Abstract
A set of equations for determining chlorophyll a (Chl a) and accessory chlorophylls b, c 2 , c 1 + c 2 and the special case of Acaryochloris marina, which uses Chl d as its primary photosynthetic pigment and also has Chl a, have been developed for 90% acetone, methanol and ethanol solvents. These equations for different solvents give chlorophyll assays that are consistent with each other. No algorithms for Chl c compounds (c 2 , c 1 + c 2) in the presence of Chl a have previously been published for methanol or ethanol. The limits of detection (and inherent error, ± 95% confidence limit), for chlorophylls in all organisms tested, was generally less than 0.1 µg/ml. The Chl a and b algorithms for green algae and land plants have very small inherent errors (< 0.01 µg/ml). Chl a and d algorithms for Acaryochloris marina are consistent with each other, giving estimates of Chl d/a ratios which are consistent with previously published estimates using HPLC and a rarely used algorithm originally published for diethyl ether in 1955. The statistical error structure of chlorophyll algorithms is discussed. The relative error of measurements of chlorophylls increases hyperbolically in diluted chlorophyll extracts because the inherent errors of the chlorophyll algorithms are constants independent of the magnitude of absorbance readings. For safety reasons, efficient extraction of chlorophylls and the convenience of being able to use polystyrene cuvettes, the algorithms for ethanol are recommended for routine assays of chlorophylls. The methanol algorithms would be convenient for assays associated with HPLC work.
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Acknowledgements
The author wishes to thank Dr Min Chen for her kind gift of preparations of pure Chlorophyll d and for providing advice on growing Acaryochloris marina in culture. The author wishes to thank Dr George F Humphrey for his critical reading of the manuscript. Access to facilities in the laboratory of Prof AWD Larkum and Dr RG Quinnell is also gratefully acknowledged.
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Appendices
Appendices
Appendix 1: Asymptotic errors
For a multiple linear equation of the form, \( Z = Av + Bw + Cx + Dy{\hbox{,}} \) where, the absorbance coefficient constants A,B,C and D all have measurable errors ±A, ±B, ±C and ±D, the asymptotic error (±Z) is,
since \( \frac{{{\hbox{d}}Z}} {{{\hbox{d}}v}}{\hbox{, }}\frac{{{\hbox{d}}Z}} {{{\hbox{d}}w}}{\hbox{, }}\frac{{{\hbox{d}}Z}} {{{\hbox{d}}x}}{\hbox{ \ }}\frac{{{\hbox{d}}Z}} {{{\hbox{d}}y}}{\hbox{ = 1,}} \) \( <$> <$>\pm Z \approx {\sqrt { \pm A^{{\hbox{2}}} + \pm B^{{\hbox{2}}} + \pm C^{{\hbox{2}}} + \pm D^{{\hbox{2}}} } } \) (Note that the error is independent of absorbance readings v,w,x or y).
For example, for a spectrophotometric equation for Chl a using absorbancesat two wavelengths, A 630 and A 664 and calculated absorbance coefficients E 630 and E 664,
The asymptotic error of a chlorophyll ratio can be calculated in a similar fashion.
For, \( Z = \frac{B} {A}{\hbox{,}} \) where B and A have errors ± B and ± A, the error is approximately, \( \pm Z \approx {\hbox{ }}{\sqrt {{\left( {\frac{{{\hbox{d}}Z}} {{{\hbox{d}}A}}} \right)}^{2} {\left( { \pm A} \right)}^{2} + {\left( {\frac{{{\hbox{d}}Z}} {{{\hbox{d}}B}}} \right)}^{2} {\left( { \pm B} \right)}^{2} } }{\hbox{,}} \) which simplifies to \( \pm Z \approx Z{{\sqrt {{\left( {\frac{{ \pm{ A}}} {A}} \right)}^{{\hbox{2}}} + {\left( {\frac{{ \pm B}} {B}} \right)}^{{\hbox{2}}} } }}. \)
A Chl b/a ratio can therefore be expressed as,
Appendix 2: Matrix algebra
The example shown are for sets of spectrophotometric readings in acetone solvent where absorbances are measured at 630, 647, 664 and 691 nm which are the Qy values are for Chl c 2 and Chl c 1 + c 2, b, a and d respectively. It can be shown that all the chlorophyll equations have the same solution matrix. For a chlorophyll equation based on absorbance readings at two wavelengths, Chl = E 647.A 647 + E 663.A 664, for a chlorophyll equation requiring measurements at three wavelengths, Chl = E 630.A 630 + E 647.A 647 + E 664.A 664,
Appendix 3: Published chlorophyll formulae used in the present study
The equations of Smith and Benitez (1955) have been recalculated using the following extinction values: εChl a,663 = 101 l g−1 A cm−1, εChl a,688 = 1.848 l g−1 A cm−1, εChl d,663 = 12.83 l g−1 A cm−1, εChl d,663 = 110.23 l g−1 A cm−1). *Inherent errors calculated on the assumption that the relative errors of the absorbance coefficients were ± 1.0%. **Inherent errors quoted here are ± 2 × standard deviations of the extinction values found by Porra et al. (1989). The acetone equations for Chl a and b by Porra et al. (1989) are for 80% acetone and so have not been included in the present study.
Authority | Solvent | Chlorophyll | Formulae (µg ml−1) | Inherent error* (µg ml−1) |
|---|---|---|---|---|
Humphrey and Jeffrey (1975)* | 90% acetone | a | 11.93 × A 664 − 1.93 × A 647 | 0.1209 |
b | −5.5 × A 664 + 20.36 × A 647 | 0.2108 | ||
a | 11.43 × A 664 − 0.40 × A 630 | 0.1144 | ||
c 2 | −3.80 × A664 + 24.88 × A 630 | 0.2516 | ||
a | 11.47 × A 664 − 0.40 × A 630 | 0.1148 | ||
c 1 + c 2 | −3.73 × A 664 + 24.36 × A 630 | 0.2464 | ||
100% methanol | a | 16.29 × A665–8.54 × A652 | 0.6056 | |
b | −13.58 × A 665 + 30.66 × A 652 | 1.1438 | ||
Rowan (1989)* | 100% ethanol | a | 13.70 × A665 − 5.76 × A649 | 0.1486 |
b | −7.60 × A 665 + 25.8 × A 649 | 0.2690 | ||
Modified from Smith and Benitez (1955)* | 100% diethyl ether | a | 9.92 × A 663 − 1.15 × A 688 | 0.0999 |
d | −0.166 × A 663 + 9.09 × A 688 | 0.0909 |
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Ritchie, R.J. Consistent Sets of Spectrophotometric Chlorophyll Equations for Acetone, Methanol and Ethanol Solvents. Photosynth Res 89, 27–41 (2006). https://doi.org/10.1007/s11120-006-9065-9
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DOI: https://doi.org/10.1007/s11120-006-9065-9