GCCCD Nitrate in Seawater Lab Report

Chem 100AS. Li
Analysis of Nitrite in Seawater
Chem 100A Lab (Week 8)
I.
Background and Goals
Nitrite (NO2-) is a key intermediate ion formed during nitrification in seawater. Its widespread use
as a meat preservative is controversial because of its transformation while heating to form
carcinogenic nitrosamines. The classical method for the determination of nitrite in seawater is
based on the Griess reaction.1 In this reaction, nitrite is converted to a highly colored azo dye,
which increases the sensitivity and selectivity of the determination. This procedure is a
modification of the method developed by Bendschneider and Robinson.2
The Greiss reaction involves reaction of nitrite in solution with sulfanilamide ion and acidic
solution. This reaction results in the formation of a diazo compound. Subsequently, the product is
reacted with N-(1-napthhyl) ethylenediamine (NED) and forms a highly colored azo dye. The azo
dye concentration is determined spectrophotometrically with a limit of detection in 1 cm cells of
0.1 ppm.
Figure 1. Chemical reactions involved in the measurements of NO2- using the Griess reaction.
Because the sulfanilamide and NED compete for nitrite in the Griess reaction, the reagents are
added sequentially, with the sulfanilamide added to the sample first, and allowed to react for 2 to
8 minutes. The NED solution is added next and allowed to react. Your UV/VIS spectrophotometer
measurements can begin 10 minutes after the addition of the NED at an absorbance of ~540 nm.
II.
Preparation Before Lab
➢ Read Chs. 4, 5, & 18 as they pertain to calibration curves, limit of detection, light
absorption, Beer’s Law, and spectrophotometry.
III.
Materials, Safety, and PPE
➢ Lab coat, safety glasses, and nitrile gloves.
Spectrophotometer (Genesys 30)
Cuvettes (inner diameter 11 mm)
Serum glass bottles x 12
Seawater
Sodium nitrite (note purity specification on the bottle)
1% sulfanilamide in 10% HCl (1.2 M HCl)
N-(10napththyl)ethylenediamine (NED) solution (0.1% in water)
1
Chem 100A
S. Li
ERPs with 12.5 mL tips
IV.
Procedure
Calibration using DI water as solvent
1. Using dried NaNO2 salt, prepare ~1 kg of a ~5000 μmol/kg-sol stock solution in DI water.
2. Starting with ~1 g of ~5000 μmol/kg-sol stock solution of NaNO2, prepare the following:
a. 100 g of a ~50 μmol/kg-sol sub-stock solution in DI water.
b. From the sub-stock, prepare five standard solutions of NaNO2 in your 60 mL serum bottles
with concentrations of 1, 2, 3, 4, and 5 μmol/kg-sol, all in 25 g amounts diluted in DI water.
3. Prepare two seawater samples: 1 sample of low nutrient seawater (LNSW, sea water from the
carboys); 1 unknown “contaminated” seawater sample from your TA. From the bottle, dispense
~25 g of water to 60 mL serum bottles; record the mass of water with the top loader balance.
4. Add ~25 g of DI water to a 60 mL serum bottle; record the mass of water using the top loader
balance.
Determination of λmax and time development curve
5. Turn on Genesys 30. Warm up for 20 min. BEST to do this as soon as you enter the lab. Two
cuvettes will be available next to the instrument.
6. From home screen, select ‘scan’. Set the λ scanning limit from 400 nm to 900 nm.
7. Fill one of the two provided cuvettes with DI H2O. Insert cuvette into the spectrophotometer
with the correct alignment.
8. Press the yellow ‘auto zero’ button. When zero is complete, remove DI H2O cuvette.
9. Add 0.5 mL of sulfanilamide solution to your 5 μmol/kg standard solution using the ERP tip
that is provided. Mix well and let the solution stand for 5 minutes before adding NED. After the
solution is full developed into a pink color, transfer some of the mixed solution to a clean test
tube cuvette and determine λmax.
