: < 1.0 mg Same magnitude or replicate measurements made the b. Micro: 1.0 to 10 mg same way c. Semimicro/ Meso: 10 to 100 mg It can lead to bias and can affect accuracy of results d. Macro: 100 to 1000 mg Sources: Instrumental Errors Constituent types by analyte level Method Errors a. Major: 1 to 100% Personal Errors b. Minor: 0.01 (100ppm) to 1% c. Trace: 11 ppb to 100 ppm c. Gross Errors d. Ultratrace: < 1 ppb Occur only occasionally, are often large, and may cause a result to be either high or low (can lead to 2. Based on extent outliers) Often the product of human errors For Crude Drugs: Proximate Assay Accuracy and Precision Total of class of plant principles (group of compounds) E.g., Total alkaloidal content in coffee beans Accuracy Closeness of an actual value to the theoretical (true) value Ultimate Assay and is expressed by error Single chemical species (specific component) Measures agreement between the result and the accepted E.g., total caffeine content in coffee beans value Absolute Error: E = │X1 – X2│ For Chemical Drugs: Relative Error: ER = │X1 – X2│ x 100 Proximate X2 Partial – selected or trace compounds Precision Closeness of 2 or more actual measurements obtained in Complete – each constituent exactly the same way Describes the reproducibility of measurements 3. Based on nature Reported as: average deviation, standard deviation, coefficient of variation or range a. Chemical/ General Methods titration, gravimetry b. Instrumental Methods UV-Vis, IR, MS, Chromatography c. Special Methods for natural products; Ash content, Water content, constants for fats and fixed oils) 4. Based on material a. Chemical chemical reagents b. Physical Boiling Point, Melting Point, optical purity, Refractive Index c. Biological potency or effectiveness of drugs: Animal models (e.g., chicken – oxytocin, Sheep – heparin); Microbial Assay - antibiotics Module 6 – Qualitative & Quantitative Analysis Page 1 of 6 RJAV 2022 Measures of Central Tendency 4. Modest cost 5. Reasonable solubility in titration medium Mean 6. Reasonably large molecular weight so that relative error in Average or arithmetic mean weighing is minimized Obtained by dividing the sum of replicate measurements by the number of measurements in the set Standardization Computation 𝑁𝑜. 𝑜𝑓 𝑒𝑞𝑢𝑖𝑣𝑞𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 Median 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 (𝑁) = 𝐿 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 Middle result when replicate data are arranged in increasing or decreasing order NOTE: 𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑡 = 𝑊𝑡 𝑜𝑟 𝑊𝑡 𝑥𝑓 𝑀𝑊/𝑓 𝑀𝑊 Less affected by extreme values (outliers) 𝑊𝑡 𝑥𝑓 II. GENERAL METHODS 𝑁 = 𝑀𝑊 𝐿 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 A. TITRIMETRY 𝑊𝑡 𝑀= 𝑀𝑊 Titrimetric (Volumetric) Analysis 𝐿 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 Method in which the volume of a solution of known concentration consumed during analysis is taken as the 𝑁=𝑀𝑥𝑓 amount of active constituent in the sample Equivalence Factors (F) A.k.a. Reaction capacity values Number of reacting entities per reagent a. Acids: f = no. of replaceable H+ Examples: HCl f=1 H2SO4 f=2 CH3COOH f = 1 H3PO4 f = 2* H3BO3 f = 1* Titrant b. Bases: f = no. of replaceable OH Aka Volumetric solution/ Standard solution Examples: Reagent