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Chromatographic determination of chlorophylls in algal cultures and phytoplankton
Deep-SeaResearch, 1966,Vol. 13, pp. 459 to 466. PergamonPress Ltd. Printedin Great Britain.
INSTRUMENTS
AND
METHODS
Chromatographic determln~tion of chlorophylls in algal cultures and phytoplankton J. C. MADOWICK*
(Received 17 November 1965) Abstract--Mixtures of chlorophyll a, b and c (total chlorophyll 1"5-10'5 ~g) were recovered quantitatively from glucose thin-layer chromatograms. Mixtures (total chlorophyll 0.9-3.0 ~g) added to crude methanol extracts of Nitzschia cioaterium were also recovered quantitatively. Spectrophotometric and chromatographic methods of chlorophyll analysis of algal cultures gave similar results for chlorophyll a (2-5 t,g), chlorophyll b (1 ~tg) and chlorophyll c (1-4 ~g). With phytoplankton the two methods gave similar results for chlorophyll a (0"3-3.0 ~Lg) but b (0-1-0.6 pg) was lower chromatographically and c (0-1-0.9 ~g) was variable. When phaeophytin was present in phytoplankton, chlorophyll a was significantly less by the chromatographic method. Cultures of the algae
Dunaliella tertiolecta, Gymnodinium, Isoehrysis galbana, Nitzschia closterium, Skeletonema costatum and an unidentified cryptomonad were analysed chromatographically for chlorophyll at different times of growth. The chlorophyll content of the cells was usually maximal between 3 and 12 days and then declined. The chlorophyll ratio (c/a or b]a) was approximately constant during the 28 day growth period, having mean values of 0.23 for Nitzschia, 0.30 for Skeletonema, 0"21 for lsochrysis, 0"26 for Dtmaliella, 0-44 for Gymnodinium and 0-54 for the cryptomonad. INTRODUCTION C m . O R O ~ in oceanic waters is usually estimated by trichromatic spectrophotometry on plankton extracts. The uncertainty of such measurements has been discussed by HUMPHREY0962). One criticism of the trichromatic method is that chlorophyll cannot be correctly estimated in. the presence of epectrally similar breakdown products. A simple way to find the effect of breakdown products (e.g. phaeophytin) is to remove them chromatographically before estimating chlorophyll. However, the introduction of chromatosraphy into the measurement raises the question of the accuracy of the various steps in the chromatographic technique. A thin-layer chromatographic method for chlorophylls has already been worked out (M~a~3WlCK1964), and shown to give accurate results on pure solutions of chlorophylls a, b and c. In the present investigation the use of the method was extended to chlorophyll mixtures and to chlorophyll determinations in extracts of algae and phytoplankton. The phaeophytin occurring in phytoplankton was separated from the chlorophylls and the subsequent chlorophyll measurement was compared with that by trichromatic spectrophotometry. Further, before the chlorophyll content of phytoplankton can be fully interpreted, more information is needed on the variation of these pigments in individualalgae during their life cycles. Therefore, the chlorophyll content of several algae was determined chromatographically at different stages in the growth of the cultures. (i)
Cultures
METHODS
free, cultures of Nitzschia closterium, Gymnodinium, Skeletonema costatum, Isochrysis £albana, Dunaliella tertiolecta, an unidentifieddiatom, and an unidentifiedcryptomonad were grown in Exdschreiber medium. A soil extract was prepared by autoclaving 1 kg soil and 1 1. of tap water for 3 hr at 10 lb pressure. After filtering through cotton wool 0-02 g NaNOa, 0~3 g NatHIK)4. 12HiO and 10 ~g thiamine HCI were added per 50 Pal filtrate. Filtered seawater (0-45 ~ Millipore® filter) was added to the enriched soil extract in the ratio 10 : 1 (v/v). Conical Uni-algal, but not b a c t ~
*Division of Fisheri~ and Oceanography, CSIRO, CYonulla, N.S.W.
459
460
Instruments and Methods
flasks (100 ml) containing 35 ml o f the above medium were then autoclaved at 5-10 Ib pressure for 15 rain. To inoculate, 5 ml of a 3-5 day culture was added per flask and illuminated at 380--420 ft-c and at a temperature between 20--25 °. Gymnodinium and Dunaliella, however, were grown at 150 It-(: and at 18-20 °. (ii)
Chromatography
One dimensional ascending glucose thin-layer chromatography was used. Preparation of the glucose slurry was described previously (MADGWXCK, 1964). Layers were formed on 5 × 10 cm glass sheets according to LeES and D~ MURtA (1962). Algae were centrifuged at 2,000g for 10 rain in a 1.5 × 9 cm glass tube. The supernatant was decanted and the 20-50 tLl packed cells were dried for 15 rain under nitrogen. Drying the cells before extraction was necessary since chlorophyllase in some of the algae might be active in methanol containing more than 2 % water. The cells were extracted with 1 mi methanol containing 0.1 ~oo water (v/v). Debris was removed by centrifugation at 2000 g for 5 rain. The residue was no longer green after this one extraction. Pigment extract was streaked on the glucose layers using nitrogen to evaporate the methanol. Application of the pigments took 5-10 rain, and the resulting zone measured 1 × 4 era. Pigments were separated by ascending chromatography using 30 ml 1 : 1 (v/v) diethyl ether and 60-80 ° petroleum spirit. During development the layer rested 2 m m from an 8 × 12 cm sheet o f filter paper saturated with the developing solvents. The developing solvents had a depth o f 1.0 cm in the glass developing chamber. Chlorophyll zones were separated after 10 rain development. Chromatograms were developed in darkness and the light intensity during all the operations was no greater than 40 ft.-c. The separated zones were removed one at a time, keeping the remaining zones moist with solvent by covering them with a 5 × 10 cm sheet of glass. The zones o f pigment were eluted and the concentrations determined as described by M ~ w I c x (1964), except that when algal and phytoplankton extracts were used, chlorophyll c was calculated using 2-component equations to correct for traces of contaminants. (iii)
