ee ——_f.Plant Plsiol. Val, 146 pp 307-313 1994) = | <. od The Spectral Determination of Chlorophylls a and b, as well cl as Total Carotenoids, Using Various Solvents with i ; af Spectrophotometers of Different Resolution* of A Atan R. Wettaurn Divison of Biological Sciences, nsiute of Environmestal and Biological Sciences, Lancaster University, Lancaster LAL se 4YQ,UK. e ™ Received February 1, 1994 » Accepted March 8, 1994 of the of “i Summary ca Specific absorption (a) coefficients for individual carotenoids and chlorophylls and b, as well a the Eld, values for combined carotenoids, have been (ce)estimated using 6 solvents (80% acetone, chloro- on form, diethyl ether, dimethyl formamide, dimethyl sulphoxide, and methanol) using 2 different types of a spectrophotometer (0.1~0.5 nim and 1—4nm band pass resolstian). From these values, 2sets of equations a 10 calculate concentrations of chlorophyll (C,), chlorophyll (C,) and total carotenoids (C,..) in ws gm" for the different instrument types were freshly drived or confirased from earlier publicsions. a These were then tested with 3 different types of spectrophotometers (the two variable types plus 2nm fixed resolution diode array) vsing equal aliquots of a mixed extract in the 6 different solvents. These a showed thatthe concentrations and ratios derived by the 2set of equations were comparsble when used ing with their own type of spectrophotometer but less so if the inappropriate equations were used. Measure- ments taken with the diode aray spectrophotometer, however, did not give accurate concentrations or he ratios of chlorophylls and carotenoids. oe Key words: 80% Acetone, Chloroform, Dietbylether, Dimethylformamide, Dimethyl-sulphoxide, Meth la anol, Xanthophylls. es Introduction by Stokes (1864) had already suggested that green plants Sy probably contained more than one type of chlorophyll as ood The first practical demonstration of chromatography and well as different yellow pigments. Meanwhile, Sorby (1873) confirmation that there were two forms of chlorophyll was by Mikhail Tswet, at a meeting of Warsaw Society for Nat- ural Science in 1903, when he separated leaf pigments using a stalk column although Day (1897) had previcushy separated hydrocarbons in petroleum using finely-divided clay and had starzed to partition chlorophylls becween immissibe sol- vents such as carbon disulphide or beazene and aqueoss ab cohol and was the first to sce the bluegreen colour of chlorophylla which he called . Well over a hundred years later from some of these events, itis sill frequent practice to measure the amounts of individ- ual chlorophylls (and roral carotenoids) specerophorometric. ally in mixed extracts rather than separate them by HPLC which is both costly in time and materials and often difficult 10 correct for measurements at single wavelengeh aad foe losses during the extract manipulations. The much-quoted equations of Arnon (1949) to determine individual levels of chlorophyll and & in 80% (v/v) acetone in water are still used by many researchers despite the fact that they are inac- curate and that particular solvent mix has many disadvan- tages. Arnon’s equations were originally derived from the specific absorption (oF extinction) coefficients (a) of Mackin- ney (1941) and always underestimate the ratio of chloro: phiyila to b (Chla/2). There are many reasons for this inac curacy including che poor resolution of the spectrophotome- ters of the 1940s but che main problem is the solvent itself, 80% acetone in water. Most importantly, it doesnot extract all the major pigments completely (Lichtenthaler, 1987). Some of the less polar chlorophyll and 8-carotene are al- ‘ways left behind especially in fibrous and other difficult plant tissues. Nevertheless, efforts to improve the 80% ace- fone equations have been made (Vernon, 1960; Ziegler ard Egle, 1965; Licheenthaler and Wellburn, 1983; Inskeep and Bloom, 1985; Lichtenthaler, 1987; Porza, Thompson, and Kriedemana, 1989; Barnes ea, 1992), iacluding 4 graph 10 recorrect old Arnon (1949) ~ derived Chla/b ratios (Porra