10. Continue measuring the %A at λmax as a function of time. Record measurements frequently
in the first 2 minutes, and less frequently afterwards until your %A reading is stable. The time to
reach a stable result is relevant for the analysis of the nitrite solution. Record and save the data
on a USB drive.
Measurement of standards and water samples
11. Add the sulfanilamide and NED reagents to the remaining standard solutions, the two seawater
samples, and the one sample of DI water, following steps 9 and 10. Allow the solutions to develop
after adding the NED for an amount of time determined in step 10.
2
Chem 100A
S. Li
12. Measure the %A of your developed standard solutions and both “contaminated” seawater and
DI water at the wavelength of maximum absorbance. Measure each sample for three times.
Remember to pre-rinse the cuvette with a small amount of solution before you fill it. Adjust the
%A of the instrument using DI water as a reference (blank) for the solutions. Record the DI water
reagent blank absorbance (with added Greiss reagents) at λmax at least 5 times.
13. Discard the solutions in the cuvette after measuring the %A; do not pour them back into the
serum bottle.
14. At the end of the lab, discard your solutions in the appropriate waste container.
V.
Data Analysis
1. Construct calibration curves for your standards prepared in DI water.
2. From your calibration curve, determine the concentration of the unknown “contaminated”
seawater samples.
3. Determine the limit of detection: LOD = 3.3*σb, where σb is the standard deviation of the blank,
for the two different reagent blanks. Does the signal of LNSW sample pass the LOD?
3. Determine the minimum concentration that can be analyzed based on the limit of Quantification:
LOQ = 10*σb, where σb is the standard deviation of the signal from the blank. Does the
concentration of LNSW pass the LOQ?
References
Bendschneider K., Robinson R. J. “A new spectrophotometric method for the determination of
nitrite in sea water”, ONR Technical Report No. 8, 1952.
Hansen H. P., Koroleff, F. “Determination of nutrients” Chapter 10 of Methods of Seawater
Analysis Third Edition, edited by Grasshoff K., Kremling K., and Ehrhardt M. Wiley VCH 1999.
Harris, D. Chapter 4 “Statistics”, and Chapter 5, “Quality Assurance and Calibration Methods” in
Quantitative Chemical Analysis Ninth Edition, W.H. Freeman and Co., 2016.
Shinn M. B. “Colorimetric method for determination of nitrite”, Ind. Eng. Chem. An. Ed. 1941,
13 33-35.
Strickland J. D. H., Parsons T. R. A Practical Handbook of Seawater Analysis, Fisheries Research
Board of Canada, Bulletin 167, 1971. http://www.dfo-mpo.gc.ca/Library/1507.pdf
3
Measurement Limits
Review: Statistical Analysis
Confidence Interval
4.9
• Table 4.12 gives the
confidence intervals for
several values of z.
• 95% confidence level is a
common choice in analytical
chemistry.
Confidence Intervals
Normal distribution curve
Area under the curve represents the probability
of finding a member of the population within a
particular range of values.
Limit of Detection
Question: What is the lowest concentration of the sample that we can tell apart from the blank
Probability
Blank
𝜎
Blank
Signal
Limit of Detection
Question: What is the lowest concentration of the sample that we can tell apart from the blank
Probability
Blank
Low Concentration Sample
𝜎
Blank
Low Concentration Sample
Signal
Limit of Detection
Question: What is the lowest concentration of the sample that we can tell apart from the blank
Probability
Blank
Low Concentration Sample
Sample is misrecognized as
Blank
Blank is misrecognized as
Sample
Blank
Low Concentration Sample
Signal
Limit of Detection
Question: What is the lowest concentration of the sample that we can tell apart from the blank
Blank
Low Concentration Sample
95% accuracy is what we need in
analytical chemistry.
Probability
Question: How large separation do
we need?
