Fluorometric Determination of Chlorophyll

By

Osmund Holm-Hansen, Carl J. Lorenzen, Robert W. Holmes

and John D. H. Strickland
Institute of Marine Resources

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Scripps Institute of Oceanography
University of California
La Jolla, California

The concentration of chlorophyll in laboratory grown cultures of marine

phytoplankton and in oceanic samples has been determined both by measure-
ment of fluorescence and by measurement of light absorption. The lower limit
for detection of chlorophyll by fluorescence with the instrumentation described
is about 0-01 ug chlorophyll a, which is about 5% that required for a spectro-
photometric determination. Through choice of appropriate filters, the amount
of fluorescence reflects either the chlorophyll a concentration or the sum of
chlorophylls a and c. By measurement of fluorescence before and after acidifica-
tion, the ratio of chlorophyll to phaeophytin can be readily determined.
Dilute HC1 is superior to oxalic acid for acidification of pigment extracts.
As the fluorometric determination of chlorophyll and phaeophytin is fast,
reliable and sensitive, it will be very useful in field studies of productivity.

Introduction*
Determination of phytoplankton in any body of water is most commonly
done by measuring the chlorophyll a content. The phytoplankton carbon may
be estimated by applying appropriate arithmetic factors. Chlorophyll a and
other pigments are usually determined by extracting them into an organic
solvent and determining the absorption values at specific wavelengths; these
values are then used in empirically derived formulae (PARSONS and STRICKLAND,
1963). Another method of measuring chlorophyll, which depends upon
measurement of fluorescence, has been described by KALLE (1951) and more
recently by YENTSCH and MENZEL (1963). This method has several important
advantages over light absorption methods. Firstly, it is far more sensitive,
permitting chlorophyll determinations to be made on samples of one litre or

*) Abbreviations: F, conversion factors which equate amount of fluorescence to con-

centration of the pigment as determined by light absorption values; O.D., optical density;
Rb, amount of fluoresced light before acidification of a pigment extract; Ra, amount of
fluoresced light after acidification; RbIRa, value of the acid factor.

I i. Cons. perm. int. Explor. Mer I 30 I No. 1 I 3-15 I Copenhague, Decembre 1965 |
4 O. HOLM-HANSEN, C. J. LORENZEN, R. W. HOLMES and J. D. H. STRICKLAND

less of open-ocean water, compared with the 5-10 1 required for a light ab-
sorption measurement in many such areas. Secondly, measurement of fluores-
cence is much quicker than determining extinction values at several wavelengths.
Thirdly, the instrument required for measuring fluorescence does not depend
upon critical wavelength alignment as does a spectrophotometer.
Jn our initial studies, however, the concentration of chlorophyll a as
determined by fluorescence, showed considerable variation when compared
with values obtained with a spectrophotometer. The factors for converting
units of fluorescence to corresponding optical density values varied by as much

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as 50%. Both laboratory-grown cultures of several different species of phyto-
plankton and ocean samples were used in these determinations. The fluctuations
seemed to be caused by differences in the biological material and were not due
to technique of the operator, as almost identical results were obtained by three
different operators. Since such deviations between fluorescence and light
absorption measurements were in contrast to the results of YENTSCH and
MENZEL (1963), thefluorescencemethod was studied in respect to factors which
might influence the ratio of fluorescence to light absorption by the algal
pigments. The results of these investigations are described below.

Materials and Methods

Pigments were extracted from phytoplankton collected from both coastal
and open-ocean water in the Pacific and also from cultures grown in the
laboratory. The phytoplankton was harvested by filtering through glass paper
filters (Whatman GF/C, 4-25 cm), one ml of a 1 % suspension of MgCO3 in
distilled water being added to each suspension as it was being filtered. The
filter was then ground in a Potter-Elvehjem tissue grinder for one minute at
room temperature with 4 or 5 ml of 90% acetone (STRICKLAND and PARSONS,
1965). The suspension was transferred to a centrifuge tube and the volume taken
to 120 ml with 90% acetone. After shaking, the tube was placed in the dark
for 10-30 minutes to allow complete extraction of the pigments. The suspension
was then centrifuged at 15,000 X g for five minutes and the supernatant
carefully decanted into 10 cm pathlength cells for optical density measurements
in a Beckman DU spectrophotometer. When optical density values at 665 mu
were 0-15 or less, the extract was poured directly into small tubes for determina-
tion of fluorescence on the fluorometer; with O.D. values greater than 0-15,
the extract was first diluted with 90 % acetone.
The amount of chlorophyll a in the extract was calculated from the extinction
values obtained on the spectrophotometer, using the formula given by STRICK-
LAND and PARSONS (1965). The amount of chlorophyll c separated from the
hexane layer was calculated from the formula of PARSONS (1963). The following
equation was used to relate chlorophyll a concentration to fluorescence:
chlorophyll a (ug)
dilution factor = {F) X {RbX

