Table Of ContentTSR ESTIMATION AMD CHARACTERIZATION OF PLANKTON BY
SPECTROPHOSTUOHMETRIC ANALYSIS OF PIGMENT EXTRACTS
hy
FRANCIS ASBURY RICHARDS
.QHtelAiSs * 'if
A thesis submitted in partial fulfillment for the degree of
DOCTOR OF PHILOSOPHY
UNIVERSITY OF WASHINGTON
1950
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UMI Number: DP16805
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ACKNOWLEDGMENTS
lost of the work reported herein was done as a
part of Contract N8onr-520/lll between the Office of Naval
Research and the UniverkL ty of Washington. Baring prelim
inary phases* the author was a Teaching fellow in the
Department of Chemistry of the university.. Acknowledgment
of these sources of financial aid is gratefully made.
Thanks are due to Professor Thomas 0. Thompson,
Director of the Oceanographic laboratories, who directed
the work, and to Professor fi. Weston Blaser of the Botany
Department, University of Washington, who was responsible
for interesting the writer in the general subject and was
of material help in the botanical phases of the research.
Many stimulating ideas have come fro® staff members
and visiting investigators at the Oceanographic Laboratories
of the University of Washington. Drs. C. A. Barnes, W. T.
Edmondson, A, W, Hutchinson, M. W. Johnson, B. H. Ketehum,
A* C. Redfield, and J. P. Tully have been especially helpful.
Dr. E. f, Swan, Resident Scientist at the Field Laboratories,
collected many of the samples analyzed and was a sympathetic
laboratory companion during much of the work.
Without the inspiration and encouragement of the
writer*s wife, the work would not have been possible.
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TABLE OF CONTENTS
Page
LIST OF TABLES iv
LIST OF FIffQBXS Vil
Chapter
I IHTRODUCTION 1
Alms and Requirements 3
II HISTORICAL A
III EXPERIMENTAL: Til ARALTTISAL METHOD 10
Objectives and Requirements of
the Analytical Method 10
Methods and Materials 11
Sample Collection 11
Removal of Plankton fro®
Water Samples 12
Extraction Methods 1%
SpeetrophotometricM ethods 19
Procedure 19
Expression of Pigment
Concentration 21
Chlorophylls 21
Carotenoids 27
Discussion of Analytical Results
and Calculations 3?
Beer Lambert Law 37
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ii
TABLE OP CONTENTS (continued) ??
Chapter Page
III (.continued)
Chlorophyll a_ 38
Chlorophyll b 38
Chlorophyll c 38
Carotenoids 41
Materials Analized 42
Discussion of the Analytical Method 43
1? EXPERIMENTAL: APPLICATIONS QW PIGMENT
A1ALT8IS 44
Photosynthesis Experiments 44
Methods and Results 45
Discussion 47
pigment Distribution In East Sound -
Barney Channel legion, July and
August, 19%9 48
Methods . 49
Results 51
Discussion • 51
Resume of Observations _ 51
Generalisations 66
Hypothesis Concerning the
Production and .Distribution
of plankton in the San loan
Archipelago 66
Examination of Hypothesis 67
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I l l
TABLE OP CONTENTS (continued)
'Chapter Page
1? (continued) .
Pigment Observation of Surface
Waters at Friday Harbor
August 19%9 ** April 1950 72
Methods and Results 72
Discussion 72
f CONCLUSIONS, FINDINGS, AID SUMMARY 77
Conclusions and Findings 77
Summary 79
APPENDIX
A ADSORPTION SPECTRA OF SOW PLANT PIGMENTS 8l
Chlorophyll a 82
Chlorophyll £ 87
Xanthophylls 96
Beta Carotene 121
B. OPTICAL METHODS AND TERMINOLOGY 130
C INVESTIGATION OF A CHROMATOGRAPHIC
METHOD FOR THE ESTIMATION OF PLANKTON
PIGMENTS 133
D SUMMARY OF PROCEDURE AND CALCULATIONS
FOR TUI ESTIMATION OF CHLOROPHYLLS AND
CAROTENQIDS IN PLANKTON EXTRACTS 139
RSFStlllGIS CITED 1A3
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i¥
■LIST OF TABLES
fable Fage
1 Changes in Optical Density at Several
Wave Length® With Time of Extraction
of Plankton With 90 Per Cent Acetone 16
2 Optical Densities of Plankton Extracts
Showing Effects of Method® of Centri
fuging, Grinding, and Washing IT
3 Specified Absorption Coefficients of
Plankton Pigment® 22
4 Specific and Specified Absorption
Coefficients of Some plankton Pigments 24
5 Absorption Spectrum of 90 Per Cent
Acetone Solution of Astaein Type Pigment
attracted from Crustaceans, Corrected
for Chlorophyll a Absorption 31
6 Concentrations of -Figment and Pigment
Groups In 90 Per Cent .Acetone. Extracts
of Plankton Samples Calculated from
Spectrophotoaetrlc Data 34
7 Observed pigment Concentration® and
Photosynthetic Bates In Clear and Opaque
Bottles of tea Water Containing Mixed
Plankton: 46
■a Milligrams of Oxygen Produced per Day
per Liter of Water 50 Cm Below the
Surface by On® Milligram (or 103SU)
of Chlorophyll 4?
