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Unit 3 of 4 · B.Sc MLS Sem 1

Unit 3: Volumetric technique and instrumentation

Basics of Biochemistry notes · PTU syllabus (BMLS103-18)

3 min read4 topics10 exam questions
On this page
  1. Unit summary
  2. Volumetric glassware and its calibration
  3. The analytical balance
  4. pH and the Henderson–Hasselbalch equation
  5. pH paper and the pH meter
  6. Key terms
  7. Quick revision
  8. Important questions

Unit summary

Volumetric glassware must be calibrated, balances must weigh precisely and pH must be measured correctly. This unit covers calibration of flasks, pipettes, burettes and cylinders, the analytical balance, pH, the Henderson–Hasselbalch equation, pKa, pH paper and the pH meter.

After this unit you can

  • Calibrate volumetric glassware
  • Use and maintain the analytical balance
  • Explain pH, pKa and the Henderson–Hasselbalch equation
  • Use, calibrate and maintain a pH meter

PTU syllabus topics

  • Calibration of flasks
  • pipettes
  • burettes and cylinders
  • the analytical balance — principle
  • working
  • maintenance
  • concept of pH
  • Henderson-Hasselbalch equation
  • pKa
  • pH paper and pH meter (principle, working, maintenance, calibration)
Key formulaspH and buffers

  • pH

    −log [H⁺]

  • Henderson-Hasselbalch

    pH = pKa + log ([A⁻] / [HA])

  • pKa

    pH at which acid is half dissociated

  • Normal blood pH

    7.35 to 7.45

1

Topic 1

Volumetric glassware and its calibration

ComparisonVolumetric glassware
Use
Accuracy

Volumetric flask

Preparing solutions of exact volume (TC — to contain)

High

Volumetric (transfer) pipette

Delivering one fixed volume (TD — to deliver)

High

Graduated pipette

Delivering variable volumes

Moderate

Burette

Titration — variable delivered volume

High (read to 0.05 mL)

Measuring cylinder

Approximate volumes

Low

Micropipette

µL volumes

High when calibrated

ProcessGravimetric calibration
  1. 1Clean and dry the glassware; equilibrate water to room temperature
  2. 2Weigh an empty container
  3. 3Deliver distilled water and weigh again
  4. 4Volume = mass ÷ density of water at that temperature (about 0.9982 g/mL at 20 °C)
  5. 5Repeat and compare with the stated volume and tolerance
  • Reading: read the bottom of the meniscus at eye level; class A glassware has tighter tolerances than class B.
2

Topic 2

The analytical balance

  • Principle: compares the mass of an object with known masses; modern balances use electromagnetic force restoration — a coil generates a force to balance the load, and the current needed is proportional to the mass. Reads to 0.1 mg.
Key termsUsing and maintaining the balance
Placement
Level, vibration-free bench away from drafts
Levelling
Centre the bubble before use
Calibration
Internal or external standard weights regularly
Weighing
Close doors; tare the container; let readings stabilise
Samples
At room temperature; never weigh chemicals directly on the pan
Cleaning
Brush spills immediately
3

Topic 3

pH and the Henderson–Hasselbalch equation

  • pH = −log₁₀[H⁺]; neutral 7, acidic below 7, alkaline above 7. Blood pH is 7.35–7.45.
  • pKa: pH at which an acid is half dissociated; a buffer works best within pKa ± 1.
Key formulasHenderson–Hasselbalch equation

  • Equation

    pH = pKa + log ([A⁻] ÷ [HA])

  • When [A⁻] = [HA]

    pH = pKa

  • Blood (bicarbonate buffer)

    pH = 6.1 + log ([HCO₃⁻] ÷ (0.03 × pCO₂))

Example

Plasma bicarbonate 24 mmol/L and pCO₂ 40 mmHg: 0.03 × 40 = 1.2; 24 ÷ 1.2 = 20; log 20 = 1.3; pH = 6.1 + 1.3 = 7.4.

  • Buffers resist pH change on adding small amounts of acid or base — bicarbonate, phosphate and protein buffers in the body.
4

Topic 4

pH paper and the pH meter

  • pH paper (indicator paper): litmus shows only acid or base; universal indicator paper gives an approximate pH by colour (± 0.5–1 unit).
ProcesspH meter: principle and working
  1. 1A glass electrode develops a potential proportional to H⁺ activity across a thin glass membrane
  2. 2A reference electrode (calomel or Ag/AgCl) gives a constant potential
  3. 3The meter measures the potential difference (about 59 mV per pH unit at 25 °C)
  4. 4The reading is displayed as pH, with temperature compensation
ProcessCalibrating a pH meter
  1. 1Rinse the electrode with distilled water and blot
  2. 2Immerse in pH 7.0 buffer and set (standardise)
  3. 3Rinse; immerse in pH 4.0 or 9.2 buffer and adjust slope
  4. 4Rinse; measure the sample
  5. 5Store the electrode in storage solution (3 M KCl), never dry or in distilled water

Key terms

Meniscus
Curved surface of a liquid in a tube
Tare
Setting the balance to zero with the container on it
pKa
pH at which an acid is half dissociated
Buffer
Solution resisting pH change
Glass electrode
pH-sensitive electrode of a pH meter

Quick revision

  • TC vs TD glassware; class A and B; gravimetric calibration; meniscus.
  • Analytical balance principle, placement, calibration, use.
  • pH = −log[H⁺]; buffers; pKa; Henderson–Hasselbalch; blood example.
  • pH paper vs pH meter; electrodes; two-point calibration; electrode storage.

Important exam questions

Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).

Short-answer questions

  1. Q1.Distinguish TC and TD glassware.
  2. Q2.How is a pipette calibrated?
  3. Q3.Why must the balance be levelled?
  4. Q4.State the Henderson–Hasselbalch equation.
  5. Q5.What is the normal pH of blood?
  6. Q6.How should a pH electrode be stored?

Long-answer questions

  1. Q1.Describe the calibration of volumetric glassware.
  2. Q2.Describe the principle, working and maintenance of the analytical balance.
  3. Q3.Explain pH and the Henderson–Hasselbalch equation.
  4. Q4.Describe the principle, working and calibration of a pH meter.

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