C83 7045 Printed in U.S.A.

TURBIDITY STANDARDSTechnical Information Series Booklet No. 12

By: Mike Sadar

2In memory of

Clifford C. Hach(1919-1990)

inventor, mentor, leader and, foremost,

dedicated chemist

3Introduction 4Importance of Primary StandardsDefinition of Primary StandardTurbidity StandardsStandard Methods Clarification of Primary vs Secondary Standards

Primary Turbidity Standard Development5Definition of TurbidityTurbidity Units of MeasureFormazin as a Primary Turbidity Standard

Using Formazin Standards 7Preparation of Formazin Stock SuspensionPreparation of Formazin Calibration StandardsStorage of Formazin Stock Suspensions

StablCal Stabilized Formazin Standards 8Theory Behind StabilityStability DataComparability

Secondary Standards 11Application of Secondary Turbidity StandardsLatex Secondary Turbidity StandardsGelex Secondary Turbidity StandardsUsing Gelex Turbidity StandardsCare and Storage of Gelex Turbidity Standards

Alternative Standards 12Definition of Alternative StandardApplication of Alternative Standards

Appendix I. Preparation of Formazin 12

Appendix II. Preparing Formazin Dilutions 13

Appendix III. Preparing StablCal Standards 13

Appendix IV. Using Low Range StablCal Standards for Verification of Calibration 15

Appendix V. Apparatus and Reagents 18

4IntroductionImportance of StandardsAll scientific work depends on precise measurement toassure meaningful results. British mathematician andphysicist Lord Kelvin expressed the necessity of accuratemeasurement over 100 years ago: If you can measurethat of which you speak, and you can express it innumber, you know something of your subject; but if you cannot measure it, your knowledge is meager and unsatisfactory.

Standards are the foundation of scientific measurement,the fundamental units in which we describe our work.They provide the terms we need to express conditions,procedures, and results, serve as a reference againstwhich other measurements are compared, and establisha basis for compatibility in communication of scientificwork throughout the world.

Definition of Primary StandardPhysical measurements such as length, weight, mass, andvolume are based on primary standards. Primarystandards, once defined, require no further reference.The primary standard of length, the meter unit, is definedas being equal to 1,650,763.73 wavelengths of theorange-red light emitted by Krypton-86, and the primarystandard of volume, the liter, is defined as the spaceoccupied by 1 dm3=10-3 m3.

Some primary standards are by agreement. For instance,in an alternative definition above of the standard forvolume, another unit the kilogram is used. Aspecific bar of platinum-iridium metal is used as theworlds accepted definition of a kilogram, the unit ofmass. In chemistry, primary standards are often definedby repeatable experiment. For instance, a primarystandard can be a compound that can be preparedrepeatably with acceptable purity and stability. As theterm primary implies, its preparation results inestablishment of the reference, the basis by which allother measurements are taken. Once the primarystandard is defined, no other reference is necessary.

Turbidity StandardsThe subject of standards in turbidimetric measurement is complicated partly by the variety of types of standardsin common use and acceptable for reporting purposes by organizations such as the USEPA1 and StandardMethods, and partly by the terminology or definitionapplied to them.

In the 19th edition of Standard Methods for the Examinationof Water and Wastewater, clarification was made in definingprimary versus secondary standards. Standard Methodsdefines a primary standard as one that is prepared by the userfrom traceable raw materials, using precise methodologiesand under controlled environmental conditions. In turbidity,the only standard that can be defined as primary is formazinthat has been prepared by the user on the bench.

Standard Methods now defines secondary standards asthose standards a manufacturer (or an independenttesting organization) has certified to give instrumentcalibration results equivalent (within certain limits) toresults obtained when an instrument is calibrated withuser-prepared formazin standards (primary standards).Various standards that are suitable for calibration areavailable, including commercial stock suspensions of4000 NTU formazin, stabilized formazin suspensions(StablCal Stabilized Formazin Standards, also referred to as StablCal Standards, StablCal Solutions, or StablCal),and commercial suspensions of microspheres of styrenedivinylbenzene copolymer.

Calibration verification items supplied by instrumentmanufacturers, such as sealed sample cells filled with alatex suspension or with metal oxide particles inpolymer gel, are used to check a calibration and are notto be used in performing instrument calibrations.

If there is a discrepancy on accuracy of a standard or aninstrument, the primary standards (i. e., user-preparedformazin) are to be used to govern the validity of theissue. In turbidity, formazin is the only recognized trueprimary standard and all other standards are traced backto formazin. Further, instrument algorithms andspecifications for turbidimeters should be designedaround this primary standard.

Currently, the USEPA designates user-prepared formazin,commercial stock formazin suspensions, and commercialstyrene divinylbenzene suspensions (sometimes referredto as alternative standards) as primary standards andusable for reporting purposes. The term secondary isused for those standards that are used only to check orverify calibrations. Under this USEPA definition, primarydoes not have anything to do with traceability, only toacceptability for USEPA reporting purposes.

Primary standards are also used for measuring anddetermining the value of all other standards. Under theUSEPA definition, secondary standards, once their valuesare determined versus primary formazin, are used toverify the calibration of a turbidimeter. However, thesestandards are not to be used for calibrating instruments.Examples of these standards include the metal oxidegels, latex, and any non-aqueous standards that aredefined to monitor calibrations on a day-to-day basis.This usage depends on the design of the standard. Onthe other hand, formazin, StablCal standards and Amco AEPA-1 Alternative Standards are designed to calibratethe instruments.

In the discussion that follows, we will examine themeaning and importance of primary and secondarystandards of turbidity, various commercial forms andtheir proper use, and alternative standards. We willconsider the advantages and disadvantages associatedwith each.

1United States Environmental Protection Agency

5Development of the Primary Turbidity StandardDefinition of TurbidityBy definition, turbidity is an optical property that causeslight to be scattered and absorbed rather thantransmitted in straight lines through the sample.2

Scattering and absorption are caused when undissolvedparticles such as silt, clay, algae, organic matter, andmicroorganism suspended in a sample interfere withlight passing through. Their effect is to impart a haze orcloudiness to the sample. Simply defined, turbidity is theopposite of clarity.

