Folin-Wu Method For Estimation of Blood Glucose
Learn the Folin-Wu method for blood glucose estimation: step-by-step procedure, sodium tungstate role, spectrophotometric measurement (680 nm), advantages/disadvantages, and modern comparisons. Essential for clinical labs and history.

The Folin-Wu method is a colorimetric assay developed in 1919 by Otto Folin and Hsien Wu to quantify blood glucose. It involves:
- Deproteinization: Removing proteins using sodium tungstate and sulfuric acid.
- Reduction: Glucose converts alkaline copper tartrate to cuprous oxide, reacting with phosphomolybdic acid to produce a measurable blue color.
This method was pivotal in early clinical biochemistry but has been largely replaced by enzymatic assays.
Principle of Folin-Wu Method
The principle of Folin-Wu method involves a two-step biochemical reaction to estimate blood glucose:
- Deproteinization: Proteins in blood are precipitated using 10% sodium tungstate and 2/3N sulfuric acid, creating a protein-free filtrate.
- Reduction & Color Development:
- Step 1: Heating the filtrate in an alkaline medium converts glucose to enediol, which reduces cupric ions (Cu²⁺) to cuprous oxide (Cu₂O).
- Step 2: Cuprous oxide reacts with phosphomolybdic acid, forming phosphomolybdenum blue (intense blue color).
- Measurement: The blue color’s intensity, measured at 680 nm, correlates with glucose concentration.
Role of Sodium Tungstate in Folin-Wu Method
Sodium tungstate acts as a precipitating agent. Combined with sulfuric acid, it denatures blood proteins, ensuring they don’t interfere with glucose measurement. This step is critical for achieving a clear, analyzable filtrate.
Features
- Folin-Wu Tubes: Constricted design prevents atmospheric oxygen from reoxidizing cuprous oxide, ensuring accuracy.
- Non-Specificity: Reacts with other reducing sugars (e.g., fructose), yielding slightly higher glucose values than true levels.
What is Folin-Wu Tube?
A Folin-Wu tube is a specialized glass tube designed for the Folin-Wu method, a historic biochemical technique for blood sugar analysis. Its primary uses include:
- Preventing Reoxidation: The tube’s constricted design minimizes contact with atmospheric oxygen, avoiding reoxidation of cuprous oxide (a critical intermediate in glucose estimation).
- Ensuring Precision: Equipped with calibrated markings, it ensures accurate measurement of reagents and filtrates during the glucose reduction process.
Specifications
- Durability: Made from heat-resistant glass to withstand boiling during the reaction.
- Measurement: Final glucose levels are determined by measuring the blue color intensity at 680 nm, proportional to blood sugar concentration.

Requirements
1. Essential Equipment
- Folin-Wu Tubes: Constricted design to prevent reoxidation of cuprous oxide by atmospheric oxygen.
- Colorimeter/Spectrophotometer: Measures absorbance at 680 nm for quantifying phosphomolybdenum blue.
- Centrifuge or Filter Setup: Whatman No. 1 filter paper for obtaining protein-free filtrate.
2. Reagents & Preparation
A. Deproteinization Reagents
- 2/3N Sulfuric Acid (H₂SO₄): Mix 2 mL concentrated H₂SO₄ with 50 mL distilled water (D/W), then dilute to 100 mL.
- 10% Sodium Tungstate: Dissolve 10 g sodium tungstate in 100 mL D/W.
B. Reduction & Color Development Reagents
- Alkaline Copper Tartrate:
- Solution A: Dissolve 40 g sodium carbonate + 7.5 g tartaric acid in 400 mL D/W.
- Solution B: Dissolve 4.5 g copper sulfate in 100 mL D/W.
- Mix A + B and dilute to 1,000 mL with D/W.
- Phosphomolybdic Acid:
- Dissolve 35 g molybdic acid + 5 g sodium tungstate in 200 mL 10% NaOH.
- Boil for 45 minutes to remove ammonia. Add 125 mL 89% phosphoric acid, then dilute to 500 mL with D/W.
