How to Calculate Total Soluble Sugars: A Comprehensive Guide

October 24, 2025 •

4 min read

Scientific research shows that soluble sugars play a critical role as signaling molecules and osmoprotectants in plants under stress. This guide will detail how to calculate total soluble sugars, covering the precise, lab-based Anthrone method and the rapid, field-friendly refractometry technique for accurate measurements in various samples.

Quick Summary

An explanation of two key methods for determining total soluble sugar content: the chemical Anthrone assay for precise laboratory quantification and the refractometry (Brix) method for quick field estimations.

Key Points

Spectrophotometric Accuracy: The Anthrone method uses a spectrophotometer to precisely measure the color intensity produced by sugars reacting with anthrone reagent, allowing for accurate lab quantification.
Quick Field Readings: The refractometer measures the total soluble solids (°Brix) by assessing light refraction, providing a fast, on-site estimate of sugar concentration in liquid samples.
Standard Curve Necessity: For the Anthrone method, a standard curve created with known sugar concentrations is critical to convert absorbance readings into accurate sugar content.
Temperature Matters: Refractometry readings are temperature-dependent, so measurements must be taken at a standard temperature (e.g., 20°C) or corrected accordingly.
Hydrolysis is Key: The Anthrone method uses concentrated sulfuric acid to break down all complex carbohydrates into simpler sugars, ensuring the "total" soluble sugar is measured.
Method Choice: Choose the Anthrone method for high-precision laboratory work or the refractometer for rapid, practical estimations in the field or for quality control.

Soluble sugars, primarily mono- and disaccharides like glucose, fructose, and sucrose, are crucial components in living organisms, serving as energy sources, metabolic intermediates, and signaling molecules. In the food industry, they determine the sweetness and quality of products, while in plant biology, their concentration can fluctuate significantly in response to environmental stress. Accurate quantification of these sugars is essential in numerous scientific and industrial applications. This article explores two prominent methods for how to calculate total soluble sugars: the Anthrone method, a precise chemical assay, and refractometry, a rapid field technique.

The Anthrone Method: A Spectrophotometric Approach

Principle of the Anthrone Assay

The anthrone test is a colorimetric assay used for the quantification of total carbohydrates, which are first hydrolyzed to monosaccharides. The reaction proceeds as follows:

First, concentrated sulfuric acid in the anthrone reagent hydrolyzes polysaccharides and disaccharides into their component monosaccharides.
Next, the concentrated acid dehydrates the monosaccharides to form furfural derivatives (e.g., hydroxymethylfurfural from hexoses).
Finally, the furfural derivatives condense with two molecules of anthrone to form a blue-green colored complex. The intensity of this color, which is proportional to the total sugar concentration, is measured using a spectrophotometer.

Detailed Procedure for the Anthrone Method

To perform the anthrone assay, follow these general steps:

Sample Extraction: Begin by extracting the soluble sugars from your sample material. For solid samples like plant tissue, grind the material and mix it with a solvent, typically 80% ethanol. A common protocol involves mixing the ground sample with the solvent, heating the mixture in a water bath, and then centrifuging to collect the supernatant containing the soluble sugars.
Standard Curve Preparation: A standard curve is essential for accurate quantification. Prepare a series of test tubes with known, graded concentrations of a standard sugar, most commonly glucose. This will allow you to plot absorbance against concentration.
Reagent and Sample Mixing: Take a measured aliquot of your extracted sample and a blank (containing only distilled water) and add them to separate test tubes. Carefully add the anthrone reagent (prepared by dissolving anthrone in concentrated sulfuric acid) to each tube, ensuring the process is carried out on ice to control the exothermic reaction.
Heating and Cooling: Mix the contents thoroughly, then place the tubes in a boiling water bath for a specific time, such as 7.5 to 10 minutes. Immediately after heating, cool the tubes rapidly in an ice bath to stop the color development reaction.
Spectrophotometer Measurement: Allow the tubes to reach room temperature. Read the absorbance of the standards and your samples using a spectrophotometer at a wavelength of 620 nm or 625 nm, using the blank to zero the instrument.
Calculation: Plot the standard curve with absorbance on the Y-axis and glucose concentration on the X-axis. Using the linear regression equation derived from the standard curve, calculate the sugar concentration in your unknown sample based on its absorbance reading. The final calculation should account for the initial sample weight and any dilution factors from the extraction.