It is possible to quantify turbidity by measuring theamount of light scattered away from the direction ofincident light, or the amount of light absorbed from theincident beam. In order for either of these measurementsto be meaningful, however, it is necessary to have aframe of reference, or standard, against which thenumbers can be compared.

Turbidity Units of MeasureEstablishing a standard for turbidity measurement anddefining its units is not as simple a process as it is formany chemical measurements. Turbidity is a qualitativeproperty, usually caused by a wide variety of substances.Light-scattering properties of a given particle depend onits size, shape, and refractive index (see Figure 1).Extremely small particles scatter short wavelength lightdifferently than long wavelength light. As particle sizeincreases, the effect is the opposite. The complex natureof the scattering effect and the number of variablesinvolved make absolute comparisons difficult.

Early efforts to quantify turbidity were done by Whippleand Jackson in 1900. Their work involved theformulation of a standard suspension of 1000 parts permillion of diatomaceous earth in distilled water.Although the suspension could not be formulatedrepeatedly using materials from different sources, thiswas the first relative scale of comparison for turbiditymeasurements. Compared to the standard suspension, asample could be described as having the same turbidityas X parts per million suspended silica in distilled water.Units of parts per million, silica turbidity were used,and are still found in references today. Because this wasan equivalent scale and did not relate to a specificquantity of matter in a sample, the unit of measure waslater redefined as a Turbidity Unit (TU), the basic unit ofmeasure accepted today.

Turbidity Units are usually stated with a qualifier thatspecifies the method of measurement. A nephelometermeasures light scattered by a sample in the direction thatis 90 degrees from the incident light path (see Figure 2);turbidity measured in this way is stated in NephelometricTurbidity Units (NTU). Visual extinction methods ofmeasurement such as the Jackson Candle Turbidimetermethod measure attenuation of light in the direction ofthe incident beam; these methods report in JacksonTurbidity Units (JTU). Note that although both unitshave the same basis, results derived by the twomeasurement techniques can differ substantially for thesame sample.

Formazin as a Primary Turbidity StandardDefining a unit of measure and a relative calibration scalewas a key development in turbidity measurement. Themajor remaining difficulty was that the standardsuspension could not be formulated repeatably whenusing natural materials from different sources. Manyorganic and inorganic substances were proposed for usein preparing a primary turbidity standard. Formazin, apolymer suspension, was proposed for the role as earlyas 1926, but little research resulted. It wasnt until theearly 1960s that Johns-Manville Corporation representativescontacted Hach Company and also suggested formazin.

2Standard Methods for the Examination of Water and Wastewater, 19thedition, 1995, page 2-8.

IncidentBeam

IncidentBeam

IncidentBeam

Size: Smaller Than 1/10the Wavelength of LightDescription: Symmetric

Size: Approximately 1/4 theWavelength of LightDescription: Scattering Concentratedin Forward Direction

Size: Larger Than the Wavelength of LightDescription: Extreme Concentrated of Scattering in ForwardDirection; Development of Maxima and Minima of ScatteringIntensity at Wider Angles

(A) Small Particles (B) Large Particles

(C) Larger Particles

GlassSample Cell

TransmittedLight

90 ScatteredLight

Detector

Aperture

Lamp

Lens

Figure 1: Scattering is a function of light wavelength andparticle size. Assuming a wavelength of approximately 600 nm, the figure shows angular patterns of scatteredlight from particles of three relative sizes: A) < 60 nm, B) 150 nm, and C) 6000 nm. From Brumberger et al,Light Scattering, Science and Technology, November,1968, page 38.

Figure 2: A nephelometer quantifies turbidity by measuringlight scattered by suspended particulate material. This con-figuration detects light scattered at 90 from the incident beam.

6Pursuing their suggestion, Hach chemists found thatformazin, prepared by combining solutions of hydrazinesulfate and hexamethylenetetramine, could indeed meetall the requirements of a primary standard. Under thecorrect conditions, formazin could be prepared as a purecompound, its preparation was reproducible to within 1%, and it was relatively stable and easy to use.

Additional work led Hach researchers to define a particularformazin suspension, equal volumes of a 1 gram/100 mLsolution of hydrazine sulfate and a 10 gram/100 mLsolution of hexamethylenetetramine, as a 4000 NTUstandard. Later, American Public Health Association(APHA) and American Water Works Association (AWWA)committees accepted this system. Formazin wasspecified as the primary turbidity standard beginningwith Standard Methods, 13th edition, 1971. The UnitedStates Environmental Protection Agency (USEPA) has alsospecified formazin as the primary turbidity standard.

Formazin has several desirable characteristics that makeit an excellent analytical standard. First, it can bereproducibly prepared from assayed raw materials.Second, the physical characteristics make it a desirablelight-scatter calibration standard. The formazin polymerconsists of several different length chains which fold into random configurations. These result in a wide array of particle shapes and sizes ranging from 10 microns. Studies on the particle distributionindicate irregular distributions among different lots ofstandards, but the overall statistical nephelometricscatter is very reproducible. This wide array of particlesizes and shapes analytically fits the wide possibility ofparticle sizes that are found in real world samples. Dueto the statistical reproducibility of the nephelometricscatter of white light by the formazin polymer,instruments with the traditional tungsten filament whitelight optical designs can be calibrated with a high degreeof accuracy and reproducibility. The randomness ofparticle shapes and sizes within formazin standards yieldstatistically reproducible scatter on all makes and modelsof turbidimeters.

Since real-world samples have a wide distribution ofparticle sizes and shapes, the ideal turbidity standardshould encompass this variability. Formazin, with itsbroad particle distribution, is the one turbidity standardthat will encompass the particle shapes and sizes of mostaqueous samples. Other standards that have very narrowparticle distributions and the same particle shapes mayvery well be outside the constraints of many real-worldsamples. This feature can lead to unsuspected error inturbidity measurement. Attempts have been made toreplicate the size and distribution of formazin standardsand have had only limited success. Further, theproduction of such standards is complex, timeconsuming and costly.