Chlorophyll standards
Spectroscopically pure chlorophylls a and b were prepared from spinach leaves o n columns of sucrose (S'rRAI~, 1958). The chlorophylls were eluted in ether and stored at -- 20 °. To ensure freedom from breakdown products these pigments were transferred to 60-80 ° petroleum spirit and rv-chromatographed before use if they had been stored longer than two days. Spectroscopically pure chlorophyll c solutions were prepared from the kelp Eklonia using paper chromatography (IEF~EY, 1961). Chlorophyll c was ehited from paper with methanol, added to an equal volume of ether and washed with 10 volumes [20% (w/v)] NaCI solution. The upper pigmented layer was removed and dried over anhydrous sodium sulphate. The chlorophyll c solution was kept at -- 20 ° before use. When mixtures of these chlorophylls were separated on thin layers a n d eluted in 90% acetone, the absorption spectra were not significantly altered. (iv)
Recovery experiments
(a) Mixtures of purified chlorophylls. The concentration in the standard solutions was determined using extinction coefficients given previously for ether solutions (MADCWXCK, 1964). Known concentrations, in mixtures, were then applied to the layers, chromatographed, and eluted in 90% acetone. The concentration recovered was calculated using the extinction coefficients given previously for 90% acetone solutions (MADGWICK, 1964). (b) Mixtures of purified chlorophylls added to crude methanol extracts. Known mixtures of chlorophylls a, b and c in ether were added to crude methanol extracts of Nitzschia closterium in the proportion of one volume o f ether to six of methanol. The concentration of chlorophyll in the crude Nitzschia extract was determined chromatographically before adding the mixture of standard solutions. The recovery of chlorophyll was then found by difference. Chlorophyll c elutes were often contaminated with a 663 m p absorbing substance. This was probably due to slight contamination with chlorophyll a, chlorophyllide a or phaeophorbidc a. The contamination did not significantly alter chlorophyll c recoveries provided elute extinctions were greater than 0-03 at 630 rn~. The contamination was corrected for by using 2-component equations for mixtures o f chlorophyll a and c (HUMPHl~Y and J~FVREY, unpublished) i.e. it was assumed that the contaminants has spectra similar to chlorophyll a.
461
Instruments and Methods
(v) Comparison of spectrophotometric and chromatographic methods using algal cultures and phytoplankton Seawater was taken from inshore and coastal regions at Port Hacking, Sydney. Phytoplankton were harvested in a continuous centrifuge (DAviS, 1957), before resuspending in filtered seawater. Half of the sample was analysed chromatographically. The remaining half was passed through a 47 m m Millipore® 0.45/~ filter covered with MgCOa. The filter was then ground with 90% acetone according to YEwrSCH and MENZEL (1963). The extinctions of the extract were read at 750, 663, 645 and 630 n ~ and were corrected for turbidity by subtracting the extinction at 750 m~. The concentrations o f chlorophylls were calculated using simultaneous trichromatic equations described by HUMPHREY and JEFFR£Y (unpublished). Algae were treated similarly except that they were harvested at 2000 g in a swing-out centrifuge before resuspending, and the concentrations were calculated for the spectrophotometric method using 2-component equations (Hu~HREY and JEFWEV unpublished). RESULTS (i)
Chromatographic recovery of mixtures of chlorophylls a, b and c
(a) Mixtures of spectrophotometrically pure chlorophylls. Table 1 shows that chlorophyll a recovery was quantitative at all the concentrations tested, but chlorophyll c was recovered with losses up to 22%. Chlorophyll b was over-estimated by 10-20% in all cases.
Table 1. Recover), of spectroscopically pure chlorophylls a, b and c from mixtures Chlorophyll a, b and c were applied together in ether, and eluted with 90% acetone. Concentrations were calculated with extinction coefficients for ether and 90 % acetone respectively.
Chlorophyll a
Chlorophyll b
Chlorophyll c
Applied (~e)
Recovered (%)
Applied b'g)
Recovered (%)
Applied O'g)
Recovered (%)
0"62 0"82 0"92 1"1 ! 1.84 2"94 4'10
99 97 98 97 98 98 100
0"33 0'49 0"55 0.67 1"10 1"77 2'44
1i 2 I 18 120 120 121 116 116
0.50 0-74 0"82 0"97 1"88 2"81 4"00
99 90 90 92 78 84 82
(b) Mixtures of purified chlorophylls added to crude methanol extracts. Table 2 shows that when mixtures of spectroscopically pure chlorophylls a, b and c were added to crude methanol extracts o f Nitzschia closterium good recoveries o f a and c were obtained. Recoveries o f chlorophyll b were poor when only 0.08 ~g was used.
Table 2. Recovery of chlorophylls a, b and c added to Nitzschia extracts Mixtures of spectroscopically pure chlorophyll a, b and c were added to crude methanol extracts of Nitzschia closterium, separated, and eluted with 90 % acetone. The concentration of chlorophyll a and c in the crude extracts was determined first on separate chromatograms, and the recoveries found by difference. Chlorophyll a Applied (~g)
Chlorophyll b Recovered
(%)
Extract
Added
0-50 0'56 1.00 "1"18 I "51 2"53
0.27 0.66 0.23 0.66 0.23 0.66
Applied (#g)
0"08 0"29 0.08 0.29 0"08 0.29
Applied (#g)
Recovered
(%)
Added 92 106 106 102 I01 101
Chlorophyll c
62 86 63 1 I0 75 101
Recovered
(%)
Extract
Added
O" 13 0"12 0-19 0-I0 0.34 0.61
0"57 2.06 0.57 2-06 0.57 2.06
96 95 I00 II0 91 96
462
Instruments and Methods
(ii) Comparison of speetrophotometric and chromatographic methods using algal cultures and phytoplank ton Table 3 shows that in algal cultures, chromatography gave 30--40% higher values for chlorophyll a than spectrophotometry. Chlorophyll b, from the one comparison made, was 21% greater by chromatography. Chlorophyll c was 22-28 % lower by chromatography except with the cryptomonad where it was I 1% higher.
