Thin, layer chromatographic method for plant pigments in ...

Ggochemical Journal, Vol. 5, pp. 23 to 37, 1971

Thin, layer chromatographic method for

plant pigments in marine particulate and ecological significance of the

determining

matter,

results

KATSUMI YANAGI and TADASHIRO KOYAMA

Water Research Laboratory, Faculty of Science,Nagoya University, Chikusa, Nagoya, Japan

(Received February 13, 1971; in revised form July 3, 1971)

Abstract-Thirty to 100 liters of sea water sample containing MgCO3 powder was filtered through a glass fiber filter in order to collect particulate matter. Soon after the filtration, this glass fiber filter was kept in a deep freezer (-20°C) for later use. The following

pigment analysis was carried out within a week. Plant pigments in the collected particulate matter were extracted with 90% acetone in a dark refrigerator. Pigments in the acetone extract were separated by thin layer chromatography using a mixture of cellulose powder and soluble starch as an adsorbent. The developing solvent was 1.2% n-propanol in

petroleum ether. According to this procedure, 9 kinds of pigments were fractionated. Each of the fractionated pigments was eluted with 90% acetone and determined by measuring fluorescence of the acetone extracts after acidification with a small amount of hydrochloric acid. The analyses of sea water samples from the central Pacific showed 'the following facts: (1) Vertical distribution of chlorophyll a had. a peak at a depth of 100m or 150m and its amount remarkably decreased with depth in parallel with the total pigments. (2) The percentage of chlorophyll a in the total pigments was more than 60% for the sea water samples examined, even at the depth of 1,000m. (3) The absence of phaeophytin a was made clear for the samples examined,

I NTRODUCTION

The spectrophotometric method proposed by RICHARDS and THOMPSON (1952) and that modified by CREITZ and RICHARDS (1955) were widely employed for determining

planktonic pigments in sea water. PARSONS and STRICKLAND (1963) reported a number of discrepancies in the spectrophotometric determination of plant pigments with the Richards and Thompson method and presented a revised set of equations for the

determination of chiorophylls as well as a new equation for the approximate estimation of carotenoids. GOODwIN (1947) and KREY (1958) proposed a sensitive method for chlorophyll determination by measuring fluorescence. YENTSCH and MENZEL (1963) applied the fluorometric method for the sensitive determination of phytoplanktonic chlorophyll and phaeophytin. The principle of this method is based on the decrease in

23

24 K. YANAGI and T. KOYAMA

fluorescence of chlorophyll by acidification. YENTSCH (1965) reported a modified fluorometric method in which hydrochloric acid was used for acidification instead of oxalic acid. This method was carried out with the assumption that the ratio of fluo

rescence of the unmodified sample to that of the acidified one is 2.3 for pure chlorophyll a and 1.0 for pure phaeophytin a. HOLM-HANSEN et al. (1965) and SAIJO and NISHIZAwA (1969) discussed the effect of excitation spectra on the fluorometric determination.

A number of errors are inherent in these indirect methods to determine the

individual pigments as pointed out by HUMPHREY (1962). Therefore, for the accurate

determination, a direct method by which the phytoplanktonic pigments are separated

and individually determined is highly required. JEFFREY (1961) determined chlorophyll a, b, and c, and thirteen kinds of

carotenoids in some marine algae by using a modified two-dimensional paper chromato

graphic method of LIND et al. (1953). PARSONS (1961) determined chlorophyll a, b, and c of marine phytoplankton by the spectrophotometric method and the individual carotenoids by a column chromatography similar to that described by STRAIN et al.

(1964). MADGWICK (1965) separated chlorophylls from phytoplankton by thin layer chromatography (T.L.C.) using glucose as an adsorbent. JEFFREY (1968) isolated and determined photosynthetic pigments. by T.L.C.

The present paper will describe a thin layer chromatographic method combined

with fluorometry, which is useful to determine individual pigments of phytoplankton

in marine particulates, and the ecological significance of some results obtained by

this method.

