10514011 Atomic Absorption Spectroscopy

Atomic Absorption Spectroscopy (AAS) Reading: Rouessac and Rouessac; Chapter 14 Introduction The phenomenon of atomic absorption (AA) was first observed in 1802 with the discovery of the Fraunhofer lines in the sun's spectrum. It was not until 1955 however that Walsh proposed that atomic absorption be used for quantitative chemical analysis. Atomic absorption analysis involves measuring the absorption of light by vaporized ground state atoms and relating the absorption to concentration. The incident light beam is attenuated by atomic vapor absorption according to Beer's law. To obtain the best results in AA, the instrumental and chemical parameters of the system must be geared toward the production of neutral ground state atoms of the element of interest. Conversion of the sample from its native state to the atomic state can be achieved using a flame (flame-AAS) or an electric furnace (electrothermal or graphite furnace AAS). The latter will be studied herein. In the furnace, the sample undergoes a number of pretreatment steps prior to analysis. First the sample is dried by evaporating the solvent (in this case the water). Second the organic matrix is decomposed by heating of the sample (often to temperatures exceeding 1000ºC). Care must be taken during these steps not to lose any of the analyte through evaporation processes (e.g., inclusion in a plume from flammable organics). Finally, the furnace is rapidly heated to temperatures around 2400ºC to produce vaporized neutral atoms with as many as possible in their in electronic ground states. The absorption spectrum of the gas phase atoms is extremely narrow ( ≤10-2 nm). Thus the light source used for absorbance measurements must be of precisely the correct wavelength and of narrow line width for Beer’s law to remain valid. The light source used in AAS is a hollow cathode lamp in which light is emitted from excited atoms of the same element which is to be determined. This ensures that the radiant energy corresponds directly to the wavelength, which is absorbable by the atomized sample. This method provides both sensitivity and selectivity since other elements in the sample will not generally absorb the chosen wavelength and thus, will not interfere with the measurement. However, molecular species may also be formed during the atomization step. This can alter the spectral characteristics of the analyte metal or can cause spectral interference at the wavelength being monitored. To reduce background interference, the wavelength of interest is isolated by a monochromator placed between the sample and the detector. Additional techniques such as D2 or Zeeman background correction may also be used for complex matrices such as beer.

Chem 313 Instrumental Analysis

Experiment Summary In this experiment: •

a graphite furnace atomic absorption spectrometer will be used

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the ashing and atomization processes will be examined

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the GF-AAS will be calibrated for copper using a series of standard solutions

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copper in food products will be determined using 1) calibration curve and 2) standard additions methods

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the copper content will be compared to the specifications on the products

Instrumentation The instrument used in this lab is the Varian SpectrAA-880Z AA spectrometer. It is a single beam instrument which consists of four main building blocks or modules: a hollow cathode lamp turret and power supply, a graphite furnace equipped with Zeeman background correction, a monochromator, and an electronic readout system (computer and software). Some aspects of these modules are discussed below. i. Hollow Cathode Lamp Insulating disk Quartz or glass window

+ Hollow cathode

Anode

Figure 1. A typical hollow-cathode lamp (HCL) Hollow cathode lamps are used as the light source for AA measurements. A typical hollow cathode lamp is shown in Figure 1. The majority of HCLs are single element lamps with the hollow cathode coated with the desired element. The lamps are generally filled with neon under reduced pressure (1-5 torr), are operated at a few hundred volts (150 - 400 volts) and require currents anywhere from approximately 3 - 25 mA. When a neon filled tube is operating the hole in the cathode has an orange glow due to Ne emission.

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Chem 313 Instrumental Analysis ii. Electrothermal Atomizer (Graphite Furnace) Electrothermal atomizers generally provide a number of advantages compared to other atomization methods: enhanced sensitivity and lower detection limits, the capability to deal with extremely small samples (0.5-10µL), the capability to directly treat solid samples, and lower noise associated with the atomization process. Electrothermal atomizers work by heating the sample to the point of atomization. The heating profile is essential to the proper performance of the instrument. Typically there are three major stages to an atomization process: 1) evaporation of solvents-this step usually 30-60s with temperatures slowly ramping up to 100-120ºC; 2) ashing- this step usually lasts a 515s and involves heating the sample to 700-900ºC, at these temperatures most of the organic matrix is removed; and finally 3) atomization-this step involves actual atomization of the sample, the temperature of this step may approach 2900ºC. The exact choice of heating profiles depends on the matrix and the analyte. These profiles are often determined empirically by systematically varying the times and temperatures for each of these steps. This particular graphite furnace is fitted with a Zeeman Background correction assembly. The details of this method can be found in the text in section 9-C (pages219220). Essentially, a magnetic field is used to cause Zeeman splitting of the energy levels of the atomized analyte. The absorbance signal without the field and with the field may be compared to remove unwanted background noise from the signal. This is especially useful in complex matrices.

