The extraction and measurements of chlorophyll are procedures with several steps, each of which can be performed in several slightly different ways. It is further complicated by the presence of several types of chlorophyll (a, b, c) and pheopigments with similar absorption peaks in the spectrophotometric analysis.
In literature several formulas for chlorophyll concentration are available (UNESCO 1966, Stickland and Parsons 1968, Jeffrey and Humphrey 1975). For the calculation of chlorophyll a the differences between the equations are insignificant (<2.5%). For the calculation of chlorophyll b and c the differences between the equations are more noticeable.
Till the end of 1995, according to the recommendation of BMB (1979) our measurements were mainly carried out with the use of acetone-extract method. Since 1996 ethanol has been used as an extraction solvent. Some details, such as filter type, spectrometer, formula, were different form cruise to cruise. These details are given in each data file.
The procedure was as follow:
Filtration: The samples were filtered immediately after sampling, but when a lot of samples were taken, the total filtration time could take about 3 hours. The volume of filtered water was 0.5-5l, depending on the chlorophyll contents. The suction pressure was smaller than 0.5 atm. After filtration the filters were dried and wrapped in a piece of clean filter paper.
Storage: Till the end of 1997 the filters were stored frozen. The analyses were carried out from 2 weeks to 3 months after filtration. None preservation substances were added. Since 1998 the filters have been frozen only in exceptional cases. Usually the analyses are carried out directly after filtrations.
Extraction: Till the end of 1995, the 90% acetone was used as an extraction solvent. The works were carried out in subdued light (lately green light). Till 1987 the filters were homogenised according to the current procedure. Later the filters were flooded with the 90% acetone and were kept for 24 hours in darkness in a fridge (such extraction gives satisfactory results). The extract was centrifuged (~4000 rev/min, 10 min). The tubes were shaken before centrifugation. Since 1996 the extraction solvent has been changed for ethanol. The filters are flooded with ethanol and are kept for 24 hours in darkness in a room temperature. Then the extract is centrifuged (~4000 rev/min, 10 min). The tubes were shaken before centrifugation.
Spectrophotometric reading: When possible, the absorption spectra in the range 400-750nm were collected. In 1994 - 1997 our spectrophotometer SPECORD M40 was connected with the PC computer. Some of the earlier data were registrated on the paper. Since 1998 new spectrophotometer UNICAM UV4-100, placed on s/y Oceania board has been used. The length of cuvettes and the extract volume were chosen according to the chlorophyll concentration range (usually 1-2cm and 8-10ml, respectively).
Formulae (90% acetone):
The equation of Jeffrey and Humphrey (1975) [JH]:
Ca [mg m-3] = (11.85 D663-665 - 1.54 D647 - 0.08 D630) v l-1 V-1
The equation of Lorenzen (1967) [L]:
Ca [mg m-3] = 26.7 (D663-665b - D663-665a) v l-1 V-1
The equation of Parsons and Strickland (1968) [PS]:
Ca [mg m-3] = (11.6 D665 - 1.31 D645 - 0.14 D630) v l-1 V-1
The equation of SCOR/UNESCO (1968) [SU] :
Ca [mg m-3] = (11.64 D663 - 2.16 D645 - 0.1 D630) v l-1 V-1
D - absorbance at wavelength indicated by subscript, after correction by the cell-to-cell blank and subtraction of the cell-to-cell blank corrected absorbance at 750nm
D663-665b - absorbance at 663-665nm, after correction by the cell-to-cell blank and subtraction of the cell-to-cell blank corrected absorbance at 750nm, before acidification.
D663-665a - absorbance at 663-665nm, after correction by the cell-to-cell blank and subtraction of the cell-to-cell blank corrected absorbance at 750nm, after acidification.
v - volume of acetone [ml]
l - cell (cuvette) length [cm]
V - volume of filtered water [l]
The description of the procedure is given above.