of known concentration Na(OH) f=1 Mg(OH)2 f=2 Titrand Al(OH)3 f=3 Aka Analyte/ Active constituents NH3 f=1 Sample being analyzed + H2O → NH4OH → NH4+ + OH- Indicators c. Salts: f = total (+) or (-) charges Compounds capable of changing colors near or at the end Examples: point NaCl f=1 MgO f=2 Equivalence Point End Point MgSO4 f=2 Aka Stoichiometric point Actual point at which Ca3(PO4)2 f = 6 Theoretical point at equivalent amounts of the which equivalent amounts analyte and titrant have d. Oxidizing Agents: f = no. e gained of the analyte and titrant reacted Examples: have reached Point where a physical Permanganate: MnO4- → Mn2+ f = 5 N1V1 = N2V2 or change occurs that is M1V1 = M2V2 associated with the Dichromate: Cr2O72- → 2Cr3+ f=6 condition of chemical Bromate” BrO3- → Br f=6 NOTE: Molarity is used when the equivalence Ceric: Ce4+ → Ce3+ f=1 stoichiometric ratio between titrant Iodine: I2 → 2I- f=2 and analyte is 1:1 Ex. Analyte: 10mL, 0.1M HCl e. Reducing Agents: f = no. e lost Titrant: 0.1M NaOH Examples: Equivalence point = 10mL End point: Ferrous: Fe2+ → Fe3+ f=1 1) 9.9mL Oxalate: C2O42- → 2CO2 f=2 2) 10.1mL Thiosulfate: 2S2O32- → S4O62- f=2 Ave = 10mL Arsenite: AsO2- → AsO3- f=2 Titanous: Ti3+ → Ti4+ f=1 Standardization Process of determining the exact concentration of a solution Classification based on the Number of Titration Primary Standard Secondary Standard 1. Direct Titration – 1 titrant/VS Substance of high degree Standard solutions whose 𝑀𝑊 𝑊𝑡 of purity purity has been 𝑁𝑥𝑉𝑥 𝑥𝐹 𝑓 𝑥 1000 Serves as a reference determined by chemical %𝑃 = 𝑥 100 𝑁 = 𝑀𝑊 material (standard) in analysis 𝑊𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 𝐿 titrations Used in indirect Used in direct standardization purposes 2. Residual Titration – 2 titrant/VS standardization purposes A.k.a. Back titration 1st VS added in excess; Important requirements for a Primary Standard: 2nd VS used to titrate the excess (unreacted) 1st VS (back titrant) 1. High purity Indications: 2. Atmospheric stability Insoluble sample Volatile sample 3. Absence of hydrate of water (so that composition of solid will Resection too slow not change in variations of humidity) It does not give a sharp end point Module 6 – Qualitative & Quantitative Analysis Page 2 of 6 RJAV 2022  𝑀𝑊 Iodimetry (𝑁1𝑉1 − 𝑁2𝑉2) 𝑥 𝑓 𝑥 1000 Assay for RA: Rxn: I2 + RA(sx) → 2I- %𝑃 = 𝑥 100 𝑊𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 VS: I2 1° std: As2O3 (Arsenic trioxide) Residual Titration with Blank Indicator: starch (appearance of blue) 𝑀𝑊 𝑁 𝑏𝑎𝑐𝑘𝑡𝑖𝑡𝑟𝑎𝑛𝑡 𝑥 (𝑉𝑏 − 𝑉𝑎) 𝑥 Example Assay 𝑓 𝑥 1000 - Direct: Vit C. %𝑃 = 𝑥 100 𝑊𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 - Residual: NaHSO3, methionine Iodometry Blank Determination Assay for OA: Rxn: 2I- + OA(sx) → I2 For correction I2 + 2S2O32- → 2I- + S4O62- To enhance the reliability of the end point VS: Na2S2O3 (Sodium thiosulfate) 1° std: K2Cr2O7 (K Dichromate) Indicator: starch (disappearance of blue) Classification based on the Reactions involved Example Assay: 1. Acid-Base (Neutralization) - Direct: CuSO4, NaOCl 2. Oxidation-Reduction (Redox) - Residual: Phenol, resorcinol, Thyroid hormones, SeS2 3. Complexation 4. Precipitation Cerimetry VS: Ce(SO4)2 ACID-BASE TITRATION 1° std: As2O3, Fe fillings (old) Indicator: o-phenanthroline (Ferroin) Endpoint: red to blue Acidimetry