Table 3. Comparison of results of estimating chlorophylls in algal cultures using spectrophotometry and chromatography Cultures used were less than one week old. Two-component equations were used in the spectrophotometric method
Chromatographic
Method Alga
Chlorophyll
Nitzschia
a
Spectrophotometry Chromatography (l~g/Sample) (ag/Sample) 3.41 1.32 2"06 1.22 3.05 1.30 1.60 1-40 2"50 1.36 4.33 3.75 4.22 1.08
c a c a c a c a c a c
Skeletonema Isochrysis Gymnodinium Diatom (unidentified) Cryptomonad (unidentified) Dunaliella
a b
3.51 1"01 2"34 0.88 3.28 1-02 2-24 1.08 2.60 I "03 5-15 4.15 5.21 1-31
value as % of
Spectrophotometric value 103 77 I 14 72 108 78 140 77 104 76 119 111 124 121
Table 4. Comparison of results of estimating chlorophylls in seawater by spectrophotometry and chromatography Five litres o f seawater were used for each experiment, except 3, where 20 l. were used for chromatography.
Method Chromatography
Chromatography value as a % of speetraphotoraetry value
0.29 0"~ O"10 0"39 0.04 0-14 0"20 0"02 0"11 0.48 0"00 0"24 0.06 0"00 0-03 0'25 0.00 0.18
107 1 200 91 57 280 51 18 85 87 -109 86 -1~ 41 -138
(pg/1.)
Experiment
Chlorophyll
1
a b c a b c a b c a b c a b c a b c
2 2*t 3 4 5?
Spectrophotometry 0"27 -- ve 0"05 0"43 0"07 0"05 0"39 0.11 0"13 0"55 0"10 0-22 0"07 0.01 0"03 0-61 0"04 0.13
*Duplicate centrifuged samples were stored in the dark for 3 days at room temperature. tPhaeophytin a was found chromatographically.
Instruments and Methods
463
The results in experiments I, 2, 3 and 4 in Table 4 show good agreement between chromatography and spectrophotometry for chlorophyll a. Phaeophytin a was found in experiment 2" (aged suspension) and 5. Experiment 2* is one where the sample of phytoplankton was aged by standing in the dark for three days at room temperature. The phytoplankton in experiment 5 contained phaeophytin a even though it was analysed at once, i.e. without storage. In both of these experiments the chromatographic value for chlorophyll a was about half that found spectrophotometrically. Phaeophytin a was identified from its position on the chromatograrn and its absorption spectrum. In only two cases (Table 4) was chlorophyll b found chromatographically. The two methods agreed reasonably (85-138%) for chlorophyll c, except in experiments 1 and 2 (200 and 280%) which had very low spectrophotometric values. (iii)
Chlorophyll content of algal cultures
Table 5 shows that the maxima for chlorophyll per million cells occurred at 3 days in Nitzschia for chlorophyl a and in the cryptomonad for a and c. For chlorophyll c in Nitzschia and for both chlorophylls in Skeletonema, Isoehrysis and DunalieUa, the maximum per million cells came at one day's growth. In Gymnodinium, the maximum chlorophyll per million cells came at 15 days. The maxima in cell numbers for Nitzschia, Dunaliella, Skeletonema, Isochrysis and the cryptomonad came after the maxima for chlorophyll concentration at 7, 23, 7, 7 and 14 days respectively. In Gymnodinium the maximum in cell numbers, at 7 days, was before the maximum for chlorophyll concentration. Disregarding the initial and final values, where the concentrations of the minor chlorophylls were close to the lower limit of the sensitivity o f the method, the ratios b/a and c]a varied little during the periods of observation. The results are represented in Fig. 1.
Table 5. Chlorophyll in algal cultures Analyses were done using glucose thin-layer chromatography
Alia Nitzsehia
Cryptomonad
Skeletonema
Isochrysis
Gymnodinium
Dunaliella
Age (days) 1 3 7 14 21 1 3 7 14 1 3 7 14 21 1 3 7 14 21 7 12 15 23 27 1 2 7 12 23 30
Chlorophyll Chlorophyll Chlorophyll a b c
(~g/10ecells) (/t.g/10ecells) (~g/10ecells) 0.83 0.90 0"31 0.I1 0.10 1.00 1.27 0.70 0.35 0.71 0.17 0.10 0-17 0"03 0"25 0"15 0"12 0"07 0"03 0"47 2"58 4"64 1"78 1"75 1"72 1.15 0.88 0.80 0.60 0.54
*Extinctions below range of method.
0.25 0.21 0.07 0"02 0.02 0-57 0"70 0.44 0-15 0.29 0-04 0-03 0.04 0.00* 0-08 0"02 0"03 0.01 0.00* 0"22 1"05 2.00 1"00" 1"38" 0"35 0.30 0.22 0.20 0.14 0.19
Ratio
Cell No.
c/a or b]a (10e/mlculture) 0.30 0.23 0.23 0.18 0.20 0.57 0"55 0.63 0.43 0.41 0.24 0-30 0-24 -0"32 0"13 0"25 0"14 m 0"47 0"41 0"43 N -0"20 0.26 0.25 0.25 0-23 0.35
0.12 0.82 2.36 2-14 0"59 0.14 0-40 0.50 0.60 0-07 1.51 3.37 1.15 0.00 0"65 2"50 5"80 5"06 3"18 0"49 0"19 0"08 0"09 0"08 0"30 0.33 2.28 2.50 3.12 2.89
464
Instruraents and Methods I0
130
2'0
I'0
C r yptomonod
I 0 "~/
05
0"~
0
L 0
0
IO
I0-0
@
ua $totum r'-
~sochr ysis
cJal bona
z~
r-
c
Z c
o s 0 c 0 J z
~0 3
•,
]
O..
~ ~0,. ~ ~'
•.,..:.-2:.'., .... ,...:..%.