where F is the conversion factor for any particular slit setting on the Turner
fluorometer and Rb is the reading on the dial which indicates the amount of
light received by the phototube. An estimate of the amount of phaeophytin
present was obtained by measuring the amount offluorescencebefore and after
 Fluorometric Determination of Chlorophyll

Table 1
Conversion factors and acidification values for determination of chlorophyll a
by light absorption or by fluorescence using both laboratory cultures and natural
populations (See text for details)
Depth of Conversion factor (F) Average Average
sample for slit 3 factor acid
Organism in metres (X 10-*) (x 10-0 factor
Thalassiosira rotula - 1-7, 2-3, 2-4, 2-5 2-2 2-9
2-5,2-7 2-6 2-6

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Thalassiosira fluviatilis .... -
Coccolithus huxleyi 20, 2 0 20 30
Amphidinium carteri 20 20 31
Syracosphaera elongata . . . - 2-2,2-2 2-2 2-2
Skelelonema costatum .... - 2-5,2-9, 3 1 , 3 1 2-9 1-6
Monochrysis sp 30, 3 0 , 3-1,3-1 30 2-5
Chlorella pyrenoidosa 2-9,2-9 2-9 1-8
Ocean sample 0 2-3 2-3 _
Ocean sample 10 _ _ 2-3
Ocean sample 15 2-6 2-6 2-3
Ocean sample 0 2-5 2-5 2-3
80 2-1 2-1 1-7
Ocean sample 0 2-9 2-9 20
10 2-9 2-9 20
70 2-7 2-7 1-7
90 3-8 3-8 1-2
Ocean sample 10 2-4 2-4 2-2
25 2-5 2-5 21
50 2-5 2-5 2-2
Ocean sample 2500 _ 0-9

Table 2
Comparison of light absorption and fluorescence of chlorophylls a and c.
Fluorescence data obtained from Turner with standard lamp, high sensitivity door,
and the red 25 filter (Other details in text)
Organism from Conversion factor Acid factor
Pigment which isolated for slit 3
Chlorophyll a Macrocystis sp. 3-6 X 10-2 2-4
Chlorophyll a and carotenoids Thalassiosira rotula 3-5 X 10-2 2-3
Chlorophyll c Macrocystis sp. 4-1 x 10-2 61
T. rotula 4-1 x 10-2 5-6
Chlorophylls a (1 -52 |ig) and
c (0-22 Mg) Macrocystis 2-6 x 10-2 2-9
Faecal pellet extract Calanus helgolandicus - 1-05

Table 3
Fluorescence before and after acidification of chlorophylls a and c when mixed
in various proportions. Data obtained from Turner fluorometer with high-output
lamp, standard door and slit 3 (Details in text)
Chlorophyll Chlorophyll I
a c Chlorophyll With filter CS-2-60 With filter CS-2-54
R
(Mg) (Mg) (Mg) Kb b'Ra R
b R
blRa
0-364 0 0-364 710 21 550 21
0-337 0038 0-375 710 2-2 500 20
0-291 0100 0-391 710 2-4 45-5 21
0-255 0150 0-405 720 2-7 42-5 21
0-218 0-200 0-418 71-5 30 37-5 2-2
0109 0-350 0-459 720 40 270 2-2
0 0-501 0-501 740 6-4 16-5 2-4
6 O. HOLM-HANSEN, C. J. LORENZEN, R. W. HOLMES and J. D. H. STRICKLAND

adding acid. Acidification was achieved by adding 5 drops (or 0-2 ml) of 85%
acetone saturated with oxalic acid to the extract in the fluorometer tube or two
drops of dilute HC1 (1-0 N or 0-5 N). After shaking to affect complete mixing,
readings were taken at short intervals up to 15 minutes.
The data in Tables 2 and 3 were obtained by extracting pigments from a
freshly picked frond of the Brown alga Macrocystis pyrifera, from a laboratory-
grown culture of Thalassiosira rotula, or from copepod faecal pellets. Chloro-
phyll c was separated from other photosynthetic pigments by hexane/acetone
fractionation as described by PARSONS (1963). The hexane layer, which contained

