Homework answers / question archive / Experiment 4 The Determination of Manganese in Coconut Water Using Atomic Absorption Spectroscopy Objectives In this experiment, the student will be introduced to the basic operation of the Pinnacle PerkinElmer atomic absorption spectrophotometer (AAS)

Experiment 4 The Determination of Manganese in Coconut Water Using Atomic Absorption Spectroscopy Objectives In this experiment, the student will be introduced to the basic operation of the Pinnacle PerkinElmer atomic absorption spectrophotometer (AAS)

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Experiment 4

The Determination of Manganese in Coconut Water Using Atomic Absorption Spectroscopy

Objectives

In this experiment, the student will be introduced to the basic operation of the Pinnacle PerkinElmer atomic absorption spectrophotometer (AAS). The student will determine the concentration of manganese in a commercial can of coconut water using method of standard addition following the Beer Lambert Law. Both multipoint graphical and single point standard addition data handling approaches will be used to quantify the final concentration of manganese (mg / 330 mL carton) in the coconut water sample. Statistical data handling methods will be employed to evaluate the quality of the final results. The experimental results will then be compared to the manufacturer’s labels in order to deduce the identity of the unknown coconut water sample.

Introduction

I- Coconut Water

Minerals are important for humans to stay healthy. The body uses minerals for many different reasons, including building bones, synthesizing hormones and regulating heartbeats.

There are two kinds of minerals: macro minerals and trace minerals. Macro minerals are minerals that the body needs in large amounts. These include calcium, phosphorus, magnesium, sodium , potassium, chloride and sulfur. The body also needs small amounts of trace minerals. These include iron, manganese, copper, iodine, zinc, cobalt, fluoride and selenium.

The best way to obtain the minerals that the body requires is to drink water and eat a wide variety of foods. Coconut water is a clear liquid found in the fruit’s interior cavity or endosperm of young, green coconuts about 5-7 months. Each nut may contain about 200 to 1000 mL of water depending on type and size. The clear liquid is sweet, and sterile and composed of unique chemicals such as sugars, vitamins, minerals, electrolytes, enzymes, amino acids, cytokine, and phyto-hormones.

A recent health craze claims that coconut water is natures ‘energy drink’ due to endorsements by celebrities and athletes. Coconut water contains natural sugars and fiber with fewer calories than most commercial energy drinks and has far more potassium 60 mg compared to 6 mg per ounce and less sodium 5 mg rather than 14 mg per ounce. It is also a good source of essential Bcomplex vitamins such as riboflavin, niacin, thiamin, pyridoxine, and folates, which are important for overall health and should be consumed by diet. Coconut water also contains a small amount of vitamin C, which possesses antioxidant properties.

The mineral content of coconut water along with other commercial beverages must be verified for quality control purposes. As of December 14, 2016; amendments to nutritional labelling under the Canadian Food and Drug Regulations Act, requires all companies to list and accurately label the concentration of all ingredients and food colours for all products sold for consumption. Atomic absorption spectroscopy provides a rapid way to analyze many minerals in commercial food and beverage products at trace and levels (ppm or ppb).

II-Atomic Absorption Spectroscopy

The easiest way to determine the chemical composition of steel is to use either atomic absorption (AAS) or flame emission spectroscopy (FES). The equipment for both techniques is similar except that for AAS, light absorption is measured while for FES, intensity of light emission is measured.

To understand the theory of the technique, it is necessary to review the physics of the atom. The atom is made up of a positively charged nucleus surrounded by electrons. Every element has a specific number of electrons which are associated with the nucleus in an orbital arrangement that is unique to each element. The electrons occupy the orbital positions in an orderly and predictable way; lowest energy orbitals being filled first. The lowest energy or most stable electronic configuration of an atom is known as the ground state. If energy of the right magnitude is absorbed by the atom, an outer valence shell electron will be promoted to an orbital position higher in energy. This is known as an excited state and is unstable in comparison to the ground state. As this state is unstable, the atom will immediately and spontaneously return to the ground state. In doing, so the electron will return to its initial and stable orbital configuration or ground state. This will result in the release or emission of radiant energy. The amount of the energy of released when the electron returns to the ground state is equal to the amount of the energy initially adsorbed in the excitation process. This process is illustrated in Figure 1.