ecal,, 1989). Even so, the basic problems with the extractant ‘mix remain (ce incomplete extraction and variable evapora- tion of acetone during maceration, centrifugation, filtration, and spectrophotometric reading). Changes in acetone to wa ter ratios are important because the specific absorption cock ficients of chlorophyll and b vary with acetone content (ve. those for 79% are slightly different from those of 80%) but toa lesser extent with pH or mineral content. For example, accurate equations derived with 80% acetone in distiled wa ter (Lichtenthaler, 1987) are only slight different to those for 80% acetone in buffered water (Porra ct al. 1989). “The use of alternative solvents to 80% acetone for spectro- photometric determinations of pigments has heen prompeed for a variety of reasons. Firstly, accurate ax coefficients can only be determined in a reliable reference solution. BY com: vention, this has devolved to diethylether but a beccer case could have been made for methanol. As Lichtenthaler (1987) has shown, a coefficients derived in ultrapure or waterfrce cor water-sicurated diethyhecher are different anc moreover, diethylether evaporates readily during volumetric and spec twophotometrie operations despite the greatest of care Nevertheless, there is general agreement that the ax coeff cients of chlorophyll and b derived by Smith and Benivez (1955) for diethylether (ultra-pure) are accurate and all other ‘coefficients in other solvents should be derived relative to these values in the manner fully described by Porra et al. (1989). Second, each type of plant tissue often suits one type of ex- tractant better than another. Some of these are inefficient (eg. 80% acctone), some are highly efficient but still require maceration or grinding and centrifugation o filtration, aq, tome will allow immersion ofthe inact dase in wary sible solvents whea the pigments will eventually come avy often after shaking. Dimethylformamide and dimethyl sulphoxide are examples of this last type of solvent and anulkancous equatioas for determination of chlorophylls both have gradually been refined (dimethyLforamide ~ Mo. ran and Porath, 1980; Moran, 1982; Inskeep and Bioow 1985; Porra et al. 1989; dimethyt-sulphoxide - Barnes et 3)” 1992) although, with the exception of Porra et al (1989) none of these have used the approved procedure described jy the paragraph above. The others used comparisons to 80% acetone and commercial sourees of chlorophyll and which are sill contaminated and partially degraded rather than freshlyseparated chlorophyll and chlorophyll from an approved procedure. Of the two solvents, dimethy/-forry amide appears to have advantages over dimerhylsuphoxid, for most plant tissues. The latter is slid up to 18°C: and re. crystallizes slowly but has advantages for delicate tissue, such as lichens although care bas 10 be taken wher heating during exeraction (Barnes etal, 192). Sometimes, however, maceration or grinding and fitr tion or centrifugation has to be used for tough plint mate rial (eg, certain conifer needles). In the past, the procedure of Quai, Gallager and Wellbura (1976) using a 1:3 mature (9/4) of methaaol chloroform has been found to remove al trace of greenness from macerated plant tissues and, upon centrifugation, all the pigments pass into the lower chloro form layer which can then be washed and the concentrations of chlorophyll and & determined by spectrophotometry (Wolfenden et al, 1988). The simultaneous equations used were derived by crosscomparison of a coefficienss in chloroform wih those in ultrapure diethylether in the ap proved manner (see above). These equations, like those of Lichtenthaler and Wellbuen (1983) and Lichtenthaler (1987), also have an additional feature in that wich one extra spectro. ‘photometric determination they also permit the determize tion of total carotenoid content (Cy.,) in the same solution as the chlorophylls using ES, valves in a procedure de scribed fully by Lichtenthaler (1987). Finally, a renewed warning about the different types of spectrophotometers which may be