Preparation of Standards for Dye Studies Using …

1 fluorometric Facts998-5111 (11/28/95)Page 1 of 6 Preparation of Standards for Dye StudiesUsing Rhodamine WTCONTENTSv WHAT IS A STANDARD? v UNITS OF MEASUREMENT v LINEARITY v ACTIVE INGREDIENT OR 20% SOLUTION? v MEASURING BY WEIGHT OR VOLUME v THE VOLUMETRIC FLASK v THE PIPETTE v MAKING STANDARD DILUTIONS v MAKING MORE THAN ONE CONCENTRATION v pH, TURBIDITY, AND CHLORINE v STORING SAMPLES AND STANDARDSF luorometric Facts998-5111 (11/28/95)Page 2 of 6 WHAT IS A STANDARD?A standard is a known concentration of the dye you areinjecting. It is used to calibrate the Turner Designs Model 10 Fluorometer to the desired sensitivity.

2 The fluorometer reading ofthe standard will be compared with the readings of unknownsamples to obtain their concentrations. A known concentration ismade by weighing or measuring a sample of tracer andprecisely diluting many cases, the standard will be a known dilution of dye, nota known concentration. For example, in flow rate measurementsyou are only interested in how much a stream dilutes the dye,not what the actual concentration is. Thus, you do not need toknow the concentration of the dye (in parts per billion or otherunits) when you calibrate your fluorometer. You need only knowthe dilution factor for the standard and the fluorometer reading,which you will then compare with the fluorometer readings foryour unknown samples collected after dye has been injected intothe stream.

3 This will allow you to calculate the extent to which thestream dilutes the : For more information on dye Studies and whether aknown concentration or dilution is required, see the monographs A Practical Guide to Flow Measurement and FlowMeasurements in Sanitary Sewers by Dye Dilution, availablefrom Turner OF MEASUREMENTUse whatever units suit your purpose best. The EPA guidelineson allowable levels will typically be in micrograms per liter (1).Other Studies are described in milligrams per cubic meter(mg/m3) or parts per billion (ppb). The simplest to use whenmaking dilutions is grams per gram, which, for all practicalpurposes, is the same as grams per easiest way to record and to think of these units is inexponential notation.

4 If you are not used to this, and are notcomfortable with it, move your decimal point around. It is veryeasy, however, to think in exponential grams dissolved in 100 ml, makes a g/gsolution. In exponential notation this is x 10-2. If 1ml is diluted to 100 ml (100 ml is 102 ml), then the newconcentration is x conversion factors:1 gram/liter1 part per thousand (ppt) 10-31 milliliter/liter1 part per thousand (ppt) 10-31 milligram/liter1 part per million (ppm) 10-61 microgram/liter1 part per billion (ppb) 10-91 milligram/cubic 1 part per billion (ppb) 10-9meterLINEARITYNote: Rhodamine WT comes as a 20% solution in water(meaning it is 20% active ingredient).

5 The EPA guidelines are interms of active ingredient. Thus, one microgram of the 20%solution in one liter is ppb active ingredient (or 20% of 1ppb).Instrument readings for fluorescent dyes are proportional toconcentration (linear) from the lowest detectable level up to acertain concentration. Above this concentration, a multipointcalibration curve may be used to obtain concentrations. Then, ata certain concentration (somewhere at about five to ten times theupper limit for the linear range) the curve flattens out andeventually takes a nose-dive. This critical concentration is afunction of the compound and of the path length of the flow cell practical purposes ( Using the Turner Designs Model 10 Fluorometer equipped with the 25-mm flow cell or cuvetteholder), Rhodamine WT is linear to ppm (500 ppb,5 x 10-7, or 500 micrograms/liter).

6 In terms of active ingredient(the 20% solution of Rhodamine WT), it is linear to ppm (or100 ppb, 1 x 10-7, or 100 micrograms/liter).ACTIVE INGREDIENT OR 20% SOLUTION?Since Rhodamine WT comes as a 20% solution, a decisionshould be made at the outset about whether to make thestandard as 100% tracer or in terms of active ingredient. Thefirst impulse is to do all calculations on the basis of the activeingredient. However, even to meet the EPA guidelines, youneed only keep in mind that 20% of the original solution is activeingredient. For other purposes, it is immaterial whether you takethe 20% into account.

7 If you discharge 20 pounds of RhodamineWT solution, or inject a 10-fold dilution, it doesn t matter that theoriginal material was only 20% pure. If you consider theRhodamine WT solution to be pure tracer, then all dilutions arerelative. For example, if you have made a 100 parts per billiondilution based on 100% tracer, your final dilution will be 20 partsper billion active ingredient. Whatever method you choose, besure to clearly mark your dilutions as to 100% tracer or Facts998-5111 (11/28/95)Page 3 of 6 MEASURING BY WEIGHT OR VOLUMEFor flow measurements and other Studies where dye is to beadded to a body of water, if you plan to add dye in pounds,grams, or other weight measure, then your standard must bemade by weighing.

8 If you are adding by volume, then thestandard must be made by volume measurements. Theimportant thing is to make your standard the same way youmake the dye concentration to be , however, that most of the literature cites the specific gravityof Rhodamine WT as (sometimes ). Recent literatureaccompanying the dye says Thus, one gram equals1/( ) milliliter. If you add by volume, you could be adding asmuch as 20% more dye than if you add by weight. Therefore, ifyou are concerned with absolute concentrations (as with anEPA study), you should make an initial 100-fold dilution byweight (or compensate for the specific gravity).

9 A 100-folddilution by weight has a specific gravity of For the vastmajority of Studies , errors of in doing further dilutions,whether by weight or volume, are you weigh, it is best to weigh directly into a volumetric flask.(See discussion of flasks below.) This avoids the problem ofhaving to rinse whatever vessel is used for weighing. All of thematerial must wind up in the flask. Since it will be diluted withwater, the rinsing can be done with you measure by volume, an accurate method is to use alarge-tip pipette of at least 10-ml capacity and measure dye intoa 1-liter volumetric flask (a 100-fold dilution).

10 Even moreaccurate is to measure dye with a 20-ml pipette and dilute to 2liters. The larger the pipette, the smaller the surface area withrespect to the volume, hence the smaller the error due toincomplete drainage. The error from a 10-ml pipette willprobably be negligible for most work. It certainly should be lessthan 1%.The most accurate way to measure the tracer by volume is to filla 10-ml volumetric flask to the mark, then rinse the tracer into a1-liter volumetric flask. You can t rinse a pipette, as it is made todeliver the stated volume. Volumetric flasks contain the statedvolume, so if you want the stated volume out of the flask, youmust , however, that there is not much point in preparing yourstandards to an accuracy greater than the means by which thetracer will be added to the VOLUMETRIC FLASKPut your measured sample into the flask, then add water up tothe line.