Sample is misrecognized as
Blank
Blank is misrecognized as
Sample
Blank
Low Concentration Sample
Signal
Limit of Detection
Question: What is the lowest concentration of the sample that we can tell apart from the blank
Blank
Question: How large separation do
we need?
Shade area < 5% Probability Blank is misrecognized as Sample Blank Low Concentration Sample Signal Recall: Confidence Intervals Normal distribution curve Area under the curve represents the probability of finding a member of the population within a particular range of values. Limit of Detection Question: What is the lowest concentration of the sample that we can tell apart from the blank Separation >
2 X 1.65 𝜎 = 3.3 𝜎
Probability
Error < 5% Blank Low Concentration Sample Signal Limit of Detection Definition: Limit of Detection (LoD) is the limit where you can accurately (>95%) distinguish the sample from Blank
LoD = 3.3
Separation >
2 X 1.65 𝜎 = 3.3 𝜎
Probability
Error < 5% Blank Low Concentration Sample Signal Limit of Quantification Question: What is the limit of the concentration that we can accurately determine from the measured signal? Probability There are additional error in linear regression analysis: We need a higher limit Blank Low Concentration Sample Signal Limit of Quantification Definition: Limit of Quantification (LoD) is the minimum signal required to accurately determine the concentration of an unknown sample LoQ = 10 Probability 10 Blank Low Concentration Sample Signal LoD vs LoQ When to use LoD LoD vs LoQ When to use LoD For example, the minimum concentration standard solution need to give a signal larger than the LoD LoD vs LoQ When to use LoD For example, the minimum concentration standard solution need to give a signal larger than the LoD When to use LoQ For example, the unknown sample you want to determine the concentration need to pass the LoQ LoD vs LoQ When to use LoD For example, the minimum concentration standard solution need to give a signal larger than the LoD When to use LoQ For example, the unknown sample you want to determine the concentration need to pass the LoQ Question: Does the LNSW sample pass the LoQ? Nitrite in Seawater Background: Nitrate in Seawater Nitrogen Cycle in the Sea • Nitrate is considered to be the only thermodynamically stable oxidation state of nitrogen in the presence of oxygen in seawater • In many sea areas nitrate is considered to be the micronutrient controlling primary production in the euphotic surface layer (biolimiting element). • Concentration of nitrate in these layers is governed by the advective transport of nitrate into surface layers, micobial oxidation of NH3, and uptake by primary producers Background: Nitrate in Seawater Nitrogen Cycle in the Sea • If light penetration into the water is sufficient, the uptake rate is usually faster than the process of transferring nitrate to the surface layers, thus surface concentrations of nitrate are near zero • Nitrate content may be as high as, Pacific ≤ 40 µmol/L Atlantic ≤ 32 µmol/L Indian ≤ 45 µmol/L Background: Nitrite in Seawater Nitrogen Cycle in the Sea • Nitrite in an intermediate compound in the microbial reduction of nitrate or in the oxidation of ammonia • Nitrite may also be excreted by phytoplankton especially during periods of excess feeding (i.e. surplus nitrate and phosphate stimulates a heavy bloom of plankton) • Natural levels of nitrite are very low < 0.1 µmol/dm3 But in the transition zone (oxic  suboxic) thin layers of high nitrite concentrations may occur (> 2 µmol/dm3) together with low concentrations of
dissolved oxygen (< 0.15 cm3/dm3) • Analysis of Nitrite: Griess Reaction The photometric determination of nitrite is based on the two step Griess reaction: Step 1: Nitrate react with an aromatic amine leading to the formation of a diazonium ion, which couples with a second aromatic amine to form an azo dye (Griess Reaction). aromatic amine sulfonamide Step 1 (2~8 min) diazonium Analysis of Nitrite: Griess Reaction The photometric determination of nitrite is based on the two step Griess reaction: Step 1: Nitrate react with an aromatic amine leading to the formation of a diazonium ion Step 2: The diazonium ion couples with a second aromatic