Limit tests, UV and I R Spectroscopy

 

⭐ LIMIT TESTS — DETAILED NOTES (B.K. SHARMA, CHATWAL & ANAND, GUNDU RAO)

1. Definition

A Limit Test is a semi-quantitative procedure to identify and control trace or small amounts of impurities that may be present in a substance.
It compares the impurity in the test sample with a standard limit under identical conditions.

👉 It only checks whether the impurity is below or above the prescribed limit—not the exact quantity.

2. Purpose / Need for Limit Tests

Impurities affect:

• Safety (toxic metals like Pb, As)
• Therapeutic activity
• Stability of drugs
• Colour, clarity, appearance of pharmaceuticals

Hence limits are set by IP/BP/USP.

3. General Principle

1. Prepare Test Solution (sample + reagent).
2. Prepare Standard Solution containing the maximum allowable impurity.
3. Compare colour/turbidity intensity of test vs. standard in Nessler cylinders.
4. The test should not be more intense than standard → passes the test.

4. General Reagents Used

Purpose	Reagent
Oxidizing agent	Potassium permanganate, persulphate
Reducing agent	Hydrazine, sodium sulphide
Complexing agent	Sodium citrate, ammonium citrate
Colour developing agents	Rhodamine-B, silver nitrate
pH adjusters	Acetic acid, ammonia solution

5. Limit Tests in IP (Important for CSI/DI/Pharmacist)

A. LIMIT TEST FOR ARSENIC (As)

Principle:
Based on formation of arsine gas (AsH₃) when arsenic reacts with zinc + acid.
Arsine reacts with silver diethyldithiocarbamate, producing yellow to brown colour.

Apparatus:
Gutzeit apparatus (important MCQ).

Procedure:

1. Sample → treated with acid + potassium iodide + stannous chloride → reduces arsenic to arsenite.
2. Add granulated zinc → arsine gas forms.
3. The gas passes through lead acetate cotton (removes H₂S).
4. Then through silver diethyldithiocarbamate paper.
5. Colour intensity is compared with a standard.

End point:
Yellow stain on filter paper should not be more intense than standard.

Reagents:

• HCl
• SnCl₂ (reducing agent)
• KI
• Lead acetate cotton
• Silver diethyldithiocarbamate (colour forming)

Mnemonic (for reagents):
👉 "H-S-K-L-S" = HCl, SnCl₂, KI, Lead acetate, Silver salt

B. LIMIT TEST FOR HEAVY METALS

(Primarily Lead)
Principle:
Metal impurities react with H₂S in alkaline medium → produce brown/black metallic sulphides.

Reagent:
Sodium sulphide / Hydrogen sulphide solution.

Procedure:

1. Sample dissolved in water or acid.
2. Adjust pH with ammonium acetate buffer.
3. Add H₂S.
4. Compare with a lead standard (prepared by Pb(NO₃)₂).

Observation:
Black/brown coloration of PbS should not exceed standard.

Mnemonic:
👉 "P-A-S-H": Pb standard + Acetate buffer + Sulphide + Hydrogen sulphide

C. LIMIT TEST FOR CHLORIDE

Principle:
Chloride reacts with silver nitrate → forms white turbidity (AgCl) in the presence of nitric acid.

Procedure:

1. Sample + 2 mL dilute HNO₃.
2. Add AgNO₃ solution.
3. Compare turbidity with standard (NaCl).

End point:
Turbidity ≤ standard.

Mnemonic:
👉 “HAg-Cl” = HNO₃ + AgNO₃ for chloride

D. LIMIT TEST FOR SULPHATE

Principle:
Sulphate reacts with barium chloride in acidic medium → produces white barium sulphate turbidity.

Procedure:

1. Sample + HCl (to acidify)
2. Add BaCl₂
3. Compare turbidity with known sulphate standard (K₂SO₄).

End point:
Turbidity should not exceed standard.

Mnemonic:
👉 “BaS”: Barium for Sulphate

E. LIMIT TEST FOR IRON

Principle:
Iron reacts with thioglycolic acid in ammonia buffer → forms purple/violet colour of ferrous-thioglycolate complex.

Reagents:

• Ammonia buffer
• Thioglycolic acid
• Oxidizing agent if needed

Observation:
Purple colour of test ≤ standard.

Mnemonic:
👉 “Iron loves Purple” (for violet ferrous-thioglycolic complex)

F. LIMIT TEST FOR LEAD (Specific)

Principle:
Lead reacts with dithizone (diphenylthiocarbazone) → red-pink complex.

Steps:

1. Sample dissolved + pH adjusted.
2. Add dithizone.
3. Compare colour intensity vs lead standard.

Mnemonic:
👉 “Lead loves Dithi-RED-zone” (leads to red colour)

G. LIMIT TEST FOR SILVER

Principle:
Silver forms brown colour with H₂S or chromate reagent.

Reagents:

• HCl
• Potassium chromate

End point:
Brown colour ≤ standard.

6. Summary Table (Rapid Revision)

Test	Principle	Reagent	Observation
Arsenic	Arsine gas → coloured stain	Silver diethyldithiocarbamate	Yellow stain
Heavy metals (Pb)	Sulphide formation	H₂S / Na₂S	Brown/black
Chloride	AgCl precipitation	AgNO₃ + HNO₃	White turbidity
Sulphate	BaSO₄ precipitation	BaCl₂ + HCl	White turbidity
Iron	Ferrous-thioglycolate complex	Thioglycolic acid + NH₃	Purple/violet
Lead (specific)	Pb-Dithizone complex	Dithizone	Pink/red
Silver	Ag₂CrO₄ formation	K₂CrO₄	Brown/red

⭐ 7. Typical Exam Questions (IP/Drug Inspector/Pharmacist)

1. Explain limit test for arsenic with diagram.
2. Why is lead acetate cotton used in arsenic limit test? (To remove H₂S).
3. Why is acetic acid-ammonium acetate buffer used in heavy metals test?
4. Why HCl is added in sulphate test? (To prevent precipitation of carbonates/phosphates).
5. What is the role of thioglycolic acid in iron test? (Forms coloured complex).
6. Difference between limit test and assay.
7. Reagents used in limit test of chloride/sulphate.