Alkalimetry Measurement of a base by a Measurement of an acid by Example Assay standard acid standard base - Direct: FeSO4, FeSO4 tab, Hydroquinone, Menadione Indicators (Aqueous) – uses water as solvent COMPLEXATION TITRATION SA + SB = Phenolphthalein, Methyl red/orange Compleximetry / Chelometry pH acid base VS: EDTA P (8-10) Colorless Pink/ red 1° std: CaCO3 MO/ MR Red Yellow Indicator: (3.2-2.4) eriochrome blact T (EBT) – Mg, Zn (4.2-6.2) hydroxynaphthol blue (HNB) -Ca dithiozone (DT) – Al, Bi WA + SB = Phenolphthalein WA + SA = methyl red/ orange Example Assay WA + WB = not employed - Direct: ZnO, Bi content of Glycobiarsol (Non-aqueous) – nonpolar solvent; NOTE: very weak acid/ base In all EDTA complexation reaction, ratio of EDTA to metal is 1:1 Non-aqueous Acidimetry – Crystal violet [EDTA] = molarity Non-aqueous Alkalimetry – Thymolthalein, Thymol blue, Azoviolet Special Technique: Acidimetry Aqueous Non-aqueous Masking Determination of a metal in the presence of another metal VS: HCl/ H2SO4 VS: HClO4 (perchloric acid) in GAA 1° std: Na2CO3, TRIS/THAM 1° std: K biphthalate (KHP) 2° std: NaOH VS 2° std: - Masking Agents: Triethanolamine: Fe, Mn, Al Example Assay Example Assay Thioglycols: Hg, Cu, Pb, Bi - Direct: NaOH, KOH, - Direct: Methacholine, K CN: Cu, Co, Ni, Zn Ca(OH)2, NaHCO3 acetate, Diphenoxylate F- (NH4F): Ca, Mg, Al - Residual: ZnO, NaKC4H4O6 Diazepam - Special Tech: Double indicator for mixed alkali PRECIPITATION TITRATION Alkalimetry Argentometric methods – determination of halides Aqueous Non-aqueous Volhard Mohr Fajans VS: NaOH VS: Na methoxide (in EtOH) or Li 1° std: K biphthalate (KHP) methoxide (in MeOH) Titration Residual Direct Direct 2° std: HCl/ H2SO4 VS 1° std: Benzoic acid VS NH4SCN AgNO3 AgNO3 2° std: - 1° std AgNO3 NaCl NaCl Example Assay Indicator Ferric ammonium K2CrO4 TS Dichlorofluorescein, - Direct HCl, H2SO4, H3PO3, Example Assay sulfate Eosin Y, TEE H3BO3 - Direct: Phenytoin, (adsorption - Residual: Aspirin & Aspirin Ethosuximide, Amobarbital indicators) tab, Parabens End point Reddish brown Brick red ppt Green to Pink (dcf) REDOX TITRATION B. GRAVIMETRY Permanganometry Sx + precipitant → ppt → dry wt → %P sample VS: KMnO4 1° std: Na2C2O4 (Sodium Oxalate) NOTE: Dried to constant wt = 2 consecutive weighing should nmt Indicator: none (self-indicating) 0.5mg/g of substance taken (USP) End point: Slight pink color Jenkins: nmt 0.0002 g or 0.2mg Example Assay: - Direct: H2O2 Gravimetric Factor (GF) - Indirect: Malic acid content of cherry juice - Residual (Oxalic Acid VS-back titrant): KNO2, NaNO2, KMnO4 𝑀𝑊 𝑠𝑎𝑚𝑝𝑙𝑒 𝐺𝐹 = 𝑀𝑊 𝑝𝑝𝑡 Module 6 – Qualitative & Quantitative Analysis Page 3 of 6 RJAV 2022 Examples: NaCl + AgNO3 → AgCl ↓ + NaNO3 NaCl: AgCl 1:1 𝑀𝑊 𝑁𝑎𝐶𝑙 (𝑠𝑎𝑚𝑝𝑙𝑒) 𝐺𝐹 = 𝑀𝑊 𝐴𝑔𝐶𝑙 (𝑝𝑝𝑡) BaCl2 + AgNO3 → 2AgCl ↓ + Ba(NO3)2 BaCl2: AgCl 1:2 𝑀𝑊 𝐵𝑎𝐶𝑙2 (𝑠𝑎𝑚𝑝𝑙𝑒) 6. Period (p) – the time required for one cycle to pass a fixed 𝐺𝐹 = point in space 𝑀𝑊 𝐴𝑔𝐶𝑙 (𝑝𝑝𝑡) 𝑥 2 7. Frequency (v) – the number of cycles which pass a fixed III. INSTRUMENTAL AND SPECIAL METHODS point in space per second A. SPECTROSCOPY The Wave Equation A branch of science that studies the interaction between 𝑐 = λ / 𝑝 or 𝑐 = λ 𝑣 electromagnetic radiation and matter. Since the speed of light in vacuum is constant, there is an inverse relationship between wavelength and frequency. Principle