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chlorophyll a plus carotenoids, was washed with 90% methanol and taken to
dryness with a stream of nitrogen. It was then redissolved in hexane containing
0-5% n-propanol and run through a powdered sugar column, using the same
solvent as the eluting agent. The fraction of eluate containing chlorophyll a was
taken to dryness with nitrogen and redissolved in 90% acetone. The column
chromatography was omitted for the chlorophyll-carotenoid containing hexane
layer obtained from the extract of T. rotula. It was simply taken to dryness with
nitrogen and redissolved in 90% acetone. The faecal pellets were from the
copepod Calanus helgolandicus which had been reared in the laboratory on a
diet of T.fluviatilisand Dunaliella tertiolecta. The pellets were separated from
the culture medium by filtration through a phytoplankton net of 50 micron
mesh size, and rinsed several times with millipore filtered sea water. The pellets
were extracted by 90% acetone; no attempts were made to separate the pigments
in this extract. The purity of the isolated pigments was checked by comparing
the absorption curves obtained on a Bausch and Lomb 505 recording spectro-
photometer (400 to 700 mu) with published absorption curves of the individual
pigments. There appeared to be little or no contaminating pigments in the
separated solutions of chlorophylls a or c.
The spectrophotometric determination of chlorophyll is critically dependent
upon proper wavelength alignment of the instrument. Proper alignment of the
DU was achieved by setting the wavelength reading to the 6563 A line emitted
from a hydrogen lamp. Most of the fluorometric determinations were made in
a Turner fluorometer equipped with a F4T4-BL lamp, a high sensitivity door,
a Wratten 47B blue filter for the excitation light, and a Wratten red 25 filter to
screen emitted light. Some results were obtained with a Turner fluorometer
equipped as described by YENTSCH and MENZEL (1963); it had the high-output
lamp No. 110-853, the standard door, and Corning filter CS-5-60 for light
excitation and either CS-2-60 or CS-2-64 for light screening. The spectral
transmission characteristics of thefivefiltersmentioned above were determined
in the Bausch and Lomb 505 recording spectrophotometer. Emission and
excitation spectra of the extracted pigments were obtained in an Aminco-
Bowman recording spectrofluorometer.

Results
Preliminary work indicated that readings obtained on the Turner fluorometer
are very reproducible, and that dilutions of any one solution yield a straight
line when the O. D. values at 665 mu are plotted against fluorescent units (see
also YENTSCH and MENZEL, 1963). There did not, however, seem to be great
consistency between the conversion factors (F values) when extracts of different
 Fluorometric Determination of Chlorophyll

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20 40 60 80 100
FLUORESCENCE
Figure 1. Relationship between chlorophyll a concentration as determined by light absorp-
tion and by fluorescence. All surface samples from tropical Pacific taken between 11 ° N -
10°S and 112°W-137°W. See text for details. Curve A, fluorescent readings with slit 3;
curve B, readings with slit 10.

species were compared. Data in Table 1 show both the conversion factors and
the acidification ratios for different species of phytoplankton as well as for
various natural populations. With a few exceptions, the value of the conversion
factor for slit 3 on a Turner fitted with the high sensitivity door was between
2 x 10~2 and 3 X 10"2. The acid factors varied from 0-9 in the ocean sample
from 2,500 m to 3-0 in a laboratory culture of Coccolithus huxleyi.
Figure 1 shows the relationships between chlorophyll a concentrations
determined on the spectrophotometer and readings of fluorescence obtained
on a large number of surface samples collected in the eastern tropical Pacific
between H°N-10°S and 112 O W-137°W. The fluorometer used for this work
was equipped with the high-output lamp and filter CS-2-60; its sensitivity was
about 5 times greater than the instrument fitted with the high sensitivity door.
It can be seen that there was about a 15-20% variation above and below the
lines drawn for slit 3 and slit 10. This variation is just a little less than that found
in the laboratory work summarized in Table 1. It should be noted that when
different slits are used with any one solution, the amount of fluoresced light is
not necessarily in the ratios of 1, 3, 10, and 30 as would be expected from the
nominal slit sizes. The exact relationships must be determined with any
particular instrument.
The various conversion factors obtained (Table 1) and the scatter about the
lines in Figure 1 might be reflecting different quantum yields for fluorescence
of the various photosynthetic pigments. The fluorescence of isolated pigments
was therefore examined. The values obtained for light absorption and for
fluorescence of solutions of chlorophylls a and c in 90% acetone are shown
in Table 2. It may be seen that the amount of light fluoresced by chlorophyll c
is only 88 % of that emitted by chlorophyll a per unit dry weight of each pigment
(fluorescent intensities are inversely proportional to conversion factors). When
chlorophylls a and c were mixed in the ratio of 7 to 1 (by dry weight), the
 O. HOLM-HANSEN, C. J. LORENZEN, R. W. HOLMES and J. D. H. STRICKLAND

