NOTES AND COMMENT 343

DETERMINATION OF CHLOROPHYLL AND PHEO-PIGMENTS :

SPECTROPHOTOMETRICEQUATIONS~

It has been shown that chlorophyll deg- fere with the determination since, unlike
radation products may at times constitute the other two chlorophylls, a and c, b shows
a significant fraction of the total green pig- an increase in fluorescence upon acidifica-
ments present in seawater (Yentsch and tion. Algae containing chlorophyll b gen-
Menzel 1963; Lorenzen 1965; Yentsch 1965). erally have an acid factor slightly less than
These degraded forms, or inactive chloro- those without chlorophyll b, which will re-
phyll, absorb light in the red part of the sult in a slight underestimation of chloro-
spectrum; if they are present in concentra- phyll a and a slight overestimation of pheo-
tions significant relative to chlorophyll a, pigments. In taxonomically diverse popula-
a serious error may be introduced into tions this would not be a serious problem.
chlorophyll data using the present spectro- On the other hand, all three chlorophylls
photometric techniques ( Richards with show a reduction in absorbancy upon acidi-
Thompson 1952; Parsons and Strickland fication; that is, the pheophytin absorbs less
1963), because the absorption of light by light per unit weight than its respective
the degraded forms is not distinguished chlorophyll.
from that absorbed by active chlorophyll. The method proposed is similar to that
Chlorophyll a can readily be converted described by Vernon (1960) and others
to pheophytin simply by the addition of a reviewed by Holden ( 1965), but absorb-
weak or dilute acid, either oxalic acid or ancies are measured at only two wave-
1 N HCl, and when the reaction is carried lengths, 750 and 665 rnp, before and after
out on a specific sample the absorbancy of acidification of the sample, and only chloro-
the solution is reduced (Vernon 1960). The phyll a and pheo-pigments are calculated.
pheo forms of chlorophyll, both pheophytin An attempt to measure all three chlorophylls
and pheophorbide, do not show a reduc- and their pheo forms was deemed unwar-
tion in absorbancy when treated with an ranted for routine measurements because
acid, although the chlorophyllide apparently eight absorbancies would have to be deter-
does ( unpublished data). This reduction mined on each sample, and the uncertainty
in absorbancy by acid is probably the re- of the calculated values derived for chloro-
sult of the removal of the bound Mg atom phyll b and c and their degraded forms
in the porphyrin ring. would be too high.
This simple property, brought about by This method may find application in
the conversion of chlorophyll to pheophytin, freshwater, estuarine, and coastal environ-
could be used in a method for the deter- ments where a relatively small quantity of
mination of chlorophyll a in samples con- water can be filtered in a short time yield-
taining pheo-pigments. The fluorometric ing samples with an absorbancy at 665 rnp
technique for pigment determination (Yen- of 0.2 or greater.
tsch and Menzel 1963; Holm-Hansen et al.
EXPERIMENTAL
1965) has been used extensively in this
laboratory, and its precision and the re- Chlorophylls were purified in three
covery of both chlorophyll and pheo- ways from Dunaliellu sp., Chaetoceros sp.,
phytin from prepared solution is excellent; Macrocystis sp., and English Ivy. The lat-
but the presence of chlorophyll b may inter- ter two plants were used when a large
quantity of pigment was desired.
l Contribution from the Scripps Institution of The first purification method was two-
Oceanography, University of California, San Diego. dimensional paper chromatography accord-
This investigation was part of the Scripps Tuna
Oceanography Research program and was sup-
ing to the method of Jeffrey (1961) using 4%
ported by U.S. Bureau of Commercial Fisheries n-propanol in ligroine (bp: 65-9OC) for the
Contract No. 14-17-0007-458. first dimension and 30% chloroform in
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344 NOTES AND COMMENT