Formazin is not without its limitations. The starting rawmaterials used in the synthesis of the formazin polymer,hydrazine sulfate and hexamethylenetetramine(hexamine), are currently listed as a suspectedcarcinogen and an experimental mutagen respectively.However, in the final synthesized stock formazinpolymer solution (4000 NTU) and in any dilutions of thisstock standard, these two starting raw materials are invery low concentrations. Thus, the exposure level ofthese starting raw materials is at exponentially lowerlevels in the final calibration standards containing thesynthesized formazin polymer. When the quantities ofthese starting raw materials in the finished stock standardand subsequent dilutions are compared to theconcentration of the starting raw materials presentbefore the synthesis of the polymer, the quantity ofhydrazine sulfate is several orders of magnitude lower inthe finished standards. An independent analysis forhydrazine sulfate in formazin calibration standardsconcluded that there is between 10-2 and 10-3 times lesshydrazine sulfate present than the amount present at thebeginning synthesis step of the formazin polymer. Thereis no strong supporting evidence on the hazardousnessclaims on the finished formazin calibration standards.

A second limitation of formazin is its stability. Formazinis only stable at high concentrations. In-house stabilitystudies indicate that formazin standards above 400 NTUare stable for greater than one year, standards between20 and 400 NTU are stable for approximately one month, standards between 2 and 20 NTU are stable forapproximately 12 to 24 hours; and, standards below 2 NTU are stable for 1 hour or less. Hach does notrecommend user preparation of formazin standardsbelow 2 NTU.

When an analyst would like to calibrate or verify aninstrument calibration in the measurement range ofinterest, the preparation of a low-turbidity formazinstandard is extremely difficult. Particle contamination isusually imminent and extreme preventive precautionsmust be taken through every step of this preparationprocess. Due to the care that must be taken,preparation of low-turbidity formazin standards is a verytedious, difficult and timely process.

7Using Formazin StandardsPreparation of Formazin Stock SuspensionFormazin is an aqueous suspension of an insoluble polymerformed by the condensation reaction between hydrazinesulfate and hexamethylenetetramine (see Figure 3). Asstated previously, a 4000 NTU suspension is prepared bycombining equal volumes of a 1.000 g/100 mL solution ofhydrazine sulfate and a 10.0 g/100 mL solution ofhexamethylenetetramine. After standing at 25 1 C for24 hours, the solution develops a white particulatesuspension. Appendix II describes the completeprocedure for preparing dilute formazin standards.

The 4000 NTU formazin suspension can be reproducedwith a high degree of accuracy, and is very stable whenstored properly (see below). However, formazinpreparation is affected by a number of variables,including purity of materials, temperature and exposureto light. These factors must be accounted for to assurereproducibility and accuracy. For convenience, analystsmay wish to work with commercially-prepared 4000NTU formazin standards. Prepared under the properconditions, these standards ensure repeatability (1% lot-to-lot) for the greatest accuracy and consistency inmeasurement and reporting.

Preparation of Formazin Calibration StandardsCalibration or working standards may be prepared bydiluting a 4000 NTU stock suspension with high-puritywater. Standard Methods, 19th edition, 1995, alsodescribes procedures for directly preparing a stocksuspension equal to 4000 NTU and a standardsuspension (by dilution) of 40 NTU. Appendix IIcontains complete procedures, with dilution ratios,

for preparing turbidity calibration standards from 2 to200 NTU from an initial dilution of 1000 NTU. Turbiditystandards below 2 NTU are difficult to prepare accuratelybecause the dilution ratio is so high that even minutevariations in the volumes of formazin used and dilutionwater quality are significant.

Note that there is no sure way of producing or obtainingabsolutely pure, turbidity-free water to use for dilutions.Multiple distillations, deionization, and ultrafiltration can all leave some residual particulate contamination,which will contribute to the turbidity of the finaldilution. However, it is possible to account for lowlevels of dilution water turbidity by taking readings overa range of dilutions and extrapolating back to the puredilution water value. Thus, given an initial dilution water turbidity less than 0.5 NTU, quite accurate low-level turbidity measurements can be made without theneed to prepare extremely low-level calibrationstandards. Currently available turbidimeters are capableof automatically determining and compensating for initialdilution water turbidity values less than 0.5 NTU duringthe calibration procedure.

Storage of Formazin Stock SuspensionsChemical stability of formazin suspensions, like that ofmany other chemicals, is highly dependent on storageconditions. Exposure to heat or direct sunlight willbreak down the polymer structure, while prolongedexposure to ambient air allows the suspending fluid toevaporate and air-borne contaminants to enter. Storeformazin standards in a cool, dark place. Refrigerationwill provide extended life, but is not absolutely essential.Once a formazin standard has been removed from its

Figure 3: Preparation of formazin

(1)

(2)H

H

n C O

H H

H H

:N N:+n2

H

H

6 C O

N

N N

N

+ 6 H 2 O + 2H2SO4 + 2(NH4)2 SO4

+ n H 2 O

x

Hexamethylenetetramine

Hydrazine

(from hydrazinesulfate) Formaldehyde

Formazin

N N N

N N N

8storage bottle and used, it should be discarded. Do not leave containers open longer than necessary, and seal them tightly for storage. The pouring of thesestandards back into their storage containers willintroduce contamination and could lead to erroneousfuture calibrations.

Properly stored, a 4000 NTU formazin suspension will bestable for about one year. Dilute formazin suspensionsused as working standards are considerably less stable.As a rule the lower the concentration of a formazinstandard, the lower the stability of that standard.

StablCal StabilizedFormazin StandardsA new turbidity standard has been developed for use incalibrating or verifying the performance of anyturbidimeter. StablCal Stabilized Formazin Standardscontain the same light scattering polymer as traditionalformazin primary turbidity standards. By using adifferent matrix, the polymer in StablCal standards willnot deteriorate over time as is the case with low turbidityformazin standards. Due to this enhanced stability,StablCal standards of any concentration ranging up to4000 NTU can be manufactured and packaged in ready-to-use formats. Thus, time is saved and direct exposureto the standard is minimized.

In trying to find a new or improved turbidity standardthat would replace formazin, the following requirementsfor this standard must be met:

1. The standard should be stable for a long period of time.

2. The standard should be safe to handle.

3. The standard should be easy to prepare and to use.

4. The standard should have a particle size distributionthat will encompass most water samples.

5. The standard should provide the same light scatter characteristics that closely resemble that oftraditional formazin.

6. The standards should not be instrument specific. Forexample, a 2 NTU standard should read 2 NTU on anyinstrument regardless of make or model. Thesestandards should be able to be interchanged with freshlyprepared formazin standards of the same NTU value.

