Name Lab Exercise 2 - Standard Curves Nitrate, Ammonia, Phosphorus, and Chlorophyll

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1 Name Lab Exercise 2 - Standard Curves Nitrate, Ammonia, Phosphorus, and Chlorophyll For most analytical chemical procedures, we use "standard curves" to calibrate our measurements. For example, we want to measure chlorophyll concentration in some water samples. One way to do this is to take our sample and extract it in a solvent of 90% acetone. To quantify how much chlorophyll is in our extract we measure the absorbance at a particular wavelength. This wavelength usually is at the absorption peak for that compound in that particular solvent. Figure 1 shows a typical absorption spectrum for two different kinds of chlorophyll. Note how the absorption spectrum closely matches the photosynthetic action spectrum (the wavelengths of light that actually stimulate photosynthesis). In our case we would look for the wavelength of maximum absorption in 90% acetone. The peak wavelength might be different if we had extracted the chlorophyll in methanol or used 100% acetone. In the example shown in Figure 1, our measurements might be made at a wavelength of 690 nm to coincide with the chlorophyll a peak there, and that would also minimize interference from the chlorophyll b peak which is at a slightly lower wavelength. In order to be able to measure the concentrations from samples taken in the ocean, which we call our "unknowns", we have to calibrate the response of our spectrophotometer to "known samples". We therefore prepare a series of "standards" which have known concentrations. For this example, our known samples have concentrations of 0.1, 0.25, 0.5, 0.75, 1 and 1.5 mg / liter. We put each of these in the spectrophotometer and measure their absorbance. Our results are shown in the table below. Concentration Absorbance If we plot these data in a spreadsheet or other program we can see the relationship between absorbance and the known concentration (Figure 2). From this graph we could determine the concentration of unknown samples by drawing a line from the absorbance of the unknown sample on the X-axis, straight up to where it intersected a line drawn through the data points. We could then read the concentration of that sample on the Y-axis. If some unknown sample Figure 1 Chlorophyll a and Chl b absorption spectra and their relation to the photosynthetic action

2 had an absorbance of 0.035, in this example, it would have a concentration somewhere around 0.9 mg / liter. That is not as precise as we would like to be. A better way is to determine the regression line through the data and use the equation for a line to calculate the concentration. Example Standard Curve Concentration Absorbance Conc pred Linear (pred) A simple way to do this is using a spreadsheet or calculator with statistical functions. In the example here, the slope is 25.4 and the intercept is Putting these values into the equation for a straight line we get Y = 25.4*X Since our unkown had an absorbance of 0.035, the concentration would be Y = 25.4* = mg/liter The lab today will use this basic procedure to determine standard curves for nitrate, ammonia, phosphate, and chlorophyll. The instructor will give you the range of concentrations you should prepare for each constituent. After preparing the standards, use the directions for the particular chemical analysis and determine the absorbance (or fluorescence) for each sample. Prepare a graph of your data and calculate the regression equation using a spreadsheet of your choice. You will use these equations throughout the semester to determine nutrient and chlorophyll content in a variety of water samples.

3 NUTRIENT ANALYSIS LAB Oceanography Introduction We will be analyzing water for the concentration of nitrate (NO 3 - ), nitrite (NO 2 - ), ammonia (NH 3 + ), phosphate (PO 4 3- ), silicate (Si), and iron (Fe). In order to do this we will perform some wet chemistry which causes color formation in a solution. The color developed is directly proportional to the compound we are measuring. For quantitative work, we need to know the relationship between the intensity of the color and the concentration of the compound. This is the same as a regression line in statistics, that is, a correspondence between an X-variable (the absorbance of light by the solution) and a Y-variable (the concentration in the solution). 1. How might you try to figure out the relationship between concentration and color development? To obtain this relationship we will construct what is known as a standard curve. The standard curve is determined from a series of samples of known concentration. The samples of known concentrations will be made by you, by dilution of a stock solution (also known as the standard). The range of the concentrations you make up should be from zero to the maximum concentration detectable by the method. This will produce a series of solutions with different colors depending on the concentration. The amount of color development might be judged by eye, but this is not very useful. 2. Why is a visual determination not sufficient for analytical work?

4 We rely instead upon instruments like spectrophotometers to accurately assess the amount of color. The output of the spectrophotometer is in units of Absorbance or % Transmittance. We will use Absorbance as our measure. 3. Once we have the data on concentration and color, how might we put it to use? For example what if you were given some unknown sample to analyze? For our experiments we will make a graph by plotting the absorbance versus the concentration on an X-Y graph. This relationship should be linear over the range of concentrations. 4. Which axis might we use for absorbance? Why? 5. In this experiment we will use 10 known concentrations to determine the relationship between concentration and color development. Why should we use 10, can we not make a straight line between two points? 6. How might you then use this graph to determine the concentration of unkown samples? What if the concentration of the unknown falls between those of the known standards you made up?

5 One could fit a line through the data points by eye. 7. Is this a satisfactory solution? Explain. 8. What could be a better way to fit a line to the data? Hint: remember the computer lab we did? 9. Once we have the line fitted, what do we need to do in order to determine the concentration of our unknown samples?

