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.  

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. 

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.

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.