of Spectroscopy: 𝑣 ∝ 1/ λ The higher the frequency, the shorter the wavelength the intensity of radiant energy transmitted, reflected, or The longer the wavelength, the lower the frequency emitted is related to the concentration of the chemical species that absorbs energy Planck’s Equation Chromophore – functional group that absorbs maximum radiation in the UV or visible regions The energy of EMR is given by this equation: Auxochrome – functional group which does not give rise to 𝐸 = ℎ𝑣 an absorption band by itself, but upon being attached to a chromophore where h = Planck’s constant (6.6 x 10-34 joule sec) v = frequency (Hz) Absorption and Intensity Shifts Energy is directly proportional to frequency Energy is inversely proportional to wavelength Electromagnetic Spectrum Electromagnetic Radiation (EMR) A form of energy that has both wave and particle properties Described by means of a classical sinusoidal wave model Region Wavelength Parts and properties of a Wave Ultraviolet 180 – 380 Visible 380 – 780 Near IR 780 – 3,000 Medium IR 3,000 – 15,000 Far IR 15,000 – 300,000 Units for λ: nanometer (nm), micrometer (μm), angstrom (Å) 1 Å = 0.1 nm 1. Crest – point with the max upward (+) displacement Fundamental Laws of Spectrometry 2. Trough – point with the max downward (-) displacement 3. Amplitude (A) – the maximum height of a wave 1. Beer’s Law 4. Wavelength (λ) – the distance between 2 identical adjacent Transmittance decreases exponentially as the point in a wave concentration of the solution increases arithmetically 2. Lambert’s Law Transmittance decreases exponentially as the thickness of the solution increases arithmetically 3. Beer-Lambert Law 𝐴 =ℇ𝑏𝑐 where A = absorbance = log 1/T ℇ = molar absorptivity (L/ mol-cm) b = thickness (cm) c = concentration (mol/L) 5. Wavenumber (k) – the number of cycles per unit distance Module 6 – Qualitative & Quantitative Analysis Page 4 of 6 RJAV 2022 Sample Problem: Particles should be as small and Using a spectrophotometer to measure the concentration of a homogenous as possible sample, the following data were obtained: 2. Mobile Phase (MP) Absorbance of the standard solution was 0.55 Fluid that moves through or over Absorbance of the sample was 0.58 the surface of the stationary Concentration of the standard used was 16.5 mcg. phase May be liquid or gas The concentration of the sample was? 3. Solute Mixture 𝐴 𝑠𝑡𝑑 𝐴 𝑠𝑥 0.55 0.58 Components must be in solution or = → = 𝒙 = 𝟏𝟕. 𝟒𝒎𝒄𝒈 vapor state 𝐶 𝑠𝑡𝑑 𝐶 𝑠𝑥 16.5𝑚𝑐𝑔 𝑥 The relatively affinity of the solutes for each of the Instrumentation phases must be reversible Classification of Chromatographic Methods Based on Nature of SP 1. Normal-Phase – SP is polar 2. Reverse-Phase – SP is nonpolar 1. Source of radiation Based on equilibrium process Generates a beam of radiation 1. Adsorption a. Continuum sources (ex: deuterium lamp, 2. Partition tungsten lamp, Nernst glower) 3. Ion Exchange b. Line sources (ex: hollow cathode) 4. Pore Penetration/ Permeation 5. Affinity 2. Wavelength Selector Isolates a restricted region of the spectrum Chromatographic Methods a. Filters – dedicated to a single band of wavelength b. Monochromators – designed for spectral 1. Adsorption Chromatography scanning (ex: prisms, gratings) The SP is a solid on which the sample is adsorbed while the MP may be a liquid or a gas 