ANALYTICAL METHOD

The analytical procedure for determining individual pigments, which was develop

ed by the authors is schematically shown in Fig.l.

(1) Sample preparation To a sea water sample of 30-100Q was added 1020 ml of 1 % MgCO3 powder suspension to prevent acidification and breakdown of

chlorophylls. The sea water sample containing MgCO3 was filtered through a glass fiber

filter (984 H, Reeve Angel Co.). Soon after the filtration, this glass fiber filter was

kept in a deep freezer (-20°C) for later use. In the case of water samples from 0-150 m

depths, 30-50Q was enough for each sample, while for deeper samples, 80-100Q was

used to determine the phytoplanktonic pigments in the particulate matter.

(2) Extraction of pigments from the collected particulate matter The glass fiber filter holding particulates was cut into small pieces, which were put into a flask

(capacity: 50m1). To the flask was added 30m1 of 90% acetone. It was left to stand for 3 hr in a dark refrigerator (5'C) with occasional shaking to extract pigments from

particulates. The solution was filtered through a glass fiber filter by the aid of suction. The residue was repeatedly washed with small amounts of 90% acetone. Each washing

Thin layer chromatography for plant pigments 25

SEA WATER SAMPLE

containing MgC03 suspension

Filtered through glass fiber filter

PARTICULATE MATTER

Extracted with 90% acetone for 3 hr

ACETONE EXTRACT

Extracted with pet. ether

and 0.05M NaHCO3

PETROLEUM ETHER EXTRACT

Dried with Sephadex G-25 Evaporated to syrup

SYRUP

Separated by T.L.C.

Added one drop of 0.1NHCI

DETERMINATION of total pigments

FRACTIONS

Extracted with 90% acetone and added one drop of 0.1 N HCI Determined the fractionated pigments

DETERMINATION

of quantitative composition of the fractionated pigments

CALCULATION of each amount

of the fractionated pigments

Fig.1. Scheme of fractionation and determination of pigments

in marine particulates.

was filtered through the same glass fiber filter. Volume of the combined filtrate was adjusted to 50m1 with the same solvent.

(3) Determination of total chlorophyll pigments To 5 ml of the acetone extract was added one drop of 0.1 N HCl. After 15 min absorbance of the acidified acetone solution was measured at 666mµ by using a spectrophotometer (Hitachi Co. Model 139) and a micro-cell with 50mm of light path or the intensity of fluorescence was measured using a fluorophotometer (Hitachi Co. Model 203) at 670mµ, which was emitted by excitation at 365mµ. On measuring the intensity of fluorescence, it is important to adjust exactly the condition of the fluorophotometer to that under which the calibration curve was made.


(4) Calibration curve for the determination of chlorophyll pigments The standard solution of phaeophytin a was prepared according to the procedure presented by KOYAMA et al. (1968). Purities of the prepared chlorophyll a and phaeophytin a were calculated as 94% from their specific absorption coefficients and those presented by SMITH and BENITEZ (1955). Calibration curve of fluorescence intensity or' of absorbance against various solutions prepared from the standard solution of phaeophytin a was made according to the way which was mentioned in item 3. On measuring the fluorescence intensity, the sensitivity and selector of the fluorophotometer were previously fixed so that the meter reading indicates exactly 100 scale under the intensity of fluorescence at 515 my emitted from a standard aqueous solution of Na-fluorescein

(uranine) (0.5 ppm) excited at 435 mp. Fig.2 shows the calibration curve thus obtained. (5) Preparation of thin layers Thirty grams of cellulose powder No.C

(about 300 mesh, Toyo Roshi Co. LTD.) and IOg of soluble starch were thoroughly mixed with 100ml of a solvent (CH3OH : CHC13 = 1 : 3v/v). A glass plate of 50 by 200 mm was coated with the mixture to make a thin layer. The layer was dried in open air for a few hours. In this preparation, it was important to use a carefully cleaned glass plate and newly purified chloroform, because even trace impurities in

chloroform brought some degradation of the pigments.

