> DETERMINAGAO ESPECTROFOTOMETRICA DA CLOROFILA AL PRINCEPIO i Qs zooplanctontes de maiores dimensdes so removidos paséando « amostra de agua por uma rede de malha de 300 p. Os fitoplanctontes elo Fetidos por filtracio num filtra de fibra de vidro de onde os pignentos sto extraidas. A sua concentracko é determinada por espectrafotonetria 3B. AKOSTRAGE A anostra de agua (0,5 2 $ 1) @ colhida, filtrada por una rede de 300 @ armazenada num frasco de polietileno. Adicionea-se 2 ou 3 gotas G2 Suspensto da carbonato de maghésio para prevenir a acidificagae das clorofilas e resultante detoatosicao em feofitines. A anostra deve ser arzazenada a baixa temperatura e no escuro por um zximo de B horas. £ sconsalhavel que se proceda & filtragdc no momento da amostragen S8 Proceda 4 filtragia no momento da amos: Os filtres podem ser aruszenedos dobrados com o plancton para o interior a -20" C no escuro e de preferdacia num excicader, ms apenas per poucas semanas (este proced dificultar a extracgz0), Agitar vigorosazente o frasco cantendo a azcstra e filtr: us filtro de fibre de vidro (Vaatnan GF/C, 4.5 cm. 2. Drenar cuidadosazente o filtro antes de o remover do equipamento de filtragem. Armazenar o filtro dobrado cu preferivelsente passar A extrac¢fo, 3. Colocer o filtro num tubs’ de centrifuga cam tampa (vol. = 15 BL) e adiciamar—t SecOra a SUP. Nacerar o filtro com una vareta Ge vidro, tapar ¢ agrtarvigurosanente, Colocer 0 tubo no refrigerador ¢ as escuras durante 20 horas para que se processe a extraccho. : eee eee ce Ee we 4, Renover os tubos_do.cretrigerador e deixar atingir a temperatura axbiéaté...Ceitri Zugatvasd duraate 10 xinutos. 4 “s > a “cuvettet do Slucko contra um branco de espectictitsnstro e midir a absorpdtela da acetonad: 90%;¥a! 750," 665) 645). 630"e7480" ih: @Habscrvancia subtraindo o valor da eves exéeder. 0.02)) aos obtidos a, 665, alorzsozobtido:a.420 om, (A 750 um néo ba aveii;tamben ser feitas as devides clivettes" usadas,ng pigmento / nm? = C/V. le eu que Y ¢ o volume de Agua filtrada (1), le 6 um valor que depende da espessura da “cuvette” (1 para 10 cm, 4 para 2.5 cme 10 para lcm eC é um valor obtide pela equagdo: : © (CHL a) = 11.6 Aece - 1.31 Acas ~ 0.14 heae que 4 @ 0 valor de absorvaneia corrigida correspondente 20 comprinento de onda subscrito. 7 ADENDA: MINAGEO ESPECTROROTOMETRICA DOS FEOPIGMENTOS A absorvéncia de um extracto de pigmentos vegetais 4 medida antes @ depois de tratada com dcido dilufdo. A alteragio verificada ¢ ucada cons sedida da quantidade de feopignentes ta amostra original 1, Medir a abscrvancia do extracto a 750 ¢ 665 az. Adicis gota de acido cloridrico diluido, misturar e voltar a medir a absorvancia acs nesnos comprimentas de onda (esperar 4 a 5 para que se processe a destruicdo da clorofila em feofitina). 2. Corrigir o valer 1ido a 665 am subtraindo a valor lido a 750 me calcular a concentragdo em clorofila a e em feopignentos usando as ag3es CAL a (mg/m) = C 26.7 (6650-665n) «ev / Tx 1 PHAEO (mg/m?) = ¢ 26.7 (1.7x665.-665.) x v1 / Vx 1 am que 6655 @ a absorvancia do extracto antes da acidificacdo, 665. 6 a abscrvancta do extracto acidificado;zivié “oi volume de acetona usado na extracgio (al), V é 0 volunerde ay itrada,€) © 1 6a espessura da “cuvetto" utilizada (em, a8o APERDICE REAGEETES UTILIZADOS: 1, ACRTOWA A 90%: Pipete 100 ml de agua destilada para um baléo volunétrica @ perfaga 1 1 com acetona pura. A solugdo pode ser arnazonada nua frasco de plastico que deve ser mantido sempre quase che:o, 2, SSUSPENSAO DE CARBOMATO DE MAGHESIO: Adicione 1 g de carbonato Ge magnécio finauente pulverizado a 100 nl de agua destilada. A suspeneso deve ser agitada vigorosanente antes de ser utilizada. 