"o
OQ.
q
0
oj
5"0
-O-
~
X
x
0
/ 0
4'0 "~ O
G~mn odin~um
sp
j, / /
\ \
/
\
/
\
..'"
20
\
."X".. "'.
,(i II
//
Ounahella
\
".
0 "X
%
I I0
I
20
~.-A.--.~.A .......
O
1
0
fO
" - - 'A
20
0
25
OATS
Fig. 1. C h l o r o p h y l l c o n t e n t ofalgalcultures. (Chlorophyll a - - 0 - - ; b . . . . A-.; e . - - x . . • ; cell number
•
).
DISCUSSION
Recovery o f chlorophyll a from mixtures of spectroscopically purified chlorophylls was slightly low (1-3 ~ ) . Chlorophyll b w a s overestimated by 12-21 ~ and chlorophyll c was underestimated by 1-22 ~o. T h e decreasing recovery o f chlorophyll c with increasing concentration might be due to decreasing elution efficiency. N o satisfactory explanation can be given for the overestimation o f chlorophyll b. Recoveries from crude extracts o f Nitzschia were g o o d : -- 8 to ÷ 6 ~ for chlorophyll a, -- 14 to -~ 1 0 ~ for b, and -- 9 to ~- 1 0 ~ for c in the concentrations used, except for b, when only
Instruments and Methods
465
0.08 ~g was present. Chlorophyll b was used in much smaller amounts than a or c to simulate the concentrations present in seawater. The sensitivity of the chromatographic method is such that 0.5 ~g of each chlorophyll, or slightly less, can be estimated. Although the chromatographic method gave consistently higher results for chlorophyll a than the spectrophotometric method when using algal cultures, the agreement was sufficiently close to suggest that neither method was grossly in error. A similar suggestion can be made for chlorophyll b and c. However with seawater samples the agreement between chromatographic and spectrophotometric methods was good for chlorophyll a but not for b and c. The minor chlorophylls have little adsorption at the peak for a whereas a absorbs relatively strongly at 645 and 630 n ~ , and spectrophotometric errors would be more pronounced because of the small concentrations of c and b compared with a in these seawater samples. It is not known which, if either, of the two methods gives the correct answer. The agreement for chlorophyll a, and for c when it was greater than 0.1 ~g/1., indicates both methods are comparable. The seawater comparisons were made under conditions similar to those usually met in routine spectrophotometric determinations where low extinctions make values of b, and sometimes c, unreliable. It is quite clear that when phaeophytin a is present the chromatographic method is to be preferred.
Table 6. Comparison of chlorophyll analyses by different authors Algae were grown on Erdschreiber media except by PARSONS (1961) who used a modified Provasoli medium.
Chlorophyll
Alga
ratio
a
Nitzschia Closterium
c
c/a
Isochrysis Galbana
0-33 0.18 1'25 0-10 0"22 0"84 0.04 1.00 0.06 0.11 0-30 0"14
0-08 0"11 0"29 0.04 0-20 0"34 0-02 0.28 0"02 0-03 0"08 0.04
b
b/a
Dunaliella tertiolecta
0.56 0"50
0.26 O. 13
0.47 0.26
Gymnodinium Sp. Skeletonema Costatum
Growth conditions
(/Lg/100 ~g dry ,act.) Chlorophyll
Light
Age
(ft-c)
(days)
400 420 400 400 420 150 400 1700 420 400 400 400
14-28 7 3 14-28 7 12 14-28 log phase 7 7 14-28 7
400 150
14-28 7
0.24 0.61 0'23 0.40 0"91 0"40 0.50 0"28 0"35 0.27 0"27 0.29
Reference
J ~ R E Y (1961) HUMPHREY (1963) Present paper JEWRY (1961) HUMPHREY (1963) Present paper JEFFREY(1961) P~.soNs (1961) HuMrm~Y (1963) Present paper JEFFREY(1961) Present paper Jv~-~Y (1961) Present paper
Table 6 summarizes the present, and some previous work on the chlorophyll content of algae. The results of the different authors have been recalculated with the extinction coefficients used in the present paper, and for the results of HUMPI-I~Y (1963) dry weights were determined from cell numbers by using conversion factors foundin thepresent work. Because ofthedifferencesin cultureconditious used by the various authors, it is not possible to make direct comparisons. Nevertheless the table is important in showing the ranges of values obtained when different analytical methods and growth conditions were used. The first point which emerges is that chlorophyll c (or b) was always less than a. Secondly, chlorophyll a was never more than 1.25 % and chlorophyll c (or b) never more than 0.34% of the algal dry weight. In the present work, except for Gymnodinium, a feature of the chlorophyll content per cell was that it did not keep pace with cell division. REFERENCES DAVzS P. S. 0957) A method for determination of chlorophyll in sea water. CSIRO Aust. Div. Fish. Oceanogr. Rep. No. 7.
466
Instnm~nts and Methods
HUMPH~Y G. F. (1962) Proceedings of the Conference on Primary Productivity Measurements, Marine and Freshwater, 1961 : 121-141. ( U.S. Atomic Energy Commi~ion, Division of Technical Information, TID- 7633). HUMI'I-m~ G. F. (1963) Chlorophyll a and c in cultures of marine algae. Aust. ?. mar. Fre,shwat. Res. 14, 148--154. JeJm~Y S. W. (1961) Paper chromatographic separation of chlorophylls and carotenoids from marine algae. Biochem. Y. 80, 336-342. L ~ s T. M. and DE MUP.XAP. J. (1962) A simple methoct for the preparation of thin layer chromatography plates. J. Chromatog. 8, 108-109. MADGWXCEJ. C. (1964) Quantitative chromatography of algal chlorophylls on thin layers of glucose. Deep-Sea Res. 12, 233-236. PAgSONST. R. (1961) On the pigment composition of eleven species of marine phytoplankers. J. Fish. Res. Bd. Canada 18, 1017-1025. STRAINH. H. (1958) Chloroplast pigments and chromatographic analysis, pp. 27-28, The Pennsylvania State University, University Park, Pa. Ye~'SCH C. S. and M~Z~L D. V. (1963) A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Res. 10, 221-231.