TABLE 1. Change in absorbancy of chlorophyll a, for chlorophyll a, b, and c, and for

b, and c at the wavelengths specified when treated their respective pheophytins. Pheophorbide
with 1 N HCZ. Readings expressed as ratio of ab- was spectrophotometrically indistinguish-
sorbance before divided by absorbance after the
addition of acid able from pheophytin a but was readily
identified by chromatography, It was not
665 mp 645 mp 630 m,u possible to obtain enough pure chloro-
Chlorophyll a 1.7 1.4 2.0 phyllide a to measure an absorption spec-
Chlorophyll b 1.0 2.3 2.0 trum, and it was uncertain if the chloro-
Chlorophyll c 1.0 2.0 3.1 phyllide or pheophorbide of the other two
Chaetoceros sp. 1.67 1.91 3.57 chlorophylls were observed.
Dunaliella sp. 1.71 2.14 3.45
Gymnodinium sp. 1.66 2.03 3.00
The red absorption maximum of chloro-
phyll a and b shifted to longer wavelengths
upon acidification, and the red peak for
chlorophyll c virtually disappeared when
ligroine for the second dimension. Pigments acidified in acetone. These pigments be-
were identified by their movement relative have similarly in ether (Smith and Benitez
to the solvent front and each other, and Rf 1954)) and have been observed to behave
factors usually corresponded to published in this manner in acetone by other in-
factors. Chromatograms were run simul- vestigators (Vernon 1960; Parsons 1963).
taneously to yield enough pigment for ab- The reduction in absorbance observed
sorption spectra (obtained with a Bausch upon acidification of chlorophyll extracts
and Lomb 505 recording spectrophotom- showed some variation which seemed to
eter), and these were compared with pub- be due to the handling technique. Fresh
lished absorption spectra. extracts, and those that were not dried when
Larger quantities of pigments were transferring from one solvent to another,
handled either by column chromatography showed a reduction in absorbance at 665
or partition chromatography. Pigments were rnp of 0.58-0.61 of the initial value. Other
separated on sugar columns ( Strain 1958) extracts showed a much smaller reduction,
using a variety of solvents, but hexane was sometimes only 0.8 of the original. For
used most frequently. A good separation of the fresh extracts, the ratio before acidifica-
chlorophyll a and c was obtained by the tion to that after acidification (corrected
method of Parsons ( 1963), although this for the 750 rnp reading), 6650 : 665,,, was
did not provide a good separation of chloro- 1.7. Chlorophyll a showed a similar change
phyll c and chlorophyllide a. Chlorophyll at 665 rnp, while both chlorophyll b and c
a and carotenoids were not separated by showed essentially no change. All three
partition chromatography. At times a com- chlorophylls showed changes at the other
bination of all three techniques was used, two wavelengths normally used for pig-
but the paper chromatography was usually ment estimation, 645 and 630 rnp. The
used as a criterion of purity. magnitude of these changes is shown in
The pheo form of the pigment was either Table 1.
prepared from the chlorophyll by acidifica- PROPOSED METHOD
tion, or obtained from fecal pellets of The sample should be handled as fol-
crustaceans feeding on algal suspensions. lows: A volume of water is filtered through
The former methol produced pheophytin, a Whatman GF/C glass paper filter. These
but if chlorophyllide a was acidified, the filters have a slightly irregular effective
pheophorbide was produced. Fecal pellets pore size, so a small quantity of MgC03
yielded pheophorbide rather than pheo- suspension is placed on the filter and drawn
phytin. through before the water sample is filtered.
RESULTS After the sample has passed through the
Pigments were always transferred to 90% filter, the filter is ground in a Teflon tissue
acetone before spectrophotometric measure- grinder with acetone until it is thoroughly
ments. Absorption spectra were obtained macerated. The time will vary with the
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NOTES AND COMMENT 345

