Evaluation of Optical Detection Methods for

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optical detection metiods forwatwt lorne suspension t

Making design criteria for turbidimeters more stringent would enable an integrated approach to turbidity measurement and article detection. Virendra Sethi, Priyamvada Patnaik, Pratim Biswas, Robert M. Clark, and Eugene W. Rice

scattering and light obscuration techniques ed successfully to characterize suspended matdrinking water, beverages, and process fluids. lity and, increasingly, particle counting are used ET ect waterborne particulate matter for regulatory Lance and for monitoring the effectiveness of water treatment at various process stages. Recent changes in water quality regulations and concerns regarding waterborne cryptosporidiosisr,2 are causing the water supply industry to reevaluate the “lkrbidimeters and optical particle counters (OPCs) are used to capabilities of particle monitor particulate matter in water. The response from these detection instruments.3 instruments is governed by the optical properties of the Because of simplicity suspension and the instrument design. The recommended design and ease of operation, criteria for turbidhneters allow for large tolerances that lead to turbidimeters are used variations in measurements from different instruments. OPCs routinely to monitor finprovide size-specific information but may inaccurately size ished water quality. microorganisms or particles having optical properties different More important, turbidfrom those of the calibration particles. The authors evaluate the ity serves as a surrogate effects of optical design parameters in turbidimeters and OPCs. indicator of the presence Performance data from two OPCs are presented. A multiple-angle of microorganisms in light-scattering method was developed to obtain “optical water. The assumption of a correlation between signatures” from suspensions of monodisperse spheres and several microorganism isolates. Such “signatures” provide more turbidity and microbial optical information on waterborne particles and may also be used quality either may not be to identify specific types of microorganisms in real time. true4 or may be inadequate in preventing out-

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Copyright (C) 1997 American Water Works Association

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breaks of waterborne disFlaURE 1 Multiple-angle light-scattering (MALS) experimental setup eases. Design differences among turbidimeters make it Sample vial He-Ne laser, 5 mW, 632.6 nm Light trap difficult to compare responses from different turbidimeterss and raise the issue of a need for standardization.6~s Further, turbidity is an aggregate Linefilter (632.6+1 nm) i Frequency optical property and lacks PhlT photosensitive i reference ; signal size-specific information for DetectIony I surface optics detection of microorganisms in a specific size range. The .I. . ..I............................ recent availability of optical PMT signal Lock-in amplifier particle counters (OPCs) for liquids, however, has provided a means of acquiring size distributions for waterborne suspensions. in another study* no relationship among turbidity, Turbidimeters and particle counters operate difparticle counts, and bacteriological quality of water ferently. Turbidimeters respond to a “cloud” of partiwas found. The lack of such agreement may be cles in a sample view volume, whereas OPCs count caused by one or more reasons: (1) lack of inforand size individual particles as they pass through a mation about optical properties of suspensions and view volume. Comparative studies in performance microorganisms; (2) limitation of OPCs in detectevaluation show that because of their higher sensiing particles smaller than 0.5-1.0 pm; (3) differtivity, OPCs respond to particle breakthroughs much ences in optical design of the instruments, and (4) sooner than turbidimeters.s-1’ Ideally, given an OPCdilution errors.15 measured size distribution, it should be possible to The success of OPCs in providing size-specific obtain turbidity as a sum of the scattered light signal information tends to obscure the fact that a large response from each particle in a suspension,12 profraction of suspended matter is smaller than the vided particle characteristics such as size, shape, strucdetection limit of OPCs. The undetected particles ture, and refractive index are known. may carry nutrients that can support biological activExperimental correlations of changes in turbidity later and may also exert a disinfectant demand.4 ity with corresponding changes in particle counts This fraction of undetected particles, however, conhave been successful in some studies.ra,r* However, tributes to turbidity and is possibly missed onlv because of the lack of sense’tivity of turbidimeters at the currently recommended 900 2 Schematic diagram for turbidimeter design specifications detection angle. Jj$c;;laURE Further, the interpretation of a physical particle size from the optical response obtained from OPCs requires a known optical response for the particles that is specific to the Trap angle of detection of the sensor. In the absence of such information for the variety of inorganic, organic, and microbial particles being viewed, the sizing is at best an estimate of an optical response relative to that from calibration suspension. Previous studies that have attempted to correlate the performance of turbidimeters and OPC measurementss,i4 have not addressed the problem of optical detection with respect to optical properties of particles and the effects of instru-

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heoretical optical response from turbidimeters: nd IS0 design criteria for X and collection angle

FIQURE

USEPA

-+

100.00

of USEPA

IS0 h range

1 range

400nm

effect

+600nm

--

94Onm

+-66Onm

-920nm

I 60

I 100

A

USEPA: 0.01

90+30’