9 Salinity, Temperature, and Pigment
Concentrations at Shag Rook and
Rosario Stations, 11 July 1949 52
10 Salinity, Temperature, and Pigment
Concentrations at Shag lock and
Rosario Stations, 26 July 1949 53
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¥
LIS^T 0JP TABLES (continued)
Table Page
11 Salinity, Temperature, and Figment
Concentrations at Shag lock and
losarlo Stations, 3 August 19^9 54
. It Salinity, Temperature, and Pigment
Concentrations at Shag lock and
Rosario Stations, 11 August 1949 55
13 Salinity, Temperature, and Pigment
Concentrations at Shag look and
Rosario Stations, 19 August 1949 56
1% Salinity, Temperature, and pigment
Concentrations at President Channel
Station, 19 July 19*9 TO
15 Pigment Analyses of Plankton Samples
from 1 M Depth at Cantilever Pier,
Friday Harbor, August 1949 - April 1950 72
16 Specific Absorption Coefficients (log si®J)
of Chlorophyll a in Acetone and
Diethyl Ether 'Solutions 84
1? Specified Absorption Coefficients of
Chlorophyll c in Methanol and 90 ter
Cent Acetone,■Calculated from Data
Reported by Strain and-Hanning' (42) and
Strain, Hanning, and Hardin (I3) and from
Original Observations. ■Values of log
Tabulated lcm 88
IS Specific Absorption Coefficients (log l}f®)
of light Leaf lanthophylls at the
Yellow Absorption Maxima 98
19 Specified Absorption Coefficients of Diatom
Xanthophylls at Their Yellow Absorption
Maxima in 90 Per Cent Acetone Solutions 98
20 Specified (log sfSm) Absorption Coefficients
of Ethanol Solutions of Diatom and Blno-
flageliate Xanthophylls, from Strain
at al (44) 99
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Vi
LIST OF TABLES (continued)
Table Page
• SI Specified Absorption Coefficients of
Pitto® Xanthophylls Prepared from Mixed
Diatoms by the Method of Strain, Hanning,
and lariln (44). Values of log sf® Given
for Ethanol and 90 Per Cent Aeetone
Solutions. Uncorrected for Residual
?
Dimethyl Aniline. 10
Sif
22 Specified Absorption Coefficients (log Eieffi)
of Meofucoxanthin B and Fucoxanthin,
Prepared by Method of Pace (30), in the
Presence of Blaethylaniline, Uncorrected
and Corrected for Chlorophylls a and e.
ninety Per Cent Acetone Solution. 115
su
23' Specified Absorption Coefficients (logle }
of Three Fucoxanthin Fractions
Prepared from Diatoms by the Method of
Pace (30), Showing the Effects of
iedrying Material Preparatory to Dissolving
in 90 Per Cent Aeetone, Material for
Ethanol Solution Mot Subjected to Iedrying.
Procedure, preparations lade in the
Absence of Dimethyl Aniline. ill
2% . Specified Absorption Coefficients (log iff)
of Xanthophylls Prepared from Diatoms
by Method of Strain, Manning, and Hardin
(44), Using MgCQ3 Instead of Dimethylani-
llne. All Pigments Dried at .Reduced
Pressure for 10 lours Before faking Ip in
95 Per Cent Ethanol or 90 Per Cent Acetone 119
25 Specific Absorption Coefficients (log li|»)
of Beta Carotene, Calculated from ■
Data Reported for Hexane Solutions
by (l) Zechmelster and Folglr (50), and
(2) Eseheile, White, Beadle, and Roach
(57), for (3* "Skellysolve B" Solution
by laumgarten, Bauernfeind, and Boruff
(1), and for Petroleum Ether Solutions
by (4) Strain (40) and (5) Haagen-Smit,
Jeffreys, and Kirshner (&) 122
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vli
LIST Of TABLES (continued)
Table Page
26 Specific Absorption Coefficients (log
of a Commercial Beta Carotene In
Hexane and 90 Per Cent Acetone Solutions.
Treatments as Described in Text. Concen
trations of Solutions From Which the
Coefficients Were Determined As'Shown at
Top of lach Column. 125
27 Slit and nominal Band Width used in Waking
Absorption Measurements on the Beckman
Spectrophotometer 131
LIST OF FIGURES
.figure Page
1-A Absorption Spectrum of an Acetone
Extract of a plankton Sample 22a
1-1 Absorption Spectra of Acetone Extracts
of plankton Samples 22b
2-A Absorption Coefficients of Plankton
figments in 90 Per Gent Acetone
Solution* 350-530 ^ 25
2-B Absorption Coefficients of Plankton
Pigments in 90 Per Gent'Acetone
26
Solution, 500-700
3 Specified Absorption Coefficients of
Aataein fype Pigment Extracted 'from
Crustaceans in Plankton Tows 32
A Salinity, Temperature, and Pigment
Concentrations at Shag Hock Station,
11 July 19*9 57
5 Salinity, Temperature, and Pigment
Concentrations at Rosario Station,
11 July 19*9 §7
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