 

🌟 UV SPECTROSCOPY — DETAILED NOTES

1. Principle

UV spectroscopy is based on absorption of ultraviolet (200–400 nm) and visible (400–800 nm) radiation causing electronic transitions in molecules.

👉 When molecules absorb UV light → electrons move from ground state → excited state.

2. Types of Electronic Transitions

(Important MCQ)

Transition	Energy	Example
σ → σ*	Highest energy	Alkanes
n → σ*	Medium	Alcohols, amines
π → π*	Lower	Alkenes, aromatics
n → π*	Lowest	Carbonyls (C=O), nitro compounds

Order of energy:
σ → σ* > n → σ* > π → π* > n → π*

3. Chromophores

A chromophore is the part of molecule responsible for UV absorption.

Examples:

• C=C, C=O, NO₂, N=N, aromatic ring.

4. Auxochromes

Groups that do not absorb UV alone but shift λmax when attached to a chromophore.

Examples: –OH, –NH₂, –OR, –Cl.

5. Types of Shifts

Name	Meaning
Bathochromic shift (Red shift)	λmax ↑ (shift to longer wavelength)
Hypsochromic shift (Blue shift)	λmax ↓
Hyperchromic effect	Absorbance ↑
Hypochromic effect	Absorbance ↓

6. Beer–Lambert Law

A = ε c l
A = Absorbance
ε = Molar absorptivity
c = concentration
l = path length (1 cm)

Important: Absorbance is directly proportional to concentration.

7. Instrumentation of UV Spectrophotometer

1.     Radiation Source:

• Deuterium lamp (UV)
• Tungsten lamp (visible)

2.     Monochromator: Prism / Diffraction grating

3.     Sample cell: Quartz cuvettes

4.     Detector: Photomultiplier tube

5.     Readout: Digital display

8. Applications of UV

• Quantitative analysis (Beer-Lambert law)
• Purity check
• Kinetic studies
• Determination of transition metals
• Detection of conjugation
• Identification of aromaticity
• Dissolution testing in pharma industries

9. MCQ Highlights — UV

• Conjugation increases λmax.
• Solvent polarity affects n→π* transitions.
• Aromatics show λmax ~ 260–280 nm.
• Carbonyls: n→π* around 290 nm.

🌟 IR SPECTROSCOPY — DETAILED NOTES

1. Principle

IR spectroscopy is based on absorption of infrared radiation (4000–400 cm⁻¹) causing vibrational transitions.

Molecular bonds behave like vibrating springs → absorb characteristic IR frequencies.

2. Types of Molecular Vibrations

A. Stretching (↑ energy)

• Symmetric stretching
• Asymmetric stretching

B. Bending (↓ energy)

• Scissoring
• Rocking
• Wagging
• Twisting

Mnemonic: "SRWT" = Scissor, Rock, Wag, Twist.

3. IR Regions

Region	Range
Near IR	14000–4000 cm⁻¹
Mid IR	4000–400 cm⁻¹ (Pharma uses this)
Far IR	400–10 cm⁻¹

4. Functional Group Frequencies (Very Important)

A. O–H Stretch

• Alcohol (free): 3600–3650 cm⁻¹
• Alcohol (H-bonded): 3200–3400 cm⁻¹
• Carboxylic acid: 2500–3000 cm⁻¹ (broad)

B. N–H Stretch

• Primary amine NH₂: 3300–3500 cm⁻¹ (2 peaks)
• Secondary amine NH: 3300–3500 cm⁻¹ (1 peak)

C. C=O Stretch

• Ketones: 1715 cm⁻¹
• Aldehydes: 1720–1740 cm⁻¹
• Acids: 1700–1720 cm⁻¹
• Esters: 1735–1750 cm⁻¹
• Amides: 1640–1690 cm⁻¹

D. C≡N / C≡C Stretch

• C≡N: 2210–2260 cm⁻¹
• C≡C: 2100–2260 cm⁻¹

E. Aromatic C=C Stretch

• 1450–1600 cm⁻¹

F. Alkanes, Alkenes

• C–H alkane: 2850–2960 cm⁻¹
• =C–H alkene: 3020–3100 cm⁻¹

5. IR Instrumentation

1. IR Radiation Source:

• Nernst glower
• Globar (silicon carbide)
• Tungsten filament

2. Monochromator:

• Prism or Grating

3. Detector:

• Thermocouple
• Golay cell
• DTGS (Deuterated Triglycine Sulphate)
• Pyroelectric detectors

4. Sample Handling Methods

• Solid: KBr pellet, Mulling (Nujol) technique
• Liquid: Salt plates (NaCl)
• Gases: Gas cell

6. Fingerprint Region

Range: 1500–400 cm⁻¹

Unique for each molecule → used for identification.

7. Applications of IR

• Identification of functional groups
• Verification of drug purity
• Identification of polymorphism
• Structural elucidation
• Monitoring reactions
• Quality control tests in pharmaceuticals
• Determining hydration (water bands at ~3400 cm⁻¹)

8. Differences: UV vs IR (Important for Viva)

Feature	UV	IR
Region	200–400 nm	4000–400 cm⁻¹
Transition	Electronic	Vibrational
Purpose	Conjugation, quantification	Functional group analysis
Cuvettes	Quartz	KBr/Salt plates
Law	Beer–Lambert applicable	Not applicable in solids