Atomic Absorption Spectroscopy (AAS) Reading: Rouessac and Rouessac; Chapter 14 Introduction The phenomenon of atomic absorption (AA)

was first observed in 1802 with the dis covery of the Fraunhofer lines in the sun's spectrum. It was not until 1955 howe ver 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. T he incident light beam is attenuated by atomic vapor absorption according to Bee r's law. To obtain the best results in AA, the instrumental and chemical paramet ers of the system must be geared toward the production of neutral ground state a toms 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 fur nace (electrothermal or graphite furnace AAS). The latter will be studied herein . In the furnace, the sample undergoes a number of pretreatment steps prior to a nalysis. 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 1000C). Care must be taken during these steps not to lo se 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 2400C to produce vaporized neutral atoms with as many as possible in thei r 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 measureme nts must be of precisely the correct wavelength and of narrow line width for Bee r'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 det ermined. This ensures that the radiant energy corresponds directly to the wavele ngth, which is absorbable by the atomized sample. This method provides both sens itivity and selectivity since other elements in the sample will not generally ab sorb the chosen wavelength and thus, will not interfere with the measurement. Ho wever, molecular species may also be formed during the atomization step. This ca n alter the spectral characteristics of the analyte metal or can cause spectral interference at the wavelength being monitored. To reduce background interferenc e, the wavelength of interest is isolated by a monochromator placed between the sample and the detector. Additional techniques such as D2 or Zeeman background c orrection 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 us ed the ashing and atomization processes will be examined the GF-AAS will be cali brated for copper using a series of standard solutions copper in food products w ill be determined using 1) calibration curve and 2) standard additions methods t he 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 modul es: a hollow cathode lamp turret and power supply, a graphite furnace equipped w ith Zeeman background correction, a monochromator, and an electronic readout sys tem (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 t he light source for AA measurements. A typical hollow cathode lamp is shown in F igure 1. The majority of HCLs are single element lamps with the hollow cathode c oated with the desired element. The lamps are generally filled with neon under r educed 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 . AAS-2

Chem 313 Instrumental Analysis ii. Electrothermal Atomizer (Graphite Furnace) Electrothermal atomizers generally provide a number of advantages compared to ot her atomization methods: enhanced sensitivity and lower detection limits, the ca pability to deal with extremely small samples (0.5-10L), the capability to direct ly 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 -120C; 2) ashing- this step usually lasts a 515s and involves heating the sample to 700-900C, at these temperatures most of the organic matrix is removed; and fin ally 3) atomization-this step involves actual atomization of the sample, the tem perature of this step may approach 2900C. The exact choice of heating profiles de pends on the matrix and the analyte. These profiles are often determined empiric ally by systematically varying the times and temperatures for each of these step s. This particular graphite furnace is fitted with a Zeeman Background correctio n 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 fi eld 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 an d the detector and is used to isolate the wavelength of interest and reduce back ground interference. This is a rather typical monochromators and a description o f monochromators may be found in the textbook section 7C (pages 159-166). Concave Mirrors Reflecting Grating Entrance Slit l1 Exit Slit l2 Figure 2. Czerny-Turner Grating Monochromator AAS-3

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, pro grammed temperature profiles, lamp selection, just to name a few. This software also incorporates data acquisition and statistical analysis to determine the con centrations of unknowns, by using a number of common calibration methods. AAS-4

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 g/L copper standard for the calibr ation curve method, a 50 g/L copper standard for the standard addition method, a blank solution, and a sample solution. All solutions are to be prepared in 1% HN O3. You will be provided with a 5 g/mL stock solution and therefore you do NOT ne ed to prepare a stock solution from copper wire. 100 g/L Copper Standard Prepare 100 mL of this solution by dilution of the provided 5 g/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% HN O3 by appropriate dilution of concentrated nitric acid. 50 g/L Copper Standard (p repare this solution as the calibration curve method is running) Prepare 100 mL of this solution by dilution of the provided 5 g/mL stock solution. Add an approp riate 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 instrum ent. 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 star tup sequence. b. You do not need to wait until it is warmed up in order begin wi th the software. 4 Turn on the computer, if it is currently off. You will be pro mpted 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 <star t><programs><spectrAA><spectrAA>. a. If you see an error saying HC lamp strike fa ilure, just ignore it, it doesn't matter AAS-5