o
X
a>

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>

CHLa
Figure 2. Relation of conversion factors (F) to the ratio of chlorophyll c to chlorophyll a.
Fluorometer equipped with high-output lamp, standard door and slit 3. Curve A, with
CS-2-60 filter; curve B, with CS-2-64 filter.

conversion factor was only 2-6 X 10~2. The fact that this value is lower than
that for chlorophyll a (3-6 X 1(H) and for chlorophyll c (4-1 x 10~2) is because
the fluorescence reading is the sum of the light emitted from both chlorophyll
a and chlorophyll c, but in calculating the value for /"only the extinction values
relating to chlorophyll a were used.
Additional data concerning the dependence of fluorescence upon the ratio of
chlorophyll a to chlorophyll c in the extract are shown in Table 3 and Figure 2.
Table 3 shows the actual amounts of chlorophylls a and c in solutions prepared
by mixing samples of the isolated pigments and the amount of fluorescence
when using either the CS-2-60 or CS-2-64 filter. The F values obtained from
these data are shown in Figure 2. It is seen that both lines A and B have negative
slopes which are steepest in the region where the ratio of chlorophyll c to
chlorophyll a is between 0 and 1. The variation in F values in this region is,
however, far less for filter CS-2-64 (curve B) than for filter CS-2-60 (curve A).
It is possible to determine the ratio of chlorophyll a to phaeophytin a by
fluorescence measurements as the fluorescence of phaeophytin a is only about
42% that of the same concentration of chlorophyll a. Chlorophyll is readily
converted to phaeophytin by dilute acids. YENTSCH and MENZEL (1963) used
oxalic acid-saturated acetone for acidification of chlorophyll solutions but the
rate of conversion of chlorophyll to phaeophytin is low with this acid. Dilute
hydrochloric acid, however, resulted in a very rapid and reproducible decrease
in fluorescence. Typical results showing the effects of these two acids are shown
in Figure 3. It is seen that an acid factor of 2-0 was obtained with oxalic acid
only after 5-10 minutes, whereas with addition of HC1 an acid factor of 2-4
 Fluorometric Determination of Chlorophyll

25r

O -a A
O

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<

10 15

TIME (minutes)
Figure 3. Time curves showing decrease of fluorescence upon acidification of chlorophyll
solutions. Five ml of a 90 % acetone extract of Chaetoceros sp. treated with 0-2 ml of 85 %
acetone saturated with oxalic acid (curve A) or with 2 drops of IN HC1 (curve B). Measure-
ments made with fluorometer with the high-output lamp and filter CS-2-60.

was obtained within 1 minute. Neither the concentration nor the amount of
HC1 added to the solutions seem to be very critical. For general work, two
drops of 0-5 N HC1 gave excellent results.
The acidification factors obtained on various cultures and in ocean samples
ranged from 0-9 to 3-1 (Table 1). As YENTSCH and MENZEL (1963) used a value
of 1-7 to indicate pure chlorophyll, it was surprising to get values so much
higher than 1-7. It is seen from Table 2, however, that chlorophyll c isolated
from Macrocystis and from T. rotula has acid factors of 6-1 and 5-6, respectively.
The acid factors for chlorophyll a isolated from the same organisms were 2-4
and 2-3, respectively. When chlorophylls a and c were mixed in the ratio of 7 to 1,
which corresponds to a ratio that might be found in nature, an acidification
ratio of 2-9 was obtained. Copepod faecal pellets yielded an acetone extract
which had an acid factor of 1 -05. The absorption spectrum of this extract was
very similar to that of pure phaeophytin a (SMITH and BENITEZ, 1955).
Similar results are shown by the acid factors in Table 3. When using the
CS-2-60 filter, the acid factors ranged from 21 (all chlorophyll a) to 6-4 (all
chlorophyll c). With the CS-2-64 filter, the range in value of the acid factor
was 21 (all chlorophyll a) to 2-4 (all chlorophyll c).
Figure 4 shows the emission spectra of chlorophylls a and c when excited
with light of 420 mu, as well as the absorption curves for the five different
filters used in the present investigation. It is seen that the blue filters should be
equally good for this work as they both transmit maximally in the region of
420 mu. The three red filters differ appreciably, however, in the extent to which
they absorb the light fluoresced by chlorophyll c. At the peak of chlorophyll c
fluorescence (about 632 m|i), filters 25, CS-2-60, and CS-2-64 transmit about
80%, 40% and < 1 % respectively, of the incident light.
10 O. HOLM-HANSEN, C. J. LORENZEN, R. W. HOLMES and J. D. H. STRICKLAND