-

, I

0

I

20

40

i 60

i 120

I 140

Anglti

FIGURE

4

Theoretical optical response and IS0 design criteria for Effective 1.300

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refractive m 1.200

from effect index

turbidimeters: of refractive

effect of USEPA index variations

(m,) * 1.100


ment design. Evaluating and identifying the causes for common problems reported in previous studies may lead to improved instrument design and redefine compliance criteria. Both turbidimeters and OPCs use the single-angle detection method, which is a subset of a more broad-based multiple-angle light-scattering (MALS) measurement method. The MALS technique has been used successfully to characterize liquidborne macromoleculest6 and gasborne particles.17-l9 The availability of the electrodynamic balance made it possible to study lightscattering properties of individual particles in the micrometre size range. Such studies have been limited to gas-suspended particles or droplets; to the authors’ knowledge, there are no MALS techniques for the study of individual particles suspended in liquids. The only exception is a method used in flow cytometry, in which scattered light can be measured at one near-forward angle and also at 900. Ideally, simultaneous multiple-angle measurements from individual particles could provide more detailed information about the size and shape of individual particles in water. Such a device is not currently available. MALS measurements from a collection of particles in a small view volume (similar to turbidimeters) come closest to such an approach. Multiple-angle measurements from dilute suspensions of monodisperse spherical particles can be used to represent the behavior of a single particle of the same size. If the particles are not spherical but have other geometric shapes (cylindrical rods, ovoid shapes), random orientation of the particles in the view volume decreases the information that could have been available from sin-

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TABLE

1

Summary

and

results

of experiments

4-7 pm 8-12 pm

gle particles aligned at a specific angle with the incident beam. The averaging effect in such a case, however, may still be unique, and the technique can be used to characterize the optical properties of particles in monodisperse suspensions.20 As an extreme case, a suspension may be made up of particles of irregular geometric shapes (single cells, chains, agglomerates), different sizes (polydisperse), or both. In such a case, the optical response would represent a statistical average of the size distribution and optical properties of the suspension. In the absence of prior information about the optical properties, multiple-angle measurements can be used to infer the absolute or mean optical characteristics. Multiple-angle measurements can provide insight into the optical properties and internal structure of microbial suspensions and may also serve as unique “optical signatures” to identify specific microorganis&. MALS measurements may also enable the study of suspensions that are not TABLE 2 USEPA and resolvable under the microscope because of lack of ?ammeter refractive contrast or conLight source stant particle motion.21 ai +)Length

Objectives The objective of this study was threefold. The first objective was to evaluate the extent of variations in optical response that could result because of tolerances permitted in the rec-

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of beam

path

3.7 7.8

(A)

NE

ommended design criteria for turbidimeters. In a theoretical simulation, the effects of specification of wavelength of light and the collection angle at the detector were investigated. The effect of the refractive index of particles on turbidimeter response was also demonstrated theoretically. The second objective was to evaluate the sizing performance of OPCs for microorganisms. A theoretical simulation of a light scattering-type OPC sensor was used to show the significance of minor changes in refractive index on the sizing accuracy of spherical particles. Sizing performance of two OPCs was then evaluated experimentally using spherical homogeneous monodisperse particle suspensions of known refractive indexes and microbial suspensions with unknown optical properties. The third objective was to present a case that the single-angle detection method used in turbidime-

IS0

design

criteria

for turbidimeters

USEPA Tungsten 40

cm (c3.94

Is0 X*=860 AX=f60

lamp

2,20&3,000

K in.)

90f300 response

2.0

400-600

nm 0.02 ntu for waters with 1.0 ntu or less Formazin or polymeric standard


nm nm Parallel beam with maximum divergeno s-convergence of 2.50

QOf2.50 f&=10-2

00 Not specific ed 0.01 fnu fa ,r waters 1.0 fnu or less Formazin

with

V. SETHI

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‘ractiona I contribution A p for ti ~0 size ranges

to turbidimetric

response

as a function

lilprn

The angular distribution of scattered light intensity from a spherical particle, measured at a distance R in the scattering-^ plane, is described as24 IoA2 (Gv+ Gf.d I(0) = (1) 8n2R2

-

in which CA and C,, are the Lorentz-Mie parameters for the vertical and horizontal polarized components of the scattered intensity. C, and C,, are a function of the size parameter X, the particle refractive index m, and the scatter angle 8. They can be calculated using computer subroutines.23Js The refractive index of particles, m, is a key property that affects the calculation of C, and C,, in Eq 1. Waterborne suspensions vary widely in composition and content (silt, clay, minerals, inorganic and organic matter, and microorganisms), and their optical properties remain largely unknown. Refractive index may be real (nonabsorbing) or complex (absorbing) and may be affected by structural inhomogeneities. The value of the real part of the refractive index for waterborne particles relative to water (m,) could be expected to 8m

Angle--8

ters and OPCs is limited and that a MALS technique could be developed to better characterize nonspherical particles with unknown optical properties. An experiment was developed to obtain angular intensity distribution of scattered light from several suspensions of monodisperse spherical particles and microorganism isolates. A size inversion scheme was applied to infer particle sizes from these angular distribution measurements. e-angle

measurements can provide ht into the optical properties and internal structure of microbial suspensions and may also serve as unique “optical signatures” to identify specific microorganisms.

