Analysis Lab | Pharmaceutical Analysis

PHARMACEUTICAL ANALYSIS LAB NOTES

Experiments 5, 6, 7, 8 & 9

EXPERIMENT 5
Assay of Calcium Carbonate (CaCO₃)
Date: 04/03/2026

Physiological Roles of Calcium Ion (Ca²⁺)

1. Bone Formation and Teeth Mineralization

Calcium is a major structural component of bones and teeth in the form of hydroxyapatite:
Hydroxyapatite = Ca₅(OH)₂(PO₄)₆

When calcium intake is sufficient, blood Ca²⁺ is absorbed into bone tissue. When bone degrades,
hydroxyapatite dissociates, releasing Ca²⁺ back into the bloodstream. When calcium intake is
insufficient, bone dissociates to fill the deficit, which is why calcium supplementation is necessary to
prevent osteoporosis.

2. Muscle Contraction
Actin and myosin are responsible for muscle contraction. Ca²⁺ is essential for this binding mechanism:
• Ca²⁺ activates actin's binding site (troponin-tropomyosin complex shifts)
• Activated actin binds with myosin, causing muscle contraction
• When the stimulus is removed (during muscle relaxation), Ca²⁺ is released back into the
sarcoplasmic reticulum

3. Neurotransmitter and Hormone Release

Ca²⁺ plays a critical role in exocytosis of neurotransmitters and hormones:
• Vesicles containing neurotransmitters/hormones are released from storage
• Calcium-Sensing Receptor (CaSR) detects Ca²⁺ levels
• Ca²⁺ binds and activates CaSR, which then binds to vesicles
• Vesicle membrane fuses with plasma membrane via exocytosis, releasing hormones and
neurotransmitters

Pharmaceutical and Cosmetological Applications of CaCO₃

Cosmetic Applications
i. Cleansing Agent → Tooth Paste / Tooth Powder
Calcium carbonate serves multiple functions in dental hygiene products:
• Acts as a mild abrasive agent for mechanical plaque removal
• Works as a teeth polishing agent
• Carbohydrates from oral bacteria form debris and dental plaque, which cause Ca²⁺ loss from
enamel
 • Bacteria produce lactic acid, which erodes enamel surface and damages teeth
• CaCO₃ reacts with lactic acid to form calcium lactate, H₂O, and CO₂
• This neutralizes lactic acid, preventing enamel damage
∴ CaCO₃ helps prevent dental caries (tooth decay)

Pharmaceutical Applications

1. Nutritional Supplement
When dietary calcium intake is insufficient, bone tissue dissociates to maintain blood calcium levels.
CaCO₃ is used as a calcium supplement to:
• Replenish calcium stores
• Prevent osteoporosis
• Support bone density maintenance

2. Anti-diarrheal / Constipative Agent (Large Dose)

At large doses, CaCO₃ binds to Calcium-Sensing Receptors (CaSR) in the intestine, producing the
following effects:
• Decreases intestinal motility (slows gut movement)
• Increases fluid reabsorption from the intestinal lumen
• Decreases fluid secretion into the intestine
• Net result: reduced water content in stool, decreased stool volume
This mechanism is exploited clinically for its constipating/anti-diarrheal effect.

3. Excipients
CaCO₃ is widely used in tablet manufacturing as:
• Tablet diluent (filler/bulking agent)
• pH modifier
• Coating agent

4. Antacid
CaCO₃ neutralizes excess stomach acid via the following reaction:
CaCO₃ + 2HCl → CaCl₂ + CO₂ + H₂O
This reaction neutralizes hydrochloric acid (HCl) in the stomach, providing rapid relief from heartburn
and indigestion.

Procedure for Assay of CaCO₃

Reagents Required
• 3M HCl (approximately 3 mL)
• 200 mL distilled water (approximately)
• 1M NaOH (15 mL)
• 0.1M Na₂EDTA (titrant)
• Hydroxy Naphthol Blue indicator
Steps
1. Weigh a specified amount of CaCO₃ and place into a conical flask.
2. CaCO₃ does not dissolve in water; to dissolve it, add 3M HCl dropwise until the solution
becomes transparent (maximum 3 mL). CaCO₃ becomes water-soluble after reacting with HCl.
3. Add approximately 200 mL distilled water.
4. Add 15 mL of 1M NaOH.
5. Add indicator: Hydroxy Naphthol Blue.
6. Titrate with 0.1M Na₂EDTA from the burette.

