Phenol-Sulfuric Acid Method for Total Carbohydrates

The phenol-sulfuric acid method is a straightforward and rapid technique used to measure the total amount of carbohydrates in a sample. It’s widely regarded for its simplicity and reliability in detecting various types of carbohydrates, from simple sugars to more complex forms like polysaccharides.

Here’s how it works: concentrated sulfuric acid is used to break down polysaccharides, oligosaccharides, and disaccharides into simpler monosaccharides. These monosaccharides, whether pentoses (with five carbon atoms) or hexoses (with six carbon atoms), undergo further chemical reactions to produce compounds like furfural and hydroxymethyl furfural. These compounds then react with phenol, resulting in a distinct yellow-gold coloration.

To quantify the amount of carbohydrates present, standard curves are prepared using known sugars like xylose for pentoses or glucose for hexoses. The absorbance of the resulting colored solution is measured at specific wavelengths—480 nm for pentose-rich samples and 490 nm for hexose-rich samples. This color reaction remains stable for several hours, ensuring reliable measurements.

Overall, the phenol-sulfuric acid method stands out among colorimetric techniques for its ease of use and broad applicability in carbohydrate analysis, providing accurate results within a margin of ±2% under optimal conditions.

Contents

Objective of Phenol-Sulfuric Acid Method

Find out the total amount of carbohydrates in the sample.
Determine the total carbohydrate content of soft drinks and beers.

Principle of Phenol-Sulfuric Acid Method

The principle of the phenol-sulfuric acid method revolves around the reaction of carbohydrates with strong acid and heat, producing measurable compounds. When carbohydrates—whether simple sugars, oligosaccharides, or polysaccharides—are exposed to concentrated sulfuric acid, they break down into simpler forms.

Under the action of sulfuric acid and heat, these carbohydrates decompose into furan derivatives. Pentoses turn into furfural, while hexoses convert into hydroxymethyl furfural. These furan derivatives then react with phenol, resulting in a stable yellow-gold color.

The intensity of this color is directly related to the carbohydrate concentration in the sample. Using a spectrophotometer, you can measure the absorbance of this color at specific wavelengths (480 nm for pentoses and 490 nm for hexoses), giving you a quantitative measure of the total carbohydrates present.

This method is reliable and simple, making it a go-to technique for carbohydrate analysis. The reaction produces consistent results, with minimal variation, ensuring accurate and repeatable measurements.

Requirements for Phenol-Sulfuric Acid Method

Chemicals

D-Glucose (C6H12O6): CAS No. 50-99-7
Phenol (C6H6O): CAS No. 108-95-2, Toxic
Sulfuric Acid (H2SO4): CAS No. 7664-93-9, Corrosive

Reagents

Glucose Standard Solution: 100 mg/L
Phenol Solution: 80% wt/wt in H2O, 1 ml
- Mix 20 g deionized distilled water with 80 g redistilled reagent-grade phenol (crystals).
Sulfuric Acid: Concentrated

Supplies

Beer (light and regular, same brand)
Waste collection bottle
Cuvettes for spectrophotometer
Erlenmeyer flask, 100 ml, for dd water
Two Erlenmeyer flasks, 500 ml, for beverages
Gloves
Mechanical pipettors, 1000ml and 100ml (or 200ml) with plastic tips
Pasteur pipettes and bulb
Parafilm®
Pipette bulb or pump
Repipettor (for fast delivery of 5 ml concentrated H2SO4)
Clear-colored soft drinks, diet and regular, same brand
Twenty test tubes, 16–20 mm internal diameter
Test tube rack
Four volumetric flasks, 100 ml or two volumetric flasks, 1000 ml
Volumetric pipette, 5 ml
Two volumetric pipettes, 10 ml

Equipment

Spectrophotometer
Vortex mixer
Water bath, maintained at 25°C

Procedure of Phenol-Sulfuric Acid Method

1. Prepare Standard Curve Tubes

Use the glucose standard solution (100 mg glucose/L) and deionized distilled water.
Pipet aliquots into clean test tubes as indicated in the table below.
Each tube should contain 0–100 ml of glucose in a total volume of 2 ml.
Use duplicates for each concentration to create a standard curve.
Use the 0 mg glucose/2 ml sample to prepare the reagent blank.

mg Glucose/2 ml	0	20	40	60	80	100
ml glucose stock solution	0	0.2	0.4	0.6	0.8	1.0
ml dd water	2.0	1.8	1.6	1.4	1.2	1.0

2. Record Caloric Content

Analyze total carbohydrate content.
Choose a regular and diet soft drink of the same brand, or a regular and lite beer of the same brand.
Record the caloric content from the nutrition label before starting the sample preparation and analysis.

3. Decarbonate the Beverages

Pour approximately 100 ml of the beverage into a 500-ml Erlenmeyer flask at room temperature.
Shake gently to avoid foaming (especially if it is beer) until no CO2 bubbles appear.
Filter the sample if there is any noticeable suspended material.

4. Prepare Sample Tubes

Ensure the sample tested contains 20–100 mg glucose/2 ml.
Follow the dilution procedure and volumes to be assayed as given below.
Pipette 1.0 ml of the diluted sample into a test tube and add 1.0 ml of dd water.
Analyze each diluted sample in duplicate.