(6) Thin layer chromatographic fractionation of the extracted pigments The residual acetone extract was transferred to a separating funnel having a capacity of 100ml. Twenty milliliters of petroleum ether (b.p. 40-60°C) and 30m1 of 0.05M NaHCO3 aqueous solution were added to the funnel and then gently mixed to transfer

the pigments to the petroleum ether layer. The aqueous fraction was removed, and

80

U U N.

60 0

w w 0

40

t+ .y

`" 20

0

0.25

0.20

0.15

0.10

0.05

0

U

E 0

a

U U

cad i-1 0 y en

0 0.2 0.4 0.6 0.8 1.0

Concentration of phaeophytin a (pg/ml)

Fig.2. Calibration curve for phaeophytin a.


the petroleum ether fraction was washed with another 30m1 of NaHCO3 solution by mild shaking. After the aqueous fraction was removed , the petroleum ether fraction was passed through a column (1 cm X 1 cm2) charged with Sephadex G-25 in order to dry the fraction. A part of the fraction remained in the column was washed out with a small amount of petroleum ether. All of the dried petroleum ether fraction was transferred to a flask and condensed to a small volume (0.5 ml) by using a rotary evaporator. In this case, it is important to take precautions against dry up and increase in temperature. The condensed pigment solution was applied on the thin layer along a line drawn parallel to the lower border at an interval of 1 cm. The pigments were developed with a mixed solvent (petroleum ether : n-propyl alcohol = 500: 6v/v) for 20 min by ascending chromatography in a chromatographic chamber kept in a refrigerator (5°C). The front of the solvent moved 15 cm during the developing time . According to this procedure, 9 kinds of pigments were fractionated.

(7) Determination of fractionated pigments Nine kinds of pigments were named fractions No.1 through No.9 in the order of their Rf values. Each fraction of

the chromatogram on the thin layer was scraped into a test tube and to which was added 3-4ml of 90% acetone in order to extract the pigment. The acetone extract

was filtered through a small piece of cotton placed on the bottom of a small funnel . Since minor part of the pigment still remained in the scraped adsorbent, it was

extracted with another 1-2 ml of the same acetone and the extract was filtered through

the same cotton. Total volume of the combined acetone extract was adjusted to 5 ml

with the same solvent. Amount of the pigment in the acetone extract was measured

after acidification according to the foregoing procedure (items 3 and 4). From the

results, composition of the fractionated pigments was quantitatively determined. From

the composition and the total amount of the pigments, each amount of the fractionated

pigments was calculated.

RESULTS AND DISCUSSION

Characteristics of the fractionated pigments are shown in Table 1 . From the table, it was suggested that fraction No.1 is 0-carotene and fractions No .2 and No.3 are carotenoid pigments and that fractions No.6 through No .9 are chlorophyll derivatives. However, fractions No.4 and No.5 were identified as phaeophytin a and chlorophyll a

, respectively, from the comparison of their absorption spectra

, specific absorption coefficients, fluorescence spectra and Rf-values with those of purified phaeophytin

a and chlorophyll a.

These results are quite similar to those reported by KOYAMA et al . (1968). Fractions No.4 through No.9 emitted a reddish fluorescence under ultra violet

rays, whereas fractions No. I through No .3 did not. Therefore, according to the present method, fractions No. I through No .3 might be underestimated.

(1) Preservation conditions for particulate samples Experiment on the


Table 1. Characteristics of various pigments fractionated by T. L. C. from the particulate matter of Ise-Bay

FractionNo. Rf value

Color on chromatogram

Absorption max. (mµ)

Remarks

1

2

3

4

5

6

7

8

9.