The Refractometer Method: Quick Brix Measurement

How Refractometry Works

A refractometer measures the refractive index of a liquid, which changes based on the concentration of dissolved solids, such as sugars. As light passes through the liquid, it is bent or refracted. The degree of refraction is proportional to the density of the solution, which in many cases (like fruit juices), is primarily determined by its sugar concentration. The measurement is typically expressed in degrees Brix (°Bx).

Using a Handheld Refractometer

Calibration: Calibrate the refractometer by placing a few drops of distilled water on the prism. The reading should be zero; if not, adjust the screw to set it correctly.
Sample Application: Place a few drops of your liquid sample (e.g., fruit juice, honey, syrup) onto the refractometer's prism.
Reading: Look through the eyepiece while pointing the device toward a light source. The boundary line where the blue and white fields meet indicates the °Brix value on the scale.
Temperature Correction: For high accuracy, especially with laboratory-grade refractometers, temperature corrections may be necessary, as readings are sensitive to temperature changes. Handheld models often have automatic temperature compensation.

Anthrone vs. Refractometry: A Comparison


Feature	Anthrone Method	Refractometry (Brix)
Accuracy	High. Measures total carbohydrates after hydrolysis.	Lower. Measures total soluble solids; can be affected by other compounds besides sugar.
Equipment	Spectrophotometer, precise glassware, heating apparatus.	Handheld or Abbe refractometer.
Speed	Slow. Requires multiple steps including extraction and incubation.	Very fast. Provides an instantaneous reading.
Cost	High. Involves expensive equipment and chemical reagents.	Low. Handheld devices are relatively inexpensive.
Sample Type	Can be used for various solid or liquid samples after extraction.	Primarily for liquid samples like juices and syrups.
Application	Precise lab research, detailed analysis for complex samples.	Quick field assessments, quality control for beverages and fruits.

Conclusion

Determining how to calculate total soluble sugars depends on the application, required precision, and available resources. The Anthrone method is the gold standard for high-accuracy, quantitative analysis in a laboratory setting. This chemical assay involves specific reactions to produce a colorimetric result that is precisely measured by a spectrophotometer. The method is versatile and can be applied to a wide range of solid and liquid samples, but it is time-consuming and requires specialized equipment. In contrast, refractometry offers a quick, cost-effective, and practical estimation of total soluble solids (°Brix) for field applications and quality control. While less precise for sugar content alone, it provides instant readings suitable for routine monitoring. The choice between these methods should be guided by the specific analytical needs, balancing high precision with practicality. Learn more about total soluble sugar quantification from ethanolic plant extracts on protocols.io.

Frequently Asked Questions

The anthrone test is a colorimetric method used for the quantification of total soluble carbohydrates, including monosaccharides, polysaccharides, and carbohydrates bound to lipids or proteins, after they have been hydrolyzed.

A standard curve is created by measuring the absorbance of known concentrations of a standard sugar, like glucose. By plotting these data points, a linear regression equation is generated, which allows you to determine the unknown sample's concentration based on its absorbance.

Measured by a refractometer, °Brix is a unit of measurement for total soluble solids in a liquid. While it's a good indicator of sugar content in many solutions, it can also be affected by other dissolved compounds.

Yes, refractometers measure all soluble solids, not just sugar. In samples with a high concentration of other dissolved compounds (e.g., salts or acids), the °Brix reading may not be an accurate representation of only the sugar content.

Soluble sugars are carbohydrates that are easily dissolved in water, such as monosaccharides (glucose, fructose) and disaccharides (sucrose). They serve as important energy sources and signaling molecules in biological systems.

After heating the sample with concentrated sulfuric acid, the tubes are rapidly cooled in an ice bath to stop the chemical reaction. This ensures consistent color development and prevents over-reaction, leading to a more accurate absorbance reading.

For solid materials like plant tissue, you must first extract the sugars. A common method involves grinding the sample, mixing it with 80% ethanol, heating it, and then centrifuging to obtain the supernatant containing the soluble sugars.