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

Qualitative Analysis
• Reveals the identity of the
sample elements and
compounds in a sample
• Presence or absence of a
component
• E.g., USP ID Tests
• Due to uncontrollable variables
Quantitative Analysis • Variations in a series of observations (by the same
• Indicates the amount of observer under identical conditions)
each substance in the • Affect measure precision
sample
• Exact amount or proportion
of component (expressed in
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
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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 𝑊𝑡 𝑊𝑡
NOTE: 𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑡 = 𝑜𝑟 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
• A branch of science that studies the interaction between
electromagnetic radiation and matter.
Principle of Spectroscopy:
• the intensity of radiant energy transmitted, reflected, or
emitted is related to the concentration of the chemical
species that absorbs energy
• Chromophore – functional group that absorbs maximum
radiation in the UV or visible regions
• Auxochrome – functional group which does not give rise to
an absorption band by itself, but upon being attached to a
chromophore
Absorption and Intensity Shifts
The Wave Equation
𝑐 = λ / 𝑝 or 𝑐 = λ 𝑣
Since the speed of light in vacuum is constant, there is an inverse
relationship between wavelength and frequency.
𝑣 ∝ 1/ λ
• The higher the frequency, the shorter the wavelength
• The longer the wavelength, the lower the frequency
Planck’s Equation
The energy of EMR is given by this equation:
𝐸 = ℎ𝑣
where h = Planck’s constant (6.6 x 10-34 joule•sec)
v = frequency (Hz)
• 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:

Method Description
Fluorometry Measurement of excess
energy lost by emission
(fluorescence)
Turbidimetry Measurement of transmitted
light in a suspension
Nephelometry Measurement of reflected
light in a suspension
• If Rf is high → nonpolar
Application • If Rf is low → polar
Thiamine
Riboflavin Sample Problem
Distance traveled by solvent front = 4.5 cm
Antibiotics
Vitamins
Distance traveled by Component 1 = 2.2 cm
Distance traveled by Component 2 = 3.9 cm
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