6 Procedure I. Each one will pick a different analysis and develop a standard curve using both distilled water and a 3% salt solution as a diluent. 10. What is the significance of using a 3% salt solution? II. You will make a set of about 10 samples of known concentration (see section III) which span the range of the limits of the method. Use the standards provided for each nutrient as the stock solutions. The concentrations of the stock solutions are written on the side of the bottles. The relationship Vol 1 x Conc 1 = Vol 2 x Conc 2 should be helpful to you in making the dilutions. Where Conc 1 Conc2 Vol 1 Vol 2 is the concentration of the dilution desired. This is one of the ten values you need. is the concentration of the stock solution as written on the bottles. is the volume of sample required to perform the analysis. This is indicated on the procedure sheets for the analytical method. is the volume of the stock solution needed to make the dilution. This is the value you need to solve for in the equation. 11. Can you think of an alternative way to make up the range of ten dilution without using the above equation?

7 12. What is the compound you are analyzing for? 13. What is the concentration of the stock solution you are using? 14. What is the range (the minimum and maximum) of concentrations that you need to make up? 15. What is the volume required for the analysis you are doing? III. Make a series of dilutions first with distilled water and run the tests. The procedure for each analytical method is provided on the separate handout available in the lab. The procedure is written as if you were analyzing just one sample. Therefore you need to perform those steps for each dilution and unknown. Then repeat the procedures with the 3% salt solution as the diluent. Use the table below to organize the volume of the dilutions you need to make. Required Concentration Volume of Stock 0 IV. Record your data in the table on the following page.

8 V. Also perform the test on the unkown sample of sea water, and a sample drawn from the tap. Record the values in the table. Concentration Abs. Distilled Abs. 3% Salt 0 Seawater XXXXXXXXXXX Tap Water XXXXXXXXXXX

9 VI. Plot the curves for the two sets of standards. Remember how to do this from the computer lab? VII. VIII. Determine the lines through the data. Determine the concentrations of the two unkowns. 16. Is there a difference in using tap water or salt as the diluent in making up the standard samples? Explain why or why not. 17. Is there a difference between the tap water and the sea water? 18. Is this difference significant? How can you tell? IX. Share the results of your standard curves with the others in the class.

10 APPENDIX DILUTIONS In the laboratory you are required routinely to make accurate dilutions of various solutions to carry out a particular assay. The following gen eral formulae should assist you in understanding how dilutions can be made quickly and accurately. You have a solution of sodium chloride, NaCl, which is 1 mol/l (1 mole/liter) in concentration. Remember the definition of concentration is quantity (usually in moles) per volume (usually in liters). This can be called your "stock" solution. Let's say you want to use a part of this stock solution to make a new solution which is 0.1 mol/l NaCl in concentration. First, decide what volume of the new 0.1 mol/l NaCl you want to make. Let's say 100 ml. In this example you can see clearly that the concentration of the new solution is 1/10 that of the stock solution, and once you become proficient at diluting, you can determine the next few steps in your head. But this becomes tricky when your stock solution is, for example, 0.93 mol/1 and you wish to make a new solution which is mol/l. We can use a general equation to solve all dilution problems. In our example you will take a small subsample of the 1 mol/ l NaCl stock and add enough distilled water to give the desired volume of 0.1 mol/l NaCl. Since a concentration in this case has units of moles/liter, your subsample is still 1 mol/inacl. Therefore, by adding distilled water to the subsample, you are in effect increasing the denominator of the concentration expression of the new solution (liters) while holding the numerator constant (moles). If that's confusing, work through the example using the equation below, and then reread this section. FORMULA FOR DILUTIONS VolumeNew X ConcentrationNew = VolumeStock X ConcentrationStock Where, V Stock = the volume of stock needed to make the new solution, Cnew = the concentration of the new "desired" solution, CStock = the concentration of the stock solution, and V New = the volume of the new solution you wish to make. STEP 1 V Stock C New * V C Stock New In our example C new is 0.1 mol/ 1 NaCl, Cstock is 1 mol/1 NaCl, and V New is 100 ml. Therefore, using the equation we determine that we need 10ml of the stock solution to make the new solution.

11 0.1mol / l *100ml V Stock 10ml 1.0mol / l Notice that the units mol/l cancel. STEP 2 Now that we know how much (volume) stock is required to make the new solution, we must determine exactly how much distilled water is to be added to make the dilution. This is simply: VDistilled Water = VNew - VStock In our example, this is 100 ml -10 ml = 90 ml. So, to make the new solution you simply combine 10 ml of stock solution with 90 ml of distilled water. You now have 100 ml of NaCl which is exactly 0.1 mol/1 in concentration. In practice we would use a 100 ml volumetric flask to carry out the dilution. This would be done by pipetting 10 ml of 1 mol/l NaCl stock into the volumetric flask and then filling the flask with distilled water to the 100 ml mark on the long neck of the flask, which is designed to hold exactly 100 ml at room temperature. ADDITIONAL DILUTION PROBLEMS Use the equations above to work each problem before looking up the correct answer provided below. 1. If you have stock solution of g/l NaCl, how would you make 200 ml of a 0.1 g/l NaCl solution? 2. You have a stock solution of 5 g/100 ml P04-2. How would you make 5 ml of a seawater solution with 0.01 g/ml P0 4-2? 3. If you have a stock solution of 0.2 g/l NO 3-2, how would you make 500 ml of a 0.1 mg/l NO 3-2 solution? Be careful with units here. Answers to problems 1 through 3 above ml stock ml distilled water ml stock ml artificial seawater ml stock ml artificial seawater