0.98 ± 0.02 yellow

0.95 ± 0.01 faint yellow

0.91 ± 0.02 pink

0.81 ± 0.02 gray

0.74 ± 0.02 green

0.67 ± 0.02 yellow green

0.41 ± 0.01 faint yellow

0.35 ± 0.01 orange yellow

0.00 ± 0.00 dark green

425

410*

575

410

580

412 610

410 666

408

410 668

448*

450**

450**

505

610

428*

618

430 668

430

470

450

475

535

667*

535

663*

480

475

668

505

carotenoids redish in CS2 solution

phaeophytin a, dark green in solution asp.*** at 667mµ= 60.2

chlorophyll a asp.*** at 663mµ= 94.3

chlorophyll derivatives (unidentified)

* Principal absorption: maxima . Solvent; diethyl ether.** Broad peaks .

*** asp .; specific absorption coefficient, expressed in 1 g/cm.

problem was conducted under the following four kinds of conditions: (i) Sea water sample containing MgCO3 was filtered through a glass fiber filter. Soon after the filtration, the glass fiber filter was kept in a deep freezer (-20'C). (ii) After the. filtration of sea water sample containing MgCO3 according to the condition (i), particulate matter on the filter was first completely dried in a vacuum desiccator and then kept in a deep freezer (-20'C). (iii) Particulate matter was kept in a deep freezer

according to the condition (i) but without MgCO3. (iv) Particulate matter was kept in a deep freezer according to the condition (ii) but without MgCO3. Cultured diatoms (Phaeodactylum tricornutum) were used in the experiment.

The experimental results are shown in Fig.3. These results clearly show that the

preservation rate of total pigments of the particulate matter was not remarkably altered during storage under the different conditions mentioned above. The preser

vation rate of chlorophyll a which was isolated by the thin layer chromatography,

however, indicated a remarkable time change according to the different conditions.

The results also indicated that the preservation rates of chlorophyll a under the con

ditions of Nos. i and ii decreased to about 95% at the 4 day storage, while, under the

conditions of Nos. iii and iv they decreased to about 85 %. Under the condition No. i, even at the 8 day storage, the preservation rate of chlorophyll a remained at 95%,

while, under the other conditions, they decreased to the value lower than 85%. This fact indicates that the pigment analysis should be carried out within one week of

storage under the condition No. i which was the best among the four conditions to


100

90

80

0

100

ca

a 0

> 90

a

80

70

60

I

(A)

.........0.......

Total pigment

(i)

.. .....0..• ~~ '•0......

I

b

-0 (ii)

0

'0

(B)

m' '0.

•0.

Chlorophyll a

(i)

'0 (M•0 (iii)

0 I I 1 I 1 1 i 10 2 4 6 8 10 20 30

Time (Day)

Fig.3. Preservation rate of total pigment(A) and chlorophyll a(B) during storage of cultured sample (Phaeodactylum tricornutum) under the following conditions.

(i) Particulate matter containing MgCO3 was kept in a deep freezer. (ii) Particulate matter containing MgCO3 was first completely dried

and then kept in a deep freezer.

(iii) Particulate matter without MgCO3 was kept in a deep freezer. (iv) Particulate matter without MgCO3 was first completely dried and

then kept in a deep freezer.

keep the pigments.

Fig.4 shows the relationship between pigment contents of cultured diatoms

(Phaeodactylum tricornutum) and storage time under the condition No. i. This figure indicates that the amounts of total pigments and chlorophyll a remained constant

during the first 8 days of storage and then slightly decreased with the storage time and

those of pigments other than chlorophyll a and phaeophytin a remained constant

during the storage.


0.3

0.2

n

a

a 0 U

0.1 bq w

Total pigment

iiS.

.0

Chlorophyll a

Others

•0Phaeophytin a

---0

0

a 11... .0 40

0%. 0

Fig.4.