I Optical detection: bkkground Light scattering by single homogeneous spherical particles is described by the Rayleigh and Lorentz-Mie theories.20,22,23 The value of dimensionless size parameter X ( X = T d,lX, in which dp is the particle diameter and X is the wavelength of the incident light) is used as guiding criteria for the choice of the appropriate theory. The Rayleigh scattering theory is applicable for small particles (Xc 0.3). The Lorentz-Mie theory extends the size range to include larger particles (X > 0.3) and reduces to the Rayleigh theory for smaller partitles. The interaction of an incident light beam of intensity I, with a particle in the Lorentz-Mie regime results in a dissymmetric angular distribution of the scattered light intensity I(0) and is a unique function of particle size, shape, and refractive index.

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lie in the range between 1.05 (estimate for microorganisms) and 1.50 (inorganic minerals) .z6 Turbidimeters are calibrated in nephelometric turbidity units (ntu) using suspensions of formazin (m, unknown) prepared under controlled conditions27 or using suspensions of styrene divinyl benzene copolymer microbeadss (m, = 1.17) prepared to match nephelometric turbidity unit scales. OPCs are calibrated using monodisperse polystyrene latex (PSL) suspensions (m, = 1.195). Sensitivity to variations in refractive index is minimized in OPCs by limiting the collection angle at the photosensor to a narrow cone of light at a near-forward angle. Despite such


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design features, particles with refractive indexes that vary markedly from that of PSL may not be sized correctly, and the sizing accuracy varies from sensor to sensor.2s For turbidimetric measurements of the heterogeneous suspensions encountered in drinking water, the use of an “average” or “effective” refractive index may be applicable. Such information for raw and treated drinking waters, however, is conspicuously missing in the literature. In addition to parameters m and X for single particles, the angular scatter intensity distribution for polydisperse suspensions is a function of particle size distribution parameters. The Lorentz-Mie theory (Eq 1) may be extended for dilute suspensions of polydisperse particles. For a suspension with size distribution function n(dp!, the response at any angle is an aggregate contribution from all the particles in the suspension and can be written as Qw (0) = i;

C,

FlalJRE

ptical

sensor

assumed

as 1.05)

Experimental calibration response 0 Pregain OPC-LS response from PSL calibration

lo-*

Ill11

I

I

I

I

Ill11

1 .,.

I

I

I

I

10 Particle

III

1I loo

Diameter-pm

,’

(2)

(3)

in which (x and B are the two power law parameters. CYis a measure of the particle number concentration (normalized by the size increment) at the size 1 pm, and B reflects the relative fraction of the total count as a function of particle size. As B increases, the ratio of smaller particles to larger particles increases. The value of B ranges from 2 to 5 for most waters.iaJs The upper size limit of particles in drinking water is observed to be - 10 pm, which is reliably determined by optical methods. Determination of the lower end of the size range, however, is limited by the measurement method (e.g., optical microscopy - 0.5 pm; OPC - 0.5-1.0 urn). Ideally, it may be desirable to develop the measurement capability to detect viruses, implying a lower detection limit of 0.001-O. 1

1997

OPC-IS

Theoretical response PSL (mw= 1.197) -~ SiD, (In,= 1.10) Microorganism (m,

in which Qw is the theoretical scattering efficiency of the vertical polarized component at angle 0 with particle sizes ranging from d, to d, and n(d,) is the slope of the cumulative number concentration distribution at each value of dp. The following power law function has been found to fit the size distribution of particle suspensions in drinking water for particles in the l-lo-pm size range:9,10

FEBRUARY

for

lo4

(m, 0, A, dp) n(d,) dd,

n(d,) = a dp-P

response

urn. Rayleigh scattering from the water molecules and noise from photoelectronics, however, have limited the current practical detection capability in OPCs to -0.5 pm. The power law representation for particle distributions in drinking water has been based on particle size and count data obtained primarily from OPCs. The power law form of representation may be a subset of a more complete lognormal or Gaussian form of distribution usually found in natural systerns.9 In a study of particles in ocean waters,31 an electron microscope was used to measure particle size distributions in the 0.02~SO-pm size range; the data were found to conform32 to a power law representation. Because the contribution to light scattering from particles smaller than 0.02 pm is expected to be negligible, power law may be assumed to completely represent the optically detectable size range. The scatter intensity as described by Eq 1 is a function of the angle of detection. Dissymmetry in angular intensity distribution has been used in the past to characterize macromolecules16 and particles in the Lorentz-Mie regime. 33,34 A data inversion procedure has been used to estimate the lognormal size distribution parameters and refractive index for combustion aerosols.35,36 The procedure uses a least-square fitting technique to match scattering intensity measurements with the theoretical response for spherical