amine to form an azo dye “color sensitive” N-1-naphthylethylenediamine dihydrochloride (NED) Step 1 Step 2 (~10 min) azo dye The Determination of Nitrite in Seawater The classical method for the determination of nitrite in seawater is based on the Griess reaction.1 In the Greiss reaction, the nitrite is converted to a highly colored azo dye. This reaction increases the sensitivity and selectivity of the determination. This procedure is a modification of the method developed by Bendschneider and Robinson.2 The Greiss reaction involves reaction of nitrite in solution with sulfanilamide ion and acidic solution. This reaction results in the formation of a diazo compound. The product is subsequently reacted with N-(1-naphthyl) ethylenediamine (NED) and forms a highly colored azo dye. The concentration of the azo dye is determined spectrophotometrically. The limit of detection in 1 cm cells is 0.1 ppm. Because the sulfanilamide and NED compete for nitrite in the Griess reaction, the reagents are added sequentially. The sulfanilamide is added to the sample first, and allowed to reactor for 2 to 8 minutes. The NED solution is added next and allowed to react. Spectrophotometric measurements of the absorbance at 543 nm can begin 10 minutes after the addition of the NED. The solution is also stable for up to 2 hours. To ensure accurate NO2 quantitation, prepare a calibration (working) curve should be prepared in a matrix similar to that of the samples as the matrix can impact the sensitivity (slope) of the working curve. 1. Griess P. Berichte der Deutschen chemischen Gesellschaft 12 (1): 426–428, 1879. 2. Bendschneider and Robinson, J Marine Res.,11:87, 1952 Materials: Spectrophotometer (Spec 20) Test tube cuvettes (with 11 mm path length) Seawater (record which sample you test) Sodium nitrite (note purity specification on the bottle) 1% Sulfanilamide in 10% HCl (1.2 M HCl) N-(1-naphthyl)ethylenediamine (NED) solution (0.1% in water) Eppendorf repeating pipette (ERP) with 12.5 mL tips (×2) Serum bottles (60 mL ×12) Procedure: Before first day of lab. Do this Wed or Thurs the week prior. Weigh ~0.5 g of NaNO2 into a weighing jar. Label beaker and desiccator with your name and section. Leave the sample out on lab bench so that lab staff can collect it and dry in an oven. Day 1. I. Prepare the following: a. 500 g of a ~5 mmol/kg stock solution of NaNO2. Use the dried solid NaNO2 and DI water. b. 100 g of a ~50 μmol/kg sub-stock of NaNO2 from your stock. c. Six standard solutions of NaNO2 from your sub-stock, with concentrations 1, 2, 4, 8, 10, and 20 μmol/kg. Prepare 25 g of each standard solution in 60 mL serum bottles (glass). II. You will be assigned an unknown seawater sample. Record the number. Add ~100 g of the seawater to a plastic bottle. Then add ~25 g of seawater to three 60 mL serum bottles; record the mass of seawater using the analytical balance. Time development curve A. Turn on the Spec 20 spectrophotometer and allow the unit to warm up for 15 minutes. B. Set the wavelength to 540 nm and adjust the 0%T and 100%T of the spectrometer. C. Add 0.5 mL of sulfanilamide solution to your 10 μmol/kg standard solution using the ERP tip that is provided. Mix well and let the solution stand for 5 minutes before continuing to the next step. Do not wait more than 10 minutes. Note: the following 2 steps must be done as efficiently as possible. D. Add 0.5 mL of NED solution to the 10 μmol/kg standard solution using the ERP tip that is provided, and mix well. Start a timer once the NED has been added. E. Immediately transfer some of the mixed solution to a clean cuvette, and begin measuring the %T at 540 nm as a function of time. Record measurements frequently in the first 2 minutes, and less frequently afterwards until your %T reading is stable. The time to reach a stable result is the reaction time to be used on Day 2. Lambda max determination Measure the %T of your developed 10 μmol/kg solution from 400–650 nm with 50 nm steps. (Note: you only need to perform this step for the one solution). At each wavelength, make sure to adjust the 100%T control with DI water in a cuvette, as described in step 2. Collect additional data points at 5 nm intervals in the wavelength region where absorbance approaches the maximum (low %T). The closer spacing will improve your determination of the wavelength where absorption of the azo dye is strongest. (Just like last week) Measurement of standards and seawater samples 1. Add the sulfanilamide and NED reagents to the standard solutions and to the three seawater samples following steps C, D and E. Allow the solutions to develop after adding the NED for an amount of time determined in step E before making transmission measurements. 