The equation of Jeffrey and Humphrey (1975) [JH]:
Cb [mg m-3] = (-5.43 D663-665 + 21.03 D647 - 2.66 D630) v l-1 V-1
The equation of Parsons and Strickland (1968) [PS]:
Cb [mg m-3] = (20.7 D645 - 4.34 D665 - 4.42 D630) v l-1 V-1
The equation of SCOR/UNESCO (1968) [SU] :
Cb [mg m-3] = (20.97 D645 - 3.94 D663 - 3.66 D630) v l-1 V-1
D - absorbance at wavelength indicated by subscript, after correction by the cell-to-cell blank and subtraction of the cell-to-cell blank corrected absorbance at 750nm
v - volume of acetone [ml]
l - cell (cuvette) length [cm]
V - volume of filtered water [l]
The description of the procedure is placed at the beginning of this file.
The equation of Jeffrey and Humphrey (1975) [JH]:
Cc [mg m-3] = (-1.67 D663-665 - 7.6 D647 + 24.52 D630) v l-1 V-1
The equation of Parsons and Strickland (1968) [PS]:
Cc [mg m-3] = (55 D630 - 4.64 D665 - 16.3 D645) v l-1 V-1
The equation of SCOR/UNESCO (1968) [SU] :
Cc [mg m-3] = (54.22 D630 - 14.81 D645 - 5.53 D663) v l-1 V-1
D - absorbance at wavelength indicated by subscript, after correction by the cell-to-cell blank and subtraction of the cell-to-cell blank corrected absorbance at 750nm
v - volume of acetone [ml]
l - cell (cuvette) length [cm]
V - volume of filtered water [l]
The description of the procedure is placed at the beginning of this file.
The equation of Parsons and Strickland (1968) [PS]:
Ccar [mg m-3] = 4.0 D480 v l-1 V-1 if crop predominately Chlorophyta or Cyanophyta.
Ccar [mg m-3] = 10.0 D480 v l-1 V-1 if crop predominately Chrysophyta or Pyrrophyta.
D - absorbance at wavelength indicated by subscript, after correction by the cell-to-cell blank and subtraction of the cell-to-cell blank corrected absorbance at 750nm
v - volume of acetone [ml]
l - cell (cuvette) length [cm]
V - volume of filtered water [l]
The description of the procedure is placed at the beginning of this file.
Pheopigment a
The equation of Lorenzen (1967) [L]:
Pheopigmenta [mg m-3] = 26.7 (1.7 D663-665a - D663-665b) v l-1 V-1
D663-665b - absorbance at 663-665nm, after correction by the cell-to-cell blank and subtraction of the cell-to-cell blank corrected absorbance at 750nm, before acidification.
D663-665a - absorbance at 663-665nm, after correction by the cell-to-cell blank and subtraction of the cell-to-cell blank corrected absorbance at 750nm, after acidification.
v - volume of acetone [ml]
l - cell (cuvette) length [cm]
V - volume of filtered water [l]
Formulae (ethanol):
Ca [mg m-3] = 103(D665 - D750) v 83-1l-1 V-1 D665 - absorbance at 665nm, after correction by the cell-to-cell blank.
D750 - absorbance at 750nm, after correction by the cell-to-cell blank.
v - volume of ethanol [ml]
l - cell (cuvette) length [cm]
V - volume of filtered water [l]
83 - absorption coefficient in 96% ethanol
The description of the procedure is placed at the beginning of this file.
References:
BMB, 1979, Recommendations on methods for marine biological studies in the Baltic Sea. Phytoplankton and chlorophyll, [Ed. L. Edler], BMB, 5
Guideline for Baltic Monitoring Program, 1988, Baltic Marine Environment Protection Commision, Helsinki, 116.
Jeffrey S. W., Humphrey G. F., 1975, New spectrophotometric equation for determining chlorophyll a, b, c1 and c2, Biochem. Physiol. Pflanz., 167, 194-204
Lorenzen C. J., 1967, Determination of chlorophyll and phaeopigments: spectrophotometric equations, Limnol. Oceanogr., 12
Strickland J. D. H., Parsons T. R., 1968, A practical handbook of seawater analysis. Pigment analysis, Bull. Fish. Res. Bd. Canada, 167
UNESCO, 1966, Determinations of photosynthetic pigments in seawater, Rep. SCOR/UNESCO WG 17, UNESCO Monogr. Oceanogr. Methodol., 1, Paris