Dilution	Volume Assayed (ml)
Soft Drink Regular	1:2000
Soft Drink Diet	0
Beer Regular	1:2000
Beer Lite	1:1000

Dilution Schemes

1:2000 Dilution:
- Pipette 5 ml of beverage into a 100-ml volumetric flask, dilute with dd water, seal with Parafilm®, and mix (1:20 dilution).
- Pipette 1.0 ml of this dilution into another 100-ml volumetric flask, dilute with dd water, seal with Parafilm®, and mix.
- Alternatively, pipette 1.0 ml of beverage into a 1000-ml volumetric flask, dilute with dd water, seal with Parafilm®, and mix.
1:1000 Dilution:
- Pipette 10 ml of beverage into a 100-ml volumetric flask, dilute with dd water, seal with Parafilm®, and mix (1:10 dilution).
- Pipette 1.0 ml of this dilution into another 100-ml volumetric flask, dilute with dd water, seal with Parafilm®, and mix.
- Alternatively, pipette 1.0 ml of beverage into a 1000-ml volumetric flask, dilute with dd water, seal with Parafilm®, and mix.

5. Add Phenol

To each tube with a total volume of 2 ml, add 0.05 ml 80% phenol.
Use a mechanical pipettor for accuracy.
Mix using a Vortex mixer.

6. Add Sulfuric Acid

Add 5.0 ml of concentrated sulfuric acid to each tube rapidly.
Direct the stream of acid against the liquid surface for good mixing.
Mix on a Vortex mixer.
Let the tubes stand for 10 minutes.
Place in a 25°C bath for 10 minutes to cool to room temperature.
Vortex the tubes again before reading the absorbance.

7. Measure Absorbance

Wear gloves to transfer samples from test tubes to cuvettes.
Do not rinse cuvettes with water between samples.
Zero the spectrophotometer with the standard curve sample containing 0 mg glucose/2 ml (blank).
Retain the blank sample for later use.
Read absorbances at 490 nm.
Start with the standard curve tubes from low to high concentration, then read your beverage samples.
Wipe the outside of the cuvettes with a clean paper wipe before each reading.

8. Absorbance Spectra

Use a duplicate tube from a standard curve sample with an absorbance reading of 0.5–0.8.
Determine the absorbance spectra from 450 to 550 nm.
Read at 10 nm intervals.
Zero the spectrophotometer with the blank at each interval.

Simple Protocol of Phenol-Sulfuric Acid Method

1. Prepare Test Tubes

Use seven clean, dry test tubes for the phenol-sulfuric acid method.

2. Prepare Glucose Standards

Prepare dilutions using glucose standards with concentrations of 40, 80, 120, 160, 200 μg/200 μl.
Transfer the appropriate volume of glucose from the 1 mg/mL glucose standard solution.
Dilute with distilled water to make a total volume of 200 μl.

3. Add Phenol Solution

Add 0.2 ml of a 5% phenol solution to each test tube.

4. Add Sulfuric Acid

Add 1 ml of concentrated sulfuric acid to each test tube.
Mix thoroughly by shaking.

5. Mix and Incubate

Mix the contents of the test tubes and let them stand for 10 minutes.
Place the tubes in a water bath maintained at 25 to 30°C for 20 minutes.

6. Use Spectrophotometer

Turn on the spectrophotometer and set it to 490 nm wavelength.

7. Zero the Blank

Set the absorbance (OD) to zero using the blank sample.

8. Create Absorbance Standard Curve

Prepare an absorbance standard curve at 490 nm with glucose concentration (g/200 μl) on the Y-axis.
Record the absorbance values from the standard curve.

9. Analyze Sample

Weigh 100 mg of the sample and place it in a boiling tube.
Hydrolyze by placing in a boiling water bath with 5 ml of 2.5 N HCl for 3 hours.
Cool to room temperature and neutralize with solid sodium carbonate until effervescence stops.
Make up the volume to 100 ml and centrifuge the solution.
Pipette out 0.2, 0.4, 0.6, 0.8, and 1 ml of the working standard into separate test tubes.
Pipette out 0.1 and 0.2 ml of the sample solution into two separate test tubes and make up the volume to 1 ml with water.
Set a blank using 1 ml of water.
Add 1 ml of phenol solution to each tube.
Add 5 ml of 96% sulfuric acid to each tube and shake well.
After 10 minutes, shake the contents of the tubes again and place them in a water bath at 25-30°C for 20 minutes.
Read the color at 490 nm using the spectrophotometer.
Calculate the total carbohydrate content in the sample solution using the absorbance values from the standard curve.

Data and Calculations

Phenol-Sulfuric Acid Method for Total Carbohydrates | Source: noteshippo.com

1. Procedure and Results Summary:

Standards and Samples: The following tables summarize procedures and results for standards and samples.
Example: Below are tables showing one standard curve sample and one soft drink sample as illustrations.

