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Calibration

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ABSTRACT
This experiment will examine the concentrations of Zinc in several "samples." We will be using a Zinc lamp on the flame atomic absorption spectrophotometer. The lab assistant will demonstrate the software and the procedures for preparing the instrument.
Each group will run a series of calibration standards to generate a calibration curves. The concentrations are given below. Then, one of the "unknown" samples (A or B) should be run. The printer will generate a report of the concentration of Zinc in the sample. We have to be sure which sample we are used.
Our laboratory report should describe the functions of the AAS and describe the procedures for preparing the instrument for use. The report should include a printout graph of the calibration curve that we have made. We also needed to indicate the data points for the calibration standards and our sample.

INTRODUCTION
Atomic absorption spectroscopy (AAS) determines the presence of metals in liquid samples. Metals include Fe, Cu, Al, Pb, Ca, Zn, Cd and many more. It also measures the concentrations of metals in the samples. Typical concentrations range in the low mg/L range.
In their elemental form, metals will absorb ultraviolet light when they are excited by heat. Each metal has a characteristic wavelength that will be absorbed. The AAS instrument looks for a particular metal by focusing a beam of UV light at a specific wavelength through a flame and into a detector. The sample of interest is aspirated into the flame. If that metal is present in the sample, it will absorb some of the light, thus reducing its intensity. The instrument measures the change in intensity. A computer data system converts the change in intensity into an absorbance.
As concentration goes up, absorbance goes up. The researcher can construct a calibration curve by running standards of various concentrations on the AAS and observing the absorbances. In this lab, the computer data system will draw the curve for you! Then samples can be tested and measured against this curve.
We describe the motion of macroscopic objects using Newton's laws of physics. We can easily measure velocity, acceleration, force, kinetic and potential energies as well as quantify gravitational effects. However, when we look at atomic particles (i.e., protons, electrons, neutrons, etc.), we can no longer describe motion and energy using Newtonian physics. The characteristics of atomic particles are described using the theorems of quantum mechanics.
Quantum mechanics--or quantum chemistry--describes the geometry of atoms and molecules in terms of complex mathematical expressions. It also describes the relative states of atomic matter. The atomic absorption spectrometer uses the principals of quantum chemistry to detect the presence of certain metals (i.e., iron, aluminium, copper, etc.) and determines the concentration of those metals in samples.
All atoms and their components have energy. The energy level at which an atom exists is referred to as its state. Under normal conditions, atoms exist in their most stable states. We refer to that most-stable level as the ground state. Al though we cannot measure the precise energy state for an atom, we can usually measure changes to its energy relative to its ground state.
Certain processes can change the energy state for an atom. For example, adding thermal energy (heat) can cause an atom to increase to a higher energy state. This change in energy is written as DE. We refer to energy states which are higher than the ground state as excited states. In theory, there are infinite excited states; however there are decreasing numbers of atoms from a population that reach higher excited states.
The laws of quantum mechanics tell us that atoms do not increase their energy levels gradually. An atom goes directly from one state to another without going through intermediates. We refer to these "quantum leaps" as transitions. The transition from the ground state (written as Eo) to the first excited state (E1) requires some form of energy input. This energy is absorbed by the atom. That energy absorption is equal to DE0® 1. When this energy absorption takes place in the presence of ultraviolet light, some of that light will be absorbed. This uv absorption occurs at a specific wavelength.
Each element in the periodic table will have a specific D E that will absorb a specific wavelength of uv light. The relationship between the energy transition and the wavelength (l) can be described by:
D E=h/ l
Where h is Planck's constant. Atomic absorption uses this relationship to determine the presence of a specific element based on absorption in a specific wavelength. For example, calcium absorbs light with a wavelength of 422.7 nm. Iron absorbs light at 248.3 nm.

