Absorption of light within the aquatic medium
3.2 The measurement of light absorption
We saw inChapter 1 how the light absorption properties of the aquatic medium are characterized in terms of the absorption coefficient,a. We saw also that althougha is defined in terms of the behaviour of light passing through an infinitesimally thin layer of medium, its value can nevertheless be obtained from the observed absorptance,A, of a layer of medium of finite thickness, provided that the effects of scattering can be overcome.
Instruments specifically designed to measure the absorption coefficients of surface water bodies, marine or inland, are referred to asabsorption meters.
Laboratory methods
Commercially available equipment (spectrophotometers) used for the measurement of light absorption, do so in terms of an optical parameter known as theabsorbanceoroptical density:although there is no standard 3.2 The measurement of light absorption 53
symbol we shall indicate it byD. Although absorbance is one of the most frequently measured parameters in biochemistry and chemistry it does not, unfortunately, appear to have a rigorous definition. It is commonly defined as the logarithm to the base 10 of the ratio of the light intensity, I0, incident on a physical system to the light intensity, I, transmitted by the system
D¼log10I0
I ð3:1Þ
The meaning of the word ‘intensity’ is not usually given: we shall equate it to radiant flux,F. The incident beam is generally assumed to be parallel.
The exact meaning of ‘transmitted’ is also not stated. If we make the simplest assumption that any light that re-emerges from the system, in any direction, is ‘transmitted’, thenIincludes all scattered light as well as that neither absorbed nor scattered. Thus, Iis equal to the incident flux minus the absorbed (F0– Fa), from which, given thatFa/F0is absorp-tance,A, it follows that
D¼log10 1
ð1AÞ¼ log10ð1AÞ ð3:2Þ If we can arrange that all the scattered light is included in the measured value ofI, then scattering may be ignored (except in so far as it increases the pathlength of the light within the system), and so eqn 1.36 may be considered to apply. Therefore,
D¼0:4343ar ð3:3Þ
i.e. the absorbance is equal to the product of the absorption coefficient of the medium and the pathlength (r) through the system, multiplied by the factor for converting baseeto base 10 logarithms. The absorption coeffi-cient may be obtained from the absorbance value using
a¼2:303D=r ð3:4Þ
In a typical spectrophotometer, monochromatic light beams are passed through two transparent glass or quartz cells of known pathlengths, one (the sample cell) containing a solution or suspension of the pigmented material, the other (the blank cell) containing pure solvent or suspending medium. The intensity of the light beam after passing through each cell is measured with an appropriate photoelectric device such as a photomulti-plier. The intensities of the beams that have passed through the blank cell
and the sample cell are taken to be proportional toI0andI, respectively, and so the logarithm of the ratio of these intensities is displayed as the absorbance of the sample.
When used in their normal mode (Fig. 3.2a), however, spectrophotom-eters have light sensors with a collection angle of only a few degrees. Thus any light scattered by the sample outside this collection angle will not be detected. Such spectrophotometers do not, therefore, measure true absorbance: indeed if their collection angle is very small, in effect they measure attenuance (absorption plus scattering, see }1.4). If, as is often the case, the sample measured consists of a clear coloured solution with negligible scattering relative to absorption, then scattering losses are trivial and the absorbance displayed by the instrument will not differ significantly from the true absorbance. If, however, the pigmented mater-ial is particulate, then a substantmater-ial proportion of the light transmitted by the sample may be scattered outside the collection angle of the photo-multiplier, the value ofIregistered by the instrument will be too low, and the absorbance displayed will be higher than the true absorbance.
This problem may be solved by so arranging the geometry of the instrument that nearly all the scattered light is collected and measured by the photomultiplier. The simplest arrangement is to have the cells placed close to a wide photomultiplier so that the photons scattered up to quite wide angles are detected. A better solution is to place each cell at an entrance window in an integrating sphere (Fig. 3.2b): this is a spherical
Blank
Fig. 3.2 Principles of measurement of absorption coefficient of non-scattering and non-scattering samples. The instrument is assumed to be of the double-beam type, in which a rotating mirror is used to direct the monochromatic beam alternately through the sample and the blank cells.