2 4 6 8 10

Time (Day)

Relationship between pigment contents of

tricornutum) and storage time under

20

cultured sample the condition (i)

30

(Phaeodactylum

(2) Relationship between extraction time and the amount of pigments extracted from marine particulate matter Fig.5 shows the relationship of extraction time to the amount of pigments extracted with 90% acetone from the particulate matter of Ise Bay (A) and to that from cultured algal cell (B). The results indicate the following facts: (i) In the case of (A), the amount of total pigments increased with extraction time and reached a plateau at 5 hr, while that of chlorophyll a isolated by

thin layer chromatography (T. L.C.) reached a plateau at 2.5 hr. The amount of

phaeophytin a, which might have been formed from chlorophyll a, slightly increased with the extraction time and that of pigments other than chlorophyll a and phaeophytin a slightly decreased. (ii) In the case of (B), the amounts of chlorophyll a, phaeophytin

a and others remarkably increased with extraction time and reached their plateaus at 2hr. In the latter half of the extraction time, they slightly decreased with time. The amount of phaeophytin a slightly increased with time just as in the case of (A). From the fact, the extraction time of 3 hr was adopted to extract all pigments from particulate matter without degradation.

(3) Solvent system for re-extracting pigments from the acetone extract In


b

U aY

k N

a

00

0 a a 0

E d

0.5

0.4

0.3

0.2

0.1

0

0.3

0.2

w

0.1

0

Total pigment

f--O

Chlorophyll a -----0----0---------- o------i I--u

i Others

0 0Phaeophytin a •.....1I••••0 ........... 0...,. .

(A)

lP. 1cr

Total pigment I--o

,0___O-_-O-_-o-_-__.o--__-aChlorophyll a

0-

Others ._

.0....0 0

Phaeophytin a~0 1 I....

(B)

I0 2 4 6 8 10 12 24

Time (hour)

Fig.5. Relationship between extraction time and the amount of

pigments extracted from marine particulate matter(A) and that fromcultured sample (Phaeodactylum tricornutum) (B) .

order to know the relationship between the solvent system and the composition of

pigments recovered by T.L.C., 50m1 of an acetone solution of pure chlorophyll a or

pure phaeophytin a was transferred to a separating funnel. To the funnel were added 20m1 of petroleum ether and 30m1 of an aqueous solution and shaken to transfer the

pigment to the petroleum ether layer. The aqueous solutions examined were as follows: Distilled water, 0.1 M NaHCO3, 0.05M MgC12 and 0.1 M phosphate buffer (pH : 7.0).

After removing the aqueous fraction, the petroleum ether fraction was washed again

with the same aqueous solution. The petroleum ether fraction was dried by passing

through a column of Na2SO4 or Sepha.dex G-25. The pigments in the petroleum ether fraction were determined according to the foregoing procedure Nos. 3 through 6.

Table 2 shows the relationship between the solvent system for reextracting

pigments from acetone extract and the composition of pigments recovered by T.L.C.

This table indicates that the aqueous solutions with an exception of 0.05 M NaHCO3


Table 2. Relation between the solvent system for extracting pigmentsfrom acetone extract and the composition of pigments recovered

by thin layer chromatography

Pigment applied Composition of pigments recovered by T .L.C.Solvent system

Reagent for drying

Chl.a Phaeo. a Others (%) (%) (%)

Chlorophyll a

Chlorophyll a

Chlorophyll a

Chlorophyll a

Chlorophyll a

Chlorophyll a

Chlorophyll a

Chlorophyll a

Phaeophytin a

Phaeophytin a

91.4 73.8

82.0

98.5 98.1

83.1 92.8

90.4

79.0

90.6 91.0

85.5

0.0 0.0

0.0

7.9 23.0

6.8

0.0 0.7

10.4 5.6

5.4

11.3

2.7 1.3

10.5

100 99.5

100

0.8 3.2

11.2

1.5 1.2

6.5 1.6

4.2

9.7

6.3 7.7

4.0

0.0 0.5

0.0

Petroleum ether and dist. water

Petroleum ether and 0.1M NaHCO3


Petroleum ether and 0.05M MgCl2

Petroleum ether and OA M phosphate buffer (pH = 7.0)