APPARATUS
Volumetric flasks, Pipette and Atomic Absorption Spectrometer
CHEMICALS
Zinc Sulphate, Deionised water and Water sample
PROCEDURE
A. PREPARATION OF STOCK SOLUTION (1000 PPM Zn) 100 ml of 1000 ppm Zn solution was prepared by weighing 0.4407 g ZnSO4.7H2O. Then the salt was dissolved in some deionised water. Next the solution was quantitatively transferred into a 100 ml volumetric flask. Deionised water was added to the mark. Lastly the exact concentration (ppm) of Zn was calculated in the stock solution.
B. DETERMINATION OF Zn USING THE LINEAR CALIBRATION METHOD Standards was prepared by calculating the volume of the stock solution required to prepared a series of 50 ml standards of Zn (0.1, 0.2, 0.3, 0.4, 0.5 ppm). A serial dilution was performed by pipetting the calculated volumes into 50 ml flasks. Deionised water was added to the mark. Next 10 ml of the unknown sample was pipeted into a 50 ml volumetric flask. Distilled water was added to the mark. After that, absorbance was measured by recording the absorption readings of each samples followed by the sample. Distilled water was used as Blank. Lastly Zn in the sample was determined by using ‘Excel Spreadsheet’. Calibration graph of absorbance versus concentration of Zn was plotted by using ‘Excel Spreadsheet’. The linear equation and the R² value was generated. The concentration of Zn in the sample was determined by using the linear equation of the calibration graph.
C. DETERMINATION OF Zn USING THE STANDARD ADDITIONS METHOD Standards and sample were prepared by calculating the volume of the Zn stock solution needed to prepare a series of five 100 ml standards with concentrations of (x+0.00), (x+0.25), (x+0.50), (x+0.75), (x+1.00) ppm. The appropriate volume of stock solution as required was pipetted into each 100 ml volumetric flask. Next 20 ml of the water sample was pipetted into each flask. Deionised water was added to the mark. After that, absorbance was measured. Distilled water was used as Blank. Calibration graph of absorbance against concentration of Zn was plotted by using ‘Excel Spreadsheet’. The concentration of Zn (x) from negative intercept of the x-axis was determined.

Results and Calculation:
Preparation of Stock Solution (10 ppm Zn)
Weight of Zinc Sulphate for standard: 0.4407
Dilution Factor: 1000/10: 100
Concentration of Zinc: 10ppm Determination of Zn using the Linear Calibration Curve Method
Since Blank equal to zero, Absorbance Reading already calibrated Sample | Concentration(ppm) | Calibratted Absorbance Reading | Blank | 0.00 | 0.0000 | Standard 1 | 0.10 | 0.0413 | Standard 2 | 0.20 | 0.0851 | Standard 3 | 0.30 | 0.1556 | Standard 4 | 0.40 | 0.1697 | Standard 5 | 0.50 | 0.2541 |
Table 1: Calibrated reading of the blank and standard sample of zinc concentration
Atomic Absorbance for sample A: -0.0610
Linear Method
Using the linear equation,
: Atomic Absorbance Value
: Concentration of Zinc Sample (diluted)

Rearrange the equation:
: -0.0610

= -0.06276
Sample A: 10 ml in 50 ml volumetric flask
Dilution Factor: 50/10 ml = 5
So, the concentration of zinc in the sample

=0.00 ppm
Determination of Zn using the Standard Addition method
Since Blank equal to zero, Absorbance Reading already calibrated Sample | Concentration | Standard Concentration added to sample (ppm) | Blank | 0.0000 | 0.00 | X+0.00 standard | 0.0208 | 0.00 | X+0.25 standard | 0.2655 | 0.25 | X+0.50 standard | 0.4477 | 0.50 | X+0.75 standard | 0.7039 | 0.75 | X+1.00 standard | 1.0186 | 1.00 |
Table 2: Calibrated reading of the blank and standard concentration added to the zinc sample
Calibrated linear standard Method
Sample A: 20 ml in 100 ml volumetric flask
Dilution Factor: 100/20 ml = 5
: Atomic Absorbance Value
: Standard Added + Zinc Sample (diluted)
So, the concentration of Zinc Sample when extrapolated at y=0:

, thus
So, the concentration of zinc in the sample
Standard Addition method

Discussion In this experiment, the concentration of zinc in the sample was being determined. There are two type of method applied here. First method is using linear calibration graph and second method is using standard addition method. So, by comparing the concentration obtained from two methods the concentration can be checked whether correct or not. By using atomic absorption spectroscopy (AAS), the concentration of metal element or mineral in a sample can be determined. This technique use absorption or emission spectrometry to assess the concentration of analytes in sample. To conduct the experiment, a range of standards must be prepared containing varying amounts of the analyte. The amount of analyte present in the sample can be calculated by plotting a calibration curve of signal against amount of analyte in the sample analyzed. The linear equation can be generated and the equation can be used to calculate amount of zinc in the unknown water sample A. The second method was known as standard addition method.; this method often used to reduce the problems of matrix effects. To find the amount of zinc from the water sample, certain amount of sample was mixed with known standard concentration. From the graph, a line can be extrapolate and value of x represent the concentration of the zinc in the sample at y=0. In part A of the experiment, 100 ml of 1000 ppm Zn solution were made by weighing 0.4407 g Zn SO4.7H2O in 100 ml volumetric flask. To make the process of producing the standards, 1000 ppm Zn were diluted to 10 ppm by taking 10 ml of 1000 ppm Zn solution made into another 100 ml volumetric flask. To determine the concentration of zinc in water sample, the standard sample and unknown water sample A must be analyzed under atomic absorption spectrometer. The standard sample series was ranged from 0.1 ppm to 0.5 ppm. The reading of the standard sample was tabulated into table 1.Based on the data obtain a calibration graph can be plotted in figure 1. Since the blank does not show any reading, thus the instrument already calibrated and the point can be used without subtract the blank value. The concentration must be measured from low to high to avoid previous Zinc concentration affect the sample afterwards. Based on the graph of Linear equation of the calibration graph 1, a linear equation was generated. By using this equation, y = 0.9736x + 0.045, x equal to concentration of the analyte in the zinc water sample and y is the reading of the absorbance value. So, from the calculation, the calculated concentration of zinc in water sample equal to -0.06276. This is not the real concentration of the sample, the concentration of the sample must be times with it dilution factor 5 times giving value of -0.3138. However, this value is incorrect since the concentration cannot be negative. So, it can be conclude that the concentration of the sample should be zero ppm or less then the limit of detection of the instrument. The second method is through the standard addition technique. The advantage of using this technique was that the effect of the matrix on the analyte can be reduced. As the results, more accurate result can be obtained. To determine the concentration of zinc in the water sample, 20 ml of water sample A was mix known amount of standard zinc sample. The series ranged from 0 to 1.00 ppm. The result was tabulated into table 2 and a calibrated graph 2 was plotted based on the data in table 2. The analysis of the data of the second method give that the concentration of the zinc in the water sample was -0.0578 after times with its dilution factor of 5. The concentration was obtained by measuring the value of x when extrapolated. Since x cannot be reading from the graph 2 easily, the generated linear equation was used instead by set y = 0. Based on the equation, y = 0.4932x – 0.0057, x can be find by equating y = 0. So, the value of x was 0.01156 but the concentration equal to –x. So, the concentration of the sample after dilution equal to -0.01156 and in real sample equal -0.0578. Same as method 1, the result give negative value, since concentration cannot be negative, so the concentration of zinc in water sample A must be zero ppm or less than the detection limit of the instrument. There are few precaution need to be taken to avoid errors during the experiment. Firstly, the concentration of the standard sample must be tested from lower concentration to high concentration. Meanwhile the sample must be tested first to check whether the sample reading within the standard range. Secondly, the sample must be diluted first if the concentration is higher than the standard range. The concentration of the sample A calculated equal to zero. However, the concentration might not accurate because of the instrument. If the concentration of the zinc was to low pass the limit of detection the concentration cannot be measured.
Conclusion
The concentration of sample A was identified to zero ppm. By using the same method atomic absorption spectrometer give out negative value. For linear graph method it give -0.3138 ppm and standard addition method it gives -0.0578 ppm. The concentration of sample A is not measured correctly either the problem was the sample or the instrument.
References
1- Galen Wood Ewing (1997), Analytical Instrumental handbook 2nd ed. Pg 257 – 266, USA 2- R.S. Khandpur (2006), Handbook of Analytical Instruments, Pg 32 – 34, Tata Mc Graw Hill…...

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