motor used, but a regular hand drill with TABLE 2. Recovery of chlorophyll a and pheo-pig-
about 1,000 rpm is satisfactory and will ment from known solution using the proposed
equations. Concentrations of pigments expressed in
macerate the filter in about 1 min. The pg/10 ml of 9070 acetone solution. Also shown are
macerated sample is placed in a centrifuge the values of chlorophyll a that would have been
tube with the required rinses of the mortar calculated if the sample had not been acidified and
and pestle, and the final volume is made a second reading taken, and the ratio of absorbance
up to 10 ml plus the volume of the filter. before and after acidification, 6650 : 665,
The pigments are eluted for 30-60 min and Chlorophyll Chloro- Pheo-pigment
then centrifuged. The centrifuge tubes can Recov-
uhvll
with&t
be shaken once or twice during extraction. Added ered correction Added Rz;l;- 665, : 665,
The absorbance is read at 750 and 665 rnp 1.70 1.70 1.69 0.00 0.03 1.69
in a spectrophotometer before and after 1.57 1.59 1.58 0.15 0.06 1.67
acidification with 2 drops of 1 N HCl. The 1.42 1.36 1.55 0.30 0.39 1.54
absorbance can be controlled to some ex- 1.29 1.32 1.53 0.45 0.44 1.52
tent by adjusting the amount of water fil- 1.16 1.14 1.49 0.60 0.67 1.44
1.02 0.92 1.42 0.75 0.92 1.35
tered, volume of acetone used to extract the 0.88 0.88 1.41 1.02 0.97 1.33
sample, and the length of the cuvette. The 0.75 0.79 1.37 1.05 1.04 1.30
greater the first and last of these factors, 0.61 0.67 1.33 1.20 1.19 1.26
and the smaller the volume of acetone used, 0.47 0.55 1.30 1.35 1.33 1.20
the greater the absorbance will be. It is 0.00 0.09 1.16 1.88 1.88 1.02
important to keep the absorbance greater
than 0.2 for the initial reading, but it is
probably not necessary for it to be above CONCLUSIONS
0.5. The cuvette should be shaken after The proposed method is intended to en-
the acid is added. Most cuvettes, including able one to estimate both chlorophyll a
the l-cm cell, hold at least 4 ml and the and pheo-pigments. Since the change in
quantity of acid added does not appreciably absorbance brought about by treatment
alter the final readings. with 1 N HCl is measured, it in fact prob-
The readings obtained at 665 rnp before ably discriminates between chlorophyllous
and after acidification, corrected for the compounds containing Mg atoms and those
750 rnp reading and cell to cell differences, which are Mg-free. Chlorophyll a and
are entered in the following equations. chlorophyllide a, if present, are measured
AxKx (665,-665,) xu as “chlorophyll a” and both pheophytin and
Chl a (mg/m”) = , pheophorbide are measured together as
Vf x 1 “pheo-pigments,” The calculation of the
A x K(R [ 665,] - 6650) x v pheo-pigments assumes that all this pig-
pheo (mg/m3) = , ment is pheophytin, which probably is not
Vf x 1 the case (Patterson and Parsons 1963), but
where the absorption coefficient of pheophorbide
A absorption coefficient of chlorophyll is unknown.
a = 11.0, An estimate of the recovery of both
K factor to equate the reduction in ab- chlorophyll a and pheo-pigments was made
sorbancy to initial chlorophyll con- by mixing a known solution of chlorophyll
centration, 1.7 : 0.7, or 2.43, a and pheophytin in varying proportions
6650 absorbance before acidification, ( Table 2). The amount of chlorophyll a
665, absorbance after acidification, that would have been calculated if no acid
V volume of acetone used for extrac- was added and the ratio 6650 : 665,
tion (ml), are included in Table 2. The recovery
Vf liters of water filtered, of either chlorophyll a or pheo-pigment
1 path length of cuvette ( cm) is high if the concentration is high,
R maximum ratio of 665, : 665, in the and the error of estimation increases when
absence of pheo-pigments, 1.7. the concentration decreases. The difference
 19395590, 1967, 2, Downloaded from https://aslopubs.onlinelibrary.wiley.com/doi/10.4319/lo.1967.12.2.0343 by Kalyani University, Wiley Online Library on [29/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
346 NOTES AND COMMENT

between the value calculated for chloro- HOLM-HANSEN, O., C. J. LORENZEN, R. W.