7. Preparation of the standard should be reproduciblewhen using assayed starting materials. Accuratemeasurement apparatus and techniques must also beused. The preparation of these standards should bedemonstrated to be repeatable.

8. Volumetric dilutions of a concentrated standardshould be accurate and linear.

Several different materials were tested to determine ifthey met these requirements. Research into the area offinding a suitable replacement for formazin has been metwith limited success. Formazin turbidity standards metmany of these requirements, but still lacked the majorfeature of stability (particularly at low turbidity levels).

StablCal Stabilized Formazin Standards do meet all ofthese criteria. Two of the most important criteria arethat the standards must be stable over time, regardless ofconcentration, and the standards provide comparablecalibration response on any turbidimeter. Research thatled to the development of the StablCal standards focusedon these two areas of improvement over traditionalformazin standards.

The development of a new stable turbidity standard(StablCal) is described below. This is followed by thecomparability study performed after the stability was proven.

Stability ResearchThe key to stabilizing diluteformazin solutions is based on two points: Le ChateliersPrinciple and proper standard dilution. Le ChateliersPrinciple states: When more reactant is added to, orsome product is removed from, an equilibrium mixture,thereby changing the concentration of the reactant orproduct, the net reaction will move from left to right togive a new equilibrium and more products beingproduced. In applying this principle to stabilizingformazin standards, it is known that there is anequilibrium that forms between reactants and productsbecause residual hydrazine, the theoretical limitingreagent in the condensation reaction to produce theformazin polymer, is still present in parts per millionamounts. This indicates that this reaction does proceedto near completion (>99%) but not to full completion.Formazin standards of low turbidity have the productsremoved through dilution. When a dilution is made, theequilibrium shifts back to the left toward the reactantsand away from the products. Thus, the formazinpolymer is more likely to degrade in order to compensatefor this equilibrium shift. (The mechanism of thisdegradation is thought to be hydrolysis of the formazinpolymer from the residual protons in solution.) In orderto stabilize the formazin polymer, the reaction equilibriumneeds to be pushed to the right as far as possible.

The second point to stabilizing the formazin polymerwas based on changes that are made to the polymer andthe matrix relative to a dilution. When a stock standardis diluted, the dilution is traditionally performed usingultrapure water. During this dilution, both the turbidity-causing polymer and any other components in thematrix are diluted. Theoretically, only the turbidity-causing formazin polymer should be diluted. Theconcentrations of all other components in the matrixshould remain unchanged. Thus, when a concentratedformazin stock solution is traditionally diluted to make alow-turbidity standard using ultrapure dilution water, allcomponents in the matrix are also diluted. The

equilibrium then shifts slightly back to the left, and theproducts are less stable. Thus, the matrix of a formazinstandard is significantly different in a low-turbidity standardversus the highly concentrated 4000 NTU stock standard.

To verify this theory, some low turbidity formazinstandards were prepared using two different procedures.In the first procedure, a 2-NTU standard was preparedusing traditional techniques and ultrapure dilution waterwas used as the standard diluent. In the secondprocedure, a 2-NTU formazin standard was preparedusing the same traditional techniques, but dilutions wereperformed using an ultra-filtered solution that replicatedthe matrix present in the stable 4000 NTU formazinconcentrate. The standards were then monitored forstability for over 2+ years. This stability data issummarized in the graph in the table on the right and inFigure 4 . This graph shows the 2-NTU standardprepared using dilution water remained stable for only abrief period of time. However, the other standard,prepared using the ultra-filtered solution that replicatedthe 4000 NTU matrix, continued to remain relativelyunchanged throughout the duration of the test. Thisstandard is the StablCal Stabilized Formazin Standard.

9

Time in Days 2 NTU 2 NTU Since The Standards Traditional StablCal

Were Prepared Formazin Formazin0 2.03 1.968 1.95 1.9616 1.94 1.9524 1.80 1.9631 2.02 1.9641 1.66 1.9755 1.64 1.9665 1.62 1.9573 1.70 1.9784 1.46 1.9294 1.27 1.96105 1.35 1.94120 1.03 1.93133 0.88 1.93141 0.75 1.94233 0.39 1.90369 0.58 1.91478 0.05 1.96576 0.27 1.96602 0.13 1.96671 0.09 1.93716 0.08 1.91734 0.08 1.95827 0.07 1.95

2.50

2.00

1.50

1.00

0.50

0.000 100 200 300 400 500 600 700 800

Time in Days Since the Standards Were Prepared

Turb

idity

Rea

ding

in N

TU's

2 NTU Traditional Formazin

2 NTU StablCal Formazin

Figure 4: Stability Comparison Between Turbidity Standards Prepared at 2 NTUTraditional Formazin and StablCal Stabilized Formazin

10

Comparability:Once the StablCal standards were demonstrated to bestable, the next test was to determine and compare thefunctional aspects of StablCal standards to traditionalprepared formazin standards.

The comparability research involved determining theperformance of several turbidimeters that had beencalibrated on StablCal standards to other formazinturbidity standards that had been prepared usingtraditional methodologies. The testing involved severalmakes and models of instruments. These instrumentswere calibrated on StablCal standards in the reportingrange of 0 to 40 NTU. Then, fresh formazin standardswere prepared from a primary stock standardconcentrate using ultrapure water as the diluent. Thetraditional formazin and StablCal Standards were bothread versus the StablCal based calibration on eachinstrument. The data was collected into Table 1.

Table 1 shows the instrument performance when usingStablCal Stabilized Formazin Standards for calibration.The instruments accurately performed to within 1 percentof the theoretical value of the standards that were readin this study. This accuracy was based on severalinstruments, which further enhances the instrument-to-instrument comparability and accuracy of StablCalstandards. Since the particle distribution of the StablCalStabilized Formazin Standards matches that of formazin,the standard has a universal usage. Any standard can beused on any turbidimeter or nephelometer. Instrument-specific StablCal standards are not necessary.