INSTRUMENTS
AND
METHODS
Chromatographic determln~tion of chlorophylls in algal cultures and phytoplankton J. C. MADOWICK*
(Received 17 November 1965) Abstract--Mixtures of chlorophyll a, b and c (total chlorophyll 1"5-10'5 ~g) were recovered quantitatively from glucose thin-layer chromatograms. Mixtures (total chlorophyll 0.9-3.0 ~g) added to crude methanol extracts of Nitzschia cioaterium were also recovered quantitatively. Spectrophotometric and chromatographic methods of chlorophyll analysis of algal cultures gave similar results for chlorophyll a (2-5 t,g), chlorophyll b (1 ~tg) and chlorophyll c (1-4 ~g). With phytoplankton the two methods gave similar results for chlorophyll a (0"3-3.0 ~Lg) but b (0-1-0.6 pg) was lower chromatographically and c (0-1-0.9 ~g) was variable. When phaeophytin was present in phytoplankton, chlorophyll a was significantly less by the chromatographic method. Cultures of the algae
Dunaliella tertiolecta, Gymnodinium, Isoehrysis galbana, Nitzschia closterium, Skeletonema costatum and an unidentified cryptomonad were analysed chromatographically for chlorophyll at different times of growth. The chlorophyll content of the cells was usually maximal between 3 and 12 days and then declined. The chlorophyll ratio (c/a or b]a) was approximately constant during the 28 day growth period, having mean values of 0.23 for Nitzschia, 0.30 for Skeletonema, 0"21 for lsochrysis, 0"26 for Dtmaliella, 0-44 for Gymnodinium and 0-54 for the cryptomonad. INTRODUCTION C m . O R O ~ in oceanic waters is usually estimated by trichromatic spectrophotometry on plankton extracts. The uncertainty of such measurements has been discussed by HUMPHREY0962). One criticism of the trichromatic method is that chlorophyll cannot be correctly estimated in. the presence of epectrally similar breakdown products. A simple way to find the effect of breakdown products (e.g. phaeophytin) is to remove them chromatographically before estimating chlorophyll. However, the introduction of chromatosraphy into the measurement raises the question of the accuracy of the various steps in the chromatographic technique. A thin-layer chromatographic method for chlorophylls has already been worked out (M~a~3WlCK1964), and shown to give accurate results on pure solutions of chlorophylls a, b and c. In the present investigation the use of the method was extended to chlorophyll mixtures and to chlorophyll determinations in extracts of algae and phytoplankton. The phaeophytin occurring in phytoplankton was separated from the chlorophylls and the subsequent chlorophyll measurement was compared with that by trichromatic spectrophotometry. Further, before the chlorophyll content of phytoplankton can be fully interpreted, more information is needed on the variation of these pigments in individualalgae during their life cycles. Therefore, the chlorophyll content of several algae was determined chromatographically at different stages in the growth of the cultures. (i)
Cultures
METHODS
free, cultures of Nitzschia closterium, Gymnodinium, Skeletonema costatum, Isochrysis £albana, Dunaliella tertiolecta, an unidentifieddiatom, and an unidentifiedcryptomonad were grown in Exdschreiber medium. A soil extract was prepared by autoclaving 1 kg soil and 1 1. of tap water for 3 hr at 10 lb pressure. After filtering through cotton wool 0-02 g NaNOa, 0~3 g NatHIK)4. 12HiO and 10 ~g thiamine HCI were added per 50 Pal filtrate. Filtered seawater (0-45 ~ Millipore® filter) was added to the enriched soil extract in the ratio 10 : 1 (v/v). Conical Uni-algal, but not b a c t ~
*Division of Fisheri~ and Oceanography, CSIRO, CYonulla, N.S.W.
459
460
Instruments and Methods
flasks (100 ml) containing 35 ml o f the above medium were then autoclaved at 5-10 Ib pressure for 15 rain. To inoculate, 5 ml of a 3-5 day culture was added per flask and illuminated at 380--420 ft-c and at a temperature between 20--25 °. Gymnodinium and Dunaliella, however, were grown at 150 It-(: and at 18-20 °. (ii)
Chromatography
One dimensional ascending glucose thin-layer chromatography was used. Preparation of the glucose slurry was described previously (MADGWXCK, 1964). Layers were formed on 5 × 10 cm glass sheets according to LeES and D~ MURtA (1962). Algae were centrifuged at 2,000g for 10 rain in a 1.5 × 9 cm glass tube. The supernatant was decanted and the 20-50 tLl packed cells were dried for 15 rain under nitrogen. Drying the cells before extraction was necessary since chlorophyllase in some of the algae might be active in methanol containing more than 2 % water. The cells were extracted with 1 mi methanol containing 0.1 ~oo water (v/v). Debris was removed by centrifugation at 2000 g for 5 rain. The residue was no longer green after this one extraction. Pigment extract was streaked on the glucose layers using nitrogen to evaporate the methanol. Application of the pigments took 5-10 rain, and the resulting zone measured 1 × 4 era. Pigments were separated by ascending chromatography using 30 ml 1 : 1 (v/v) diethyl ether and 60-80 ° petroleum spirit. During development the layer rested 2 m m from an 8 × 12 cm sheet o f filter paper saturated with the developing solvents. The developing solvents had a depth o f 1.0 cm in the glass developing chamber. Chlorophyll zones were separated after 10 rain development. Chromatograms were developed in darkness and the light intensity during all the operations was no greater than 40 ft.-c. The separated zones were removed one at a time, keeping the remaining zones moist with solvent by covering them with a 5 × 10 cm sheet of glass. The zones o f pigment were eluted and the concentrations determined as described by M ~ w I c x (1964), except that when algal and phytoplankton extracts were used, chlorophyll c was calculated using 2-component equations to correct for traces of contaminants. (iii)
Chlorophyll standards