used, Some modern mi croprocessar-contralled spectrophocameters have a spectrd resolution of 0.1-0.51nm over the visible spectrum and these ‘were the type of instrument used by Lichtenthaler (1987) and Porra ec a. (1989) to derive their equations. Earlier it struments capable of 1~$.nm resolution were used up co and including the determinations of Lichtenthaler and Weilbura (1983). Flowever, iis often the case that routine laboratory inscruments more similar to the latter type are sill used. Re cently, instrument manufacturers have tended to emphasize speed of scanning over resolution. Often this means the we of diode-array spectrophotometers and for technical reasons the resolution of such machines is still 2am. All ofthis ha implications for spectral measurements of mixed solutions! containing both chlorophyll and b plus total carotenoids! because all spectral peaks are much sharper ar smaller reso ‘ions, This means that, although che peak heights at wav) length (X) maxima of chlorophylisa or b will be similar 02 different types of instrument, because all cx coetfcients a)tack-corrected to those of Smith and Benetiez (1958) in di ethyl ether, the value of chlorophyll under the Nn of hlorophylla and, more significantly, that of chlorophyll tnder the Nowe of chlorophyll & will be higher using a spec- trophotometer with a resolution of 1-4nm than one wich 0.1-0.5nm. In other words, the equations of Lichtenthaler (1987) and Porra et al. (1989) cannot be used to achieve greater accuracy than some of the carler equations (eg. Tichtenthaler and Welburn, 1983) on an instrument which his a resolution of 1=4nm. On the contrary, by using these later equations on such machines, further inaccuracy is pos sibly introduced. If doubt remains, valid equations should be recalculated for each new instrument according to the meth- ods described by Lichtenthaler (1987) and Porra et al. (1989). The same principles apply to the determination of the to- tal carotenoids, the £}2, values remain the same but the spread of the Soret bands of the chlorophylls, especially chlorophyll into the 470-480 nm measurement region in 1-4nm instruments requires use of the total carotenoid equations derived for 1—4nm resolution instruments rather than those calculated from 0.10.5 nm machines This introduction sets the scene for this paper which de scribes the parallel use of three spectrophotometers of the types described (0.1-0.5nm variable, 1~4nm variable and diode-array 2nm fixed resolution) to (re)determine a values for chlorophylla and chlorophylld, as well as appropriate E13, values lor total carotenoids, using a range of 6 solvents From these it was possible to (re)derive appropriate simulta- neous equations for different types of spectrophotometers suding equations to those for dimethyl-formamide and di- methyl sulphoxide for the determination of total carote- noids (Cyd where none existed before, Material and Methods Purification of chlorophylls and carotenoids A bulk extract of total lipids from macerated tree mallow (Lane tora oa L) leaves was made in semidarkness using the methe rol:chloroform (1:3 v/») procedure of Quail «tal. (1976). The washed chloroform layer was then taken to dryness under reduced pressure, a small volume of mrhexane war added and the extract was Stored under Ovfree No at -20°C until required. Immediately be- fore the speetrophotometric measurements, aliquots of this extract, (075ml) were chromatographed as lines on precoated silcs gel 20320cm thinlayer chromatography (TLC) plates (Type 69 with ‘ut indicator, Merck) using 209% (v/s) exhy] acetate in diethylether. The various pigments separated rapidly (< 20min) with this sol vent mix and, once removed frezm the tank, the TLC plate dried ‘quickly. Specific bands were then ploughed up with a small screw: river (3mm blade width) nota sparula, while the TLC plate was flaton the bench, The fragments ofthe silica gel were then collected by Banging the edge of she TLC plate vertically onto glazed paper, before eluting them with acetone through a cone of Whatman No. ‘flee paper