Instrument 40 NTU 40 NTU 20 NTU 20 NTU 10 NTU 10 NTU 5 NTU 5 NTU 2 NTU 2 NTU 1 NTU 1 NTU Make and StablCal Standard StablCal Standard StablCal Standard StablCal Standard StablCal Standard StablCal Standard

Model Formazin Formazin Formazin Formazin Formazin Formazin Formazin Formazin Formazin Formazin Formazin Formazin

Hach 39.7 39.4 20.1 19.9 10.0 9.91 5.15 5.02 2.03 2.07 1.10 1.052100AN

(NonRatioMode)

Hach 40.0 40.3 19.7 19.9 9.78 10.1 4.94 5.00 1.97 2.06 1.07 1.032100AN

Ratio Mode

Hach 39.6 39.5 19.9 19.7 9.95 9.76 5.15 4.99 2.00 2.06 1.06 1.042100AN ISNonRatio

Mode

Hach 39.8 40.3 19.7 19.7 9.80 9.89 5.03 4.98 1.97 2.06 1.06 1.032100AN IS

RatioMode

Monitek 38.3 39.0 19.7 19.8 9.90 9.87 4.97 4.83 1.95 1.97 1.05 1.01Model 21

LaMotte 41.0 40.4 20.0 20.0 10.1 10.1 4.78 5.00 2.00 2.04 0.96 1.00Model2008

Hach 41.5 39.6 20.0 19.5 9.95 10.0 5.00 5.12 2.20 2.15 1.10 1.082100A

Hach 39.0 40.3 19.4 19.8 9.70 10.1 4.94 5.04 1.97 2.08 1.09 1.072100P

Table 1: Instruments calibrated on traditional formazin. Both StablCal Stabilized Formazin Standards andtraditional formazin standards were read relative to traditional formazin. The results are displayed below:

11

Conclusion:The StablCal Stabilized Formazin Standards have beenshown to be both stable and read comparable totraditional, freshly prepared formazin standards.Standards in the range of 0.30 to 4000 NTU have beendemonstrated to remain within 5 percent of their originalpreparation values for a minimum of two years. From acomparability standpoint, StablCal Standards can beinterchangeably used as calibration standards on anyturbidimeter with only very minimal differences in theinstrument response.

The stabilization of formazin has resulted in thedevelopment of the StablCal standards. These standardsserve as the solutions to those problems that wereassociated with traditional formazin standards. Thisstabilization allows for these standards to be packaged instructures that greatly reduce any kind of potentialexposure to the user from the standard. Further, whencomparing StablCal to traditional formazin standards ofequal concentration, studies have shown that residualhydrazine sulfate in StablCal is reduced by two to threeorders of magnitude. The stabilization of formazin in theStablCal standards provides the user with ready-to-usestandards, and the large quantity of time required toprepare low turbidity traditional formazin standards isnow eliminated. Users can take these stabilizedstandards and use them in the field, and at the same timebe confident that the standards are accurate andrepeatable in these non-laboratory settings.

Secondary StandardsDefinition of Secondary StandardPrimary standards may be prepared directly, and theirvalues are defined. The value of a secondary standard is derived by reference to a primary standard. Bymeasuring against a primary standard, and in many casesadjusting to match the primary, a secondary standard isestablished as having a known value to be used forsubsequent comparisons.

In physical measurement, secondary standards arerequired because there may be only one master standarddefined and accepted as the standard unit (e.g. kilogram),and copies must be made for actual use. Thesesecondary standards are often referred to as transferstandards, and are usually certified. Their derived valueis traceable to the original primary standard. It isaccepted that copies cannot be made to absolute perfectionand contain probability for error.

In turbidity measurement, anyone with the proper materialsand equipment can directly synthesize the primarystandard. In this case, the need for secondary standardsis more a matter of convenience, due to the instability ofdilute formazin suspensions and the need to prepare freshformazin dilutions for each use. Secondary standardsare used to check instrument calibration stabilityand are not to be used to perform a calibration.

Application of Secondary Turbidity StandardsHach's secondary turbidity standards are particulatesuspensions formulated to give results that match or readwithin a specified turbidity range. The value of asecondary standard is defined by comparison with adilute formazin suspension. These types of secondarystandards are used to verify instrument performancebetween primary standard calibrations, providing a stablereference and eliminating the need to prepare freshformazin dilutions for routine calibration checks. Thesestandards are not to be used to perform calibrations.

Gelex Secondary Turbidity StandardsGelex secondary turbidity standards are a recent innovationin the field of turbidity measurement. Gelex standardsare suspensions of metal oxide particles in a rubber-likepolymer gel. Locked in place by the gel, the particlescannot settle, drift, or coagulate. The gel medium alsoprotects the metal oxide particles from additionaloxidation or contamination.

With care, Gelex secondary turbidity standards offer anearly indefinite shelf life and very stable turbidity value.For many years, Hach provided secondary standardsmade from suspensions of polystyrene latex spheres.Because their shelf life was limited to about a year, Hachdiscontinued production of latex standards in favor ofGelex standards.

Using Gelex Turbidity StandardsThe light-scattering properties of any particle are afunction of its size and shape, its refractive index, and the wavelength of the incident light. Differentturbidimeters, using different light sources, detectors,and optical configurations, will respond differently to any standard other than formazin, the standard to whichtheyve been calibrated. Gelex secondary standards, nothaving exactly the same light-scattering characteristicsas formazin, must be specifically formulated for theturbidimeter model with which they will be used.(Understanding Turbidity Science, Technical InformationSeries Booklet No. 11, published by Hach Company,contains additional information about light scatter.)

Each Gelex secondary standard is supplied in an individualglass vial, and is formulated to provide a nominal turbidityvalue for a specific turbidity range on a specific Hachturbidimeter model. Turbidity ranges are marked on theindividual vials. Actual values will vary slightly due tovariations in glass vials and individual instruments. Allsecondary standards must be assigned a value on theturbidimeter with which they will be used, immediatelyafter the instrument has been calibrated using formazinprimary standards. The assigned value should be markedon the vial for use in verifying instrument calibration inday-to-day use. The Gelex standards should be re-measured each time the instrument is recalibrated withformazin, and new values reassigned as necessary.

12

Care and Storage of Gelex StandardsCare should be taken in handling Gelex standards toavoid scratching the glass vial. While the stability of thegel medium minimizes the effects of heat, light andvibration on shelf life or value, it is important to protectGelex standards from temperature extremes. Exposureto freezing or to temperatures greater than 50 C (122 F)for long periods may alter or damage the standards.