Spectroscopically pure chlorophylls a and b were prepared from spinach leaves o n columns of sucrose (S'rRAI~, 1958). The chlorophylls were eluted in ether and stored at -- 20 °. To ensure freedom from breakdown products these pigments were transferred to 60-80 ° petroleum spirit and rv-chromatographed before use if they had been stored longer than two days. Spectroscopically pure chlorophyll c solutions were prepared from the kelp Eklonia using paper chromatography (IEF~EY, 1961). Chlorophyll c was ehited from paper with methanol, added to an equal volume of ether and washed with 10 volumes [20% (w/v)] NaCI solution. The upper pigmented layer was removed and dried over anhydrous sodium sulphate. The chlorophyll c solution was kept at -- 20 ° before use. When mixtures of these chlorophylls were separated on thin layers a n d eluted in 90% acetone, the absorption spectra were not significantly altered. (iv)
Recovery experiments
(a) Mixtures of purified chlorophylls. The concentration in the standard solutions was determined using extinction coefficients given previously for ether solutions (MADCWXCK, 1964). Known concentrations, in mixtures, were then applied to the layers, chromatographed, and eluted in 90% acetone. The concentration recovered was calculated using the extinction coefficients given previously for 90% acetone solutions (MADGWICK, 1964). (b) Mixtures of purified chlorophylls added to crude methanol extracts. Known mixtures of chlorophylls a, b and c in ether were added to crude methanol extracts of Nitzschia closterium in the proportion of one volume o f ether to six of methanol. The concentration of chlorophyll in the crude Nitzschia extract was determined chromatographically before adding the mixture of standard solutions. The recovery of chlorophyll was then found by difference. Chlorophyll c elutes were often contaminated with a 663 m p absorbing substance. This was probably due to slight contamination with chlorophyll a, chlorophyllide a or phaeophorbidc a. The contamination did not significantly alter chlorophyll c recoveries provided elute extinctions were greater than 0-03 at 630 rn~. The contamination was corrected for by using 2-component equations for mixtures o f chlorophyll a and c (HUMPHl~Y and J~FVREY, unpublished) i.e. it was assumed that the contaminants has spectra similar to chlorophyll a.
461
Instruments and Methods
(v) Comparison of spectrophotometric and chromatographic methods using algal cultures and phytoplankton Seawater was taken from inshore and coastal regions at Port Hacking, Sydney. Phytoplankton were harvested in a continuous centrifuge (DAviS, 1957), before resuspending in filtered seawater. Half of the sample was analysed chromatographically. The remaining half was passed through a 47 m m Millipore® 0.45/~ filter covered with MgCOa. The filter was then ground with 90% acetone according to YEwrSCH and MENZEL (1963). The extinctions of the extract were read at 750, 663, 645 and 630 n ~ and were corrected for turbidity by subtracting the extinction at 750 m~. The concentrations o f chlorophylls were calculated using simultaneous trichromatic equations described by HUMPHREY and JEFFR£Y (unpublished). Algae were treated similarly except that they were harvested at 2000 g in a swing-out centrifuge before resuspending, and the concentrations were calculated for the spectrophotometric method using 2-component equations (Hu~HREY and JEFWEV unpublished). RESULTS (i)
Chromatographic recovery of mixtures of chlorophylls a, b and c
(a) Mixtures of spectrophotometrically pure chlorophylls. Table 1 shows that chlorophyll a recovery was quantitative at all the concentrations tested, but chlorophyll c was recovered with losses up to 22%. Chlorophyll b was over-estimated by 10-20% in all cases.
Table 1. Recover), of spectroscopically pure chlorophylls a, b and c from mixtures Chlorophyll a, b and c were applied together in ether, and eluted with 90% acetone. Concentrations were calculated with extinction coefficients for ether and 90 % acetone respectively.
Chlorophyll a
Chlorophyll b
Chlorophyll c
Applied (~e)
Recovered (%)
Applied b'g)
Recovered (%)
Applied O'g)
Recovered (%)
0"62 0"82 0"92 1"1 ! 1.84 2"94 4'10
99 97 98 97 98 98 100
0"33 0'49 0"55 0.67 1"10 1"77 2'44
1i 2 I 18 120 120 121 116 116
0.50 0-74 0"82 0"97 1"88 2"81 4"00
99 90 90 92 78 84 82
(b) Mixtures of purified chlorophylls added to crude methanol extracts. Table 2 shows that when mixtures of spectroscopically pure chlorophylls a, b and c were added to crude methanol extracts o f Nitzschia closterium good recoveries o f a and c were obtained. Recoveries o f chlorophyll b were poor when only 0.08 ~g was used.
Table 2. Recovery of chlorophylls a, b and c added to Nitzschia extracts Mixtures of spectroscopically pure chlorophyll a, b and c were added to crude methanol extracts of Nitzschia closterium, separated, and eluted with 90 % acetone. The concentration of chlorophyll a and c in the crude extracts was determined first on separate chromatograms, and the recoveries found by difference. Chlorophyll a Applied (~g)
Chlorophyll b Recovered
(%)
Extract
Added
0-50 0'56 1.00 "1"18 I "51 2"53
0.27 0.66 0.23 0.66 0.23 0.66
Applied (#g)
0"08 0"29 0.08 0.29 0"08 0.29
Applied (#g)
Recovered
(%)
Added 92 106 106 102 I01 101
Chlorophyll c
62 86 63 1 I0 75 101
Recovered
(%)
Extract
Added
O" 13 0"12 0-19 0-I0 0.34 0.61
0"57 2.06 0.57 2-06 0.57 2.06
96 95 I00 II0 91 96
462
Instruments and Methods
(ii) Comparison of speetrophotometric and chromatographic methods using algal cultures and phytoplank ton Table 3 shows that in algal cultures, chromatography gave 30--40% higher values for chlorophyll a than spectrophotometry. Chlorophyll b, from the one comparison made, was 21% greater by chromatography. Chlorophyll c was 22-28 % lower by chromatography except with the cryptomonad where it was I 1% higher.