Figure 1: Absorption and Emission.

In Figure 1, it can be seen that in the first step; excitation of the electron to an excited state, occurs as a result of absorption of a quantized amount of energy (photon). The second step, the emission process, produces light and occurs spontaneously as the electron falls down to its original ground state orbital.

The wavelength of the absorbed or emitted energy is directly related to the electronic transition which has occurred. In addition, the quantized amount of light emitted and absorbed is directly proportional to the concentration of the excited atoms in the solution. As every element has a unique electronic structure, the wavelength of the absorbed or emitted energy will be different for every element. It is important to note that the orbital configuration of a large atom is complex and that this will result in the production of many electronic transitions each with its own characteristic wavelength of light. Thus, an atom will emit and absorb light of more than one frequency as shown in Figure 2.

Figure 2: The Process of Excitation (AAS) and Decay (AES) Measured for Analytical Purposes.

Atomic absorption spectroscopy (AAS) is an analytical method that measures that energy absorbed by the atom resulting in the promotion of a valance electron to a higher electronic orbital whereas Atomic emission spectroscopy (AES) measures the energy emitted by the excited electron as it returns to a ground state (valence shell). Both processes, emission or absorption of energy by electrons in atoms of elements, provides a basis for an analytical method as the frequency or wavelength of the light adsorbed is specific and quantized for each element and the amount of light adsorbed (absorbance = A) or light emitted (relative intensity of light emitted = I_E) is proportional to the concentration (C) of the element in a solution.

?? ?? ?? ?? (AAS)

or ??_?? ?? ?? ?? (AES)

To analyze a sample containing metal analytes utilizing the principles of atomic absorption (AAS), the sample solution must first be heated to a temperature that is sufficient to dissociate the analyte compound into free metal gaseous atoms. The sample solution is first drawn up into a nebulizerspray chamber system- burner assembly. An aerosol mist of the sample is formed by nebulization, and the fine sample droplets are mixed with flame gases and forced up into the burner head. In the high temperature flame, the sample mist undergoes an atomization process. During this process, the intense heat of the flame causes the solvent droplets surrounding the analyte sample to evaporate ‘desolvate’ yielding solid metal salt particles which further melt, dissociate and vaporize into metal cations. Consequently, any metal compounds in solution are broken down during evaporation and subsequently undergo a liquid melt, vaporization and atomization as illustrated in Figure 3. Reducing species from the incomplete combustion of the flame gas mixture, add electrons to the gaseous metal cations resulting in the formation of free gaseous ground state metal atoms thereby completing the atomization process.

Figure 3: Flame Atomization Process.

In turn, the gaseous ground state metal atoms formed can then absorb their characteristic wavelengths of light emitted from the hollow cathode lamp to produce causing the valence electrons to be promoted from the ground state to higher electronic energy levels (excited state). Due to the fact that each metal element in the periodic table has its own discrete electron configuration, the wavelengths (resonance lines, or energies) at which each element absorbs is unique. As a result of this absorption, the power of the original light beam "P_o", (from the hollow cathode lamp), is reduced "P" (i.e. P_o > P and T= P/P_o). The amount of the light absorbed is directly proportional to the concentration of the metal analyte atoms present in the flame.

In Figure 4, five major components of an AA spectrophotometer are shown including a line radiation source, atomizer, monochromator, detector and readout. Ground state atoms formed in the flame absorb their characteristic wavelengths of light from the hollow cathode lamp. A single wavelength is isolated by the monochromator and its power is monitored with the photomultiplier tube (PMT).

Figure 4: Major Components of a Double Beam Atomic Absorption Spectrophotometer

Almost all atomic absorption instruments use a hollow cathode lamp (HCL) as the external radiation source. It is important that the light source produce narrow lines of radiation of distinctive energies and possess adequate intensity and exhibit stability for long periods of time. An ordinary monochromator is incapable of yielding a band of radiation as narrow as the peak width of an atomic absorption line (approximately 0. 2 to 0.7 nm). In order for Beer's law to be obeyed, the radiation source must emit a line of light (resonance line) that is the same wavelength as that characteristically absorbed by the analyte atoms.