Petroleum ether and dirt. water


Petroleum ether and OA M phosphate buffer (pH = 7.0)



Sephadex

Sephadex

Sephadex

Sephadex

Sephadex

Na2SO4

Na2SO4

Na2SO4

Sephadex

Na2SO4

G-25

G-25

G-25

G-25

G 25

G-25

brought some degradation of chlorophyll a. This fact suggests that the solvent system

of petroleum ether and 0.05M NaHCO3 combined with Sephadex G-25 as a drying

reagent is the best for extracting pigments from acetone extract.

(4) Recoveries of *chlorophyll a, phaeophytin a and others by T L. C (i) In order to check the degree of degradation and disappearance of pigments during their developments by T.L.C., the first T.L.C. was conducted for the single pigment of chlorophyll a or phaeophytin a and for the other pigments. The results are summarized in Table 3. This table shows that the recoveries of chlorophyll a, phaeophytin a and

others were over 90%. (ii) The next T.L.C. was conducted for the composite samples of chlorophyll a, phaeophytin a and others. Table 4 shows that the recoveries of total pigments were generally over 90%, those of chlorophyll a and phaeophytin a were usually over 85% and those of other pigments were nearly 100%. These facts suggest that the T.L.C. generally accompanies unavoidable loss of about 10% for

determining chlorophyll a, phaeophytin a and total pigments. (iii) Therefore the foregoing analytical procedure (Procedure No.2 through No.7) (Fig.1) was applied to the composite samples of pigments. Table 5 shows that the recoveries of total

pigments, chlorophyll a, phaeophytin a and other pigments were generally 100% with some exceptions of phaeophytin a which was lower than 0.006µg/ml.

Thin layer chromatography for plant pigments

Table 3. Recovery of chlorophyll a, phaeophytin a,and others by thin layer chromatography

33

Chlorophyll a

Applied* (19)

Recovered (jig)

Recovery (%)

7 4 3 2 1

02 65 12 40 76

6 4 3 2 1

50 55 04 40 62

92.5 98.0 97.5

100 92.3

Phaeophytin a

12.4 6.603.50 0.50

11.06.20 3.20 0.60

89.0 94.0 91.5

120

Others

7 6 4 3 1

69 37 99 35 96

7 6 4 3 1

52 13 91 35 88

98.0 96.3 98.5

100 96.0

* 0 .1m]. of petroleum was applied.

ether solution

Table 4. Recovery of pigments from composite samples by thin layer chromatography

Pigments applied* Pigments recovered Recovery

Expt. No.

Total (lug)

Chl. a (ig)

Phaeo. a (lug)

Others (jig)

Total (jug)

Chl. a (Jug)

Phaeo. a (jig)

Others Total (lig) (%)

Chl a (%)

Phaeo.a (%o)

Others (%)

1

2

3

4

5

6

23.0

18.9

13.2

13.5

11.2

16.6

9.55

2.37

4.74

0.00

4.74

9.55

7.02

10.4

3.50

7.02

0.00

7.02

6.45

6.45

4.97

6.45

6.45

0.00

22.8

17.6

12.9

13.2

10.9

15.3

9.65

2.13

4.08

0.00

4.12

9.47

5.89

8.65

3.43

6.13

0.00

5.80

7.25

6.85

5.39

7.10

6.77

0.00

99.2

93.2

97.8

97.8

97.3

92.2

101

90.0

86.2

100

88.0

99.4

83.8

83.3

98.0

87.3

100

82.6

112

106

108

110

105

100

* 0 .2 m1 of petroleum ether solution was applied.

Table 5. Recovery of pigments from composite samples by the present procedure

Pigments applied* Pigments recovered RecoveryExpt.