Alternative StandardsDefinition of Alternative StandardPrimary formazin standards are accepted by the USEPAfor turbidimeter calibration when results are beingreported. Secondary standards such as Gelex, defined bycomparison with formazin, are then used to verifycalibration in day-to-day use. An alternative type ofstandard has recently been accepted by the USEPA forcalibration purposes when reporting results. Currentlyavailable alternative standards cannot be prepared directly,requiring special facilities and controls, and still must be compared and traced to primary formazin duringproduction in order to determine or adjust their value.

Application of Alternative StandardsAlternative standards provide convenience in field use or other situations where the preparation of formazindilutions is not practical. They are stable at low turbidityvalues and can be used for both calibration purposes andday-to-day verification.

Current alternative standards use a blend of styrenedivinylbenzene (SDVB) cross-linked copolymermicrospheres suspended in an ultrapure, aqueousmedium. The spheres range from about 0.1 to 0.3microns in diameter with a mean diameter of about 0.2microns. This size distribution is narrower than thatfound in formazin (0.01 to 10.0 microns) and the particlesare uniform spheres, so light-scattering properties andrefractive characteristics are not the same as formazin.Alternative standards are adjusted during manufacture bycomparison with formazin on the specific instrument forwhich they are prepared, and must be considered bothinstrument- and calibration-point specific. That is, bothformazin measurements and alternative standardmeasurements will be within accuracy specifications forthe values used as calibration points on the instrumentfor which they are intended, when the accuracy of theinstrument, formazin standard, and alternative standardare all taken into consideration.

Care and Storage of Alternative StandardsAlternative SDVB standards come in a range of NTU valuesand are ready for use. They may be transferred directlyfrom bottle to sample cell if normal precautions are takento avoid contamination. Never dilute alternative standards

and never pour material back into the bottle. Neverfreeze or boil. In addition, standards should be keptaway from direct heat. Refrigeration is not recommended.

Hach Company does not guarantee thatperformance specifications for its turbidimeterswill be met if SDVB standards are used forcalibration. Specifications are guaranteed only iffreshly prepared formazin or StablCal StabilizedFormazin Standards are used for calibration.

Appendix I. Preparation ofPrimary FormazinFormazin is an aqueous suspension formed by the reactionbetween hydrazine sulfate and hexamethylenetetramine.A 4000 NTU suspension is prepared by combiningspecified solutions of both and allowing the mixture to stand for 48 hours at 25 C. Under the correctconditions, a 4000 NTU formazin suspension can bereproduced with a high degree of accuracy ( 1%) and isstable when properly stored.

Dilutions of the 4000 NTU stock suspension are lessstable than original stock. Properly stored, a 4000 NTUsuspension will last at least two years. A 400 NTUdilution is stable for only about one year. Hach chemistsstrongly suggest that working dilutions be prepared froma 4000 NTU stock suspension, used immediately, andthen discarded. (See Appendix III for a complete listingof supplies available from Hach for preparing formazin.)

Storage of Formazin SuspensionsThe chemical stability of a formazin suspension is highlydependent on storage conditions. Exposure to heat ordirect sunlight and prolonged exposure to ambient aircan degrade shelf life significantly. Heat and light willbreak down the polymer structure, while exposure to airallows the suspending fluid to evaporate and air-bornecontaminants to enter.

Store in a cool, dark place. Do not store above 40 C.Refrigeration will provide extended shelf life but is notessential. Do not leave containers open longer thannecessary and seal them tightly for storage. Allowformazin standards to acclimate back to roomtemperature before being use to calibrate.

Formulating 4000 NTU Formazin StockSuspension1. Dissolve 1.000 g of ACS grade hydrazine sulfate,N2H4H2SO4, in ultra filtered deionized water and diluteto 100 mL in a Class A, 100 mL volumetric flask. Thehydrazine sulfate should be tested for purity, and onlyused if it is >99% pure.

2. Dissolve 10.00 g of analytical grade hexamethylenete-tramine, (CH2)6N4, in ultra-filtered deionized water anddilute to 100 mL in a Class A, 100 mL volumetric flask.

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3. Quantitatively combine the equal volumes of thehydrazine sulfate solution and the hexamethylenetetraminesolution into a clean, dry flask and mix thoroughly. Letthe mixture stand for 48 hours at 25 1 C. A whitepolymer suspension will form in the solution during thisperiod. The turbidity of this primary formazinsuspension is 4000 NTU. Store this suspension in abottle that filters ultraviolet light.

The recommended storage for formazin is between 5 and 25 C. Allow the standard to come to equilibriumtemperature and mix well before use.

Appendix II. Preparing FormazinDilutions1. Using a 10.0 mL TenSette Pipet, prepare a 1000 NTUformazin working suspension by transferring 25 mL of4000 NTU formazin stock suspension into a 100 mLvolumetric flask*. Dilute to the mark with turbidity-freewater; swirl to mix. See notes A, B, C, and D.

2. Using a 10.0 mL TenSette Pipet or a 1.0 mL TenSettePipet, transfer the desired amount of the 1000 NTUworking suspension to a 100 mL volumetric flask. Diluteto the mark with turbidity-free water and mix.

3. Rinse a clean turbidimeter sample cell once with theprepared formazin standard, then carefully pour theappropriate volume of standard into the cell. Insert thecell into the turbidimeter and calibrate according toinstructions in the turbidimeter manual.

Table 2 shows the amounts of 1000 NTU initial dilutionrequired to prepare 100 mL suspensions for a range ofworking standards.

Table 2

Desired NTU mL of 1000 NTUStandard Suspension

2 0.24 0.46 0.610 1.020 2.040 4.0100 10.0200 20.0

NotesA. Dilute formazin standards are stable only for a short period of time, and should be used immediatelyafter preparation.

B. Use Hach prepared 4000 NTU Formazin StockSuspension, or prepare a 4000 NTU suspensionfollowing the procedure in Appendix I.

C. Turbidity-free water for measuring turbidities as lowas 0.02 NTU may be prepared by filtering distilled waterthrough a 0.2-micron membrane filter or through areverse osmosis filtration unit. High quality demineralizedwater may also be used as long as its turbidity is below0.1 NTU.