2. Measure the %T of your developed standard solutions and seawater at the wavelength of maximum absorbance. Remember to pre-rinse the cuvette with a small amount of solution before you fill it. Adjust the 100%T of the instrument using DI water as a reference (blank) for the standard solutions, and seawater reference for the developed seawater solutions. (Consider: what possible advantage is there in using seawater as the reference solution for the developed seawater solutions? It takes only moments in the lab to check whether there is any difference in %T for DI water versus seawater. Make a note of any difference.) 3. Discard the solutions in the cuvette after measuring the %T; do not pour them back into the serum bottle. 4. At the end of the lab, neutralize your waste and pour it down the drain. Day 2. The second day is your opportunity to design and implement experiments that (1) check assumptions that were made on Day 1, and (2) explore fundamental aspects of analytical chemistry as related to the Griess test. Following each suggested experiment, we include a question as encouragement for each group to think about the fundamentals and/or the broader implications of the specific tests: 1. Devise and run experiments to determine whether matrix effects matter. Note that there are at least two rigorous methods that would reveal possible matrix effects. Know what those approaches are, even if you pursue only a single method. If you see matrix effects, what could be the causes? 2. Experimentally determine the lowest concentration of nitrite that can be reliably recorded with your test and instrumentation (limit of detection), and the highest concentration of nitrite that obeys Beer’s Law (limit of linearity). The range of concentration (ppm) between these two values is the linear dynamic range. What are the limitations to measuring highly concentrated and very dilute samples of nitrite? Note: if you choose this module, you may ask your TA whether square cuvettes are available and usable with your spectrophotometer. 3. You are instructed on Day 1 to add the two reagents in a specific order and with a specific waiting time in between. Investigate the importance of both variables (order and time). What reactions in organic chemistry do you think are responsible for any changes in the azo dye development? 4. Determine the linear dynamic range following suggestion #2, but at a wavelength different from that of maximum absorbance. Why is or isn’t the range dependent on the wavelength? Calculations and questions: 1. Your calibration curve is crucial for determining the concentration of nitrite in seawater. Use linear regression techniques that we have emphasized this quarter: you are expected to know and report the standard deviation of the regression, as well as the error in the slope and y-intercept. If you report an r2 value, don’t be surprised if your instructors then ask you to explain exactly what r2 means and how it is calculated. So, consider whether you really want to report r2 or not! 2. If the y-intercept of your calibration curve is not zero, consider possible reasons for that. 3. Note the purity of NaNO2 used to prepare samples for your calibration. Should you consider this purity when calculating the concentration of your solution? (Hint: what do you expect is the main impurity?) 4. Using the calibration curve, report the nitrite concentration in your seawater samples, and carry out a thoughtful error analysis. If you report the average and standard deviation of your three seawater measurements, that is a beginning, but you are also expected to take into account (propagate) uncertainties in the linear regression, and consider other sources of uncertainty. 