Rationale for Reagents

• HCl: dissolves CaCO₃ (converts it to soluble CaCl₂)
• NaOH: neutralizes excess HCl and creates a basic environment (required for EDTA titration;
chelation is pH-dependent)
• Note: Do not add NaOH before the solution becomes transparent, as the color change at that
step may be confused with the endpoint

Indicator: Hydroxy Naphthol Blue

Mechanism of action:
• Initial color: Reddish pink / wine red (indicator-metal complex is formed)
• Titrate with 0.1M Na₂EDTA
• EDTA has higher affinity for Ca²⁺ than the indicator
• As EDTA is added, it competes with and displaces Ca²⁺ from the indicator-metal complex
• Indicator is released (free indicator has a different color)
• End point: Deep blue color (sometimes described as deep violet)

Mechanism of EDTA Titration

When indicator and metal ion combine:
Indicator + Metal Ion → Indicator-Metal Complex (reddish pink color)

Upon addition of Na₂EDTA:

• Na₂EDTA has greater affinity for the metal ion than the indicator
• Metal ion dissociates from indicator-metal complex
• Metal ion binds to EDTA instead
• Free indicator is released and changes color
• At end point, indicator is fully free → color change to deep blue

Reactions
CaCO₃ + HCl → CaCl₂ + H₂O + CO₂
CaCl₂ + Na₂-EDTA → Ca-EDTA + 2NaCl

Stoichiometry and Calculations

From the above reactions:
• 1 mol Na₂EDTA ≡ 1 mol CaCO₃
• 1000 mL of 1M Na₂EDTA ≡ 100.008 g CaCO₃
• 1 mL of 0.1M Na₂EDTA ≡ (100.008 × 0.1) / 1000 g = 10.008 mg CaCO₃

Actual Amount (mg) = ΔV × Equivalent Weight of CaCO₃

= ΔV × 10.008 mg
 % CaCO₃ = (Actual Amount / Theoretical Amount) × 100%

Experimental Data
• Initial burette reading: 20.0 mL
• Final burette reading: 39.9 mL
• API weight: 200 mg
• ΔV = 39.9 − 20.0 = 19.9 mL

Pharmacopeial Acceptance Range: 98.0 – 100.5%

NaOH required: 4 g
EDTA required: 3.72 g
Sample weight (API): approximately 200 mg

EXPERIMENT 6 & 7
Preparation & Standardization of 0.1M HClO₄ | Assay of Diclofenac Sodium
Date: 08/04/2026

Background: Non-Aqueous Titration

This experiment involves non-aqueous titration (NAT):
• No water is used in the process
• Water would react with the reagents and/or analyte, interfering with the titration
• Organic solvent used: Glacial acetic acid

Diclofenac Sodium belongs to the NSAID (Non-Steroidal Anti-Inflammatory Drug) class of drugs. It has
basic properties (sodium salt of a weak acid), making non-aqueous titration with HClO₄ the method of
choice.

Part A: Preparation of 0.1M HClO₄ (100 mL)

Given Data
• Molecular weight of HClO₄ = 100.46 g/mol
• Density (ρ) = 1.67 g/mL
• Purity = 70–73% v/v (average = 71.5%)

Calculation
Step i: Calculate the weight of HClO₄ needed:
W = (S × V × M) / 1000 = (0.1 × 100 × 100.46) / 1000 = 1.0046 g

Step ii: Calculate the volume of HClO₄ stock solution needed:

ρ = m/V ⇒ V = m/ρ = 1.0046 / 1.67 = 0.601 mL (pure HClO₄)

Since purity is 71.5% (i.e., 71.5 mL of HClO₄ is present in 100 mL of stock solution):
Volume of stock solution = (100 × 0.601) / 71.5 = 0.84 mL
Why Acetic Anhydride is Used
HClO₄ is hygroscopic (absorbs moisture from the air), which causes it to react with water:
HClO₄ + H₂O ⇌ H₃O⁺ + ClO₄⁻
To avoid moisture absorption, acetic anhydride is added. Acetic anhydride reacts preferentially with
water:
(CH₃CO)₂O + H₂O → 2 CH₃COOH
This ensures HClO₄ does not react with water and its concentration remains accurate.

Preparation Steps
7. Rinse a 100 mL dry volumetric flask with glacial acetic acid.
8. Add 50 mL glacial acetic acid to the flask.
9. Add 3 mL acetic anhydride.
10. Add 0.84 mL HClO₄ acid solution dropwise.
11. Shake and mix well.
12. Make up to 100 mL with glacial acetic acid.

Part B: Standardization of 0.1M HClO₄

HClO₄ is a secondary standard (cannot be accurately weighed directly), so it must be standardized

against a primary standard.