V. SETHI

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FIGURE

7

Size

response

from

OPCs

for

PSL suspensions

particles using particle size (dP) and the refractive index (m) as two independent variables. Experimental methods The focus of the experimental work in this study was to establish the effect of optical properties of particles on the sizing performance of OPCs and to obtain MALS patterns for suspensions of microorganism isolates. mo commercial OPCs, one based on the principle of light scattering (OPC-LS*) and the other based on the principle of light obscuration (OPC rbidity is LO+) were used to size suspensions of PSL* (1.020, nd lacks 1.500, 2.032, 3.500, and 5.100 pm) and SiO,$ (1.100 for detection and 5.000 pm). in a specific Several suspensions of microorganisms were prepared to study the sizing response from OPCs and MALS patterns. Escherichia coli, Enterobacter cloacae, and Bacillus subtilis cultures were grown in brain-heart infusion broth for 1S-20 h at 35OC. The cultures were concentrated and washed twice in phosphate buffer.z7 The cultures were resuspended in 10 mL of buffer and further diluted for the experiments. The Streptococcusbovii and Enterococcusfaecium cultures were grown in brain-heart infusion broth

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supplemented with 1 percent (w/v) yeast extract and prepared in the same manner as the E. coli and E. cloacae cultures. The Giardia muris cysts and Cryptosporidium muris oocysts were prepared as described elsewhere.37 Diluted suspension of S. bovis, B. subtilis, C. muris, and G. muris were sized using the two OPCs. A MALS experiment was developed for studying the optical response for waterborne suspensions. A brief description of the experimental setup (Figure 1) follows. A 5.0-mW, He-Ne (A = 632.8 nm) plane-polarized laser beam of intensity I,,, chopped with a high-frequency chopper, was used as the incident light source. A vertical polarizer, a 632.&Onm line filter, and a variable aperture were used to receive only the vertical polarized component (I,) of the scattered light at the same wavelength as the incident source beam. A lock-in amplifier locked to the reference frequency from the chopper was used to eliminate background optical noise and amplify the photocurrent. The photomultiplier tube assembly (variable slit, line filter, and polarizer) was mounted on a rotator with an angular range of 30-1200 with a resolution of lo. A 35-mL cylindrical glass vial with an OD of 27 mm was used to hold the sample. The experimental setup was calibrated using standard monodisperse PSL suspensions. The angu-

an aggregate optical property size-specific information of microorganisms size range. lar distribution of scattered light from a blank sample was comparable to that reported by Black and Hannah.6 All measurements were made at 5O intervals, and the angular distribution patterns were consistently replicated at different dilutions to ensure the absence of multiple scattering. MALS measurements were made for suspensions of PSL (0.460, 0.560, 0.750, 0.993, 1.020, 1.500, and2.032


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pm) and were used to test the effectiveness of the size inversion scheme.35,36 Similar measurements were also made for SiO, ( 1.100 pm), S.

FIGURE

8

Size

response

from

OPCs

for

PSL

and

SiO,

bovis, E. faecium, B. subtilis, E. cloacae, and E. coli. A sum-

mary of experiments and results is given in Table 1. a0

Results and discussion 5 As stated earlier, this work focuses on understanding the 1 effects of optical design paras 60 meters and particle character5 0 istics on optical detection of r% waterborne suspensions. The g next two sections describe the t 3 40 results of theoretical simulaI% tions for turbidimeters and I experimental results of sizing E e from OPCs. The third section p” describes the results of the 20 MALS approach for optical characterization of particles. Turbidimeters. Manufacture of turbidimeters is guided O I by recommendations pro0 vided by the US Environmental Protection Agency (USEPA)27,38 in the United States and the International Organization for Standardization (ISO) in Europe.39 The recommendations are provided to ensure consistency among instruments for the purpose of regulatory compliance. Although all manufacturers meet the design criteria suggested by USEPA, variation is observed5 in measurements from different instruments. Such variations are likely to be a result of the tolerance’s levels permitted by the specifications. In this section, a theoretical analysis of the influence of the source light wavelength (X) and the angle of detection (0) on the scattered intensity is presented. The effect of the particle refractive index on the optical response is also simulated to highlight the importance of optical properties of suspensions. Turbidity measurements are affected by the optical, mechanical, and electronic design of the instrument.6,7J5,40 The design criteria suggested by USEPA and IS0 are listed in Table 2, and the terms are illustrated in Figure 2. The intensity of scattered light from a standard calibration suspension measured at 90” to the incident beam is used to define nephelometric turbidity units. For a suspension that can be represented by a size distribution function, the turbidity (ntu) value can be calculated using Eqs 2 and 3. The angular distribution of scattered light intensity was simulated using a typical set of values of cx (105) and p (3.82) reported in the literature2” and a relative

