Preparation of reagents and standard solutions

Dr. Arvind Kumar

Introduction

In biochemistry, reagents and standard solutions are not merely chemical mixtures—they are analytical tools that enable quantification, calibration, and reaction control. Their accurate preparation affects:

  • Precision of quantitative biochemical assays

  • Reproducibility across batches and analysts

  • Reliability of patient results in clinical labs

  • Instrument calibration and performance

  • Research validity in academic and industrial labs

An accredited biochemistry laboratory (NABL/ISO 15189) must demonstrate full traceability of reagent preparation including batch logs, calibration details, expiry, and quality control.

Fundamental Principles of Solution Preparation

Purity of Chemicals

  • Analytical Reagent (AR) grade: Highest purity; used for quantitative assays and calibrators.

  • LR/GR grade: Suitable for qualitative tests.

  • HPLC grade: Used in chromatography to avoid UV-absorbing contaminants.

Water Quality

Water must be:

  • Free of ions

  • Free of microbes

  • Free of organic contaminants

Types:

  • Type I (Milli-Q): For molecular biology, enzyme reagents.

  • Type II (Deionized): For most buffer preparations.

  • Type III (Distilled): For washing glassware.

Contaminated water alters:

  • pH

  • ionic strength

  • absorbance of colorimetric assays

Calibrated Glassware

  • Volumetric flasks: Used to make solutions to exact volume.

  • Pipettes (Class A): Used for precise volumetric transfers.

  • Burettes: Used for titrations and standardization.

Calibration certificates must be present and updated.

Labelling Requirements

Every solution must have:

  • Name of reagent

  • Concentration

  • Lot No.

  • Date of preparation

  • Expiry or validity

  • Storage condition

  • Prepared & verified signatures

 

Essential Calculations in Biochemistry Laboratory

Molar Solutions (M)

M = Wt. of solute (g) / MW×Volume (L)

Example: Prepare 1 L of 0.1 M HCl from concentrated HCl (37%, density 1.19 g/ml).
Equivalent weight of HCl = 36.5 g
Using dilution:

C1V1 = C2V2 

(12M)V1 = (0.1M)(1L)

V1 = 8.3ml

Normal Solutions (N)

Used for acid-base titrations.

N = Wt. (g) / Equivalent weight×Volume (L)

Example: 1N NaOH
EW of NaOH = 40 g
So,

1N=40g/L

Percent Solutions

Weight/Volume (% w/v)

10% NaCl = 10 g salt in 100 mL solution.

Volume/Volume (% v/v)

70% ethanol = 70 mL ethanol + water up to 100 mL.

Weight/Weight (% w/w)

Parts Per Million (ppm)

Used for trace ions and metal analysis.

1 ppm=1 mg/L

Dilution Calculations

Essential for preparing working standards and enzyme reagents.

C1V1=C2V2

Example: Prepare 10 mL of 5 mM solution from 500 mM stock.

V1 = C2V2 / C1 = (5)(10) / 500 = 0.1ml

 

Buffers solution preparation

Buffers play a crucial role in maintaining pH stability during enzymatic reactions. Enzymes show drastic activity changes with even ±0.1 pH variation.

Henderson–Hasselbalch Equation

pH = pKa+log⁡ [salt] / [acid]

Selecting a buffer:

  • pKa within ±1 of the desired pH

  • Minimal reactivity with biological molecules

  • Stable ionic strength

  • Low UV absorbance

Common Biochemistry Buffers:

Buffer pKa Applications
Phosphate 7.2 Enzyme assays, ELISA
Tris-HCl 8.1 Electrophoresis, DNA work
Acetate buffer 4.7 Enzyme assays at acidic pH
Glycine buffer 9.6 PAGE, protein electrophoresis

 

Reagent Preparation

Required Materials

  • Distilled or deionized water
  • Appropriate chemicals (solids, liquids)
  • Clean glassware (beakers, flasks, graduated cylinders)
  • Analytical balance
  • pH meter (if needed for buffer preparation)
  • Magnetic stirrer or glass rod for mixing

Preparation Steps

  1. Calculate Required Quantities: Use the molecular weight (MW) or molarity of the chemicals to determine the amount of each component needed for your solution. For example:
    • To prepare a 1 M NaCl solution, dissolve 58.44 g (MW of NaCl) in 1 L of water.
  2. Weigh Chemicals Accurately:
    • Use an analytical balance to measure the required amount of the chemical to the nearest milligram.
  3. Dissolve the Solute:
    • Place the weighed solute into a clean beaker.
    • Add about 80% of the final volume of distilled water.
    • Stir the mixture thoroughly until the solute dissolves completely.
  4. Adjust pH (if necessary):
    • If the reagent requires a specific pH, use a pH meter and add a strong acid (HCl) or base (NaOH) dropwise to adjust the pH.
  5. Transfer and Dilute:
    • Once dissolved and pH is adjusted, transfer the solution to a volumetric flask.
    • Fill the final volume with distilled water.
  6. Label and Store:
    • Label the container with the reagent name, concentration, date, and your initials.
    • Store according to the chemical’s requirements (e.g., in a fridge or at room temperature).

Example Reagents:

  • 1 M NaCl: Dissolve 58.44 g of NaCl in 1 L of water.
  • 0.1 M HCl: Dilute concentrated HCl (12 M) by adding 8.33 mL to distilled water and dilute to 1 L.

 

Standard Solution Preparation

A standard solution is a solution of known concentration used for calibration in quantitative analysis, such as titrations or spectrophotometric assays.

