Chlorophyll Quantitative Measurement: Using Equations from Japanese Water Standard Test Methods

Significance of the Topic

This application note describes a practical workflow for quantifying chlorophyll (and related pigments) in water and plant extracts by UV-Vis spectrophotometry using standardized Japanese water test methods. Accurate chlorophyll measurement is important for water quality monitoring, eutrophication assessment, and ecological studies, and implementing standard equations directly in instrument software reduces manual data handling and associated human errors.

Objectives and Study Overview

The main objective was to demonstrate how LabSolutions UV-Vis software (Ver. 1.21) can implement the equations from Japanese Water Standard Test Methods (III: Organic Materials) to calculate chlorophyll a, b, c and carotenoid concentrations directly from spectrophotometric measurements. The study compares two extraction protocols (acetone and ethanol) applied to municipal/park water samples and a cabbage leaf extract to validate instrument configuration, data processing, and reporting workflows.

Methods

Sample preparation followed the Japanese Water Standard Test Methods for acetone and ethanol extraction of chlorophyll from filtered biomass:

• Acetone extraction: Test water is amended with magnesium carbonate suspension, filtered through glass-fiber filter paper; the retained material is pulverized with acetone, several rinses are combined to a fixed acetone volume (~12 mL), left in the dark for 1 hour, centrifuged, and the supernatant is used (10 mL) as test solution.
• Ethanol extraction (alternative): Biomass on filter paper is immersed in a fixed volume (20–25 mL) of ethanol, heated briefly in a 75 °C water bath, cooled, centrifuged and the supernatant used as sample.

Spectral and photometric measurements were recorded on a UV-1900i Plus spectrophotometer. For acetone extracts absorbances were measured at 480, 630, 645, 663 and 750 nm (10 mm path); for ethanol extracts measurements were taken at 665 and 750 nm before and after acidification to differentiate chlorophyll forms.

Used Instrumentation

• Spectrophotometer: Shimadzu UV-1900i Plus
• Slit width: 1 nm
• Photometric mode: Absorbance; integration time 1.0 s
• Photometric wavelengths (acetone method): 480, 630, 645, 663, 750 nm; spectral scan: 400–760 nm (low speed)
• Photometric wavelengths (ethanol method): 665, 750 nm; spectral scan 600–800 nm (low speed)
• Lab software: LabSolutions UV-Vis Ver. 1.21 with configurable formula/factor columns

Main Results and Discussion

Key processing steps implemented in the software:

• Subtraction of baseline absorbance at 750 nm from assay wavelengths to obtain corrected absorbances (e480, e630, e645, e663).
• Implementation of chlorophyll and carotenoid calculation formulas from the Japanese standard directly within LabSolutions UV-Vis, including per-sample input fields for extracted-solution volume and filtered water volume and the ability to reference absorbance values from other rows.

Representative findings:

• Municipal tap water and public drinking-fountain water: chlorophyll a was not detected by either extraction method.
• Cabbage leaf extract: a clear absorbance peak near 663–665 nm was observed. Using the appropriate equation (acetone: linear combination of e663, e645, e630; ethanol: difference before/after acidification scaled by a constant and normalized by volumes and path length), chlorophyll a concentration was calculated.

Equations implemented (as applied in the software):

• Acetone-based chlorophyll a (µg/L): a linear combination of corrected absorbances: 11.64·e663 − 2.16·e645 + 0.10·e630, multiplied by the ratio of test-solution volume to test-water volume and a factor to convert to µg/L.
• Carotenoids: proportional to e480 with different coefficients depending on dominant algal group (e.g., ×4.0 for green/blue–green algae, ×10.0 for diatoms).
• Chlorophyll b and c: additional linear combinations of e663, e645 and e630 with coefficients defined in the standard.
• Ethanol (acidification) method: chlorophyll a calculated from (A_before − A_after-acidification) × 29.6 × (V_extracted / (V_sample × d)), where A denotes the absorbance difference (665–750 nm), V_extracted is extract volume (mL), V_sample is filtered water volume (L) and d is cell path length (cm).

By configuring these formulas in LabSolutions UV-Vis, the system produced calculated concentration values directly in the photometric results window, avoiding manual data transfer to spreadsheets. The software also supports expanded mathematical functions (power, square root, common and natural logarithms) and per-sample coefficient fields, increasing flexibility for complex or sample-specific calculations.

Benefits and Practical Applications

• Reduced manual handling and lower risk of transcription errors because the instrument software computes standardized equations and incorporates sample-specific volumes and path length variables.
• Faster reporting: direct output of concentration values alongside raw absorbance data simplifies QA/QC workflows and report generation.
• Regulatory compliance: direct implementation of Japanese Water Standard Test Methods equations ensures consistency with prescribed analytical procedures.
• Flexibility: support for inter-row reference data and expanded mathematical functions allows adaptation of the software for additional derived analytes or corrective calculations.

Future Trends and Applications

• Broader integration of standardized equations into instrument software to cover additional regulatory methods (e.g., other national water standards) will further reduce spreadsheet reliance.
• Automated QC checks and flagging of out-of-range results within the spectrophotometer software could streamline laboratory decision-making.
• Increased use of per-sample metadata (site, filter area, biomass weight) and automated LIMS integration to produce turnkey analytical reports.
• Extension to spectral-deconvolution algorithms and chemometric modules inside instrument software to improve resolution of overlapping pigment absorptions in mixed algal communities.

Conclusion

Implementing the Japanese Water Standard Test Methods for chlorophyll quantitation directly in LabSolutions UV-Vis (Ver. 1.21) demonstrates a practical way to combine standardized extraction protocols with built-in calculation capabilities. The approach reduces manual data handling, supports per-sample parameterization, and streamlines generation of concentration results for chlorophyll a, b, c and carotenoids. Application to environmental and plant-extract samples confirmed expected outcomes (no detectable chlorophyll in tested tap water samples; measurable chlorophyll in cabbage leaf extract).

Reference

1. Water Standard Test Methods 2020 Edition III: Organic Materials. Japan Water Works Association.
2. Shimadzu Application News: Quantitative Analysis of Chlorophyll a, b and Carotenoids in Cabbage: Use of Evaluation Function of LabSolutions UV-Vis, Application News No. A631.