while crushing the fragments of gel with the serew- driver blade. This procedure avoids the disadvantage of precoated plates where te feagments are apt to flyin all directions yet retains he great advantage of reproducibility, Equal aliquots of the acetone catracts were then pipetted into 10 or 20mL. volumetric flasks, taken to deyness under a stream of Oyfree Ns, and then made up to volume with the various solvents (Le. 80% acetone in distilled wa ter, dimethyl formamide, and dimethylsulphoxide, Analargrade, I determination of chlorophylls and carotennids 309 Spec BDH/Merck; chloroform, diethylether and methanol, Arise grade) before spectrophoromecry using quartz gla cefs (10mm path length, Sal volume). All these procedures were cari out in, femi-darkness. Spectrophotometry Four different specrophotometers were used in this study ll of which were checked with a holmium fier before use and, where necessary, recalibrate, The high resolution instrument wae a Us kon 941 Plus (Kontron [UK] Ltd) capable of 02am resolution ‘over the range 380- 550am of 0.5m resolution from 550-750 nm jn sean mode, and 0.1m in fixed \ mode. This latter mode was ‘used to pinpoint the \ maxima once a sean had heen done, One of the two low resolution spectrophotometers was the Pye Unicam P8000 that had been used previously by Licheenthaler and Well- born (1983) and the other was a Pye Unicam SP30 wich very similar optics (i. variable 1-4nm resolution from 400-700nm). The diode array spectrophotometer was a Hewlett Packard HIPSS52A ‘with 2 fixed resolution of 2am, Spectra of purified chlorophylls and b, carotene, lutein, vio laxanthin, neoxanthin, and the combined carotenoids were deter ‘mined on the Uvikon 941 Plus and compared to those of chloro- phyllsa and & and combined carotenoids on the Pye Unica 'SP8000 wsng all 6 solvents. Peak maxima were then established (as bore) and the specific absorption cocifcients (a), or che El val ves for the combined carotenoids at ether 470 or 482.2m, were re calelated and compared to those already published (Lichtenthaler nd Wellbura, 1983; Lichtenthaler, 1987; Porra etal, 1989; Barnes tal, 1992), Appropriate simultaneous equations were then calew Iated and then checked repeatedly using 50 L aliquots of the tree mallow lipid exteact made up to 20m in the various solvents using, the Uvikon 941 Plus, the Pye Uaieam SP3O, and the Hewlett Pak heard HPS#32A, Results Al the New of the chlorophylls at high resolution in di cthyiether, methanol and 80% acetone were found to be identical to those already published by Lichtenthaler (1987) but those for chloroform, dimethylormamide and dimet- hybsulphoxide are shown in Table 1. The long wavelength ae ate slightly higher than chose quoted by Barnes etl: (1992) for dimethylsulphoxide but the same as those for di ‘methyLformamnide given by Porra etal. (1989). Table 2 also gives the absorption maxima of the 4 principal carotenoids Table 1: Absorption raxima (\, nm) of the two chlorophylls in chloroform, dimethyformamide, and dimethyl-sulphoxide. Pigment Chloroform Dimethyl Dimethyl formamide sulphoxide Chlorophylla 6656 o68 sis. 6162 5832 5812 4315 15 4155 428 Chlorophyll b 647.6 616.8 5981 6001 460.3 4593 4352 4352 ‘Major peaks in bold eypefce.310 AtawR. Wensauan Table 2: Absorption maxima (\, nem) of the major primary carote noids in chloroform, dimethyLformamide, and dimethylsulph- oxide Carotenoid Chloroform Dimethyl. Dimethyl formamide sulphoxide BGaroene 904 W846 4958 4798 $594 478 Lucia 4840 4846 910 4560 $556 4610 B16 sh sh Violaxanthin 479.8 a4 4846 498 $506 4548 48 4258 4296 Neoxanthin 474.8 a4 818 488 4454 4534 4210 4218 sh \Mbjor peaks in bold, sh ~ shoulder but not a peak. in chloroform, dimethylformamide and dimechytsuiph oxide. Comparison of Table 2 with Table 5 of Lichtenthaler (1987) for diethylether, 80% acetone and methanol reveals thatthe ham of all 4 carotenoids in the first group of solvents (ie. those in Table 2) are shifted by over 10m to longer wavelengths than those of the second group (Table 5 in Lick tenthaler, 1987). ‘The specific absorption coefficients (a) for chlorophylls« and 5 in chloroform, dimethyhformamide and dimethy! sulphoxide a the long wavelength hu of each, 28 well as at 470 and 480 nm, for both high and low resolution spectro- pphotomecers ae shown in Table 3. The equivalent values for diethyl-ther, 80% acetone and methanol were also deter- ‘mined (not shown) but were found to be virtually identical to those for high resolution instruments given by Lichten- thaler (1987, Table 2) or for diethyl- 1247 Au -342dan > 24a 345A wiphonce G1 Boa esac CNR Rs cyan youscy/am aban! G.