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3

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4

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6

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Particle

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refractive index of 1.195 (PSL). The source wavelengths were varied in the range specified by the two design criteria listed in Table 2. The limits of integration d, and d, in Eq 2 were chosen to be 0.01 and 10 pm, respectively. The theoretical response was scaled to nephelometric turbidity units using a turbidity calibration standard with known particle concentration8 (0.269-vrn styrene divinyl benzene copolymer microbeads, 40 ntu = 4.85 x lo8 number/ml). Figure 3 shows the plot of the theoretical turbidity response as a function of the angle of detection 8 from 200 to 1300. Such an angular variation is important to ascertain the sensitivity of optical response to the detection angle (the design specification is limited to a solid angle centered at 900). The 400-600-nm allowable range of wavelength in the USEPA specification (as indicated in the spectral response range for the detector) allows for as much as 40 percent variation compared with a 15 percent variation from the 860+60-nm range allowed by ISO, both when measured at 900 and throughout the range of angles investigated. * U-21 t HRLD-

1, Met-One, Grants 150, HiaciRoyco,

Pass, Ore. Div. of Pacific

Scientific

Co.,

Silver

Spring,

Md. $ Bangs

Laboratories


Inc.,

Fishers,

Ind.

V. SETHI

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105

Despite the limitations caused by such variations, the turbidimeter method has the potential to bridge the gap between the OPC-detectable particles ( >0.5 pm) and the submicron-sized particles (0.02-0.5 urn). For p > 3, the fraction of particles in the submicron range has a greater contribution to light scattering than the larger-size fraction.9 The implication is significant, because this submicron fraction is not detectable by the OPCs but may be detected by the turbidimetric method. To investigate the relative contributions from these two size fractions, another set of simulations was made with two values of p (p = 2 when the proportion of larger particles is higher and p = 4 when the proportion of smaller particles is relatively larger). The limits of integration were set for the two size ranges as 0.01-1.0 and 1.0-10 pm, respectively. FigFurther, the size of the solid angle over which ure 5 shows the four plots for the fraction of the the scattered signal is integrated can cause additional total light scattered for the two values of l3 and the uncertainty. This is attributed to the variations in two size ranges. For the case of a suspension with p the nonlinear scattering response for the calibration = 2 and a 900 turbidity value of, say, 1.0 ntu, 99 suspension and the sample suspension. The tighter percent of the 1 .O ntu is contributed by particles in IS0 specification (9Ok2.50) is likely to cause less varithe l-lo-pm size range, and only 1 percent is conation than the larger( 90+300) angle range specified tributed by the smaller size fraction. In such a case, by USEPA. measurements made with OPCs may be sufficient to completely characterize the distribution. On the other hand, for l3 = 4, 80 percent of the 1.O success of OPCs in providing ntu is contributed by the smaller-size fraction and ze-specific information tends to obscure onlv 20 nercent bv the larger (OhC-de&able) the fact that a large fraction of suspended size range. Thus, although matter is smaller than the detection limit the OPC data would miss the many particles in the of OPCS. submicron range, the turbidimetric response may be useful for detecting the The effect of particle refractive index on the turpresence of particles in this size range. Thus the use bidimetric measurement is shown in Figure 4. A 15 of OPCs would be effective for finished water, but percent change in the average refractive index of the the influent stream with its higher proportion of suspension can change the turbidimetric response by submicron particles would still require use of the a factor of 10. The absence of prior information about turbidimetric approach. This has also been pointed the optical properties of waterborne suspensions, thereout by Kavanaugh.9 fore, makes it difficult to meaningfully compare interFigures 3 and 4 also show that the intensity of regional or interseasonal turbidimetric measurements. scattered light for a given suspension decreases with