iii. Monochromator The monochromator depicted in Figure 2 is placed between the graphite furnace and the detector and is used to isolate the wavelength of interest and reduce background interference. This is a rather typical monochromators and a description of monochromators may be found in the textbook section 7C (pages 159-166). Concave Mirrors

Reflecting Grating Entrance Slit

λ1

λ2

Exit Slit

Figure 2. Czerny-Turner Grating Monochromator

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Chem 313 Instrumental Analysis iv. Computer and Software The instrument is controlled by a software package called SpectrAA. This program allows the user to select many of the essential parameters for operation of the instrument. For example you may set the slit widths for the monochromators, programmed temperature profiles, lamp selection, just to name a few. This software also incorporates data acquisition and statistical analysis to determine the concentrations of unknowns, by using a number of common calibration methods.

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Chem 313 Instrumental Analysis

Experiments Prepare the solutions and instrument as instructed below. The TA will brief you on the AA experiment later.

I. Prepare the Solutions You are required to make four solutions, a 100 µg/L copper standard for the calibration curve method, a 50 µg/L copper standard for the standard addition method, a blank solution, and a sample solution. All solutions are to be prepared in 1% HNO3. You will be provided with a 5 µg/mL stock solution and therefore you do NOT need to prepare a stock solution from copper wire. 100 µg/L Copper Standard Prepare 100 mL of this solution by dilution of the provided 5 µg/mL stock solution. Add an appropriate amount of concentrated nitric acid to make the diluted solution 1% HNO3. Sample solution Obtain a sample of beer from your lab instructor. You will analyze the beer in its undiluted form. Blank (make-up) Prepare 100 mL of 1% HNO3 by appropriate dilution of concentrated nitric acid. 50 µg/L Copper Standard (prepare this solution as the calibration curve method is running) Prepare 100 mL of this solution by dilution of the provided 5 µg/mL stock solution. Add an appropriate amount of concentrated nitric acid to make the diluted solution 1% HNO3.

II. Prepare the Instrument Follow this sequence to turn on the AAS. 1 Turn on both of the gases, nitrogen (200 kPa) and argon (200kPa). These tanks are located to the right of the instrument. Make sure that the pressure is set to 200kPa for each. 2 Turn on the water for cooling, this faucet is located behind the computer. 3 Turn on both switches in front of the instrument a. At this point, the instrument will go thru a startup sequence. b. You do not need to wait until it is warmed up in order begin with the software. 4 Turn on the computer, if it is currently off. You will be prompted for a username and password. You can use the username “Chem313” with password “-7chem313”. 5 Once you have logged in. You can access the “SpectrAA” software via <start><programs><spectrAA><spectrAA>. a. If you see an error saying “HC lamp strike failure”, just ignore it, it doesn’t matter

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Chem 313 Instrumental Analysis III. Quantitative Analysis of Cu by the Calibration Curve Method 1

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3

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In order to do analysis, you will need to start a “Worksheet” a. A Worksheet is where you determine the parameters and conditions of operation, you control the instrument, it is also where you will perform the analysis, etc… b. Then, it will ask you to either create a “NEW” worksheet, create “New from” worksheet from one that was already developed but that you want to make a couple of changes or “Open” an already existing worksheet. c. Select “New From” and then select the worksheet named “Chem 311-Calibration Curve”. This sheet is already set up for quantitatively measuring copper for a number of samples (loaded on the auto sampler) by first performing a calibration curve. The solutions for the calibration curve are all mixed by the auto sampler by combining the make-up and the bulk solutions. d. You will need to type in a worksheet name for your new version of that template. Use your complete names separated by a hyphen. i) For the number of samples, it doesn’t matter because you can change it later as often as you like. Select the develop tab. a. Select the edit method button. b. Examine the parameters and their values, although you should not alter any of these values, it is useful to verify that you know what they do. c. Note the concentration values on the standards tab. i) In this experiment the auto sampler will automatically mix the standard solutions for the calibration curve, 10, 20, 30, 40, 50, 100µg/L. d. Next note the information on the sampler tab. i) The sample volume should equal the total volume which should be 20µL. ii) When you’re done click on OK You can go in the “Label” page to name each one of your sample a. Also, this is where you can add or delete the number of sample you want. b. It is also where you would set up you autosampler carousel “Set-up PSD carousels” c. You will need 8 autosampler vials. 1 for the 100ng/ml bulk solution to be placed in position 51, 1 for the 1% nitric acid makeup solution to be placed in position 52, and 1 vial for each of the samples to be analyzed, these should be placed in positions 1 through 6 on the auto sampler. Each of these vials should be rinsed with the appropriate solution prior to use. To start your analysis go in the Instrument tab a. First, you will need to Optimize the method i) Just click on optimize button and follow instructions given on screen. You may have to wait a minute before it will produce readings. The signal should be ~0.98 ii) If you have any error message, just start again until it works iii) To get out of that, you need to press Cancel when you’re done b. Alignment of the autosampler i) select the instrument menu item and then select furnace facilities from the menu list. ii) type in vial number 52 AAS-6