= 1672A1-¥.bdase C= 1868Au- 73445 Gy = 09 Ano 15.28Auss Chas = (1000 e= 1.63C,~108.960)/221 Table 5: Optieal densities, amounts (ig ml), and ratios of ehloro- piylls and carotenoids in differen solvents sing similar extracts from tree mallow (collected in mid-wincs) in high (in bold) and low resolution spectrophotometers. The vals shown in italics be low each were achieved using the apposite (ie inappropriate) equa Saket ax Ray Ang Gla CH wh Tend Rin roe G8 Dishyhher 9442 Q47S Ge) WAT MSL 257 sot 43 ison Mos 25 hee har These extracts were also interesting in that they showed traces of a third chlorophyll which co-chromatographs on TLC with chlorophyll acc (Wellburn, 1970, 1976). Gy = 27.05Ag— Tha, (000A wo~2 86C,~ 129.2,)/221 Discussion ‘This study, like many others beforehand, demonstrates that accurate equations to determine concentrations of chlo- rophyllsa and , as well as total carotenoids, in mixed ex- tracts can be derived and used with confidence with a ange of spectrophotometers. If, however, a large number of meas- ‘urements of this type are to be made regularly then there is no real substitute for deriving the precise er coefficients for that instrument and working out the individual equations in the manner described by Lichtenthaler (1987) and Porra et al, (1989). The important point is that there must be fixed and agreed starting comparison values. Both Lichtenthaler (1987) and Porra et al. (1989), when using high resolution in- struments, returned to the e coefficients of chlorophyll and d given Smith and Benitez (1958) for diethylether which were derived using a speetrophotometer with a poorer spec- ification. Even then they produced slightly differene equa- tions because the former took a value of 101 for chloro phylla while the atte used 100.9 from the same source, The problem with the a values quoted by Barnes etal (1992) for dimethybsulphoxide and 80% acetone, apart from the fact thit the quoted values for chlorophyll bin 80% acetone are inverted, was that they had no fixed starting point (eg. di cthylether). What they have are two sets of equations for di- methylsulphoxide and 80% acetone that give corresponding concentrations and ratios to each other which match their conditions but, given the other problems of 80% acetone (see introduction}, it may be difficult to reconcile these with values obtained wich other solvents. The coefficients given for chlorophyll and bin dimethyl. formamide given in Table 3 differ slightly from those quoted312 AuawR. Wetaavew by Porra ec al. (1989) and would produce the following equa tions for high resolution instruments: C= 1174 Aas = 265 Asus Gy= 2191 Ais — 453 Ass However, as these are so similar to those of Porra et al (1989), those ofthe later researchers were retained in Table 4, Similarly, the equations for 0% acetone for a low resol tion spectrophotometer given by Lichtenthaler and Well burn (1983) were used. However, due to an error, these do rot match the quoted «coefficients in the same paper. This scudy was able to show that the e coefficients given in Lich- tenthaler and Wellburn (1983) for 80% acetone were incor- rect. They should have read 86.99 at 663nm and 21.71 at 646m for chlorophylla and 12.13 at 663nm and 52.71 at 646nm for chlorophyll. The a values at 470nm for 80% acetone, however, remain unchanged. Spectra of combined and individual carotenoids in chloro- form, dimethyLformamide and dimethyl sulphoxide, show- ing a spectral shift of over 101m to longer wavelengths, also prompted the use of 480nm rather