5. Report all findings from your experiments on Day 2. As encouraged in the preceding page, be prepared to speak about the fundamentals and/or broader implications related to your work on Day 2. 6. Suppose that your spectrophotometer gives reliable absorption values to a lower limit of A=0.05 (T = ?) over a 1 cm pathlength. What is the expected limit of detection for the Griess test in this case? Relate the minimum absorbance and the minimum concentration using a very familiar law. References: Bendschneider K., Robinson R. J. “A new spectrophotometric method for the determination of nitrite in sea water”, ONR Technical Report No. 8, 1952. Hansen H. P., Koroleff, F. “Determination of nutrients” Chapter 10 of Methods of Seawater Analysis Third Edition, edited by Grasshoff K., Kremling K., and Ehrhardt M. Wiley VCH 1999. Shinn M. B. “Colorimetric method for determination of nitrite”, Ind. Eng. Chem. An. Ed. 1941, 13 33-35. Strickland J. D. H., Parsons T. R. A Practical Handbook of Seawater Analysis, Fisheries Research Board of Canada, Bulletin 167, 1971. http://www.dfo-mpo.gc.ca/Library/1507.pdf ~~., .. " ~»(['tE.1AlNO- 0.520 ren z w o --I Identical optical densities were obtained for m'hr-ita solutions of equal eoncentra t~.on in either med.1.um" Liko'tiise aalinity was found to have no effec·t; with the reagents now currently used if the ~-naphthylamine was added after However~ it these the sulfanilic acid" reagents were added as a mixture ~ a slightly lesser optical dens! ty was noted a t the lower salinities~ Table IT.o With the range of salinities normally en.... countered in marine work~ this e.t:f'ect is practically insignificant except in very critical work o Calibr!tion Cy;,m Figure V shows calibration curves with both the proposed and accepted res~ntso Since Curve A is linear, the color produced is directly proportional to the nitrite concentration in conformity with Beerlls law o Within the llmits noted abov8 p the slope of the curve is constant under ditterent conditiona ot pHs reagent concentrat1on~ time interval between reagents1} and standard solutions prepared with distilled water or DitriteEDfree see water" ~.BLE II The Effect of Sal11'lity upon the Maximum Color Intensity [-:roduced gy the Accept~d Procedure __.oLQQ Optical Density with Mixed Heo.gents .-.Jleage:n~f!....._ 34.. 3 O~208 0 0 210 24:;0 ,204 .,209 17 0 2 (1204 ~209 10 0 6 0201 0209 :305 .,199 . . 209 01,6 0193 0)208 00 0 0192 ;,209 Sample Salinity ~ _ I Opti~~G.l Deneity wi·th Separate Density Observations at 520 Jl1).l.0 Samples contained 0050 FE atQm of nitrite-nitrogen per liter of solution o As iridica ted in Figure Vs some 8 bsorpt:i:jn occurs at zero nitrite concantration u This probably is due to nitrite imptn."ity in the reagents or in the dist111ed water since neither reagent causes absorption at wave length 543 ~ when added alone to distilled watero Redistillation of the Via tar ~ made alkaline with sodium hydroride 9 usually reduced the observed optical density Q Distilled watf'~ plus reagents may be used in the reference cell to compensate· tor the blank, but this is not recommended since it introduces the possi- bility of variations in the absorption characteristics of the reference cell due to decomposition or contamination o In Figure V the greater slope of Curve A in comparison with Curv·e B indicates that the proposed reagents are more sensitive than those now used o to Thus with the proposed reagents~ smaller amounts ot nitrite may be estimated and smaller differences in nitrite concen... tration may be detected o It 'Was also noted that somewhat greater consistency was obtained with the proposed reagents o Definite variations in the slopes or calibration curves were obtained occasional~ ~1th the , mixed sulfanilic and d.~naphthylam1ne reagents o o . >en
z
w
.-.
•
4.
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•
0.600
I-
o
…J
c:{
D
0
t Q.
i=
00.200
o
0.20
0.60
NITRITE-NITROGEN
1.00
(~G. ATOMS PER LITER)
FIQ11BE V
Ce)4bration CurveS o
Cam A. Proposed method B t 543 muG
Carve B. Aaaapte4 method at 520 BlU.
1.40
CONCLUSIONS
By the proposed method full color is developed much more
rapi