As seen in Figure 5, cathodes are constructed from the metallic element of interest (metal analyte being analyzed). (ie. Manganese (Mn) cathode for Mn analyte solutions). Hollow cathode lamps are filled with inert neon gas at low pressure, which ionizes when a voltage is applied across electrodes. The Ne gas ions bombard the cathode, heating it and causing the solid metal to sputter into gaseous metal atoms within the cathode chamber as well as become excited. The excited metal atoms emit resonance wavelengths of light characteristic of the metal element and the metal redeposits itself back onto the surface of the cathode. Figure 5 illustrates the hollowcathode tube process consisting of sputtering, excitation emission and re-deposition of the metal back onto the cathode.

Figure 5: Schematic Cross-section of a Hollow Cathode Lamp.

The emitted light “P_o” is then directed to the sample that is now in the form of free gaseous metal atoms in the optical path within the flame. Since the cathode element and the analyte in the sample are the same element, the emitted resonance wavelengths of light from the lamp excite the gaseous metal atoms in the flame. A monochromator positioned after the flame is necessary to select the correct resonance wavelength of light to be used for a particular analysis. Figure 6 shows the double beam design for an atomic absorption spectrophotometer and the two optical pathways that the light alternatively travels; namely the reference path and the sample path.

Figure 6: Double Beam Optical Diagram of an AAS showing the Radiant Pathway of P and P_o

The detector, a photomultiplier tube (PMT) senses the decrease in the radiant power “P” (attenuation) from the cathode lamp due to the absorption of light by the sample analyte metal atoms. The PMT converts the radiant signal to an electrical signal which is proportional to the power of light at the wavelength which has been isolated by the monochromator. Figure 7

illustrates the conversion of a photon signal to an electron signal which is amplified using a series of dynodes adjusted at 90V more positive than the previous dynode. These secondary electrons accelerate sequentially through a series dynodes releasing a cascade of electrons at each until they finally reach the anode. One photon is capable of producing 10⁷ secondary electrons of photocurrent. Figure 6 shows the double beam design of the spectrometer with the PMT receiving alternative pulses of P/2 and P_o/2 due to the rotating beam chopper and half silvered mirror. The incident and attenuated signals are collected and ratioed by the signal processor (tuned electronics) to produce transmittance “T” and absorbance “A”.

Figure 7: Conversion and Amplification of the Radiant Signal by the Photomultiplier Tube

III-Beer-Lambert Law

For most atomic absorption measurements, the Beer-Lambert law is obeyed:

?? = −?????? = ??????

??_??

where a = absorption coefficient (absorptivity) b = length of absorption path - width of burner slot/flame c = concentration of analyte atoms

When experimental conditions are optimized, the absorbance is directly proportional to the concentration of the element for a given absorption pathlength at a resonance wavelength. Various experimental parameters such as aspiration uptake rate, flow rate of gases entering the flame, flame stoichiometry, burner height, radiation source etc.; influence the absorption coefficient "a". Since the value of "a" for a particular element at a specified wavelength varies with fluctuations in experimental conditions, it is difficult to reproduce accurately. For this reason, Beer's Law equation is never used directly to calculate the concentration of an analyte from a single absorbance measurement. Instead, a calibration curve must be generated by either external standard or standard addition methods.

IV-Method of Standard Addition

In this experiment, manganese will be analyzed using method of standard addition. In the event standards cannot be prepared identical to the unknown sample solution matrix (matrix matching) or that it is difficult to suppress matrix interferences that are inherent in the unknown sample, the technique of standard addition, is the method of choice. In this method, known quantities of pure analyte stock solution are added to the unknown sample solution and the increase in absorbance signal is measured for each standard addition in the series. Normally additions which are equal to twice and one-half the original amount of analyte in the unknown sample are optimum statistically. All solutions in the series contain the same amount of unknown sample solution and must be diluted to the same final volume as shown in Figure 8. Any interferent present in the unknown sample solution matrix, will affect the absorbance signal for all the solutions in the standard addition series equally since each solution contains the same amount of unknown sample solution thereby nulling out any signal due to the interferent. The resulting analyte signal will be due to the analyte only.