D. Mix turbidity standards by swirling or gently inverting.Shaking may introduce bubbles and cause erroneousreadings. Vigorous shaking will also introduce oxygenand cause oxidation of the formazin polymer chains, andwill also cause fracturing of the polymer chains.

Appendix III. Preparing andUsing StablCal StabilizedFormazin Standards: Do not transfer the standard to another containerfor storage.

Store standards between 5 and 25 C. Avoidprolonged exposure to temperatures exceeding 25 C.

Do not pour standard from the sample cell back intoits storage container (Bulk Solutions).

For long-term storage, refrigeration at 5 C isrecommended. Do not store above 25 C.

Always allow the standard to acclimate to ambientinstrument conditions before use (not to exceed 40 C).

Store away from direct sunlight. Vials should be storedin their respective kit with the cover on.

For all StablCal standards greater than the diluentsolution, mix the standard well before transferring to asample cell. Never mix the diluent solution.

The required amount of 1000 NTU working suspension to be used may bedetermined by using the formula:(volume of dilution to be prepared) X (desired NTU value) = required amount

(NTU of working suspension)For example, to prepare a 100 mL, 4 NTU suspension starting with a 1000NTU working suspension, find the required amount of 1000 NTU suspension:

(100 mL) X (4 NTU) = 0.4 mL(1000 NTU)

To prepare the working standard, transfer 0.4 mL of 1000 NTU suspensionto a flask and dilute to 50 mL with ultra-low turbidity water.

* For greatest accuracy, procedures may be performed with Class Avolumetric glassware.

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Instructions are for two conditions. Follow the first setof instructions if your standards have been sittingundisturbed for longer than one month, or if you need to prepare your standards. Follow the second set ofinstructions if the standards have been prepared(transferred into vials) and are used frequently.

Preparing StablCal Standards(After Long-Term Storage):(Bulk or Vial Standards):Note: If using bulk standards, perform step 1 (below)before breaking the seal on the bottle. If your standardshave been sitting undisturbed for longer than onemonth, shaking the standard will breakup thecondensed suspension into its original particle sizes.

Note: If you are using the

1. Thoroughly clean andrinse the sample cell.

Note: For laboratory and portableinstruments, see Note a. For a processturbidimeter, see Note b.

2. Rinse the outside of theStablCal Standard bottlewith water. This removesany dust or particulatematter that couldcontaminate the standard.Dry the bottle with a lint-free cloth or tissue.

Note: If necessary, allow the standardto come to ambient temperaturebefore use.

3. Gently invert the Low-Range StablCal Standardbottle at least 50 times tothoroughly mix and re-suspend the formazinpolymer. See Note c.

Note: Do not shake or invert rapidly.Entrained air bubbles and erroneousreadings could result. See Note d .

4. Using a clean knife,carefully remove the heatseal from the StablCalStandard bottle. Afterremoving the heat seal,always keep the containertightly capped in order tominimize contamination.

Note: Uncontaminated standardwill retain its turbidity value for atleast one month after removing theheat seal.

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5. Fill a sample cell at leasthalf full with StablCalStandard. Cap and invert the sample cell severaltimes. Immediately discardthe rinsings. Repeat thisrinsing procedure at leastone more time.

6. Slowly fill the samplecell with StablCal Standard.Immediately cap both thesample cell and the bottle ofstandard. See Notes e and f.

Note: For a process turbidimeter, fill the turbidimeter body withstandard and immediately re-position the turbidimeter head tobegin taking readings.

7. Wipe the cell with asoft, lint-free cloth to removewater spots and fingerprints.

8. Apply a thin film ofsilicone oil to the samplecell. Wipe with a soft clothto obtain a thin and evenfilm over the entire surface.

Appendix IV. Using Low Range StablCal Standards for verification of calibration(Standards between 0.10 and 1.0 NTU)

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9. Turn the instrument on.Allow it to warm up per themanufacturers instructions.

10. Place the sample cellin the instrument cellcompartment using theproper indexed orientation.Close the lid.

11. Wait at least 5 minutesto allow entrained bubblesto vacate the standardmatrix. Take measurementbetween 5 and 60 minutes.

Note: During the waiting period, theinstruments digital display readingmay fluctuate. As bubbles dissipatefrom the standard, the display valuewill become more stable.

12. Record the stablereading, which will remainunchanged (to the nearest0.003 NTU) for at least 15 seconds.

Note: Spikes can occur and aregenerally displayed as a suddenincrease in turbidity, followed by aslower decrease back to the base value.Stray bubbles, uncharacteristicallylarge particles, or electronic noise cancause spiking. Ignore spikes.

13. The instrumentaccuracy is verified if thereading is within thetolerances on the StablCalStandard Certificate ofAnalysis. If the reading is outside of the statedspecification for this test standard, seetroubleshooting section.

0.297 NTU

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TroubleshootingLaboratory and Portable InstrumentsFor high readings, first inspect the sample cell forscratches, abrasions or flaws. Re-apply a thin and evenfilm of silicone oil to the sample cell. Measure thestandard again. If the reading is still high, clean the insideof the cell and rinse the cell several times with ultra-filtered deionized water. (Ultra-filtered water may beproduced by filtering deionized water through a 0.2-mfilter or smaller. This water will have a turbidity between0.020 and 0.050 NTU.) Rinse the cell twice with thestandard and repeat the measurement. If theturbidimeter still reads out of specification, thenrecalibrate the instrument.

Process InstrumentsIf the process instrument reading is high, clean theturbidimeter body and bubble trap again. Rinsethoroughly with ultra-filtered deionized water. (Ultra-filtered water may be produced by filtering deionizedwater through a 0.2-m filter or smaller. This water willhave a turbidity between 0.020 and 0.050 NTU.) Repeatthe test with fresh standard. The previously usedstandard may have been contaminated from a dirtyturbidimeter body and should not be reused.

Notesa. Use one of the following cleaning procedures forsample cells. The first procedure involves filling thesample cells with 1:1 hydrochloric acid. Cap the cellsand place them in a sonic bath for 5 minutes. Then,allow the cells to stand for another 30 minutes. Followby rinsing both the cells and caps at least 10 times withultra-filtered deionized water. Immediately cap afterrinsing in order to prevent contamination. Dry theoutside surface of the capped cell with a soft cloth.