phyll without adding acid and that cal- HOLMES, AND J. D. H. STRICKLAND. 1965.
Fluorometric determination of chlorophyll. J.
culated using the above equation is small Conseil, Conseil Perm. Intern. Exploration
when the pheo-pigment concentration is Mer, 30: 3-15.
low, but it increases with increasing con- JEFFREY, S. W. 1961. Paper-Chromatographic
centrations of pheo-pigments. This Yis the separation of chlorophylls and carotenoids
case when one samples at increasing depths from marine algae. Biochem. J., 80: 336-
342.
in the open ocean (Lorenzen 1965) and LORENZEN, C. J. 1965. A note on the chloro-
in coastal areas where wave action may phyll and phaeophytin content of the chloro-
either resuspend particles from the bottom phyll maximum. Limnol. Oceanog., 10: 482-
or keep particles in suspension (unpub- 483.
PARSONS, T. R. 1963. A new method for the
lished data). In the Sacramento River delta, microdetermination of chlorophyll c in sea
chlorophyll samples usually show a rather water. J. Marine Res., 21: 164-171.
low acid ratio at 665 rnp, which would AND J. D. H. STRICKLAND. 1963. Dis-
indicate that pheo-pigments frequently form c&ion of spectrophotometric determination of
a large fraction of the green pigments ab- marine-plant pigments, with revised equa-
tions for ascertaining chlorophylls and carot-
sorbing light at 665 rnp (T. E. Bailey, per- enoids. J. Marine Res., 21: 155-163.
sonal communication). PATTERSON, J ., AND T. R. PARSONS. 1963. Dis-
This method may be useful if high con- tribution of chlorophyll a and degradation
tamination of samples with pigments other products in various marine materials. Limnol.
than chlorophyll is suspected. This could Oceanog., 8 : 355-356.
RICHARDS, F. A. WITH T. G. THOMPSON. 1952.
be determined by acidifying a few of the The estimation and characterization of plank-
regular samples after the normal procedure ton populations by pigment analyses. II. A
and again observing the absorbancies at spectrophotometric method for the estima-
665 and 750 mp. If the ratio of the cor- tion of plankton pigments. J. Marine Res.,
11: 156-172.
rected readings at 665 rnp is less than 1.6, SMITH, J. H. C., AND A. BENITEZ. 1954. Ab-
the above procedure should be followed. sorption spectra of chlorophylls. Carnegie
Inst. Wash., Yearbook, 53: 16S-172.
CARL J. LORENZEN~ STRAIN, H. H. 1958. Chloroplast pigments and
Institute of Marine Resources, Chromatographic analysis. 32nd Ann. Priest-
Scripps Institution of Oceanography, ley Lectures, Penn. State Univ., University
Park, Penn.
La Jolla, California 92038 VERNON, L. P. 1960. Spectrophotometric deter-
mination of chlorophylls and pheophytins in
REFERENCES plant extracts. Anal. Chem., 32: 1144-1150.
HOLDEN, M. 1965. Chlorophylls, p. 461-488. YENTSCH, C. S. 1965. Distribution of chlorophyll
In T. W. Goodwin red.], Chemistry and bio- and phaeophytin in the open ocean, Deep-
chemistry of plant pigments. Academic, New Sea Res., 12: 653-666.
York. -, AND D. W. MENZEL. 1963. A method
for the determination of phytoplankton chloro-
2 Present address : Woods Hole Oceanographic phyll and phaeophytin by fluorescence. Deep-
Institution, Woods Hole, Massachusetts 02543. Sea Res., 10: 221-231.

THE USE OF FILAMENT TAPE IN RAISING LONG CORES FROM SOFT SEDIMENT

This note describes the advantages of the variety of heavier piston samplers suit-
using filament tape to line the inside of a able for work in soft sediment in water of
sediment coring tube. Such tape has been any depth (Wright, Cushing, and Living-
used with a hand-driven aluminum sampler stone 1965 ) .
(Livingstone 1955) weighing 10 or 20 kg It is desirable for cores from the soft
and suitable for use by one man from a sediments of lakes or the ocean to be long,
skiff in 10 or 20 m of water. It should be complete, and undisturbed. The principal
equally valuable when used with any of barrier to obtaining such cores is the fric-