3. Sample cell a. Thin Layer Chromatography Holds the sample to be analyzed b. Column Chromatography a. Quartz cuvette – UV or visible region c. HPLC b. Silicate glass or plastic cell – visible region d. Gas Chromatography c. NaCl or KBr plates – IR region a. Thin Layer Chromatography 4. Radiation Detector SP: solid adsorbent spread thinly on a glass or plastic plate Converts radiant energy into a usable electrical signal Ex: silica gel, alumina CaCO3 a. Phototubes MP: organic solvent which is less polar than the SP b. Photomultipliers Ex: hexane, ethyl acetate c. Photodiodes – more sensitive TLC Analysis 5. Signal processor and readout Based on the determination of retention factor 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 Displays the transduced electrical signal into numbers 𝑅𝑓 = Ex: charge transfer devices 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡 Analytical Methods Method Application Mass Spectrometry For analysis of gaseous ions NMR Spectroscopy For structure elucidation UV-Vis Spectrometry For quantitative and qualitative analysis Infrared Spectroscopy For determination of functional groups Atomic Absorption For quantitative determination of metals Spectrometry Other Methods For normal-phase chromatography: If Rf is high → nonpolar Method Description Application If Rf is low → polar Fluorometry Measurement of excess Thiamine energy lost by emission Riboflavin Sample Problem (fluorescence) Distance traveled by solvent front = 4.5 cm Turbidimetry Measurement of transmitted Antibiotics light in a suspension Vitamins Distance traveled by Component 1 = 2.2 cm Nephelometry Measurement of reflected Distance traveled by Component 2 = 3.9 cm light in a suspension What are the Rf values for Components 1 and 2? B. CHROMATOGRAPHY 2.2 𝑐𝑚 3.9 𝑐𝑚 𝑅𝑓1 = = 𝟎. 𝟒𝟗 𝑅𝑓2 = = 𝟎. 𝟖𝟕 4.5 𝑐𝑚 4.5 𝑐𝑚 Aa procedure by which solutes are separated by a differential migration process in a system consisting of 2 or b. Column Chromatography more phases Used to separate compound from mixture SP: solid adsorbent packed in a vertical glass column Components Ex: silica gel, alumina CaCO3 MP: organic solvent added to the top and flows down 1. Stationary Phase (SP) through the column Fixed bed of large surface area Ex: hexane, ethyl acetate May be a porous or finely divided solid or a liquid that has been coated in a thin layer om an inert supporting material Module 6 – Qualitative & Quantitative Analysis Page 5 of 6 RJAV 2022 c. High Performance Liquid Chromatography (HPLC) 4. Iodine Value Widely used and preferred current assays of biological and grams of iodine absorbed by 100g of sample pharmaceutical product quantitative measure of unsaturated fatty acids SP: column packed with glass or plastic beads coated with silica derivatized with nonpolar functional group Classification MP: liquid pumped through the column with high pressure Drying oil Semidrying oil Nondrying oil Reverse-phase: water, methanol, acetonitrile Iodine Value > 120 100-120 < 100 Normal-phase: hexane, diethyl ether Examples Linseed oil Cottonseed oil Olive oil Cod liver oil Sesame oil Almond oil d. Gas Chromatography Methods: Used to analyze volatile substance USP Method I: Hanus Method – iodobromide TS SP: either a solid adsorbent or liquid ion an inert support USP Method II: Wijs Method – iodochloride TS MP: chemically