3. ACIDO CLORIDRICO DILUEDO: Dilua 50 ml de Sido cloridrico soncentrado em Agua destilada suficiente para parfazer 100 el de solugde, LIZADO Garrafa da colheita de agua de 900 p de malha esces de plastico itros Vaatman GF/C 4.5 cm pamento de filtragem incluindo tomba manual ou eléctrica bcs de ce ga com tampa tri fuga, ‘ofotémetro "Cuvattes" com 1 cm de espessura Pizets autozstica a! be spread out & atmospheric sense of the from the air al weighings, the “blank” <3) are taken. ed in 3, treated in ing, ‘ike the 2 same box +100 mg), oe 2 very variable; de (The factor for converting’ chloropty about 25 and 100.) The method described below determines the thrée’chlorophytls ‘commonly found in planktonic algae, chlorophylls a, b, and c, The carotenoid pig~ nents. (the: carotenes and xanthophylls)vareonly estimated collectively in somewhat arbitrary units. If the plant population céatains rriany myxophycede some forms of ‘phycobilin pigments may extract and interfere With all determinations except that of chlorophyll 4."Fortunately this occurs only tately i truly marine waters. “The following techniques taken largely front the method described by Richards (Richards with Thompson, J. Marine Res:, 11: 156, 1952) with later modifications (Creitz and Richards, J. Marine Res., 14: 211, 1955) and a few minor changes by the present. authors. The “specific plant unit” (SPU) defined by Richards has now attained almost international acceptance. This unit, used for chlorophyll ¢ and the carotenoids, approximates to 1 g of dry pigment. The Richards equations, however, are capable of improvement in the light of more recent research, ‘As well as the original equations we include modified versions given by Parsons and Strickland (J. Marine Res., 21: 155, 1963), and the scor/uNEsco Working Group én photo- synthetic pigments (Monographs on oceanographic methodology, Publ. Unesco, 1966). These lead to somewhat lower values for chlorophyll'a and’express the con- centration of chlorophyll c in terms of milligrams of pigment rather than “speciid: - plant units.” It must be stressed that these and other equations are still liable to change according to which specific extinction values in the literature are considered most authoritative and whether or not values for “dried” or “undried” chlorophyll are used.(refer to Parsons and Stricklind, J. Marine Res., 21: 153, 1963). Deter- minations of chlorophyll ¢ by a trichromatic method will never be satisfactory when dealing with a low standing crop of phytoplankton, and a Separate routine method for this pigment is desirable.(see Sect. IV.3.1II): The equations given by Partons and. Strickland also expréss ¢aroterioid pigments;in arbitrary units but factors are” chosen according to -whéthier-the plant Population is predominantly composed of + members of, the’ Chlorophyta and/or: Cy: o-"Werhaye not included ‘any ealctl Suggested initially: by Richards::In-our. expetiéate>o 186 A. PRACTICAL HANDBOOK OF SEAWATER ANALYSIS ‘am extaction with 90% acetone under the conditions described in the rhethod which Follows has been considered satisfactory by most workers for many Yeu We wncke his stil to be the case for most seawater samples, having regard to the ecuracy considered adequate for most investigations of marine ecology. However, asults are undoubtedly low in many instances because of the presets of plant cells Teatro not fully extracted, With certain species 30% or more of the pigments may eect behind in the cell A change of solvent may be beneficial but wil rarely ensure Complete extraction so itis probably not worth the trouble t0 re-establish extinction complete efor other solvents or solvent mixtures. The use of 2 sonic disinteat=iot Foo ieen recommended but we have not found suficient improvement 12 MN the pplication of such equipment on a routine basis. The use of @ tissue grinder, such sere cacommended by Yentsch and Menzel (Deep-Sea Res. 10+ 23), 1963), is Si aiely convenient and improves results on many narural populations but even this approach fails to give complete extraction in 2 reasonable Cts ‘with certain Species Fortunately bad cases are generally found only among the Chlorophyta and sea pentnic species, and reasonable results will be obtained with open-s68 samples rar ch of the time, even without grinding. For work in lakes or shallow estuasicS the adequacy of extraction must be carefully checked ey lg of the method described below is adequate except where sie volumes are restricted of where the chlorophyll conteat of the water is below about’ D2 mg/m!, The precision decreases appreciably with concentrations below this Teveleecoming very poor if Tess than 0.1 mg/m is present