C. Standards & Controls
- Stock Glucose Standard (1 g/dL): Dissolve 1 g glucose in 100 mL 0.3% benzoic acid.
- Working Standard (10 mg/dL): Dilute stock 1:100 with benzoic acid.
3. Critical Requirements for Accuracy
- Protein-Free Filtrate: Ensures no interference from blood proteins.
- Standardized Heating: Boiling water bath for 10 minutes to fully reduce cupric ions.
- Calibration: Use a Blank (distilled water) and Standard (glucose solution) to validate results.
Procedure
Step 1: Protein-Free Filtrate Preparation
- Sample Dilution: Mix 1 mL of whole blood with 7 mL distilled water to lyse cells.
- Precipitation:
- Add 1 mL of 10% sodium tungstate.
- Slowly add 1 mL of 2/3N sulfuric acid (H₂SO₄) while mixing.
- Filtration: Let the mixture stand for 5 minutes, then centrifuge or filter using Whatman No. 1 filter paper to obtain a clear, protein-free filtrate.
Step 2: Glucose Reduction & Color Development
- Test Setup: Prepare three Folin-Wu tubes (constricted to prevent oxygen interference):
Reagent Blank Standard Test Distilled Water 1 mL – – Glucose Standard – 1 mL – Protein-Free Filtrate – – 1 mL Alkaline Copper Tartrate 1 mL 1 mL 1 mL - Heating: Place tubes in a boiling water bath for 10 minutes to reduce cupric ions (Cu²⁺) to cuprous oxide (Cu₂O).
- Color Reaction:
- Cool tubes, then add 1 mL phosphomolybdic acid to each.
- Shake to eliminate bubbles and dilute to 12.5 mL with distilled water.
- Measurement: Read absorbance at 680 nm using a spectrophotometer. Calibrate using the Blank tube.
Calculation
- Blood Sample: A 1:10 dilution (1 mL blood + 9 mL diluent) is used. The test employs 1 mL of diluted sample, representing 0.1 mL of actual blood.
- Glucose Standard: 1 mL of working standard (10 mg/dL) contains 0.1 mg glucose, ensuring precise calibration.
Interpreting Folin-Wu Method Results
Results are calculated using a standard calibration curve. Normal fasting blood glucose ranges between 70–110 mg/dL. Values outside this range may indicate hypoglycemia or diabetes. Fasting levels are categorized as:
- Normal: 70–110 mg/dL
- Prediabetes: 111–125 mg/dL
- Diabetes: ≥126 mg/dL
Folin-Wu Method vs. Modern Techniques
While the Folin-Wu method laid the groundwork for glucose estimation, newer methods like glucose oxidase and hexokinase assays offer faster, automated solutions. However, Folin-Wu’s role in historical and educational contexts remains significant.
Folin-Wu Method Advantages and Disadvantages
Advantages
- Specificity: Minimizes interference from non-glucose substances.
- Reliability: Proven consistency in clinical laboratories.
- Cost-Effective: Uses readily available reagents.
Disadvantages
- Time-Consuming: Requires multiple steps and heating.
- Toxic Reagents: Sodium tungstate and sulfuric acid demand careful handling.
- Outdated: Modern methods like glucose oxidase are faster.
FAQs
What is the reagent used in Folin-Wu method?
Why is sodium tungstate used in Folin-Wu method?
Is the Folin-Wu method still used today?
Reference(s)
- J., Ochei., et al. “Medical Laboratory Science: Theory and Practice.”, Tata McGraw-Hill, 2008, isbn: 9780074632239.
- Naigaonkar, A. V.. “A Manual Of Medical Laboratory Technology.” 9th ed., Nirali Prakashan, 7 July 2008, isbn: 9788185790299.
- Damodaran K, Geetha. “Practical Biochemistry.” 2nd ed., Jaypee Brothers Medical Publishers, 15 June 2010, isbn: 9789351529941.
Cite this page:
- Posted by Dayyal Dg.