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increasing angles, making the choice of 90” as the angle would make the turbidimeters more sensitive angle of detection appropriate to prevent errors to concentration changes and at the same time caused by vibrations. The cost of such a choice, howbridge the current gap between the lower detection ever, is severe. A loss of an order of magnitude in siglimit of turbidimeters and the upper concentration nal strength implies a loss of detection capability for limits of OPCs. concentration changes bv a factor of 10. As discussed earlier, for the range of particles encountered in ty in comparing turbidity responses drinking water sources, the different instruments may arise from turbidity measurement is still useful, motivating the the tolerances permitted in the design investigation into increasing the sensitivity of turcriteria and the lack of information about bidimeters by using smaller optical properties of the suspensions. detection angles. The maximum allowable concentration to prevent coincidence error in OPCs is currently below the detection It follows that the difficulty in comparing turbidrange of turbidimeters. A change of 0.02 ntu, the ity responses from different instruments may arise least detectable limit of turbidimeters, is estimated to from the tolerances permitted in the design criteria be equivalent to a concentration change of -3.5 x and the lack of information about the optical proplo4 number/ml of l.O-pm PSL monodisperse parerties of the suspensions. The turbidimeter design titles. The maximum allowable concentration to criteria seem to have evolved by agreement among prevent coincidence error in the OPC-LO sensor is instrument manufacturers, by technical and cost con1.8 x lo4 number/ml and 3.5 x IO3 number/ml in straints, or both at the time of their inception.5 Given the OPC-LS sensor. Both concentrations are lower the analysis presented in this article and the advances than the least detectable concentrations in turin laser and photodetection technologies, it may be bidimeters. Simply changing to a smaller detection appropriate to reevaluate these features of the design criteria with the intention of upgrading detection capability to meet increasingly strin:omparison of MAISinferred PSI. diameters with manufacturergent demands. #pecified sizes ”

1

Optical

1’160”

0.60

1.00

Manufacturer-smcified

FEBRUARY

1997

Particle

Dial

particle

coun-

ecs. OPCs use the princilies of light obscuration and ight scattering to size and ount particles individually. I ‘he performance of two par~, ,’ / ‘,..‘, icle counters (OPC-LS and )PC-LO) was evaluated in his study to size monodislerse PSL, SiO,, and several nicroorganisms. To illustrate the effect of _,” :” Jetection angle and the particle refractive index on instrument response, light-scat.” “.,^,,...,.. tcring intensity distribution was simulated theoretically for he OPC-LS, which collects ,_ “.“, tattered light over an angle ange of 5-33O. The Lorentz/lie scattering coefficient C, vas integrated over these ngles as a function of particle .iameters and three possible alues of refractive indexes to ,,,;;f =~; ““;_:, ~_” 1 ‘, lbtain the sensor response ;;& ;,: ,, ,,;,;!,,,,,,, “i, “! =>,.~~ ~ye,;),, Figure 6). Calibration mea~!!$~h+:‘m~; ,r:; ,,,:=~ ;‘m_ _I I”” surements using various PSL


V. SETHI

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FIGURE

11

Angular

distribution

of scattered

intensity

sizes are also plotted with the theoretical curves (Figure 6). The plots indicate that particles in the 0.5 to -4pm size range with a refractive index less than that of PSL are likely to be undersized by the OPC. A loss in resolution in the -2.5 to -5.0-pm size range for the OPC-LS with PSL is predicted theoretically and is also observed in the calibration data. The principle of an obscuration-type sensor (OPC-LO) was reported by Sommer.41 The theoretically obtainable obscuration signal intensity is restricted by the size of the photodetector, which collects a narrow but finite cone of scattered light in the forward direction. The forward cone also causes a loss of resolution similar in nature to that for the OPC-LS sensor. Scattering intensity at forward angles, however, is relatively insensitive to the refractive index. Therefore, the response from an obscuration-type sensor is less likely to be influ-

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enced by the refractive index of the suspension particles. The OPC-LO sensor has a lower detection limit of 1.0 pm and a coincidence-limiting concentration of 1.8 x 1O4 number/ml. The sensor’s responses for 1.500-, 2.032-, 3.500-, and 5.100-pm PSL suspensions are plotted in Figure 7 as a cumulative percentage concentration. The use of a 50 percent cutoff size (50 percent particles less than the cutoff diameter), an often-used criterion for aerosol-sizing instruments,42 underestimates the actual particle size, possibly because of optical noise at the lower end of the detectable range. A 90 percent cutoff size was found to better match the actual sizes. The OPC-LS sensor has a lower detection limit of 0.5 pm and a coincidence-limiting concentration of 3.5 x 103 number/ml. Two monodisperse suspensions of similar sizes but different refractive indexes-l.020 pm PSL (m, = 1:195) and 1.100 pm SiO, (m, = l.lO)-were analyzed using this sensor (Figure 8). The sensor responded with clearly defined peaks for both the samples. The response from OPC-LO for 5.100~pm PSL and 5.000-pm SiO, suspensions is also shown in Figure 8. OPC response was next investigated for suspensions of B. subtilis, S. bovis, and C. muris oocysts on both OPCs and additionally for G. muris cysts on the OPC-LO (Figure 9). The curves for S. bovis on both instruments and B. subtilis on the OPC-LO remained unresolved, as indicated by the least size being greater than the 50 percent cutoff line. Table 1 shows the microscopically determined sizes for the suspensions investigated in this study and the 90 percent cutoff sizes determined from the two OPCs. As anticipated, the OPC-determined sizes for PSL samples match well with the microscopic sizes reported by the manufacturer. It is interesting to note that whereas the optical sizing of 1.020 urn was accurate, the larger 1.lOO-urn SiO, particles (with a refractive index lower than that for PSL) were sized as 0.9 urn (Figure 8). The reverse was observed for the larger 5.100-pm PSL and 5.000-pm SiO, particles spheres