Figure 8: Standard Addition Dilution Scheme

Once absorbance measurements are complete for the standard addition series, a linear plot of absorbance as a function of analyte concentration added can be made. Figure 9 displays a typical standard addition plot where the x axis represents the concentration of analyte standard added to each unknown sample solution in the series while the y axis is instrumental response or absorbance. The graph does not pass through the origin as in the case of external calibration methods, but instead is shifted backwards by an amount corresponding to the analyte concentration in unknown diluted sample solution

Figure 9: Standard Addition Calibration Plot

This multipoint plot can be treated mathematically

where A_unk = b (intercept) and

m = slope

By setting Absorbance ‘A’ = 0, one can solve the linear equation:

?? = ????_??????+ ??

Rearranging the equation, one can solve for C_unk

?? ????????

??_??????= ?⁻_?? ? ???? ?⁻_???

In addition to the multipoint graphical approach, the concentration of analyte in an unknown sample solution may also be calculated using a single point method as shown in the formula below:

???????? ?????????? × ????????

???????? = (??(?????? ??????????&??????)−???????? )

It is advisable to check the calculated unknown analyte concentration with two or more standard additions assuming linearity. Single point method and multipoint standard addition method applies only when the standard addition solutions are linearly related to analyte concentration.

Prelab Questions

Calculate the Mn²⁺ stock solution concentration, if 0.303 g of MnSO₄*H₂0 salt needed to weigh, dissolved and transferred to a 200.00 mL volumetric flask and made to the mark with diluent.
Calculate the Mn²⁺ substock solution concentration, if 000 mL of stock solution (question 1) is transferred to a 50.00 mL volumetric flask and made to the mark with diluent.
Calculate the Mn²⁺ standard addition solution concentrations, if 1.000, 1.500, 2.000 and 2.500 mL of substock solution (question 2) are transferred to 50.00 mL volumetric flasks and made to the mark with diluent.
Determine the total volume of diluent (1% v/v HNO₃) that you need to make in order to prepare a stock, substock manganese solution as well as the two series of standard addition calibration solutions. Assume that you need a further 50 mL as a blank to zero the AAS.
Describe the preparation of 800 mL of 1% v/v HNO₃diluent.
State which solutions must be used to analytically rinse the volumetric flasks and volumetric pipets.
Using a diagram, outline the steps for the preparation of the unknown coconut water sample solution prior to standard addition.
State the dilution factors for the unknown coconut water for this entire procedure.
Diagram the dilution scheme for the preparation of the Mn²⁺standard additions for this analysis
State the analysis wavelength in nm that will be used for this experiment and the type of spectrophotometer that will be used.

State the purpose of the blank solution.

Procedure

NOTE: Use ASTM Type 1 water throughout the entire procedure to make all of your solutions. The diluent in this experiment is 1% v/v HNO₃. You must prepare your own diluent using Type 1 ASTM water and concentrated HCl.

A – Preparation of Mn²⁺Stock and Substock Solution

Using the analytical balance, weigh accurately 0.14 - 0.15 g (to the nearest 0.0001 g) of MnSO₄* H₂O salt into a clean dry 50 mL beaker. Record the actual mass to 4 decimal places in your hardcover notebook.

To the 50 mL beaker containing the Mn salt, add approximately 20 mL of 1% v/v HNO₃diluent at the lab bench under the ventilation trunk. Swirl gently to dissolve. If it doesn’t dissolve, add a few drops of concentrated HNO₃.

Analytically transfer the dissolved Mn salt solution to an analytically rinsed 50.00 mL volumetric flask. Rinse the beaker several times with small volumes of 1% v/v HNO₃ and transfer the rinses to the volumetric flask. Make the solution up to the mark with 1% v/v HNO₃Invert and shake 15 times. This is your manganese salt stock solution.

To prepare a Mn²⁺ substock solution, using a 0.500-5.000 mL micropipette, transfer 2.000 mL of Mn²⁺ stock solution into a 50.00 mL volumetric flask. Make this solution up to the mark with 1% v/v HNO₃ diluent. Invert and shake 15 times.

B – Preparation of Unknown Coconut Water Sample

Obtain an unknown coconut water sample from the professor. Record the sample number in your hardcover notebook.

To prepare a coconut water substock solution, using a 0.5 - 5.0ml micropipette, transfer 3.000 mL of coconut water sample solution into a 50.00 mL volumetric flask.

Make this solution up to the mark with 1% v/v HNO₃ diluent. Invert and shake 15 times. This is your coconut water substock solution.