(a) Operation of spectrophotometer in normal mode for samples with negli-gible scattering. (b) Use of integrating sphere for scattering samples.
3.2 The measurement of light absorption 55
cavity, coated with white, diffusely reflecting material inside. By multiple reflection, a diffuse light field is set up within the sphere from all the light that enters it, whether scattered or not. A photomultiplier at another window receives a portion of the flux from this field, which may be taken to be proportional to I or I0 in accordance with whether the beam is passing through the sample, or the blank cell. With this system virtually all the light scattered in a forward direction, and this will generally mean nearly all the scattered light, is collected.
Another procedure, which can be used with some normal spectropho-tometers, is to place a layer of scattering material, such as opal glass, behind the blank and the sample cell.1211 This ensures that the blank beam and the sample beam are both highly scattered after passing through the cells. As a result, the photomultiplier, despite its small collection angle, harvests about the same proportion of the light trans-mitted through the sample cell, as of the light transtrans-mitted through the blank cell, and so the registered values of intensity are indeed propor-tional to the true values ofIandI0, and thus can give rise to an approxi-mately correct value for absorbance.
With all these procedures for measuring the absorbance of particulate materials, it is important that the suspension should not be so concen-trated that, as a result of multiple scattering, there is a significant increase in the pathlength that the photons traverse in passing through the sample cell. This would have the effect of increasing the absorbance of the suspension and lead to an estimate of absorption coefficient that would be too high. Even with an integrating sphere, some of the light is scattered by the sample at angles too great for collection: a procedure for correction for this error has been described.701
An instrument based on completely different principles to any of the above is the integrating cavity absorption meter,357,418,419
(ICAM). The water sample is contained within a cavity made of a translucent, and diffusely reflecting, material. Photons are introduced into the cavity uniformly from all round, and a completely diffuse light field is set up within it, the intensity of which can be measured. The anticipated advan-tages are, first, that since the light field is already highly diffuse, add-itional diffuseness caused by scattering will have little effect; second, because the photons undergo many multiple reflections from one part of the inner wall to another, the effective pathlength within the instrument is very long, thus solving the pathlength problem. A prototype instrument gave very satisfactory results.419In an alternative version of the ICAM – the point source integrating cavity absorption meter(PSICAM) – the cavity
is spherical, and a uniform light field is achieved by introducing the photons from a centrally placed point light source.715,783In this case also prototype instruments have given excellent results.1150
Most seawater, and many freshwater, samples have absorption coeffi-cients (above those of pure water) too low to measure accurately in laboratory spectrophotometers An approach pioneered by Yentsch (1960), applicable to the particulate fraction is to pass the water through a filter – usually glass fibre – until enough particulate matter has accumu-lated, and then measure the spectrum of the material on the filter itself without resuspension, using a moist filter as a blank. To correct for the additional attenuation due to scattering by the particles on the filter the value of apparent absorbance at some selected wavelength (715–750 nm) in the near infrared, where it is assumed that true absorption is negligible, is subtracted from absorbance at all other wavelengths.
Implicitly it is assumed that the substantial attenuation due to scatter-ing by the filter itself is the same for both the sample and blank filters.