and ovoids


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measured with the obscur FIGURE 1P Angular distribution of scattered intensity for rod-shaped tion (OPC -LO) sensor. microorganisms The agreements betwec OPC-measured sizes and tl microscopic sizes vary for the B different microorganisms on each OPC because of the effects of shape and refractive index. The OPC-LS ind cated a size of 1 .O pm for tl rod-shaped B. subtilis (tl microscopic size being 0.5pm x 1.2-5.0 urn). The G. muris response on the OPCLO indicated a peak at 7 pm at the 90 percent cute (the expected size range B. subtilis 7-12 pm). A comparison the responses for C. mu? from the two OPCs highligh the influence of optical se] sor design and particle cha acteristics on sizing of part cles. For the OPC-LO, tl E. co/i peak appeared in the -4-p: range, as was also reports by Rossi. The OPC-L: however, responded with peak at about 2 pm as 01 posed to the microscopical observed 4-7-pm diametr The difference may haT been a result of the influenl I of angular dependence I I I I I I I 20 40 60 60 100 120 140 optical response for tf oocysts, the loss of size rest Angle-0 lution in the -2.5 to -5-p: range, or both, as has bee discussed previously. Detail< information on the angular scatter intensity distribution from such microorfor determining sizes more preferable than others, as ganisms is essential for accurate sizing. In the folhas also been reported by Kerker et al.44 lowing section, the results of MALS measurements The MALS patterns for a 1.02-pm monodisperse for the microorganisms are presented. PSL suspension, a 1. loo-pm monodisperse SiO, susMALs. The objective of this MALS study was first pension, and five suspensions of microorganism isoto develop and validate the experimental technique lates are shown in Figures 11 and 12. The plots repand the data inversion method using PSL suspensions resent an average of several replicates run at various and then to measure angular scatter intensity distribconcentrations to ensure absence of multiple scatutions for monodisperse suspensions of known and tering. The intensity scale is arbitrary, and the plots are unknown optical properties. The experimental setup displaced vertically for clarity. The change in angular described previously was calibrated using 0.993-pm distribution because of refractive index differences PSL suspensions and was used to obtain multiple-angle for similar-sized PSL and SiO, suspension is evident measurements for 0.460-, 0.560-, 0.750-, 1.020-, in Figure 11. The patterns for S. bovis and E. faecium 1.500-, and 2.032-pm PSL suspensions. The data inver(both ovoid) are also shown in Figure 11 for comsion scheme referred to previously was used for these parison with the spherical PSL and SiO, suspensions. measurements at 40-500, 50-60°, and 60-700 angle Figure 12 shows the response for rod-shaped microorpairs to estimate the particle sizes. The inferred sizes ganisms. The random orientation of the many rods in were plotted against the sizes determined by the manthe view volume tends to “wash out” the finer strucufacturer using electron microscopy (Figure 10). The tures (peaks and valleys in the case of spheres). Howhigh sensitivity of the scatter intensity to angular posiever, the patterns in part B still reflect distinct shapes tion at some angles makes the choice of some angles for the three microorganisms. These “optical signa-

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V. SETHI

ET AL

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FIGURE

13

MALS

measurements

for

formazin

and

tures” offer a potential approach for real-time characterization for specific microorganisms. Additionally, such angular distributions of scatter intensities integrated over the band of collection angles of any given OPC photosensor design could provide a more valid scheme for accurate sizing. A summary of previous studies on light scattering from microorganisms has been presented elsewhere.32 The data inversion scheme was applied to the angular distribution data for the microorganism suspensions with the assumption that the microorganisms can be approximated as homogeneous (uniformly distributed refractive index) spherical particles. The assumption is crude and was made only to test the applicability of the size inversion scheme for the measurements obtained for microorganisms. The results were within the range of the reported microscopic sizes and are listed in Table 1. A theoretical simulation with these diameters and the average refractive index assumed for inversion did not, however, reproduce the experimentally obtained patterns. To accomplish such a matchup, it is necessary to include effects of cell shape and variations in the spatial refractive index, which remain largely ill understood. A pattern recognition scheme from a library of known signatures may provide a more fea-