C – Mn Standard Additions to the Unknown Coconut Sample (DUPLICATE SERIES)

NOTE: DUPLICATE SERIES requires 10 flasks - 2 sets of five for each coconut water sample

(U_i, U_ii).

Analytically rinse ten 50.00 mL volumetric flasks with diluent. Label five 50.00 mL volumetric flasks with U_i, U_i+1, U_i+2, U_i+3 and U_i+4. Repeat for the second series U_ii.

Using an analytically rinsed 0.005-5.000 mL micropipette, transfer 2.000 mL of coconut water substock solution step 6-7 - procedure to each of the five flasks. Repeat for the U_iiseries.

To prepare the duplicate series standard addition solutions, using an analytically rinsed micropipette, transfer the following volumes of Mn ²⁺ substock solution (prepared in step 4- procedure) to the 50.00 mL volumetric flasks:

Table 1: Standard Addition Scheme for Unknown Coconut Water Samples

Flask ID	Coconut Water Substock Volume (mL) Added	Mn²⁺ Substock Volume (mL) Added
Ui	2.000	0
U_i+1	2.000	1.000
U_i+2	2.000	1.500
U_i+3	2.000	2.000
U_i+4	2.000	2.500

Make all the solutions prepared in step 10- procedure up to the mark with 1% v/v HNO₃. Invert and shake 15 times.

To prepare the blank solution, pour 50 mL of 1% v/v HNO₃ diluent into a 100 mL beaker. This solution will be used to zero the AAS.

For each of the manganese standard addition solutions, calculate the actual Mn²⁺concentration in ppm (3 decimals) (NOT the salt!) added to the each standard addition flask. These values need to be determined before you use the instrument!

D - Analysis Using Perkin-Elmer Atomic Absorption Spectrophotometer

To analyze your standard addition samples, follow the operating procedure for the Atomic Absorption Spectrometer found in next to the PinAAcle 900F spectrophotometer. The analysis wavelength for Mn²⁺ absorbance measurements is 279.8 nm.

NOTE: See the professor or technologist for assistance setting up the instrument.

Measure the absorbance for each standard addition series.

Download and save your instrumental output as a PDF file. Before leaving the lab, download the PDF file including the raw data for each series, calibration plots and page 1 method editor and e-mail it to yourself, partner and professor.

NOTE: Some professors do not want the pdf’s e-mailed but instead, downloaded to Experiment 4- Slate – Assignments Raw Data at the end of the lab period. See your professor for advice.

Dispose all solutions in the designated waste container for this experiment as instructed by the professors or technologist.

Laboratory Report Outline

A - Operating Parameters and Standard Addition Solution Concentrations

a) Record in a table the AAS spectrometer instrumental settings (e.g. λ, slit width, gas flow, etc) used for the analysis of manganese (see the Method Editor printout).

b) Record the sample unknown number.

Calculate the actual Mn²⁺concentration (ppm) in the stock solution prepared in step 1-3 - procedure. Show the calculation.

Calculate the actual Mn²⁺concentration (ppm) in the substock solution prepared in step 4 - procedure. Show the calculation.

Calculate the actual Mn²⁺concentration in ppm added to each of the standard addition solutions based on the volume of Mn²⁺substock pipetted to each 50.00 mL flask step 10-11 procedure. Show only one sample calculation for Addition 2 only. Record your results in table format and include the corresponding Mn²⁺ absorbance values (raw data- AAS output).

Using the computer, diagram the standard addition dilution scheme used to prepare the solutions for Mn²⁺ analysis in the unknown coconut water sample. Include the size of volumetric flasks and volumes pipetted as well as actual Mn²⁺ concentrations added.

NOTE: Draw only ONE diagram; you do not need a diagram for each of the standard addition series.

B - Multipoint Standard Addition: Graphical Approach

You should have two (duplicate series) calibration curves of absorbance versus actual Mn²⁺ concentration (ppm) added. Examine each curve and report in table format the slope value and correlation coefficient as well as the x- intercept value, which corresponds to the concentration of Mn²⁺ in the diluted coconut water sample (Abs=0, see AAS summary output or graph for the x – intercept value).

For each replicate diluted coconut water sample, back- calculate the concentration of concentration of Mn²⁺(mg / 330 mL carton) in the original coconut water sample. Show your calculations for replicate #1 only. Record your results a new table. (You should have 2 final results; one result for the U_i series and one result for the U_ii series.)