EMISSION SPECTRA EXCITATION SPECTRA

(EXCITATION AT 420 my) (EMISSION AT 670 my)
a

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0.0
700 600 500 400 300

WAVELENGTH

Figure 4.
(A) Emission and excitation spectra of chlorophylls a and c extracted from Macrocystis sp.
Curve a, chlorophyll a; curve b, chlorophyll c; curve c, mixture of chlorophylls a and c.
(B) Absorption spectra of the red and blue filters used in the fluorometers.
 Fluorometric Determination of Chlorophyll 11

Discussion
The value of the fluorescence method of chlorophyll determination is that it
•enables one to get a quick and sensitive estimate of the concentration of both
chlorophyll and phaeophytin. In order to estimate these pigments, it is necessary
to know (1) the conversion factor for equating a unit of fluoresced light to a
specific amount of chlorophyll, (2) the ratio of the fluorescence of chlorophyll
to that of an equivalent weight of phaeophytin. If one were working with pure,
isolated pigments, the values for the above factors would be constant. In

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acetone extracts of phytoplankton, however, there is a mixture of chlorophylls
and carotenoids, all of which can affect the magnitude of the factors mentioned
above. These effects are discussed later in this section.
The conversion factor which is used in the calculations below (2-5 x 10~2
for slit 3 of the Turner equipped with Lamp No. F4T4-BL and high sensitivity
door) was obtained by taking the average value of conversion factors obtained
from laboratory-grown cultures and also from surface or near-surface samples
of oceanic phytoplankton. It is seen from the data that many factors were
obtained which were larger than 2-5 X 10~2, but these can be correlated with
the presence of phaeophytin. When a solution of chlorophyll a is acidified
the extinction value of 665 m|i decreases by about 40%, while the amount of
light fluoresced decreases by about 58 %. A large amount of phaeophytin in a
sample (before acidification) will therefore be associated with less fluorescence
than would be expected on the basis of light absorption at 665 mu. As the
conversion factor is obtained by dividing the amount of chlorophyll as
determined by light absorption by the amount of fluorescence, a greater
proportionate decrease in the divisor will result in high conversion factors.
The acidification ratios of phytoplankton extracts ranged from 0-9 to 3-1
(see Table 1), while for pure chlorophyll a and c they were about 2-4 and 6-1,
respectively. As pure phaeophytin would yield a ratio of 1-0, the low ratios
obtained in natural populations must be associated with a large ratio of
phaeophytin to chlorophyll. Ratios between 2-5 and 3-1 indicate the presence
•of significant amounts of chlorophyll c or some other pigment which shows a
high acidification number. As natural populations will often contain con-
siderable amounts of accessory chlorophylls, the acid factor for chlorophyll a
(2-4) can not be used. The value used (3-0) in the calculations below was chosen
as it represents the higher limit of acid factors obtained from healthy samples of
phytoplankton; it also is in close agreement to the value of 2-9 obtained when
chlorophylls a and c were mixed in the ratio of 7 to 1 (Table 2).
Using the above values, the equations for obtaining the concentrations of
•chlorophyll a and phaeophytin a are as follows:
Chlorophyll a = (1-5) X (F) X (Rb - Ra) (1)
Phaeophytin a = (1-5) X (F) X [3-0 (Ra) - Rb] (2)
Tt should be noted that if a sample shows an acidification ratio of greater than
3-0, then equation (2) would yield a negative value and equation (1) would give
too high a value for chlorophyll a. This merely reflects the fact that the value
used for the upper limit of the acidification ratio will be dependent upon the
ratio of chlorophyll a to accessory chlorophylls. Occasionally one may obtain
12 O. HOLM-HANSEN, C. J. LORENZEN, R. W. HOLMES and J. D. H. STRICKLAND