This assumption is not necessarily correct. The particles collected on the sample filter, as well as adding to total scattering may change its angular distribution and thus alter the proportion of transmitted light captured by the photomultiplier. To reduce this error, Tassan and Ferrari (1995) have developed a variation on the procedure – the ‘transmittance–reflectance’
method – in which, as well as measuring absorbance in the usual manner, spectral reflectance is also measured and used to correct for backscatter-ing. This procedure has been found to reduce the variability of the measurements of absorption coefficients of particulate matter in the somewhat turbid coastal waters of the Adriatic.1346
The most fundamental problem with the filter method, however, is that, as a consequence of multiple internal reflection within the particu-late layer, there is very marked amplification of absorption, the amplifi-cation factor being commonly indicated by b. To calculate the true absorption coefficients that the particulate material has, when freely suspended in the ocean or lake, the absorption amplification factor must be determined reasonably accurately, and this is not easy. The approach has generally been to find correction factors by growing phytoplankton species in the laboratory and comparing optical densities measured on filters with those obtained on suspensions in cuvettes.245The most com-monly used algorithm in recent years makes use of a quadratic relation-ship, first arrived at by Mitchell (1990)
ODðlÞ-corrected¼C1ODðlÞmeasuredþC2OD2ðlÞmeasured ð3:5Þ 3.2 The measurement of light absorption 57
whereC1andC2are empirically determined coefficients.Equation 3.5implies that the value ofb, the amplification factor, varies with optical density.
Roesler (1998) points out that if, as seems plausible, a completely diffuse light field is established within the glass fibre filter, then the average cosine of the forward-transmitted photons should be 0.5, which would correspond to a pathlength amplification factor of 2.0. Her investi-gations suggest that much of the variability encountered in using this method arises from the fact that the optical properties of the filter pad itself are markedly affected by the volume of water filtered through it, and it is typically the case that a much larger volume is passed through the sample filter than the blank filter. With the appropriate filter preparation, the assumption thatb¼2.0 gave satisfactory results in this study.
Allaliet al. (1995) have developed a method that, while still making use of filtration to concentrate the particles, avoids the optical problems associated with the filter itself. In their filter–transfer–freezemethod the samples are filtered through a polycarbonate filter with 0.4mm pores, so that the particles collect on the surface. The loaded filter is then trans-ferred, particle side down, onto 5ml of sea water on a glass microscope slide and quickly frozen using liquid nitrogen. The filter is then peeled off, leaving the frozen layer of particle suspension attached to the slide, and replaced with a glass cover slip. After thawing, the absorption spectrum is measured using an integrating sphere.
A different experimental approach has been developed by Iturriaga and Siegel (1989), who use a monochromator in conjunction with a micro-scope to measure the absorption spectra of individual phytoplankton cells or other pigmented particles. If sufficient particles are measured, and their numbers in the water are known, then the true in situ absorption coefficients due to the particles can be calculated. While laborious, this does avoid the uncertainty associated with estimation of the amplification factor in the filter method.
For measurements of soluble colour, scattering problems can be largely eliminated by filtration, so that measurements over long pathlengths can be used. D’Sa et al. (1999) used a 0.5 m long, 550mm internal diameter, capillary waveguide (see reflective tube principle, below) to measure the absorption spectrum of dissolved organic matter in seawater samples.
As we shall see later, the absorption coefficients of the different con-stituents of the aquatic medium can be determined separately. The value of the absorption coefficient of the medium as a whole, at a given wavelength, is equal to the sum of the individual absorption coefficients of all the components present. Furthermore, providing that no changes in
molecular state or physical aggregation take place with changes in concentration, then the absorption coefficient due to any one component is proportional to the concentration of that component (Beer’s Law).
In situmethods
The total absorption coefficient of a natural water at a given wavelength can be determined from a measurement made within the water body itself.
To make accurate measurements at those wavelengths where marine, and some inland, waters absorb only feebly, quite long pathlengths (~0.25 m or more) are needed. The simplest arrangement is to have, immersed in water, a light source giving a collimated beam and, at a suitable distance, a detector with a wide angle of acceptance. Most of the light scattered, but not absorbed, from the beam is detected: diminution of radiant flux between source and detector is thus mainly due to absorption and can be used to give a value for the absorption coefficient.