than 470nm as the Cav measuring wavelength for these solvents. This has the dow ble advantage of being further away from the Soret band of chlorophyll and avoids measuring total carotenoids in the trough between the 2 main absorption bands of the carote- noids. This procedure therefore improves on those equa tions for chloroform previously given by Wolfenden et al. (1988). This study has also demonstrated that diode array spectro- photometers, with their fixed 2nm band pass across the spectrum, are not suitable for use with equations that have been derived by spectrophotometers with variable resoha- tion across the spectrum. Measurements at 470-480m, as compared to those from 640-670nm, were generally lower in the diode acray instrument. One of the problems appears to be linked both to differences in dynamic response of the photodiodes compared to photomultiplier across the whole spectrum and that the wider band widths at longer wave- lengths in variable band pass inseruments permit proportion- ately more light to fall on the detecting system. Morcover, it is quite disconcerting to find that such an instrument always, rounds up an input fixed wavelength value before measure- rent. This means, for example, that measurements using di methyl-sulphoxide are made at 666 and 650nm instead of 1665 and 649 nm. I is conceivable that a third set ofc coef cients and simultaneous equations could be derived for use with diode array instruments given that currently over 50% of spectrophotometers now sold are of the fixed 2am band pass type (Kontron (UK) Ltd,, personal communication). However, most laboratories also have at least one of the other type which can be used immediately so the effort may not be worthwhile. In summary, a ringe of relatively robust equations are now available for use with 6 different solvents for both high and low resolution spectrophotometers. A computer pro- gram written in Microsoft Quickbasie™ using these equa- tions, which can be scanned using optical character recogni- tion into any PC as an ASCII file, is provided in the Appen- dix, Acknowledgements ‘This acl is dedicated to Prof. De. Hartmut K. Lichtenthalee con the ossation of his 60th birehday in recognition of his contriva tions to isoprenoid biochemistry and speetrophotometty. am aos tefl co the saff of Kontron (UK. Led, fr the extended loan of the Uvikord 941 Pls Rel Anwox, D. Li Copper enzymes in isolated chloroplasts: Polyphe olde in Ben eularis lane Physiol. 24,115 1985). ‘Bass, J.D, L.Basacbes,E- Marasgue, . Evens and A, W.Ds. ‘son: A reappratl ofthe use of DMSO forthe extraction and determination of chlorophylls and & in lichens and higher plants, Environ, 8 Exp. Bot. 32, 85~100 1992). Das, D. Tu A suggestion as to the origin of Pennsylvania peso eum, Pros. Amer Phil Soe. 35 112-115 (1897). Ist, W. P. and P._R. Boos Estnetion coefficients of chlor pylla and b in N.Nedimethyliormamide and 40% acetone Plan Physiol 7, 483~485 (1985) Licieriaien, H. Ks Chlorophyils and carotenoid: Pigmens of photosynthetic membranes Math. Enaym. 148, 350-382 (1987) Lecmeenuuan, HLK and AR. 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Menunons, and AR, Weuuntan: Use of carotenoid ratios, ethylene emissions and buffer capacities forthe eaely diag. hosi of forest decline, New Phytol. 10%, 85-95 (1988). Appenatx FEM Progam cla catenoeadtoopyi a in fie solves REM «sWoenby Ala R Welburn Mand te fo geass denes by" Het Lain Wn) Le (9, Pa 9" ‘Re sVanbien Ad thepet dta-D the aon"* REM “ERP ue aati of rp nb ea Rata™™ RIM ““G8l ae te anne nl tye & carn ‘G2SUB ie IA Sfecrop mee Hee gs" PRINT 80% Aceoge ook on TPRINT FAINT "Dine stphode 3 oa 11"PRIT PRINT "Mena PRINT IRFU toga. ay mer eae NINO) GoTo Act Coenen, Dimetir Dee Me, ‘eea.Cvowa bets Don Dine ata INDUTBIN AGAIN? CZ AS IPLEFTS (AS3) =p Cow sare RETURN IPLEETS (AS) =e HEN WIDTAROEND INPUT‘OD. 42"™A:PRINTINPUT “0, 64688: GOSUR cas Brana asm "0 Foe ais atsitD sito" G2 dy -050 +9159 * COTO ae TNPOT “OD. G&S 6A: PRINT: INPUT “OD. 6476": GOSUB cas? Brarnwe a?" 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