Plain white, 47 -mm., Type HA Millipore 

 filters (Millipore Filter Corp., Bedford, Mass.) 

 were used for the filtration of chlorophyll 

 samples. Filtration was generally carried out 

 in the laboratory or some other shady, pro- 

 tected place. 



The vacuum at the pump was not monitored 

 routinely. On TEMPO cruise, when a vacuum 

 gauge was placed on the vacuum pump, the 

 negative pressure generally ranged from 18to 

 Z5 lbs/in.^ These values are probably typical 

 of the negative pressures, since the same 

 vacuum pumps have been used throughout the 

 investigation. 



Pigment Extraction and Determination of 

 Chlorophyll a Concentration 



Following filtration, the filter edges were 

 trimmed, and the filters were placed in per- 

 forated numbered plastic petri dishes in a 

 darkened vacuum desiccator with activated 

 silica gel for at least 24 hours. The dried 

 material on the filter was then extracted in 

 glass -stoppered centrifuge tubes with 3 ml. of 

 90-percent glass -redistilled acetone for 18 to 

 24 hours in the dark and cold (ca, 8° C). The 

 extract was centrifuged for 20 to 30 minutes 

 at about 3,000 r.p.m.; the supernatant was 

 decanted into a glass -stoppered 10-ml. gradu- 

 ated cylinder and placed in the dark. The 

 residue remaining in the centrifuge tube was 

 shaken up in an additional 2 ml. of 90-percent 

 acetone and recentrifuged for 20 to 30 minutes. 

 This supernatant was combined with that pre- 

 viously obtained; if no turbidity was visible 

 in the combined extract, it was diluted with 

 90-percent acetone to 5.5 or 6 ml. If turbidity 

 was apparent, the extract was recentrifuged 

 until clear. 



The spectrophotometric determinations were 

 carried out, by the Richards with Thompson 

 (1952) technique. Semimicro absorption cells 

 of 10 cm. with a volume of about 5.5 ml. were 

 substituted for the 1-cm. cells used by Rich- 

 ards with Thompson. Percentage transmission 

 (against a 90-percent acetone blank), con- 

 verted to extinction, was measured at 750, 665, 

 645, and 630 m^ with a modified Model DU 

 Beckman Spectrophotometer (Holmes and Linn, 

 1961), at a slit width of 0.04 mm. The ex- 

 tinction at 750 mix was subtracted from that 

 observed at the other wavelengths, and the 

 calculations of chlorophyll a concentration 

 (milligrams per cubic meter) were carried 

 out by the Richards with Thompson formula: 



(15.6E,,5 - 2.0E,^5 - 0.8E^3„) x 



vol. extract (ml.) 



Wavelength and Blank Corrections 



The wavelength calibration on the spectro- 

 photometer was checked periodically on each 

 expedition or cruise by using the chlorophyll a 

 peak (Richards with Thompson, 1952) as the 

 reference. Occasionally the peak read 2.5 m^ 

 high or low. The wavelength dial reading was 

 shifted accordingly for all wavelengths. 



Regardless of the apparent visual clarity of 

 the acetone extracts, there was always some 

 residual extinction at 750 m^ which could not 

 be attributed to chlorophyll a, b, or c absorp- 

 tion. In more than 95 percent of the chlorophyll 

 determinations made, this extinction value 

 fell between 0.03 and 0.08 (about 82-93 percent 

 transmission) for the 10-cm. path length. No 

 apparent relation existed between this value 

 and chlorophyll a, concentration, and the ex- 

 tinctions were highest with chlorophyll a con- 

 centrations of less than 0.03 mg. per cubic 

 meter. This "blank" value was subtracted from 

 the extinction at the above-mentioned wave- 

 lengths under the assumption that the value 

 remains essentially constant over the wave- 

 length range (i.e., 750 - 630 m^) used in the 

 determinations. This turbidity presumably re- 

 sults from one or more of the following: (1) an 

 insoluble fraction of the filter; (2) the mag- 

 nesium carbonate; (3) sea salt retained in the 

 filter; (4) material contained in the sample 

 itself. 



The possible effects of (1) and (2) above 

 were examined as follows: a prefiltered sea- 

 water sample of 400 ml. was divided into 

 four equal aliquots: three containing mag- 

 nesium carbonate (Y-1, Y-2, Y-3) and one 

 without (Y-4). These samples were filtered 

 through an HA Millipore filter, the filters were 

 dried, the samples extracted in 90-percent 

 acetone, and transmission of the extract meas - 

 ured. The extinction data are presented in 

 table 11. The fact that the extinctions were 

 generally lower than those obtained at sea 

 indicates that some of the absorption at 750 m^ 

 must be due to material contained in the sea 

 water itself. Although the extinction in these 



Table 11. — Optical extinction values per 10 cm. of 90 



percent acetone extracts of HA Millipore filters, 



with and without magnesium carbonate, 1958 



[Filters pretreated by passing through 100 ml. of 



fresh prefiltered sea water] 



Sample Magnesium 



No. carbonate y^Q 



mil 



Wavelength 



665 

 m/j 



6A5 

 m/j 



630 

 m// 



vol. sea-water (1.) x absorption 

 cell path length (cm.) 



Y-1 

 Y-2 

 Y-3 

 Y-A 



Present 0.0300 0.0315 0.03';0 0.0335 



Present .0285 .0350 .0375 .0-415 



Present .0170 .0205 .0225 .0245 



Absent .0210 .0300 .0360 .0395 