a sample which is unusually high in chlorophyll c relative to chlorophyll a,

which would lead to the negative value for phaeophytin when calculated as in
equation (2). If such samples are encountered in nature it would be of interest
to determine the pigment composition of the algae to verify that they do indeed
have a low amount of chlorophyll a relative to chlorophyll c, or that they might
have other pigments which are fluorescing red light.
In their equation for estimation of phaeophytin, YENTSCH and MENZEL (1963)
used an acidification ratio of 1-7. This was obtained by acidifying with oxalic
acid-saturated acetone (85%) and reading the fluorescence after 3 minutes.

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From Figure 3 it is seen that a similar number was obtained when we repeated
their method. However, when dilute HC1 was used, phaeophytinization was
complete within a minute. If one waits 10-15 minutes before reading the
fluorescence on the oxalic acid treated sample, it is significantly higher than
after 3 minutes. Figure 3 shows that the final value is about 83 % of that obtained
in the sample treated with HC1. For most samples of natural phytoplankton,
the final values obtained (after 10-15 minutes) with oxalic acid or HC1 have
agreed within 10%. In all these cases, however, the value obtained with oxalic
acid after only three minutes was significantly lower. While dilute acids remove
the Mg from chlorophyll (to form phaeophytin), strongly acidic conditions can
degrade the phaeophytin to phaeophorbide by removal of the phytol group.
The concentration of HC1 added to our samples is not, however, sufficient to
cause removal of the phytol group. It therefore seems that HC1 is preferable to
oxalic acid in this method as it produces a reliable and rapid conversion of
chlorophyll to phaeophytin.
It should be recognized that photosynthetic pigments are not the only
biological materials which will emit red light upon absorption of blue light.
The dihydroporphyrin compounds (chlorophyll derivatives) are the most active
porphins in regard to fluorescing light of wavelengths greater than 580 mu,
but many other porphyrins and metalloporphyrins also can fluoresce in this
region upon excitation with blue light (FALK, 1964). In most field work, the
concentration of such porphyrins will be insignificant in the amounts of water
filtered for a chlorophyll'determination. When 16 1 of water from a depth of
2500 m was filtered, however, there was no detectable absorption at 665 mu,
but the fluorometer gave a reading of 52 with slit 30. The acidification ratio of
this sample was 0-9; that is,fluorescenceincreased upon adding acid. Without
a spectral analysis of the pigment(s) responsible for thisfluorescence,it is not
possible to ascribe the fluorescing material to plant or animal origin. As
phaeophytins a and b and phaeophorbides a and b have been detected in marine
sediments and fossil fuels (VALLENTYNE, 1960) it seems likely that the observed
fluorescence is due to degradation products of chlorophyll, though compounds
of animal origin can not be ruled out at the present time.
There are various sources of errors inherent in the fluorescence method of
chlorophyll determination. (1) Reabsorption offluorescedlight by one or more
of the photosynthetic pigments will result in low estimates of chlorophyll a.
This effect would be most pronounced in pigment-rich extracts, and apparently
is the reason why readings with slit 1 of the fluorometer are considered to be
unreliable (YENTSCH and MENZEL, 1963). (2) MURTY and RABINOWITCH (1964)
showed that when chlorophyll a is excited by blue light, the presence of B-
carotene quenches the red fluorescence of chlorophyll. This effect, which was
 Fluorometric Determination of Chlorophyll 13

not present when the chlorophyll was excited with light at 660 mu, was attributed
to intermolecular quenching of the second excited state of chlorophyll a.
(3) Allomerization of chlorophyll (oxidation of carbon no. 10 in the cyclo-
pentanone ring) can occur when chlorophyll is in solution and exposed to
oxygen, and is accelerated by the presence of certain inorganic ions or oxidizing
agents (RABINOWITCH, 1951, p. 613). The effect of allomerization on fluorescence
differs in magnitude for the various chlorophylls (RABINOWITCH, 1951, p. 754),
and hence might be responsible for changing the amount of fluorescence per
unit chlorophyll. (4) As the conversion factors were obtained by relating