inert carrier gas (helium or nitrogen) Unofficial Method: Hubl Method – HgCl2 + I2 Formula: 2. Partition Chromatography 𝑁 𝑥 (𝑉𝑏 − 𝑉𝑎)𝑥 0.1269 𝐼𝑉 = 𝑥 100 Based on partition between 2 immiscible solvents 𝑊𝑡𝑠𝑎𝑚𝑝𝑙𝑒 Both SP and MP are in liquid form Sample Problem a. Paper Chromatography Determine the iodine value of an unknown sample of oil weighing SP: water molecules bound to the cellulose of the filter paper 0.17g if 36mL and 17mL of 0.1100N of sodium thiosulfate are MP: nonpolar or hydrophobic solvent required for the blank and residual titration respectively 0.11 𝑥 (36 − 17)𝑥 0.1269 3. Ion Exchange Chromatography 𝐼𝑉 = 𝑥 100 = 𝟏𝟓𝟔 0.17 Used for the separation of charged molecules (ions) based their binding to fixed charges on a support C. ASH AND MOISTURE CONTENT DETERMINATION Uses: Most effective method for water purification 1. Ash Content Separation of amino acids Residue left after incineration of an organic material which SP: employs either a cationic or an anionic exchanger which represents the amount of inorganic impurity is capable of exchanging counter ions in the surrounding medium in a reversible process Types: Total Ash 4. Permeation Chromatography Residue after incinerating at 325 ± 25℃ Molecules are separated according to their size by their Acid-Insoluble Ash ability to penetrate a sieve-like structure Residue after boiling the total ash with 3N HCl and igniting Ex: Size Exclusion Chromatography the remaining insoluble matter Water-Soluble Ash 5. Affinity Chromatography Difference in weight between total ash and residue after Utilized highly specific interactions between one kind of treatment of total ash with water solute molecule and a second molecule covalently attached to the SP Temperature Equivalence: Ex: protein affinity chromatography Flame Color Temperature ℃ Very dull red 500-550 IV. SPECIAL METHODS Dull red 550-700 Bright red 800-1000 A. ASSAY OF VOLATILE OILS Yellow red 1000-1200 White 1200-1600 1. Alcohol acetalization method 2. Moisture Content Methods: 2. Aldehyde and Ketone Method I – Karl Fischer Titrimetry bisulfite method (Cassia flask) or hydroxylamine method Method II – Azeotropic Distillation (titration) Method III – Gravimetry 3. Phenol D. NITROGEN CONTENT DETERMINATION KOH method (Cassia flask) 𝑉𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑉𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 Kjeldahl Method %𝑃ℎ𝑒𝑛𝑜𝑙 = 𝑥 100 For quantitative determination of nitrogen in organic 𝑉𝑠𝑎𝑚𝑝𝑙𝑒 substance. 4. Volatile Oil in Spirit Babcock bottle Conversion of Organic N into NH4+ by adding H2SO4 Digestion Sample Problem In phenol content determination of a volatile oil, the insoluble layer in Conversion of NH4+ into ammonia the graduated neck of the Cassia flask reached 3.1mL which is Distillation obtained from a sample of 10mL after treatment with KOH solution. The percentage of phenol sample is: Titrating with sulfuric acid 10𝑚𝐿 − 3.1𝑚𝐿 Titration %𝑃ℎ𝑒𝑛𝑜𝑙 = 𝑥 100 = 𝟔𝟗% 10𝑚𝐿 B. ASSAY OF FATS AND FIXED OILS 1. Acid Value mg of KOH needed to neutralize free acids in 1g of sample 2. Ester Value mg of KOH needed to saponify the esters in 1g of sample 3. Saponification Value/ Koettstorfer Number mg of KOH needed to neutralize and saponify the esters in 1g of sample 𝑆𝑉 = 𝐴𝑉 + 𝐸𝑉 Module 6 – Qualitative & Quantitative Analysis Page 6 of 6 RJAV 2022