Primary Standard Used: Potassium Hydrogen Phthalate (KHP)

KHP is suitable because it is stable, non-hygroscopic, and has a high molecular weight (204.22 g/mol),
allowing accurate weighing.

Standardization Procedure
13. Weigh 170 mg of KHP accurately and transfer to a conical flask.
14. Add 25 mL glacial acetic acid.
15. Shake to dissolve KHP completely.
16. Add 4 drops Crystal Violet indicator.
17. Titrate from the burette using HClO₄ solution.
18. Endpoint: Color changes to Emerald Green / Crystal Green.

Reaction
KHP + HClO₄ → Phthalic Acid + KClO₄

Stoichiometry
• 1 mol HClO₄ ≡ 1 mol KHP
• 1000 mL of 1M HClO₄ ≡ 204.22 g KHP
• 1 mL of 0.1M HClO₄ ≡ (204.22 × 0.1) / 1000 g = 20.422 mg KHP

Correction Factor (CF)

The Correction Factor accounts for the difference between the prepared (nominal) concentration and
the actual concentration after standardization:
CF = Theoretical Amount of KHP / Actual Amount of KHP
= 170 / (ΔV × Equivalent Weight)
 = 170 / (ΔV × 20.422)

Interpretation of CF:
• CF = 1.0: prepared solution is exactly 0.1M
• CF > 1.0: solution is more dilute (actual concentration < 0.1M)
• CF < 1.0: solution is more concentrated (actual concentration > 0.1M)

Actual Molarity = CF × Nominal Molarity (0.1M)

Example from today's experiment:

CF = 170 / (12.5 × 20.422) = 0.66
Actual Molarity = 0.66 × 0.1 = 0.066 M
(CF < 1 indicates HClO₄ absorbed moisture and became more concentrated)

Part C: Assay of Diclofenac Sodium

Powder Preparation for Assay

Next experiment: 75 mg API required
15 tablets × 50 mg API = 750 mg API total in 2672 mg powder
1 mg API = 2672 / 750 mg powder
75 mg API = (2672 × 75) / 750 = 267.2 mg powder to weigh

Assay Procedure
19. Accurately weigh the calculated amount of powder (267.2 mg) and transfer to a dry conical
flask.
20. Add 30 mL glacial acetic acid.
21. Shake and dissolve for 2 minutes. Note: Excipients will not dissolve fully.
22. Filter using a funnel with cotton/filter paper (excipients are retained on filter paper).
23. Rinse the previous conical flask with 10 mL glacial acetic acid and filter again.
24. Add 4 drops Crystal Violet indicator.
25. Fill burette with HClO₄ and titrate.
26. Endpoint: Crystal Green color.
27. Record volume difference (ΔV).

Reaction
Diclofenac Sodium + HClO₄ → Diclofenac Acid + NaClO₄

Stoichiometry and Calculation

• 1 mol HClO₄ ≡ 1 mol Diclofenac Sodium (MW = 318.13 g/mol)
• 1000 mL of 1M HClO₄ ≡ 318.13 g Diclofenac Sodium
• 1 mL of yM HClO₄ ≡ (318.13 × y) / 1000 g
• 1 mL of yM HClO₄ ≡ 318.13y mg (equivalent weight)

% Drug Content = (Actual / Theoretical) × 100

= (ΔV × Equivalent Weight) / Theoretical × 100

Example calculation:
% Drug = (3.5 × 318.13 × 0.066) / 75 × 100 = 97.98%
 Pharmacopeial Acceptance Range: 95.0 – 105.0%

EXPERIMENT 8
Assay of Aspirin (Acetylsalicylic Acid)
Date: 15/04/2026

Reagents to Prepare
• NaOH (0.5M)
• HCl (0.5M)
• Na₂CO₃ (0.5M)

Part 1: Preparation of 0.5M HCl (500 mL)

Calculation
Using S₁V₁ = S₂V₂:
13 × V₁ = 0.5 × 500
V₁ = 250 / 13 = 19.23 mL of concentrated HCl

Steps
28. Use a 500 mL volumetric flask.
29. Add approximately 50 mL water first.
30. Add 19.23 mL concentrated HCl.
31. Make up to 500 mL with water.
32. Transfer to a 1000 mL beaker, cover with glass, and mix well.

Part 2: Preparation of 0.5M Na₂CO₃ (100 mL)

W = (S × V × M) / 1000 = (0.5 × 100 × 106) / 1000 = 5.3 g

Steps
33. Use a 100 mL volumetric flask.
34. Add approximately 50 mL water.
35. Add 5.3 g Na₂CO₃ and mix.
36. Make up to 100 mL.