An alternative approach is to have, not a collimated beam, but a point source of light radiating equally in all directions.78,850The detector does not have to have a particularly wide angle of acceptance. For every photon that is initially travelling towards the detector and is scattered away from the detector, there will, on average, be another photon, initially not travelling towards the detector, which is scattered towards the detector. Thus diminution of radiant flux between source and detector is entirely due to absorption. An absorption meter based on this principle has been used to measure the absorption coefficient of water in Lake Baikal (Russia).107A problem with this and the previous type of absorp-tion meter is that although scattering may not directly prevent photons reaching the detector, it increases the pathlength of the photons and thus increases the probability of their being absorbed, i.e. spuriously high values of absorption coefficient may be obtained. This error can be significant in waters with high ratios of scattering to absorption, but a correction can be made using the values of the scattering coefficient and average cosine of the scattering phase function if these are available for the water in question.719
To avoid the necessity of comparing readings in the water body with readings in pure water or other standard medium, a variant on the second type of absorption meter has been developed that still uses a point light source but has two detectors at different distances from the source: from the difference in radiant flux on the two detectors, the absorption coefficient may be obtained.414 By means of interference 3.2 The measurement of light absorption 59
filters, this particular instrument carries out measurements of absorption coefficient at 50 nm intervals from 400 to 800 nm.
With all these absorption meters, if the light source is modulated and the detector is designed to measure only the modulated signal, then the contribution of ambient daylight to radiant flux on the detector can be eliminated.
In the case of an absorption meter using a collimated light beam, one possible solution to the problem of distinguishing between attenuation of the signal that is genuinely due to absorption from that which is due to photons in the measuring beam simply being scattered away from the detector, is to shine the beam down an internally reflecting tube235,1496 containing the water under investigation. The principle is that photons that are scattered to one side will be reflected back again, and so can still be detected at the other end. In such an instrument, however, there is still some residual loss of photons by non-reflection at the silvered surface, and by backscattering, and so a correction term proportional to the scattering coefficient needs to be applied.713Current commercial instru-ments6have dispensed with the silvered surface and rely on the internal Fresnel reflection (100% for scattering angles up to 41) of photons that takes place in water within a glass cylinder surrounded by air.1497 The light is collected by a diffused large area detector at the far end of the flow tube. These instruments can be used for vertical profiling of absorption coefficients.163
Grayet al. (2006) have described an in-principle design of a cylindrical flow-through absorption meter, which makes use of the integrating cavity principle. Musseret al. (2009) have constructed a prototype instrument, based on this design. The sample fluid flows through a 122 cm long quartz tube, internal diameter 2.54 cm, surrounded by two cylindrical diffuse reflectors, 102 cm long, separated by an air gap. Light from light emitting diodes (LEDs) operating at six wavelengths is introduced into the air gap, leading to the establishment of a diffuse light field within the sample cylinder. The device has an operating range from 0.004 m1 to 80 m1, and like other instruments based on the ICAM principle, is essentially unaffected by high levels of scattering within the sample.
Absorption meters and other optical instruments are sometimes attached to moorings at various depths to obtain long-term time series of optical data for periods up to several months. Unfortunately, instru-ments submerged for long periods will – unless preventative measures are taken – soon have their measurements compromised by fouling – the settlement and growth of marine organisms on their surfaces.
Davis et al. (1997) arranged for a concentrated solution of bromine to diffuse into the flow tube between sampling periods, and in this way were able to prevent biofouling for 3.5 months at 11 m and 6 months at 40 m, in the Bering Sea.
Absorption coefficients of natural waters can also be determined in situ from measurements of irradiance and scalar irradiance. The instrumentation for such measurements we shall discuss in a later chapter (see }5.1). Determination of absorption coefficient makes use of the relation (see}1.7)
a¼KE
E~ Eo
where E0 is the scalar irradiance, E~is the net downward irradiance (Ed– Eu) andKEis the vertical attenuation coefficient forE. Thus~ a can be obtained from measurements of net downward and scalar irradiance at two, or a series of, depths (measurements at more than one depth are required to give KE). Absorption meters based on these principles have been built.570,1283By making use of empirical relationships derived from numerical modelling of underwater light fields, estimates of absorption (and scattering) coefficients can be derived fromin situirradiance,714,145 or irradiance and nadir radiance,1307,885,890
measurements.