No.Total

(µg/ml)Chl. a

(Jig/ml)Phaeo.a (µg/ml)

Others (llg/ml)

Total (µg/ml)

Chl, a (jig/ml)

Phaeo. a Others (µg/ml) (µg/ml)

Total (%)

Chl. a (%)

Phaeo. a Others (%) (%)

1

2

3

4

5

0.830

0.738

0.748

0.676

0.932

0.684

0.598

0.528

0.444

0.598

0.006

0.004

0.006

0.010

0.180

0.140

0.136

0.214

0.222

0.154

0.828

0.731

0.756

0.686

0.926

0.686

0.596

0.530

0.462

0.574

0.012

0.005

0.012

0.010

0.180

0,130

0.130

0.214

0.214

0.172

99

99

101

101

100

100

99

100

104

97

200

125

200

100

100

93

96

100

96

111

* 20 m1 of 90% acetone solution was applied.


(5) Comparison of results obtained by the present method with those by the conventional methods Table 6 shows the analytical results for pigment contents of cultured algal cell by the present and conventional methods. Between these results, there is no appreciable difference. However, it may be noticeable that pigments other

than chlorophyll a, which have been expressed as phaeo-pigments by the conventional and indirect methods, can be expressed as phaeophytin a and others which consist of 3 kinds of carotenoids and 4 kinds of chlorophyll derivatives. All of these findings suggest that the present analytical method is useful for the

direct determination of some individual pigments in marine particulate matters.

Table 6. Comparison of results obtained by the present method with those by the conventional methods for cultured

samples (phaeodactylum tricornutum)

Present method (by T.L.C.) Fluorimetric method* Absorption method**

Expt. No.

Total (99/ 1)

Chl. a Phaeo. a Others Total Chl. a Phaeo-pig. (lab/1) (lag/1) (lag/1) (lag/1) (Jag/1) (µg/1)

Chl. a (lag/ 1)

1

2

3

172

238

204

160

212

176

2.0

3.0

5.0

10

23

23

172

238

204

152

203

174

20

35

30

152

232

* Modified method of HOLM-HANSEN et al. (1965) ** Method by PARSONS and STRICKLAND (1963)

PLANT PIGMENTS OBSERVED IN MARINE PARTICULATE MATTER AND THEIR ECOLOGICAL SIGNIFICANCE

Plant pigments in the particulate matter from surface through 1,200m in depth

in Suruga Bay were determined according to the present method. In this case, the

concentrations of isolated pigments were determined by measuring their absorbancies. The results are shown in Table 7. This table indicates the following facts: (1) Vertical

Table 7. Vertical distribution of plant pigments in particulates collected from Suruga Bay (34° 50'N,

138'39'E) (March 14-19, 1968)

Depth Total pigment* Chl. a (m) (14g/1) (lag/ 1)

Phaeo. a (99/1)

Others (lag/ 1)

0

20

50

100

300

500

1200

1.26

2.08

1.14

0.65

0.09

0.05

0.06

1.00

1.54

0.86

0.46

tr.**

tr.

tr.

tr.

0.10

0.06

0.03

tr.

tr.

tr.

0.26

0.54

0.23

0.16

tr.

tr.

tr.

* Pigments were separated by T .L.C. and de termined by measuring their absorbancies.

** tr .; below 0.01 pg/l


distribution of the total pigments has a peak at 20m and the amount of the pigments remarkably decreases with depth. (2) Vertical distributions of chlorophyll a , phaeophytin a and others are quite similar to that of total pigments. (3) Phaeophytin a which has been suggested as the main degradation products of chlorophyll a , takes only minor part of the degradation products in all samples examined . Since the analysis was carried out after a week storage , it is probable that greater part of the determined phaeophytin a was formed during the storage. (4) Chlorophyll a takes the major part of the total pigments in all samples examined. Table 8 shows the distribution of plant pigments in particulate matter collected

from 100 Q of sea water samples from Station No. 21 in the central Pacific on the

Table 8. Vertical distribution of plant pigments from the central Pacific (KH-68-4, St. 21; 6°09'S,

in particulates collected 170°05'W, Dec. 7, 1968)

Depth 1 2+3

(m) (1Ig/m3) (Ug/m3)

Fraction numbers of separated pigments*

4 5 6 7(lg/m3) (11g/m3) (11g/M3) (ug/m3)

8 9 (4g/M3) (leg/m3)

Total (1g/m3)

0

30

50

100

150

200

500

1,000

0.8

1.0

0.4

1.0

5.0

2.4

0.7

0.6

tr. * *

3.7

tr.

tr.

