In order to analyze water samples using a spectrophotometer orplate reader, it is necessary to turn the molecules of nitrate intoa dye molecule that can be quantified. The first step in turningnitrate (NO₃^-)  into a dye moleculeis reducing it to a molecule of nitrite(NO₂^-). This is done by reacting theNO₃^- with cadmium.

After the reduction reaction, the NO₂^- isreacted with two additional reagents. The first reagent, Reagent A,is a solution of sulfanilamide and hydrochloric acid. The secondreagent, Reagent B, is a solution ofN-(1-naphthyl)-ethylenediamine, called NNED for short. Thecompounds are mixed with the water sample and produce a purplecolor. The intensity of the purple color is directly related to theconcentration of nitrite in the water sample. We can measure howpurple the water turns as absorbance on a spectrophotometer andthen convert the absorbance to concentration of nitrate.

To make Reagent A, we will need to make a solution of 10.0 g ofsulfanilamide in 1 L of 2.4 molar hydrochloric acid (HCl).

The stock solution of HCl is 6 molar HCl. Howmany milliliters (mL) of 12 M HCl would you add toproduce 0.15 liters (L) of HCl?  mLHCl

After creating 0.15 L of 2.4 molar HCl solution, how manygrams of sulfanilamide will be added?  gsulfanilamide

After reacting the nitrate with cadmium to produce nitrite, thenitrite is then reacting with sulfanilamide andN-(1-naphthyl)-ethylenediamine, to produce a purple dye moleculethat can be quantified on a spectrophotometer.

The N-(1-naphthyl)-ethylenediamine, called NNED for convenience,reagent is made by mixing 1 gram of NNED in 1 liter of water.However, we don't always want to make an entire liter of solutionbecause the NNED solution only lasts about 1 month before going badand turning brown.

How many milligrams of NNED will need to beadded to make 0.125 liters of solution?

After converting the nitrate into a purple dye, and measuringthe absorbance of the purple dye on a spectrophotometer, a standardcurve is used to convert the absorbance into concentration.

To make a standard curve, samples with known concentrations ofNO₃^- are run on the spectrophotemeter. Thesamples with known concentrations are called standards. A linearregression is then performed to relate the concentration ofNO₃^-  to measured absorbancevalues.

Here is a link to a spreadsheet containing a simulated data set.There are standards and their related absorbance values, and thereare samples from two sites that were diluted, prior to processingand measuring their absorbances. The groundwater originates fromthe upslope site, and the hope is that the microbes in the soil areremoving the NO₃^- from the groundwater beforeit reaches the downslope site.

Using the given data create a standard curve in Excel, and useTrendline to add a linear regression with the equation. Then usethe standard curve and the dilutions to determine the concentrationof NO₃^- in all the samples. Using the dataanalysis tool pack, perform the appropriate t-test to deduce if thenitrate concentration upslope is less than or greater than thenitrate concentration downslope. When performing a t-test using thedata analysis tool pack, the output will include the means for bothgroups.

What is the average NO₃^- concentration atthe upslope site?

Report your answer, from the data analysis tool pak output, to 3decimal places

What is the average NO₃^- concentration atthe downslope site?

Report your answer, from the data analysis tool pak output, to 3decimal places

Given the EPA drinking water quality standard is 10 mg/L ofnitrate, is the upslope site safe to drink based only on nitratecontent?  (Enter yes orno)

Is the downslope site safe to drink, based only onNO₃^- concentration?  (Enteryes or no)

Assuming the two sites are hydrologically well connected, thetransit time between the two sites is fast, and the two sitescannot be treated as independent samples, what kind of t-testshould be performed to show that the upslope site is greater thanthe downslope site? Enter the letter of your answer choice in theanswer blank

A. one-tailed unpaired t-test
B. two-tailed unpaired t-test
C. one-tailed paired t-test
D. two-tailed paired t-test

What is the calculated t statistic, rounded to 4 decimalplaces?

Is the calculated t statistic greater orless than the critical t value reported by thedata analysis tool pack?  (enter greateror less)

Is the nitrate concentration at the upslope site significantlygreater than the downslope site? (Enter yes orno)

Based on this statistical result, and assuming no diffusion ordilution occurs between the upslope and downslope site, do youthink microbes are removing NO₃^- from theground water?  (Enter yes orno)

DATA

mg N per L	Abs	Sample ID	Upslope Absorbance	Dilution	mg N	Downslope Absorbance	Dillution
0	0	1	0.449	0.01		0.316	0.5
0.1	0.12	2	0.243	0.01		0.251	0.5
0.2	0.225	3	0.331	0.01		0.256	1
0.4	0.432	4	0.45	0.1		0.2	1
0.6	0.585	5	0.551	0.01		0.563	1
		6	0.561	0.01		0.316	0.5
		7	0.541	0.02		0.951	1
		8	0.244	0.01		0.317	1
		9	0.532	0.01		0.2	0.5
		10	0.5	0.02		0.269	1
		11	0.332	0.01		0.2	0.5
		12	0.443	0.02		0.313	0.5
		13	0.655	0.1		0.2	1
		14	0.675	0.01		0.745	1
		15	0.5	0.1		0.119	0.5
		16	0.39	0.01		0.103	1
		17	0.5	0.02		0.149	1
		18	0.532	0.01		0.311	0.5
		19	0.5	0.1		0.918	1
		20	0.108	0.01		0.328	1
		21	0.119	0.1		0.2	0.5
		22	0.689	0.01		0.206	1
		23	0.5	0.02		0.2	0.5
		24	0.329	0.1		0.508	0.5
		25	0.753	0.01		0.256	0.5
		26	0.511	0.01		0.294	0.5
		27	0.839	0.02		0.417	0.5
		28	0.543	0.01		0.149	1
		29	0.392	0.02		0.118	0.5
		30	0.444	0.01		0.201	1