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sible route for characterizing microorganisms.45 MALS measurements were also made for two calibration suspensions used for turbidimeters: a formazin standard and a nonformazin standard.* The results (Figure 13) show the enhanced scattering response at smaller angles. The experimental results are comparable with the theoretical simulations of turbidimetric response shown in Figures 3 and 4. The full potential of the MALS technique-either to uniquely characterize polydisperse suspensions using a signal from a “particle cloud” or to characterize individual particles using multiple sensors in OPCs-has yet to be realized. Flow cytometry, a technique used frequently in the medical sciences, for example, successfully uses some of the principles of MALS.46 The application of cytometry in the drinking water industry has been limited primarily by instrument sophistication and high capital cost. The availability of low-cost lasers, photosensors, charge-coupled devices, and present-day computational capabilities, however, can lead to development of multiple-angle measurement techniques and thus make available the advantages of real-time particle characterization using the MALS method.

nonformazin

calibration

Summary and conclusions A brief review of optical particle detection methods has been presented with a focus on applications in drinking water. An integrated approach to turbidity measurement and particle detection by optical counters has been presented using fundamental elastic light-scattering principles. There is a need to reevaluate the USEPA design criteria for turbidimeters with the purpose of making the criteria more stringent, possibly following the path of ISO. In the authors’ judgment, a change to a smaller angle of detection would improve the instrument’s sensitivity without hampering its stability. No special effort was made to dampen vibrations during the experimental work in this study, and no problems were experienced with stability for angles up to 30°. Besides, commercially available ratio turbidimeters already use the second angle of detection as small as 150. * Jenway,

Felsted,


Dunmow,

Essex,

England

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Raw water feeds with power law parameter B> 3 (high proportion of particles smaller than the size detection limit of most OPCs) are candidates for characterization using the turbidity method. In their current form, a combination of the turbidity and particle counting methods is needed to obtain complete information on waterborne particulate matter. Increasing a turbidimeter’s sensitivity by changing the detection angle may bridge the lower detection limit of turbidimeters and coincidence-limited concentrations in OPCs. There is also a need to establish a database of refractive indexes of source water suspensions to permit a better comparison of interinstrument, interregional, and interseasonal turbidity measurements. The sizing performance of the two commercial OPCs-OPC-LS and OPC-LO-varied with the type of particles. The refractive index of spherical particles is a key issue in their accurate sizing. Sizing discrepancies could be explained on theoretical grounds using known optical properties of spherical particles, as in the case for SiO, and PSL. In the absence of structural and optical information (as is the case for C. muris samples), however, the observed sizing errors cannot be explained. Electromagnetic characterization of the species being sized is essential, and the application of the MALS method in one form or another may be the only method to fulfill the need. An MALS experiment was developed and calibrated using monodisperse PSL particles. Unique “optical signatures” were obtained for each pure culture of B. subtilis, S. bovis, E. faecium, E. coli, and E. cloacae. Such angular distribution contains all the information needed to uniquely characterize suspensions of pure cultures of microorganisms or to accurately size microorganisms in optical particle counters. Acknowledgment The support of Kim Fox, James Goodrich, Clifford Johnson, Larry Lobring, Richard Miltner, James O’Dell, James Owens, Lewis Rossman, and Thomas Speth of USEPA with equipment and materials is gratefully acknowledged. This work was supported in part by USEPA cooperative agreement CR-816700, W4-A. The conclusions are the views of the authors and do not necessarily represent the opinions, policies, or recommendations of USEPA. Part of this work was completed during the first author’s appointment to the Postgraduate Research Participation Program administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USEPA. The mention of commercial products is not to be construed as an endorsement of such products. References 1. MOORE, A.C. ET AL. Waterborne Disease in the United States, 1991 and 1992. Jotrr. AWWA, 86:2:87 (Feb. 1994). 2. PONTIUS, F.W. Protecting the Public Against Cryptosporidium. Jour. AWWA, 84:8:18(Aug. 1993).

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3. HARGESHEIMER, E.E.; LEWIS, C.M.; 6 YENTSCH, C.M. Evaluation of Particle Counting as a Measure of Treatment Plant Performance. AWWA, Denver (1992). 4. MCCOY, W.F. &- OLSON, B.H. Relationship Among Turbidity, Particle Counts, and Bacteriological Quality Within Water Distribution Lines. Water Res., 20:8:1023 (Aug. 1986). 5. HART, V.S.; JOHNSON, C.E.; &- LETTERMAN, R.D. An Analysis of Low-level Turbidity Measurements. Jour. AWWA, 84:12:40 (Dec. 1992). 6. BLACIC, A.P. 6 HANNAH, S.A. Measurement of Low Turbidities. Jour. AWWA, 57:7:901 (July 1965). 7. SIGRIST, W. An Assessment of the Latest Discoveries in the Measurement of Turbidity. Vom Wasser, Verlag Chemie, GmbH., Weinheim/ Bergstr (1975). 8. PAPACOSTA, I