Calculate the mean Mn²⁺ concentration (mg / 330 mL carton), standard deviation, relative standard deviation and true value at 95 % confidence interval in the original coconut water sample. Show the sample calculation for each statistic, and include a statement about the meaning of your µ value. Record your statistical results in a new summary table.

C - Single Point Equation Approach

Using the equation:

??^??????= ( ??₍?????????????? ?????????? ??????????&?????? × ??)??????−??_??????)

For flask 3 (U_x + 2) and flask U_x (unknown), calculate the concentration of Mn²⁺ in the diluted coconut water sample. Show the sample calculation. Report results in table format.

For each replicate diluted coconut water sample, back- calculate (in one calculation) the concentration of Mn²⁺(mg / 330 mL carton) in the original coconut water sample. Show the sample calculation for U_ionly. Report your results in the same as step 9.

Calculate the mean Mn²⁺ concentration (mg / 330 mL carton), standard deviation, relative standard deviation and true value at 95 % confidence interval in the original coconut water sample. Record calculated results in a new summary table.

NOTE: DO NOT SHOW THE SAMPLE CALCULATIONS FOR EACH STATISTIC.

D - Results Summary and Unknown Identification

In a final summary table, list the mean Mn²⁺concentration (mg / 330 mL carton) in the original coconut water sample, standard deviation, relative standard deviation and true value at 95 % confidence interval values for both the multipoint and single point data handling methods. Summarized results in ONE table.

Using the multipoint final results only, compare in table format your final mean Mn²⁺ concentration (multipoint method only) to ALL those posted on SLATE. STATE the IDENTITY of your unknown coconut water sample.

Based on the coconut water sample selected, calculate the relative error for your mean Mn²⁺concentration obtained using the two calibration method means (single and multipoint method). Show one calculation and report your relative errors in table format. RELATIVE ERROR MUST HAVE A SIGN!

E – Discussion (~ 16 marks)

For the multipoint method, explain whether or not the two linear equations should be the same or different for the two replicate standard addition series?

Suppose that your partner aspirated ‘U’ instead of the blank to zero the atomic absorption spectrophotometer.

1. Fully explain how this error would affect the absorbance signals for the standard addition calibration standards.

1. Explain in detail how this error would affect the resulting calibration equations and correlation coefficient.
2. Explain how this error would affect your final mean Mn²⁺ concentration for the multipoint method using the equations in part B - report.

Suppose that your partner prepared standard addition series #1 using a diluent made with tap water (1% v/v HNO₃) while you prepared series # 2 using 1% v/v HNO₃ made with ASTM water and your diluent was used to set the baseline response for the atomic absorption spectrophotometer.

1. Explain how this error would this affect the accuracy of your final result.

1. Explain how this would affect your precision.

Suppose that you were analysing Cu instead of Mn in coconut water. Outline the changes that you would need to make to the procedure to ensure that each Cu standard addition solutions in the series was within the LDR for Cu? (LDR 0. 500- 2.000 ppm at 324.8 nm). Assume that the diluted unknown coconut water sample pipetted to each volumetric flask for a series already contains 0.500 ppm of Cu.

1. Assume that you are starting with a 1000.0 ppm stock solution from pure Cu metal. Show the calculation for the preparation of the Cu stock solution using the same size volumetric flask used in this experiment. State the type of balance would you use?.

1. Using the same size volumetric flasks and approximate concentration that you prepared in this experiment for the substock, calculate the volume (mL) of Cu stock solution that you would need to make the substock. State the type and size of the pipet.

1. Using the substock solution prepared in part b), calculate appropriate volumes ( µL) that you would need to pipet to prepare a standard addition series using 5 volumetric flasks; taking into account the LDR for Cu and the unknown sample concentration.

1. 1. Make a statement regarding the concentrations for the standard additions that you wish to prepare.
  2. Show one sample calculation for U+1 and list the remaining information in table format.

Experiment 4 The Determination of Manganese in Coconut Water Using Atomic Absorption Spectroscopy Objectives In this experiment, the student will be introduced to the basic operation of the Pinnacle PerkinElmer atomic absorption spectrophotometer (AAS)

Chemistry