Tube #	Sample Identity	Dilution Scheme	ml Diluted	std or unknown A490	Glucose Equivalent
1	Blank	–	–	–	–
2	Std. 20mg	–	–	–	–
3	Std. 20mg	–	–	–	–
4	Std. 40mg	–	–	–	–
…	…	…	…	…	…
12	Soft drink, reg.	1:2000	1 ml	0.648	79 mg

2. Sample Calculation for Soft Drink, Regular:

Equation of the line: y = 0.011x + 0.1027
Given: A490 = 0.648
Calculated: Glucose equivalent = 157.95 g/L

3. Standard Curve Construction:

Construct a standard curve for total carbohydrate determinations in terms of glucose (A490 versus mg glucose/2 ml).
Determine the equation of the line for the standard curve.

4. Glucose Concentration Calculation:

Calculate glucose concentration in soft drink and beer samples:
- (a) grams/liter
- (b) g/12 fl. oz. (Note: 29.56 ml/fl. oz.)

5. Caloric Content Calculation:

Calculate caloric content (based on carbohydrate content) of soft drink and beer samples in terms of Cal/12 fl. oz.

6. Absorbance Spectra Plotting:

Plot absorbance spectra obtained between 450 and 550 nm.

Interpretation

The method of phenol sulfuric acids involves mixing a set solutions that have known glucose concentrations and the method’s phenol sulfuric acid reagent. A standard curve may be made, and the sugar concentrations of untested samples can be determined.

Advantages of Phenol-Sulfuric Acid Method

Simplicity and Accessibility:
- Straightforward Procedure: The method involves simple steps that are easy to perform, making it accessible even for beginners in biochemical analysis.
- Minimal Equipment Requirement: Requires basic laboratory equipment, reducing the barrier to implementation in various settings.
High Sensitivity:
- Detection of Low Concentrations: The method is highly sensitive, capable of detecting even trace amounts of carbohydrates in samples.
- Quantitative Analysis: Provides accurate quantification of carbohydrates, crucial for precise measurements in research and quality control.
Wide Applicability:
- Versatile in Sample Types: Suitable for a wide range of sample types, including biological fluids, food products, and environmental samples.
- Compatibility with Various Carbohydrates: Can be applied to different types of carbohydrates, from simple sugars to complex polysaccharides.
Cost-Effectiveness:
- Economical Reagents: Relies on affordable reagents such as phenol and sulfuric acid, making it a cost-effective choice for routine carbohydrate analysis.
- Batch Analysis: Allows for batch processing of samples, optimizing laboratory workflow and reducing overall costs per analysis.
Quantitative Analysis:
- Precise Quantification: Offers precise quantification of carbohydrates through calibration with standard curves, ensuring reliable and reproducible results.
- High Accuracy: Provides accurate measurements, crucial for scientific research, nutritional labeling, and quality assurance in industries like food and pharmaceuticals.

Limitations of Phenol-Sulfuric Acid Method

Specificity:
- Non-Specific Detection: The method detects total carbohydrates but does not differentiate between different types of carbohydrates (e.g., glucose, fructose, sucrose), which can be a limitation in certain applications requiring specific analysis.
Interference from Impurities:
- Effect of Impurities: Presence of impurities in samples can interfere with the reaction between phenol and carbohydrates, potentially leading to inaccurate results.
- Sample Preparation: Requires careful sample preparation to minimize interference and ensure accurate carbohydrate quantification.
Labor Intensive:
- Manual Handling: The method involves multiple manual steps, including preparation of reagents and standards, which can be labor-intensive and time-consuming.
- Precision in Handling: Requires precise handling of reagents and samples to maintain reproducibility and accuracy.
Limited Dynamic Range:
- Detection Sensitivity: While sensitive, the method may have a limited dynamic range for quantification, particularly for samples with very low or very high carbohydrate concentrations.
- Standard Curve Adjustments: Requires careful calibration and adjustment of standard curves to accurately quantify carbohydrates across different concentration ranges.
Environmental Considerations:
- Use of Hazardous Reagents: Involves the use of concentrated sulfuric acid, which is hazardous and requires proper handling and disposal protocols to ensure laboratory safety and environmental compliance.
Application Scope:
- Suitability for Specific Samples: May not be suitable for all sample matrices or types of carbohydrates, limiting its applicability in certain research or industrial settings.
- Alternative Methods: Depending on the specific requirements, alternative methods with higher specificity or automation may be preferred.

References

Viel, Marie & Collet, Florence & Lanos, Christophe. (2018). Chemical and multi-physical characterization of agro-resources’ by-product as a possible raw building material. Industrial Crops and Products. 120. 214-237. 10.1016/j.indcrop.2018.04.025.
Nielsen, S. Suzanne (2010). [Food Science Texts Series] Food Analysis Laboratory Manual || Phenol-Sulfuric Acid Method for Total Carbohydrates. , 10.1007/978-1-4419-1463-7(Chapter 6), 47–53. doi:10.1007/978-1-4419-1463-7_6
https://biocyclopedia.com/index/plant_protocols/carbohydrates/phenol_suplhuric_acid_method_for_total_carbohydrate.php
https://noteshippo.com/phenol-sulfuric-acid-method/