In these cireymsitoce® ie cco mreciewznination is recommended. Such a method requires 2 sensitive dacimmater and stitable technique is described in Sections TV.3.1V and V. ‘Healy mention should be made of chlorophyll degradation products The presence of chlorophyllide will go undetected and this pigment will be reported as ae equivalent weight of chlorophyll. If phacophytin or phacophotbide are Present Samples the extinction of 6650 A (see later) will decrease ahd these pismo will go undetected and will be reported as if about half the amount of chlorophyll were present. Some.jdea of the amaunts of phaeo-pi fi a sample may be obtained by.meesuring extinctions Cor flu cation of extracts. Chlorophyll degradation products determined by chromatograp! i routine application, The presence large amousts aj be found if bottom deposits ar ag by. Jankton, ox if saraples arc taken f ‘order of 0.02 except chloro 1, ente The cor 2. ene The co i 3. Phe ‘Theo - They -where bets accurate, | B OuTH ‘The a nylon 0 ‘a Millipo cestimatio ©, SPEC2 order of ot chlorophyll ‘Mean at n determinations 0.26/n! »g chloropbyl a. Ee 43: cattonorivLt, b PRECISION -AT_HE.0;5 46 ever’! ‘The correct value lies inthe range? - Ste sed ‘Meas of n detéimissiioss *0.21/n! rg chlorophyll b. nas net a. puawr canorexotos PREC AY THE 5 (PU LEVEE Sere ‘The correct value ies in the range: certain Mean of m determinations #0.15/n' r-sPU. “torophyte and ‘The precision of chorophy/l ¢ determinations is variable and very pooh, any, “yhere betuecn #10 and 230% of the amount being measured, and results are not ‘enesez samples allow estuati See “accurate, almost always being too high. where san e sample B, Outiine oF METHOD ae "Ths nego Zooplankters ae removed by straining a sample of sea water throush eee a aylon net of about 300-p mesh size and then the phytoplankters are Sered O90 aayestrtiad 4 aoa oe AA iter of # glass Alter. Pigments are extracted from the algae cells for indy. estimation spectrophotometrically. ects (C. SpectaL APPARATUS AND EQUIPMENT aisieeat ‘vilipore siltation equipment designed to hold 47-mm diam membrane Bters. Sean el ‘One 300-mi polyethylene wash bottle. © orang oe Stoppered graduated centtifuge tubes of 15-mi capacity having both glass anc ete ee polyethylene stoppess. fee cal “Small volume” spectrophotometer cells having 2 path Jength of 10 cm but e acid holding 10 ml or less of solution. D, SAMPLING PROCEDURE AND SAMPLE STORAGE “Adequate sampling of the euphotic zone or detrital layers for phytoplankton is de the scope.of the present method. Once obtained, the inal sample (generally 500 ml-S liters in volume) jg filtered through a small piece of clean 0.3-mm mesh nylon netting to remove ‘the larger zooplankton. For open ‘sea samples filtration of small volumes through 2 0.15-mm mesh net will stillyaot T amounts of phytoplankton. The required volume of this Strate ‘Should be measured by a polyethylene measuring cylinder into a polyethylene bottle, Two or three deops (ce. 0.1-0.2 ml) of magnesium carbonate suspension (see Sect. ochply stated. .2) are added. The sample may then be stored in a cool dark place for a maximum ‘a practice, he | of about 8 hr. It is desirable, however, that samples be filtered through a membrane rection for the i filter at the time of collection... : 7 5 to be of the _ Membrane filters ean be stofed by f6iding themin half (with the plankton ‘has been very wv the euphotic Hon of phaeo-oO 188 ‘A PRACTICAL HANDBOOK OF SEAWATER ANALYSIS, innermost) and storing them in the dark in a desiccator frozen to ~20 C but only for a few weeks. This procedure almost always leads to low results and makes the extraction of chictophiyll more difficult; filters should be extracted without delay if at alt possible, E, Srsctat Reacents 1, SPECIAL REAGENTS Distill reagent grade acetone over about 1% of its weight of both anhydrous sodium carbonate and anhydrous sodium sulphite. Collect the fraction boiling at a constant temperature near 56.5 C (uncorrected). 