Question

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

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

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

The stock solution of HCl is 6 molar HCl. How many milliliters (mL) of 12 M HCl would you add to produce 0.15 liters (L) of HCl?  mL HCl

After creating 0.15 L of 2.4 molar HCl solution, how many grams of sulfanilamide will be added?  g sulfanilamide

After reacting the nitrate with cadmium to produce nitrite, the nitrite is then reacting with sulfanilamide and N-(1-naphthyl)-ethylenediamine, to produce a purple dye molecule that 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 solution because the NNED solution only lasts about 1 month before going bad and turning brown.

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

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

To make a standard curve, samples with known concentrations of NO₃^- are run on the spectrophotemeter. The samples with known concentrations are called standards. A linear regression is then performed to relate the concentration of NO₃^-  to measured absorbance values.

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

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

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

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

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

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

Given the EPA drinking water quality standard is 10 mg/L of nitrate, is the upslope site safe to drink based only on nitrate content?  (Enter yes or no)

Is the downslope site safe to drink, based only on NO₃^- concentration?  (Enter yes or no)

Assuming the two sites are hydrologically well connected, the transit time between the two sites is fast, and the two sites cannot be treated as independent samples, what kind of t-test should be performed to show that the upslope site is greater than the downslope site? Enter the letter of your answer choice in the answer 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 decimal places?

Is the calculated t statistic greater or less than the critical t value reported by the data analysis tool pack?  (enter greater or less)

Is the nitrate concentration at the upslope site significantly greater than the downslope site? (Enter yes or no)

Based on this statistical result, and assuming no diffusion or dilution occurs between the upslope and downslope site, do you think microbes are removing NO₃^- from the ground water?  (Enter yes or no)

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

Answer 1

Note: Only Statistics part is answered in this question.