Part 3: Standardization of HCl by Na₂CO₃

Reaction
2HCl + Na₂CO₃ → 2NaCl + CO₂ + H₂O
Formula
M(HCl) × V(HCl) = 2 × M(Na₂CO₃) × V(Na₂CO₃)
M(HCl) = (2 × M(Na₂CO₃) × V(Na₂CO₃)) / V(HCl)
= (2 × 0.5 × 5) / ΔV

Procedure
37. Add 5 mL Na₂CO₃ to a conical flask.
38. Add 2-4 drops methyl orange indicator.
39. Fill burette with HCl and titrate Na₂CO₃.
40. Endpoint: Color changes from orange to pink.
41. Record ΔV of HCl used.

Part 4: Preparation of 0.5M NaOH (500 mL)

W = (S × V × M) / 1000 = (0.5 × 40 × 500) / 1000 = 10 g

Steps
42. Use a 500 mL volumetric flask.
43. Add 50% water first.
44. Add 10 g NaOH and mix.
45. Make up to 500 mL.
46. Transfer to a 1000 mL beaker, cover with glass, and stir.

Part 5: Standardization of NaOH by HCl

Reaction
NaOH + HCl → NaCl + H₂O

Formula
M(NaOH) × V(NaOH) = M(HCl) × V(HCl)
M(NaOH) = (M(HCl) × V(HCl)) / V(NaOH) = (x × ΔV) / 10

Procedure
47. Add 10 mL NaOH to a conical flask.
48. Add phenolphthalein indicator; initial color: pink.
49. Titrate with HCl from burette.
50. Endpoint: Pink → Colorless.
51. Record ΔV.

Calculation results from today:

M(HCl) = (2 × 0.5 × 5) / 13.5 = 0.37 M
M(NaOH) = (0.37 × 11) / 10 = 0.396 M

Part 6: Determination of Aspirin Content in Sample

Why Back/Indirect Titration is Used
Normally, one would directly titrate the drug with NaOH via burette. However, NaOH shows a slow
reaction with aspirin at room temperature (the ester hydrolysis is slow), making direct titration
inaccurate.

Additionally, the sodium salt of aspirin (sodium acetylsalicylate) is connected to acetate, making it
impossible to distinguish which NaOH reacted with which group.

Therefore, Back Titration (Indirect Titration) is used:

• An excess, known amount of NaOH is added to the analyte (aspirin)
• The mixture is allowed to react completely under controlled conditions (water bath, heating)
• After the reaction is complete, excess NaOH remains
• The remaining (unreacted) NaOH is then determined by back-titration with HCl (2nd reagent)
• Amount of NaOH that reacted = Initial NaOH − Remaining NaOH

Reaction
Aspirin + 2NaOH → Sodium Salicylate + Sodium Acetate (CH₃COONa)

Stoichiometry
• 2 mol NaOH ≡ 1 mol Aspirin (MW = 180.16 g/mol)
• 1M NaOH, 1000 mL ≡ 180.16 g Aspirin
• 1M NaOH, 1000 mL ≡ 180.16/2 g Aspirin (per mol NaOH)
• yM NaOH, 1 mL ≡ (180.16 × y) / (2 × 1000) g = 180.16y/2 mg (equivalent weight)

Actual Amount = Equivalent Weight × ΔV(NaOH reacted)

= Equivalent Weight × (ΔV(Blank) − ΔV(Sample))

% Drug = (Actual / Theoretical) × 100

Pharmacopeial Acceptance Range: 90 – 110%

Powder Preparation
20 tablets × 75 mg API = 2971 mg API in powder
225 mg API = (2971 × 225) / (20 × 75) = 445.65 mg powder to weigh

Procedure
Two sets of conical flasks are prepared: Blank (B) and Sample (S).

For Sample:
52. Weigh the specific amount of aspirin powder (445.65 mg) into a conical flask.
53. Add 25 mL NaOH (excess reagent).
54. Heat in water bath for 10 minutes (to complete ester hydrolysis).
55. Cool for 30 seconds, then add 4 drops phenolphthalein indicator.
56. Color: Pink (due to excess NaOH).
57. Titrate with HCl from burette until pink → colorless.
58. Record ΔV (Sample).

For Blank:
59. Add 25 mL NaOH (no aspirin).
60. Add 4 drops phenolphthalein.
 61. Titrate with HCl from burette.
62. Record ΔV (Blank).