The second cleaning procedure involves scrubbing thesample cells, both inside and out, with a mild laboratorydetergent (e.g., Liqui-nox Detergent), and a soft brush.Immediately follow by rinsing at least 10 times with ultra-filtered water. Cap to prevent contamination. Dry theoutside surface of the capped cell with a soft cloth.

b. For process instruments, both the bubble trap and theturbidimeter body must be thoroughly cleaned. Refer tothe cleaning instructions in the respective instrumentmanual for details.

c. Once the StablCal Standard has been mixed, it has areading window of between 5 and 60 minutes, excludingspikes. During this time, the standard does not need tobe re-mixed. The standard should be inverted between 3 to 5 times every hour to keep the formazin polymer suspended.

d. Do not use a sonic bath to degas the standard.Sonication will fracture micro-sized particles of glass into the standard, which will contaminate it.

e. These standards do not remain stable for long periodsof time when continuously in contact with glass surfaces.At this level of turbidity, the standard can leachparticulate matter from the glass back into the standardresulting in a slight increase in turbidity. Long-termstability requires storage in the original container. Youshould only leave the standard in a sample cell for up toone week or as experience dictates. Once the standardhas been used, discard it.

f. Do not pour the standard back into its storagecontainer or contamination of the standard will occurand future readings will be high.

Stability and Shelf-Life of Ultra-LowStablCal Turbidities StandardsLong-term storage of StablCal Standards in glass samplecells is not recommended. The matrix of the ultra-lowstandards is so clean that small amounts of glassparticulate matter can leach from the glass sample cellwalls. This leaching will result in a mild increase in theturbidity of the standard. This leaching effect is onlyobserved at ultra-low turbidity levels of

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Appendix V. Apparatus and ReagentsCat. No. Description Unit742-46 Hydrazine Sulfate, ACS grade 20 g742-26 Hydrazine Sulfate, ACS grade 100 g1878-34 Hexamethylenetetramine, analytical grade 500 g14574-42 Volumetric Flask, 100 mL, Class A each20858-56 Amber Bottle, Narrow Mouth, LPE, 250 mL, 6/pkg each

Prepared Formazin Stock Suspension:2461-49 4000 NTU Formazin Stock Suspension 500 mL2461-01 4000 NTU Formazin Cartridge for the Digital Titrator each16900-01 Digital Titrator each24018-12 Sample Cell Caps, Teflon lined, 12/pkg pkg

StablCal Vial and Bulk Solution Calibration Kits26591-05 StablCal Vial Calibration Kit for the 2100A Turbidimeter set26592-05 StablCal Vial Calibration Kit for the Ratio XR and Ratio 2000 Turbidimeters set26593-05 StablCal Vial Calibration Kit for the Ratio Turbidimeter set26594-05 StablCal Vial Calibration Kit for the 2100P Turbidimeter set26621-05 StablCal Vial Calibration Kit for the 2100N Turbidimeter set26595-05 StablCal Vial Calibration Kit for the 2100AN and ANIS Turbidimeter set26595-05 StablCal Vial Calibration Kit for the 2100A Turbidimeter set26591-00 StablCal Bulk Solution Calibration Kit for the 2100A Turbidimeter set26592-00 StablCal Bulk Solution Calibration Kit for the Ratio XR and Ratio 2000 Turbidimeters set26593-00 StablCal Bulk Solution Calibration Kit for the Ratio Turbidimeter set26594-00 StablCal Bulk Solution Calibration Kit for the 2100P Turbidimeter set26621-00 StablCal Bulk Solution Calibration Kit for the 2100N Turbidimeter set26595-00 StablCal Bulk Solution Calibration Kit for the 2100AN and 2100AN IS Turbidimeter set26596-00 StablCal Bulk Solution Calibration Kit for the 1720C Turbidimeter set26598-49 StablCal Bulk Solution, 1.0 NTU each27146-00 StablCal Low Range Calibration Verification Kit (0.30, 0.50, 1.0 NTU, 100 mL, 1 each) set27146-04 StablCal Low Range Calibration Verification Kit (0.30, 0.50, 1.0 NTU, 100 mL, 4 each) set27163-00 StablCal Low Range Calibration Verification Kit for 1720C and 1720D Turbidimeters

(0.30, 0.50, 1.0 NTU, 1000 mL, 1 each) set

For preparation of formazin dilutions, conventional glassware method:14574-42 Volumetric Flask, 100 mL, Class A each14574-41 Volumetric Flask, 50 mL, Class A each14515-40 Volumetric Pipet, 25.00 mL, Class A each14515-34 Volumetric Pipet, 0.50 mL, Class A each14515-35 Volumetric Pipet, 1.00 mL, Class A each14515-36 Volumetric Pipet, 2.00 mL, Class A each14515-37 Volumetric Pipet, 5.00 mL, Class A each14515-06 Volumetric Pipet, 6.00 mL, Class A each14515-07 Volumetric Pipet, 7.00 mL, Class A each14515-08 Volumetric Pipet, 8.00 mL, Class A each14515-09 Volumetric Pipet, 9.00 mL, Class A each14515-38 Volumetric Pipet, 10.00 mL, Class A each

Or, in place of the 0.50 through 10.00 mL pipets, use TenSette Pipets:19700-01 TenSette Pipet, 0.1 to 1.0 mL, 0.1 mL increments each19700-10 TenSette Pipet, 1.0 to 10.0 mL, 1.0 mL increments each

Gelex Secondary Standard Sets:22526-00 For Hach Model 18900 Ratio Turbidimeter set22956-00 For Hach Model 2100A Turbidimeter* set22958-00 For Hach Model 16800 PortaLab Turbidimeter set23287-00 For Hach Ratio/XR and Ratio 2000 Turbidimeter set24641-05 For Hach Model 2100P Portable Turbidimeter set25890-00 For Hach Model 2100N Laboratory Turbidimeter set25892-00 For Hach Model 2100AN Laboratory Turbidimeter set* Between regular calibration with formazin primary standards, Gelex secondary standards may be used to adjust instrument calibration before use.