100 ml of water is pipettéd into a liter volumettie flask and acetone added to make the volume to exactly 1000 mi. The redistilled acetone should be stored in a tightly stoppered dark glass bottle and the 90% reagent prepared in moderately small amounts (say 1 liter at a time) for tse. This reagent is conveniently dispensed from a polyethylene wash bottle which should be kept nearly full. If good quality reagent acetone is available, it should be shaken with a litle granular anhydrous sodium carbonate and decanted directly for 2. MAGNESIUM CARBONATE SUSPENSION Add approximately 1 g of finely powdered magnesium carbonate (light weight or “Levis” grade) of analytical reagent quality to 100 ml of distilled’ water in a stoppered Erlenmeyer flask. Shake vigorously to suspend the powder immediately before use. F. EXPERIMENTAL 1. Invert the polyethylene bottle containing the sample (Sez! D) into the fonnel of the Millipore filter equipment fitted with either a 47-mm diaii Millipore AA filter or a 4.5-cm Whatman GF/C glass filter paper (Note a)Tuié tile need not be rinsed but the contents should be shaken vigorously, before flfration. is com-. If not added previously, introduce’ about. 1 ml of ‘tiagnesium carbonate suspetision to the last few hundred milliliters of Sample. being, filteréd:(Note b). Sictidh before removing, it! from, the pligral excess, dissolve the filter by shaking the tube vigorously approximately 10 mi of 90% acetone, stopper: thet grate the paper by shaking.the tube vigorously. extracted by placing the tube in a refrigerate (Notes: é-andi) Tes: 210 ‘shia Tae Centrifuge stoppers on -contrifupatic 5. Dec meter cell d exceeding a cor L-cm cel malize then with glass values expe 6 Wi taining 905 the Richar ‘measureme the measut extinction for extinct the proced LG where Ci sea water for chloro specified ‘Richards 1 Richards gram, Th mainly fw res scon/UNt subseripts Section G R oC Ps, C SU. C0 C bit'onty and makes the” poth anhydrous ‘on boiling at a petted into a ‘actiy 1000 mi. Hass bottle and at a time) for sh bottle which 2 immediately =D) into the 2 Millipore AA sation is com- um carbonate (Note 8). al excess accessary fa Millipore se tube, and used add oe Bhd disinte- ents to be ‘or about 20 hr once more after 1 in the dark from Millipore O ml (Note g). SE. ceatrtuge the content of th Bes ‘on’ the centrifuge’ tut liquid into @ 10-cm-path-length spectrophoto- metic cel designed to hold 10 ml or less of liquid. In the event of extinction valves mele" jing about 1.3 the measucements described below should be made with 2.5610 career eels and the extinetion values multiplied by 4 oc 10, respettively, to nb oF soe them to the values expected with a 10-em cell. If 12 mil of acctone.ts used [jyith elase papers multiply. the extinction values by 4.2 to no rmalize them to the phylls & and c “yas units, Le, Owe ploued a Line Qs intercept on omaletaly, give ao ‘using Millipore lt. These filters blank. They are Du although ear fers and wouble esp commercial 9 ensure that the Millipore fiers arv3dM, Phytoplenkson sive phacophytin al with, say, the fare sched doy ated pecpteral Pash this to the ‘Menael we have 4 stirring motor. 1 subdued: light, soul be plies up Sf. doi the pete daring the exraciin, bit for nue ‘pestle should’ be, hard against-the,bottony of the tube, After. ues, the, Best Sate with a fe slit of 90% eto phic also used to transfer the eoatet ie der tabs t0'a 15 Gentifuge tobe=The ft volume in the eenrifuge fabs thould not ‘exceed 10 mi. The contents should be left in the dark for a few hours to ensure the complete ‘SShtovel ofall exrctable pigments. “c) Duiog the extrction period pigments are very photosensitive and nether énracts nor she Uicatreted filters should be exposed to strong sunlight or else chlorophy values will be ‘reduced to a small fraction of their initial level in less than 1 hr. Tubes must be stored in com- plete dafiness. : (f), The period of extraction should be about 15-20 hr. After this tims the rate of further éxiatton is too slow for an extension to be merited. Pigment extracts should preferably be kept hited but they can be kept at room temperatre for many hours without deterioration. It cell Ste pretreated tna griacee (Note €) any further extraction i slow, but for safety, tubes should ie sored for afew hours to complete the lexching of cell fragments (G) Tue