Chem 313 Instrumental Analysis iii) iv) v)

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press the align button. verify that the injection tube is inserted into the makeup vial and press ok verify that the injection tube is completely inserted into the graphite furnace. If the metal set screw does not make contact with the metal pad then there is a problem. You should also examine the placement of the injection tube within the furnace with a mirror that is supplied. Once this is verified you may press ok. c. Then you can start your method simply by clicking on Start i) If you’re doing manual injection, it will tell you that the autosampler “PSD” is off line, just click OK until it ask you to inject your sample. ii) If you want to start with something else than what they ask for than you can pause your method, click on “Select”, click on the one you want to analyze or start with. Click on “Select” again to get out. The one you selected will be highlighted in light blue. iii) Then you can go in “File > start at”, select what you want and start your analysis. When you are done and you want a printout of your results, you have a lot of choice. a. To get a clean report of your last results of your method, you need to go to the Filing tab, save and close your worksheet. This will bring you to the first page. You choose report, your worksheet and then you choose the option you want in your report. You can preview it before you print it out. b. It is also possible to print the datalog (window in the bottom left corner), but this will print out every action you did and you have no graphs i) To do that, just choose “options>datalog> print” c. You can also print any other window from the options, but just the current one

IV. Quantitative Analysis of Cu by the Standard Addition Method 1

In order to do analysis, you will need to start a “Worksheet” a. A Worksheet is where you determine the parameters and conditions of operation, you control the instrument, it is also where you will perform the analysis, etc… b. Then, it will ask you to either create a “NEW” worksheet, create “New from” worksheet from one that was already developed but that you want to make a couple of changes or “Open” an already existing worksheet. c. Select “New From” and then select the worksheet named “Chem 311-Standard Addition”. This sheet is already set up for quantitatively measuring copper for a sample (loaded on the auto sampler) by performing a standard additions with that sample. The solutions are all mixed by the auto sampler by combining the makeup, the bulk and the sample solutions. d. You will need to type in a worksheet name for your new version of that template. Use your complete names separated by a hyphen. i) For the number of samples, it doesn’t matter because you can change it later as often as you like. 2 Select the develop tab. a. Select the edit method button. b. Examine the parameters and their values, although you should not alter any of these values, it is useful to verify that you know what they do. c. Note the concentration values on the standards tab.

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Chem 313 Instrumental Analysis i)

In this experiment the auto sampler will automatically add solutions with 10, 20, 30, 40µg/L. d. Next note the information on the sampler tab. i) The sample volume should equal 5µL while the total volume equals 20µL. ii) When you’re done click on OK 3 You can go in the “Label” page to name each one of your sample a. Also, this is where you can add or delete the number of sample you want. b. It is also where you would set up you autosampler carousel “Set-up PSD carousels” c. You will need 3 autosampler vials. 1 for the 50ng/ml bulk solution to be placed in position 51, 1 for the 1% nitric acid makeup solution to be placed in position 52, and 1 vial for the sample to be analyzed placed in position 1. Each of these vials should be rinsed with the appropriate solution prior to use. 4 To start your analysis go in the Instrument tab a. First, you will need to Optimize the method i) Just click on optimize button and follow instructions given on screen. You may have to wait a minute before it will produce readings. The signal should be ~0.98 ii) If you have any error message, just start again until it works iii) To get out of that, you need to press Cancel when you’re done b. Alignment of the autosampler i) select the instrument menu item and then select furnace facilities from the menu list. ii) type in vial number 52 iii) press the align button. iv) verify that the injection tube is inserted into the makeup vial and press ok v) verify that the injection tube is completely inserted into the graphite furnace. If the metal set screw does not make contact with the metal pad then there is a problem. You should also examine the placement of the injection tube within the furnace with a mirror that is supplied. Once this is verified you may press ok. c. Then you can start your method simply by clicking on Start i) If you’re doing manual injection, it will tell you that the autosampler “PSD” is off line, just click OK until it ask you to inject your sample. ii) If you want to start with something else than what they ask for than you can pause your method, click on “Select”, click on the one you want to analyze or start with. Click on “Select” again to get out. The one you selected will be highlighted in light blue. iii) Then you can go in “File > start at”, select what you want and start your analysis. 5 When you are done and you want a printout of your results, you have a lot of choice. a. To get a clean report of your last results of your method, you need to go to the Filing tab, save and close your worksheet. This will bring you to the first page. You choose report, your worksheet and then you choose the option you want in your report. You can preview it before you print it out. b. It is also possible to print the datalog (window in the bottom left corner), but this will print out every action you did and you have no graphs