Table 3. Comparison of results of estimating chlorophylls in algal cultures using spectrophotometry and chromatography Cultures used were less than one week old. Two-component equations were used in the spectrophotometric method
Chromatographic
Method Alga
Chlorophyll
Nitzschia
a
Spectrophotometry Chromatography (l~g/Sample) (ag/Sample) 3.41 1.32 2"06 1.22 3.05 1.30 1.60 1-40 2"50 1.36 4.33 3.75 4.22 1.08
c a c a c a c a c a c
Skeletonema Isochrysis Gymnodinium Diatom (unidentified) Cryptomonad (unidentified) Dunaliella
a b
3.51 1"01 2"34 0.88 3.28 1-02 2-24 1.08 2.60 I "03 5-15 4.15 5.21 1-31
value as % of
Spectrophotometric value 103 77 I 14 72 108 78 140 77 104 76 119 111 124 121
Table 4. Comparison of results of estimating chlorophylls in seawater by spectrophotometry and chromatography Five litres o f seawater were used for each experiment, except 3, where 20 l. were used for chromatography.
Method Chromatography
Chromatography value as a % of speetraphotoraetry value
0.29 0"~ O"10 0"39 0.04 0-14 0"20 0"02 0"11 0.48 0"00 0"24 0.06 0"00 0-03 0'25 0.00 0.18
107 1 200 91 57 280 51 18 85 87 -109 86 -1~ 41 -138
(pg/1.)
Experiment
Chlorophyll
1
a b c a b c a b c a b c a b c a b c
2 2*t 3 4 5?
Spectrophotometry 0"27 -- ve 0"05 0"43 0"07 0"05 0"39 0.11 0"13 0"55 0"10 0-22 0"07 0.01 0"03 0-61 0"04 0.13
*Duplicate centrifuged samples were stored in the dark for 3 days at room temperature. tPhaeophytin a was found chromatographically.
Instruments and Methods
463
The results in experiments I, 2, 3 and 4 in Table 4 show good agreement between chromatography and spectrophotometry for chlorophyll a. Phaeophytin a was found in experiment 2" (aged suspension) and 5. Experiment 2* is one where the sample of phytoplankton was aged by standing in the dark for three days at room temperature. The phytoplankton in experiment 5 contained phaeophytin a even though it was analysed at once, i.e. without storage. In both of these experiments the chromatographic value for chlorophyll a was about half that found spectrophotometrically. Phaeophytin a was identified from its position on the chromatograrn and its absorption spectrum. In only two cases (Table 4) was chlorophyll b found chromatographically. The two methods agreed reasonably (85-138%) for chlorophyll c, except in experiments 1 and 2 (200 and 280%) which had very low spectrophotometric values. (iii)
Chlorophyll content of algal cultures
Table 5 shows that the maxima for chlorophyll per million cells occurred at 3 days in Nitzschia for chlorophyl a and in the cryptomonad for a and c. For chlorophyll c in Nitzschia and for both chlorophylls in Skeletonema, Isoehrysis and DunalieUa, the maximum per million cells came at one day's growth. In Gymnodinium, the maximum chlorophyll per million cells came at 15 days. The maxima in cell numbers for Nitzschia, Dunaliella, Skeletonema, Isochrysis and the cryptomonad came after the maxima for chlorophyll concentration at 7, 23, 7, 7 and 14 days respectively. In Gymnodinium the maximum in cell numbers, at 7 days, was before the maximum for chlorophyll concentration. Disregarding the initial and final values, where the concentrations of the minor chlorophylls were close to the lower limit of the sensitivity o f the method, the ratios b/a and c]a varied little during the periods of observation. The results are represented in Fig. 1.
Table 5. Chlorophyll in algal cultures Analyses were done using glucose thin-layer chromatography
Alia Nitzsehia
Cryptomonad
Skeletonema
Isochrysis
Gymnodinium
Dunaliella
Age (days) 1 3 7 14 21 1 3 7 14 1 3 7 14 21 1 3 7 14 21 7 12 15 23 27 1 2 7 12 23 30
Chlorophyll Chlorophyll Chlorophyll a b c
(~g/10ecells) (/t.g/10ecells) (~g/10ecells) 0.83 0.90 0"31 0.I1 0.10 1.00 1.27 0.70 0.35 0.71 0.17 0.10 0-17 0"03 0"25 0"15 0"12 0"07 0"03 0"47 2"58 4"64 1"78 1"75 1"72 1.15 0.88 0.80 0.60 0.54
*Extinctions below range of method.
0.25 0.21 0.07 0"02 0.02 0-57 0"70 0.44 0-15 0.29 0-04 0-03 0.04 0.00* 0-08 0"02 0"03 0.01 0.00* 0"22 1"05 2.00 1"00" 1"38" 0"35 0.30 0.22 0.20 0.14 0.19
Ratio
Cell No.
c/a or b]a (10e/mlculture) 0.30 0.23 0.23 0.18 0.20 0.57 0"55 0.63 0.43 0.41 0.24 0-30 0-24 -0"32 0"13 0"25 0"14 m 0"47 0"41 0"43 N -0"20 0.26 0.25 0.25 0-23 0.35
0.12 0.82 2.36 2-14 0"59 0.14 0-40 0.50 0.60 0-07 1.51 3.37 1.15 0.00 0"65 2"50 5"80 5"06 3"18 0"49 0"19 0"08 0"09 0"08 0"30 0.33 2.28 2.50 3.12 2.89
464
Instruraents and Methods I0
130
2'0
I'0
C r yptomonod
I 0 "~/
05
0"~
0
L 0
0
IO
I0-0
@
ua $totum r'-
~sochr ysis
cJal bona
z~
r-
c
Z c
o s 0 c 0 J z
~0 3
•,
]
O..
~ ~0,. ~ ~'
•.,..:.-2:.'., .... ,...:..%.
"o
OQ.
q
0
oj
5"0
-O-
~
X
x
0
/ 0
4'0 "~ O
G~mn odin~um
sp
j, / /
\ \
/
\
/
\
..'"