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fluorescence to amount of chlorophyll a, the presence of any other pigments
which absorb blue light and fluoresce red light would result in artificially high
values for chlorophyll a. These would include the chlorophylls other than
chlorophyll a, the phycobilin pigments, and various porphyrins (FALK, 1964).
As the fluorescent method of chlorophyll determination will most likely find
increasing use in oceanographic research, it is well to consider the significance
and magnitude of the errors inherent in the method. As presently used, the
fluorescent method integrates all fluoresced light which passes through the
emission filter. When using the red 25 filter, one can not distinguish between
chlorophylls a, b, or c. One way in which one could measure the fluorescence
of the individual pigments would be to use filters which transmit just a narrow
band at the peak of fluorescence of each pigment. This would, however, greatly
reduce the sensitivity of the method, and it would also be limited by the fact
that the fluorescence bands of the various chlorophylls overlap somewhat, just
as do the absorption spectra. The choice of emission filter will be dictated by
whether one wants a value for total chlorophyll or only a value for chlorophyll a.
If the former is desired, then the red 25 filter would be appropriate. The use of
this filter involves, however, the errors introduced by different F values and
acid factors for the different chlorophylls. If one wanted to know only the
amount of chlorophyll a, filter CS-2-64 would be the best of the three red
filters we had available for the present investigation. This filter absorbs only
about 10% of the light fluoresced by chlorophyll a but over 80% of the light
fluoresced by chlorophyll c. The use of this filter thus greatly reduces the error
in the determination of chlorophyll a introduced by the presence of chloro-
phyll c.
The amount of phaeophytin in laboratory grown cultures is very small and
often can not be detected (PATTERSON and PARSONS, 1963). LORENZEN has shown
(unpublished) that the ratio of phaeophytin to chlorophyll, as determined by
fluorescence before and after acidification, does not vary significantly in cultures
of Chaetoceros sp. which have been standing for 60 days. It would appear,
therefore, that a large amount of phaeophytin can best be correlated with
grazing by zooplankton (CURRIE, 1962). The faecal pellets mentioned in Table 2
showed an acid factor (1-05) characteristic for phaeophytin, and also had an
absorption curve from 400 to 700 mu that resembled published spectra of
phaeophytin. The decrease in acid ratios as one increases in depth (Table 1) is
thus most likely an indication of increasing accumulation, near and below the
pycnocline, of faecal material.
14 O. HOLM-HANSEN, C. J. LORENZEN, R. W. HOLMES and J. D. H. STRICKLAND

Acknowledgement
This research project was supported in part by the U.S. Atomic Energy
Commission Contract No. AT (1 l-l)-34 Project 108 and in part by No.
14-17-0007-221 from the U.S. Bureau of Commercial Fisheries.

Summary

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1. The concentration of chlorophyll a in extracts of natural populations and
in laboratory cultures was determined both by fluorescence and by light
absorption measurements. The chlorophyll a concentration as determined by
fluorescence varied by about ± 2 0 % of the value as determined spectro-
photometrically.
2. Much of the variation in chlorophyll a concentration as determined by
fluorescence is due to algal pigments other than chlorophyll a which can
fluoresce red light when excited with blue light. Through the use of different
filters, the error introduced by these accessory pigments (such as chlorophyll c)
can be largely eliminated. This, however, greatly decreases the sensitivity of
the chlorophyll a determination.
3. Equations are given for the calculation of the amount of chlorophyll a or
phaeophytin a from measurements offluorescencebefore and after acidification.
4. The use of dilute HC1 is preferable to oxalic acid in converting chlorophyll
to phaeophytin, as HC1 causes complete phaeophytinization within one minute.
5. The determination of phaeophytin a is complicated by the fact that the
different chlorophylls may show very different acid factors. Thus, pure
phaeophytin a fluoresced about 42 % as much light as an equivalent weight of
chlorophyll a, while pure phaeophytin cfluorescedonly about 16% as much as
an equivalent amount of chlorophyll c.
6. The acid factor (using HC1) of natural populations decreases with depth.
The factor of surface samples averages about 2-3, and decreases to about 1-0 in
deep samples. An acid factor of 1 -0 indicates all phaeophytin (or possibly other
degradation products of chlorophyll) and little or no chlorophyll, and is
suggestive of grazing by zooplankton.

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