Calculation
React Amount of NaOH = ΔV(Blank) − ΔV(Sample)

Results from today:

B = ΔV = 27 mL (Initial → 26 to 20, again 18 to 20)
S = ΔV = 20 mL; ΔV = 7 mL
M(HCl) = (2 × 0.5 × 5) / 13.5 = 0.37 M (Step 3)
M(NaOH) = (0.37 × 11) / 10 = 0.396 M (Step 5)

% Drug = (Actual / Theoretical) × 100

= (Equivalent Weight × ΔV) / Theoretical × 100
= ((180.16 × 0.396) / 2 × 7) / 445.65 × 100
≈ 110.97%

Range: 90–110% → result is borderline/slightly above range

EXPERIMENT 9
Identification of Supplied Sample Based on λmax Using UV-Visible Spectroscopy
Date: 22/04/2026

What is Spectroscopy?
Spectroscopy is the study of the interaction between electromagnetic radiation and chemical
substances.

When electromagnetic radiation interacts with a chemical substance:

• Some radiation is absorbed
• Some is emitted
• Some is reflected
Based on these interactions, we can identify and measure the amount of a substance. Spectroscopy
has four main functions:
63. Identification of compounds
64. Quantification (measuring amount)
65. Determination of physicochemical properties
66. Determination of chemical structure

Common Spectroscopic Techniques

• UV-Visible Spectroscopy (200–800 nm) – identification and quantification

• Infrared (IR) Spectroscopy – identification and functional group identification
• Nuclear Magnetic Resonance (NMR) Spectroscopy – determines the carbon and hydrogen
arrangement in a compound
• Fluorescence Spectroscopy – compound identification and quantification (uses emission of light
after absorption)
• Mass Spectrometry – exact molecular weight determination
 • X-Ray Crystallography – 3D structure of compounds

UV-Visible Spectroscopy: Principles

UV-Visible spectroscopy uses electromagnetic radiation in the UV and visible range (200–800 nm). It is
a type of absorption spectroscopy. It determines how a chemical compound absorbs radiation at
different wavelengths, and based on this, identifies and quantifies the compound.

The electronic nature of a compound determines which range of radiation it absorbs. When radiation
energy matches the energy gap between the ground state and excited state of electrons, that energy is
absorbed:
E ∝ f (frequency)
E ∝ 1/λ (wavelength)

Since different compounds have different electronic natures, they absorb at different wavelengths.

λmax (Lambda Max)

λmax is the wavelength at which a compound absorbs the maximum amount of light. It acts like a
fingerprint for identifying a compound — each compound has a characteristic λmax.

Reference λmax Values for Common Drugs

The following λmax values are used for identification (acceptance range: ±1 nm):

Drug λmax (nm) Notes

Naproxen 231–235 nm
Aceclofenac (prodrug of 275–278 nm
Diclofenac)
Paracetamol 244–248 nm
Metformin 231–233 nm Solvent differs from
Naproxen; ranges overlap

Note: Naproxen and Metformin have overlapping λmax ranges but use different solvents, allowing
differentiation.

Instrument Components (UV-Visible Spectrophotometer)

The instrument consists of the following components in sequence:

Light Source → Monochromator → Sample Cuvette → Detector → Analyzer

• Light Source: Emits electromagnetic radiation in UV and visible range (UV: 200–400 nm;
Visible: 400–800 nm). The light source emits a range of wavelengths, not a single specific
wavelength.
• Monochromator: Filters out a specific wavelength of light. Since a compound absorbs at a
specific λmax, the monochromator selects that wavelength to pass through to the sample.
 • Sample Cuvette: Contains the dissolved sample. The sample absorbs light at λmax; remaining
light passes through to the detector.
• Detector: Detects the intensity of light passing through the sample and converts it to a signal.
• Analyzer: Compares initial and final intensity of light to determine how much light was absorbed
(absorbance value).

The scan starts at 400 nm and ends at 200 nm (scans from visible to UV range). At the wavelength of
maximum absorption, the λmax is identified. The corresponding absorbance is recorded.

Procedure

67. Place drug (in tablet form — small particle size is better for dissolving) in a test tube.
68. Add appropriate solvent: water (for Metformin) or methanol (for other drugs).
69. Add approximately 10 mL solvent.
70. Mix well.
71. Transfer approximately 5 mL to a clean test tube.
72. Add 5 mL more solvent and mix (this dilutes the solution for proper absorbance range).
73. Run a trial scan.
74. Then add the correct drug to the blank solvent to confirm identification.

Initial calibration: The machine is first calibrated using the blank solvent (without drug). Then the drug
solution is scanned.

Analysis Lab — Pharmaceutical Analysis Notes | Compiled from handwritten notes