20
\
."X".. "'.
,(i II
//
Ounahella
\
".
0 "X
%
I I0
I
20
~.-A.--.~.A .......
O
1
0
fO
" - - 'A
20
0
25
OATS
Fig. 1. C h l o r o p h y l l c o n t e n t ofalgalcultures. (Chlorophyll a - - 0 - - ; b . . . . A-.; e . - - x . . • ; cell number
•
).
DISCUSSION
Recovery o f chlorophyll a from mixtures of spectroscopically purified chlorophylls was slightly low (1-3 ~ ) . Chlorophyll b w a s overestimated by 12-21 ~ and chlorophyll c was underestimated by 1-22 ~o. T h e decreasing recovery o f chlorophyll c with increasing concentration might be due to decreasing elution efficiency. N o satisfactory explanation can be given for the overestimation o f chlorophyll b. Recoveries from crude extracts o f Nitzschia were g o o d : -- 8 to ÷ 6 ~ for chlorophyll a, -- 14 to -~ 1 0 ~ for b, and -- 9 to ~- 1 0 ~ for c in the concentrations used, except for b, when only
Instruments and Methods
465
0.08 ~g was present. Chlorophyll b was used in much smaller amounts than a or c to simulate the concentrations present in seawater. The sensitivity of the chromatographic method is such that 0.5 ~g of each chlorophyll, or slightly less, can be estimated. Although the chromatographic method gave consistently higher results for chlorophyll a than the spectrophotometric method when using algal cultures, the agreement was sufficiently close to suggest that neither method was grossly in error. A similar suggestion can be made for chlorophyll b and c. However with seawater samples the agreement between chromatographic and spectrophotometric methods was good for chlorophyll a but not for b and c. The minor chlorophylls have little adsorption at the peak for a whereas a absorbs relatively strongly at 645 and 630 n ~ , and spectrophotometric errors would be more pronounced because of the small concentrations of c and b compared with a in these seawater samples. It is not known which, if either, of the two methods gives the correct answer. The agreement for chlorophyll a, and for c when it was greater than 0.1 ~g/1., indicates both methods are comparable. The seawater comparisons were made under conditions similar to those usually met in routine spectrophotometric determinations where low extinctions make values of b, and sometimes c, unreliable. It is quite clear that when phaeophytin a is present the chromatographic method is to be preferred.
Table 6. Comparison of chlorophyll analyses by different authors Algae were grown on Erdschreiber media except by PARSONS (1961) who used a modified Provasoli medium.
Chlorophyll
Alga
ratio
a
Nitzschia Closterium
c
c/a
Isochrysis Galbana
0-33 0.18 1'25 0-10 0"22 0"84 0.04 1.00 0.06 0.11 0-30 0"14
0-08 0"11 0"29 0.04 0-20 0"34 0-02 0.28 0"02 0-03 0"08 0.04
b
b/a
Dunaliella tertiolecta
0.56 0"50
0.26 O. 13
0.47 0.26
Gymnodinium Sp. Skeletonema Costatum
Growth conditions
(/Lg/100 ~g dry ,act.) Chlorophyll
Light
Age
(ft-c)
(days)
400 420 400 400 420 150 400 1700 420 400 400 400
14-28 7 3 14-28 7 12 14-28 log phase 7 7 14-28 7
400 150
14-28 7
0.24 0.61 0'23 0.40 0"91 0"40 0.50 0"28 0"35 0.27 0"27 0.29
Reference
J ~ R E Y (1961) HUMPHREY (1963) Present paper JEWRY (1961) HUMPHREY (1963) Present paper JEFFREY(1961) P~.soNs (1961) HuMrm~Y (1963) Present paper JEFFREY(1961) Present paper Jv~-~Y (1961) Present paper
Table 6 summarizes the present, and some previous work on the chlorophyll content of algae. The results of the different authors have been recalculated with the extinction coefficients used in the present paper, and for the results of HUMPI-I~Y (1963) dry weights were determined from cell numbers by using conversion factors foundin thepresent work. Because ofthedifferencesin cultureconditious used by the various authors, it is not possible to make direct comparisons. Nevertheless the table is important in showing the ranges of values obtained when different analytical methods and growth conditions were used. The first point which emerges is that chlorophyll c (or b) was always less than a. Secondly, chlorophyll a was never more than 1.25 % and chlorophyll c (or b) never more than 0.34% of the algal dry weight. In the present work, except for Gymnodinium, a feature of the chlorophyll content per cell was that it did not keep pace with cell division. REFERENCES DAVzS P. S. 0957) A method for determination of chlorophyll in sea water. CSIRO Aust. Div. Fish. Oceanogr. Rep. No. 7.
466
Instnm~nts and Methods
HUMPH~Y G. F. (1962) Proceedings of the Conference on Primary Productivity Measurements, Marine and Freshwater, 1961 : 121-141. ( U.S. Atomic Energy Commi~ion, Division of Technical Information, TID- 7633). HUMI'I-m~ G. F. (1963) Chlorophyll a and c in cultures of marine algae. Aust. ?. mar. Fre,shwat. Res. 14, 148--154. JeJm~Y S. W. (1961) Paper chromatographic separation of chlorophylls and carotenoids from marine algae. Biochem. Y. 80, 336-342. L ~ s T. M. and DE MUP.XAP. J. (1962) A simple methoct for the preparation of thin layer chromatography plates. J. Chromatog. 8, 108-109. MADGWXCEJ. C. (1964) Quantitative chromatography of algal chlorophylls on thin layers of glucose. Deep-Sea Res. 12, 233-236. PAgSONST. R. (1961) On the pigment composition of eleven species of marine phytoplankers. J. Fish. Res. Bd. Canada 18, 1017-1025. STRAINH. H. (1958) Chloroplast pigments and chromatographic analysis, pp. 27-28, The Pennsylvania State University, University Park, Pa. Ye~'SCH C. S. and M~Z~L D. V. (1963) A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Res. 10, 221-231.