7.9

tr.

tr.

tr.

undet. * * *

undet.

undet.

undet.

undet.

undet.

undet.

undet.

137

94

171

207

131

153

8.9

5.1

9.0

11

10

11

9.2

0.7

0.8

2.3

0.9 1.9

7.9 8.2

4.2 5.0

8.0 13

5.8 2.9

6.2 4.5-2 .5-0.9

1.6

3.7

1.4

2.0

1.6

1.0

1.1

tr.

151

129

192

242

163

168

14

8.9

* Pigments were separated on board by T.L.C. and determined by measuring their

fluorescent intensities.** tr .; trace, below 0.01µg/m3. *** undet.; undetected.

Southern Cross Cruise of the R.V. Hakuho Maru, Cruise KH-68-4. The determination

of pigments was carried out within a day or two after sampling, in the laboratory of

the research vessel. Concentrations of the isolated pigments were determined by using a Hitachi Fluorescence Spectrophotometer (Model 203).

Relationship between the fraction number and pigments is as follows: Fr. No. I :

0-carotene; Fr. Nos.2 and 3 : carotenoids; Fr. No.4 : phaeophytin a; Fr. No.5 : chloro

phyll a; Fr. Nos. 6, 7, 8, and 9 : chlorophyll derivatives as mentioned before. The

amounts of the carotenoids might have been underestimated by the present method.

The observed results indicate that the vertical distribution of chlorophyll a

(Fr. No.5) has a peak at 100m in depth and is quite similar to that of total pigments indicating that chlorophyll a is a major component in the total pigments. Fraction No. I has two peaks at 30 and 100m depths. The chlorophyll maximum in the oceans

was discussed by SAIJO (1969a), HANDA and YANAGI (1970) and others. In the results

of Table 8, it may be noteworthy that the absence of phaeophytin a (Fr. No.4) was

made clear and that the percentage of chlorophyll a in total pigments was more than

about 60% in all samples examined, even at 1,000m in depth. Recently SAIJO (1969b)


reported that the percentage of chlorophyll a was generally lower than 10% of the

total pigments in particulate matter from sea water below 600 m through 4,000m depths.

LORENZEN (1967) reported that the primary source of phaeo-pigments in the ocean is the result of zooplankton grazing. CURRIE (1962) reported that the phaeo

pigments produced by zooplankton feeding on phytoplankton probably do not contain phaeophytin but phaeophorbide. These reports suggest that the phaeo-pigments in the ocean are chlorophyll derivatives other than phaeophytin. It may be considered that our findings confirmed these suggestions through the absence of phaeophytin a in the

planktonic pigments from the central Pacific. LORENZEN (1967) studied the effect of bacteria on phytoplankton chlorophyll in the dark and reported that the loss of chloro

phyll was not accompanied by an increase in phaeo-pigments. It may be considered that this result was confirmed by findings in the ocean that chlorophyll a remarkably decreased with depth in water layers deeper than 100m or 150m; nevertheless it occupies a major part of the total pigments, about 60% even at a depth of 1,000m.

ACKNOWLEGEMENTS

This research was supported in part by the Scientific Research grant-in-aid of the Ministry of Education and in part by that of the International Biological Programme in Japan (JIBP). The authors wish to express their thanks to the research staffs and crews of the R.V. Hakuho

Maru and of the Tansei-Maru, Ocean Research Institute, University of Tokyo for their cooperation and assistance.

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