Chem 313 Instrumental Analysis III. Quantitative Analysis of Cu by the Calibrati on Curve Method 1 In order to do analysis, you will need to start a Worksheet a. A Worksheet is wh ere you determine the parameters and conditions of operation, you control the in strument, it is also where you will perform the analysis, etc b. Then, it will as k 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 31 1-Calibration Curve. This sheet is already set up for quantitatively measuring co pper 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 t o type in a worksheet name for your new version of that template. Use your compl ete names separated by a hyphen. i) For the number of samples, it doesn't matter b ecause 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, altho ugh you should not alter any of these values, it is useful to verify that you kn ow what they do. c. Note the concentration values on the standards tab. i) In th is experiment the auto sampler will automatically mix the standard solutions for the calibration curve, 10, 20, 30, 40, 50, 100g/L. d. Next note the information on the sampler tab. i) The sample volume should equal the total volume which sho uld be 20L. ii) When you're done click on OK You can go in the Label page to name eac h 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 Se t-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 t o be placed in position 52, and 1 vial for each of the samples to be analyzed, t hese should be placed in positions 1 through 6 on the auto sampler. Each of thes e vials should be rinsed with the appropriate solution prior to use. To start yo ur analysis go in the Instrument tab a. First, you will need to Optimize the met hod i) Just click on optimize button and follow instructions given on screen. Yo u may have to wait a minute before it will produce readings. The signal should b e ~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 facilitie s from the menu list. ii) type in vial number 52 AAS-6 2 3 4

Chem 313 Instrumental Analysis press the align button. verify that the injection tube is inserted into the make up 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 meta l pad then there is a problem. You should also examine the placement of the inje ction tube within the furnace with a mirror that is supplied. Once this is verif ied you may press ok. c. Then you can start your method simply by clicking on St art i) If you're doing manual injection, it will tell you that the autosampler PSD i s off line, just click OK until it ask you to inject your sample. ii) If you wan t to start with something else than what they ask for than you can pause your me thod, click on Select, click on the one you want to analyze or start with. Click o n 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 anal ysis. 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 nee d 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 optio n 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 c hoose options>datalog> print c. You can also print any other window from the optio ns, but just the current one iii) iv) v) 5 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 wh ere you determine the parameters and conditions of operation, you control the in strument, it is also where you will perform the analysis, etc b. Then, it will as k 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 31 1-Standard Addition. This sheet is already set up for quantitatively measuring co pper for a sample (loaded on the auto sampler) by performing a standard addition s 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 wo rksheet name for your new version of that template. Use your complete names sepa rated 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 e dit method button. b. Examine the parameters and their values, although you shou ld 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. AAS-7

Chem 313 Instrumental Analysis In this experiment the auto sampler will automatically add solutions with 10, 20 , 30, 40g/L. d. Next note the information on the sampler tab. i) The sample volum e should equal 5L while the total volume equals 20L. ii) When you're done click on O K 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 y ou would set up you autosampler carousel Set-up PSD carousels c. You will need 3 a utosampler 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 b e 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 cl ick 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 men u 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 meta l set screw does not make contact with the metal pad then there is a problem. Yo u should also examine the placement of the injection tube within the furnace wit h 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 injec tion, it will tell you that the autosampler PSD is off line, just click OK until i t ask you to inject your sample. ii) If you want to start with something else th an 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 > st art 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 re port 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 rep ort, 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 data log (window in the bottom left corner), but this will print out every action you did and you have no graphs i) AAS-8

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 g/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 g o. Create a new worksheet from the file Chem313-Ashing. Select the develop tab, un der 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 appe ar 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 th e 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 yo u go. Create a new worksheet from the file Chem313-Atomization. Select the develop tab, under furnace, note the initial atomization temperature. 1) Press the Star t button, the furnace will inject the copper solution and analyse it 2) The read ing will appear in the bottom right of the screen, beside CAL ZERO 3) Immediatel y record the value and press Stop 4) Select the develop tab, under furnace chang e the atomization temperature to 1900 oC 5) Repeat steps 1-4, changing the atomi zation temperature to the following values; 2100, 2300, and 2500 oC for each run AAS-9

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 wi th 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 som eone 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 addit ions. 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 be er sample using GFAAS? Justify your answer. 4. Many instrument companies provide instructions for performing determinations using their equipment. Varian provid es an extensive listing of application notes for various elements and matrices. These are available on-line at http://www.varianinc.com browse through spectrosc opy, atomic spectroscopy and finally to the application notes. (A link to this s ite 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. ash ing 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", Inters cience, 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. Chap ter 9. Note: References 1-3 are available at the Chem 313 web site at: http://ww w.chem.ualberta.ca/Undergrad Studies/Courses/Chem313/index.htm AAS-10