ute of 10 ml of soluton in a 1O-r-patriength cells recommended for maximom castvity, Greater sensitivity ean be obtained by sing 10m calls containing less than 10 mi Sue this is scarcely great enough to marant the increased manipulative difcltes. The sltimate Sensitivity ta practice, more dependent onthe size and reprouvetbility of blanks. Glas filters SFeitegrate to pulp, instead of dissolving in sestone and the pup retong at least | ra of solvent. Te UeRie enougt ext to fl « Ioem cel therefore, Fm of atone, instead Of 10, should be uerd. (ie) Centtifusstion should be as efficent as potsible when Milipoe fillers are weed. In ‘most small centages 30001000 rpm for about 10 minis generally saefactory but the ee ney shouldbe tested with each instrument used. Diftcutes may be encountered when cent fosiag down the glass pulp from glas filters, Tubes should be centrifuged for 1-2 min to pack test of tae bers to Une bottom, The centrifuge is then stopped, the tubes removed, and sase fers adhering tothe walls of te tubes above the level ofthe solvent are taken dowa inf the Bulk ot the ligid by gently splashing the walls by Riking the tubes. The tubes ae thea returned {othe centrifuge and spun for about tin. If this preaution isnot aken some Aber he above the solvent layer may enter the specttophatometer eel {id ‘These extracts should nat be allowed to evaporate and should be exposed only to sub- ved light for the briefest possible period. The measurement of extinction against actone {instead of agninst water) is recommended as acetone has markedly less absorption in a {0m all st 7500 A than hae itil water. (The wavelength seting of the spectrophotometer used should be checked agsinst a standard hydrogen oF neo line source asthe precision ofthe present method depends Upon set tings being correct to better than 20-30 A. With quartz prisms ai wavelengths exceeding 6000 A ‘ery slight movements of the optieal sytem, Brought about by vibration, etc, can easly result in errors of 50 A or more in wavelength settings. If a suitable lamp is not available check the txtneton ofa suitably concentrated plant extract and aust the spectrophotometer, f neeexry, ‘until a maximum extinction is obtained at 6630 A. G, DETERMINATION OF BLANK 1. CELL-TO-CELL BLANKS As the precise values of comparatively small extinctions have to be measured, corrections for all optical inequalities become important. Fill both spectrophotometer cells with 90% acetone and find the “cell-cell” blank of the sample cell against erence cella all Wavelengths used in the method. Correct all extinction values j atiount to 0.01 or more.192 ‘A PRACTICAL HANDBOOK OF SEAWATER ANALYSIS measured by the spectrophotometer reading at 7500 where there is known to be xno absorption of light from pigments. We have sometimes found a small negative blank for easons which are not clear. In any case the value positive or negative should not exceed about 0.002 and may be corrected for cell-to-cell blank and used for the extinctions at all wavelengths. ‘A certain amount of colloidal material remains after the solution of an AA ‘Millipore filter, even after centrifugation. The extinction from this material depends on the wavelength of light used, increasing at shorter wavelengths because of light seattering effects. The extinction at 7500 A is corrected for any cell-to-cell blank at this wave~ length and the resulting extinction (E,) is multiplied by a factor f to give the turbidity blank extinction to be used with spectrophotometer readings at other wavelengths. ‘Total blank correction = cell-to-cell blank + (j X Ey) where f has the values shown below: Wavelengh 6650 6850 6300 5100 4800 Ik must be stressed that these values for f are very approximate. Extinction values at 4800 A should undoubtedly be corrected by a greater blank than the one obtained at 7500 A but the value of 3 is so approximate that there is no substitute for having low E, values. If a good correction is required E, must not exceed about 0.02. Chic of the to phyll int they absc ‘from the grazing ¢ these. me ments) t following phyll ca phaeoph all chlor chromat of a lars ficient t the quar for this Oceana te the second + A. Car