Primary Standard (High Purity Solutes):

  1. Weigh the Primary Standard:
    • For example, to prepare a 0.1 M solution of a substance with a molecular weight of 100 g/mol, weigh out 10 g of the substance for 1 L of solution.
  2. Dissolve in Water:
    • Dissolve the accurately weighed solute in a portion of distilled water.
  3. Transfer and Dilute:
    • Transfer the solution to a volumetric flask and dilute to the final volume.

Secondary Standard (Concentration by Calibration):

Secondary standards require calibration against a primary standard, especially for substances that may not be pure or degrade over time.

  1. Prepare an Approximate Solution:
    • Weigh the substance and dissolve it in distilled water.
  2. Titrate or Compare Against a Primary Standard:
    • Use a primary standard in a titration to find the exact concentration of your solution.

Example Standards:

  • Glucose Standard Solution (100 mg/dL): Dissolve 1 g of glucose in 100 mL of water, then dilute to 1 L.
  • BSA Standard for Protein Assays: Weigh Bovine Serum Albumin and dissolve it in the desired volume of water.

 

MCQs

1. A solution with accurately known concentration is called:

a) Buffer solution
b) Standard solution
c) Saturated solution
d) Dilute solution

2. Primary standard should have:

a) Low purity
b) High molecular weight
c) Unstable nature
d) Hygroscopic properties

3. Which one is a good primary standard?

a) NaOH
b) HCl
c) K₂Cr₂O₇
d) FeSO₄

4. Secondary standards are:

a) Highly pure
b) Hygroscopic
c) Used to standardize solutions
d) Not used in titrations

5. The process of determining exact concentration of a solution is called:

a) Dilution
b) Standardization
c) Filtration
d) Evaporation

6. Molarity is expressed as:

a) Moles of solute per liter of solution
b) Grams per liter
c) Moles per kilogram
d) Mass per volume

7. Normality depends on:

a) Molecular mass
b) Equivalent weight
c) Density
d) Volume of solvent

8. Equivalent weight is:

a) Molecular weight × valency
b) Molecular weight ÷ valency
c) Valency ÷ molecular weight
d) Valency × density

9. A 1M NaCl solution contains:

a) 58.5 g per liter
b) 29.25 g per liter
c) 100 g per liter
d) 1 g per liter

10. To prepare 100 ml of 1N HCl from concentrated HCl (12N), volume required is:

a) 10 ml
b) 8.3 ml
c) 12 ml
d) 20 ml

11. Stock solution dilution follows which formula?

a) C = m/V
b) C₁V₁ = C₂V₂
c) N₁V₂ = N₂V₁
d) M = n/V

12. A reagent that resists pH change is called:

a) Indicator
b) Buffer
c) Standard
d) Catalyst

13. A secondary standard acid commonly used is:

a) HCl
b) Na₂CO₃
c) KHP
d) EDTA

14. Which glassware is used for preparing standard solutions?

a) Beaker
b) Conical flask
c) Volumetric flask
d) Test tube

15. The first step in reagent preparation is:

a) Filtration
b) Calculating required concentration
c) Heating the solution
d) Weighing unknown mass

16. The purity of primary standards must be:

a) <80%
b) 50–60%
c) >99%
d) 70–85%

17. A solution concentrated to known strength is called:

a) Stock solution
b) Working solution
c) Standard solution
d) Buffer

18. The process of making lower concentration solution from stock is:

a) Filtration
b) Dilution
c) Drying
d) Decantation

19. The ideal primary standard should be:

a) Hygroscopic
b) Volatile
c) Highly stable
d) Easily oxidized

20. To prepare 0.1N NaOH solution:

a) Volumetric flask is used
b) Burette is used
c) Graduated cylinder is used
d) Test tube is used

21. A reagent blank contains:

a) All reagents except sample
b) All reagents + analyte
c) Only the sample
d) Only distilled water

22. Serial dilution means:

a) Increasing concentration
b) Repeated dilution in a series
c) Mixing solutions
d) Heating reagents

23. One liter of 1M H₂SO₄ contains:

a) 98 g
b) 49 g
c) 196 g
d) 25 g

24. Volumetric pipettes are used for:

a) Rough measurement
b) Accurate transfer of fixed volume
c) Mixing solutions
d) Heating solutions

25. Standard solution used for EDTA titration is:

a) MgCl₂
b) CaCO₃
c) Eriochrome Black T
d) EDTA solution

26. A solution that changes color at endpoint is called:

a) Buffer
b) Catalyst
c) Indicator
d) Solvent

27. Reagents used in clinical biochemistry must be:

a) Sterile
b) Stable and accurate
c) Radioactive
d) Colored

28. Distilled water is used for reagent preparation because it is:

a) Cheap
b) Free of ions and impurities
c) Colored
d) Acidic

29. Working solution is prepared from:

a) Freshly weighed powder
b) Stock solution
c) Distilled water only
d) Indicator solution

30. The most accurate way to weigh primary standards is using:

a) Top pan balance
b) Analytical balance
c) Digital rough scale
d) Kitchen scale

✅ SEPARATE ANSWER KEY

  1. b

  2. b

  3. c

  4. c

  5. b

  6. a

  7. b

  8. b

  9. a

  10. b

  11. b

  12. b

  13. a

  14. c

  15. b

  16. c

  17. a

  18. b

  19. c

  20. a

  21. a

  22. b

  23. a

  24. b

  25. d

  26. c

  27. b

  28. b

  29. b

  30. b