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Chem 313 Instrumental Analysis i) To do that, just choose “options>datalog> print” c. You can also print any other window from the options, but just the current one

V. Optimization of Ashing and Atomization Ashing Curve Move the 50 µg/L copper standard solution from position 51 to 52. You will not receive a print out of this data. You must record it manually as you go. Create a new worksheet from the file “Chem313-Ashing”. Select the develop tab, under furnace, note the initial ashing temperature. 1) Press the Start button, the furnace will inject the copper solution and analyse it 2) The reading will appear in the bottom right of the screen, beside CAL ZERO 3) Immediately record the value and press Stop 4) Select the develop tab, under furnace change the ashing temperature to 700 oC 5) Repeat steps 1-4, changing the ashing temperature to the following values; 900, 1100, 1300, and 1500 oC for each run Atomization Curve You will not receive a print out of this data. You must record it manually as you go. Create a new worksheet from the file “Chem313-Atomization”. Select the develop tab, under furnace, note the initial atomization temperature. 1) Press the Start button, the furnace will inject the copper solution and analyse it 2) The reading will appear in the bottom right of the screen, beside CAL ZERO 3) Immediately record the value and press Stop 4) Select the develop tab, under furnace change the atomization temperature to 1900 oC 5) Repeat steps 1-4, changing the atomization temperature to the following values; 2100, 2300, and 2500 oC for each run

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Chem 313 Instrumental Analysis

Data Processing and Questions 1. Determine the concentration of Cu in the beer samples using your calibration line and the absorbance data obtained for the beer samples. How does this value compare to the amount of copper expected for a healthy diet (obtain this data with an online search)? Assuming that this is your only source of copper, how much of this brand of beer would you need to drink per day to remain healthy? (This is the same reasoning used in those annoying Total cereal commercials, where someone is given 20 bowls of Raisin Bran to equal 1 bowl of Total) 2. Determine the concentration of Cu in the assigned beer sample by the method of standard additions. How does this value compare to the specifications on the produce? 3. Which method of calibration is more appropriate for the determination of Cu in the beer sample using GFAAS? Justify your answer. 4. Many instrument companies provide instructions for performing determinations using their equipment. Varian provides an extensive listing of application notes for various elements and matrices. These are available on-line at http://www.varianinc.com browse through spectroscopy, atomic spectroscopy and finally to the application notes. (A link to this site is on the Chem 311 web page at http://www.chem.ualberta.ca/Undergrad Studies/ Courses/Chem311/index.htm). Your TA will assign you an element/matrix from the Varian web page. List the conditions used for this analysis. 5. Briefly (1/2 – 1 page) compare D2 and Zeeman background correction. 6. Plot i) Absorbance vs. ashing temperature ii) Absorbance vs. atomization temperature Explain the shapes of the curves observed

References [1] D.D. Smith, R.F. Bronwer, Anal. Chem., 56, 2702, 1984. [2] J.I. Dinnin, Anal. Chem., 32, 1475, 1960. [3] W. Slavin, "Atomic Absorption Spectroscopy", Interscience, New York, 1968. (call number: QC 451S63) [4] D.A. Skoog, F.J. Holler and T.A. Nieman, Principles of Instrumental Analysis, 5th ed., Saunders, 1998. Chapter 9. Note: References 1-3 are available at the Chem 313 web site at: http://www.chem.ualberta.ca/Undergrad Studies/Courses/Chem313/index.htm

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