Abstract
Spectrometry is a process used in medicine so as to identify and certainly determine the amounts of substances in biological materials including assays for Dopamine in samples, glucose in the blood besides heavy metals in tissues. The report examined the step by step procedure in conducting the process and indeed suggests that spectrometry is valuable in determining the concentration of unknown samples.
In order to achieve this, the report is segmented into five distinct parts. The first portion defines the chief terms, the primary purpose of the report encompassing the objectives as well as the hypothesis. In the second part, the step by step procedure is provided including all the equipment used to make the experiment a success. Besides, the data collection method is outlined as well as the principles used in the experiment. In addition, the assumptions of the experiment are highlighted.
The third portion of the report gives the organized data which is later analyzed in the report. The analysis involves tabulation besides calculations of the raw data provided.
In the forth portion, the analyzed data is interpreted by use of descriptive statistics whereas, the last portion gives answers to the hypothesis earlier on indicated in the introduction part. Besides, the report provides room for further research as well as the experimental errors that may influence the results.
Keywords: Spectrometry, hypothesis, data analysis
Introduction
As indicated in the abstract, spectrometry is a medical process that entails the determination of the wavelengths or frequencies of the lines in a spectrum. In chemistry, it refers to the technique based on how wavelengths within a particular portion of a given range of absorptions or radiations are designed.
A hypothesis is usually used in scientific experimentation and serves as an explanation proposed and aimed to be proven, for a given phenomenon. The explanation is further proved after recording of the observations and analysis of the observations and thereby stating whether indeed, there exists a relationship between or among the variables under experimentation or not. The statement that shows a relationship between the variables is known as an alternate hypothesis. However, experimenters and researchers are advised to outline both a positive as well as a negative hypothesis. The negative hypothesis is commonly known as the null hypothesis. In fact, only one of the two hypotheses is accepted or rejected at the end of an experiment.
In an aim to either reject or accept a hypothesis, it is tremendously paramount to collect the relevant data both qualitative and quantitative. However, this is a necessary step but insufficient for the drawing of any conclusions about the experiment. The organization of the data is in the form of tables and later analyzed by both descriptive as well as inferential statistics. Data analysis is a very important aspect in virtually all experiments and in particular those in analytical chemistry. This is merely because analytical chemistry majors in making quantitative measures rather than just qualitative measures. Quantitative measures entail manipulation of raw data so as to provide an estimate that is realistic, on the uncertainty (the hypothesis to be proved). However, in chemistry, it is defective to merely depend on quantitative measures and therefore another aspect that involves observations of changes in experiments is included.
Purpose of the Experiment
The experiment was conducted to attain the objectives as outlined. Firstly, it aimed to observe the way protons are absorbed by molecules. In this regard it has been scientifically proved that protons can absorb energy when placed in a magnetic field of strength specific to the identity of the proton. The process is known as resonance and indeed, different protons do resonate in different frequencies within a specific magnetic field. The resonance enables chemical analysts to understand the structural components of a molecule.
Secondly, the experiment aimed to measure the intensity and calculate transmittance and absorbance of protons. In chemistry, it is paramount to measure the intensity and transmittance of protons. Over the years, cost effective equipment is used to carry out this and is known as a spectrometer. The intensity, thus measured is associated to the number of transitions that occur when a radiation is absorbed. Indeed, analysts are not only interested in understanding the presence of such transitions but also the magnitude.
Thirdly, there was need to study the absorbance spectrum for a known substance. In virtually all walks of life, it is important to start with a known before heading to the unknown. This concept applies even in other fields, for instance in mathematics, where a known formula leads to an unknown answer.
In addition, the other objective was to construct the absorbance spectrum for an unknown substance. After the experimenter studies the absorbance spectrum for a known substance, the actual experiment begins and in fact this could be the crucial part carrying more weight in the experiment, the construction of the absorbance spectrum for an unknown substance.
An absorbance spectrum is an approach to determine the amount of light that a substance could absorb in relation to the wavelength of the light. This is made possible by placing a sample in the spectrometer which measures absorbance at different wavelengths. A spectrum can then be constructed with the wavelength on the X-axis while the absorbance on the Y-axis. In short, the absorbance is dependent on the wavelengths of light. In this case, the experimenter can deduce the relationship between absorbance of the substance and the wavelength of light.
Lastly, the experiment aimed to construct a calibration curve used to establish the concentration of the substance that is unknown. This is possibly the ultimate goal of the experiment and is amongst the most accurate methods when it comes to determining the concentration of a substance. In this case, a series of known concentrations of identical molecules is prepared. Consequently, the absorbance of the known sample is measured with respect to the sample of the unknown concentration. The equation for the line of the known concentration is determined. In order to determine the concentration of the unknown sample, the values for its absorbance are inserted in the equation.
Experiment Method
As above indicated, spectrometry is used to determine the concentration of an unknown substance. In order to achieve this, a number of steps are very crucial and should be conducted sequentially if accurate results remain the experimenter’s objective. At times, the presence of physical errors cannot be ruled out but the experimenters should at all times ensure that they closely follow the procedure so that erroneous results are avoided.
The basic equipments required in the experiment include; a light source, a cell, a detector, a spectrometer, a stop watch and the samples of the known and unknown substances. Before the experimenter embarks on the actual experiment, it is important to know exactly what to observe so as to note down the main observations as well as values to be used later in data analysis. In theory, the light is absorbed in virtually all substances and therefore, the intensity of light at the source is greater than the intensity reaching the detector. This implies that indeed, some photons are absorbed as they pass through the sample.
In the first place, the intensity of light at source was measured which represents the number of photons per unit second. Analysts refer to this as the intensity of light through a blank which refers to a solution that does not absorb any light photons. Apart from the fact that, light could be absorbed by a substance, some of the light could as well be scattered by the cell. This would imply a decrease in the intensity of light. The intensity of light through a blank is denoted by Io.
Secondly, it was important to measure the intensity of light after it passed through the sample. In fact, most instruments only indicate power of light rather than the intensity. Under such circumstance, the intensity of light photons per second can be calculated from the formula, power=intensity X energy per photon. Such intensity is denoted by me.
Thirdly, the data collected above is used to compute for the transmittance as well as the absorbance. Transmittance is the quotient of the intensity of light through a sample that absorbs light to the intensity of light through blank. In short, transmittance= I/Io; while Absorbance is given by the formula; A=-Log10 T.
In line with this, it would be fair to note that transmittance refers to the light that reaches the detector after passing through the sample. The remaining part is usually absorbed when passing through, by the sample. Any fraction of light absorbed by the sample is denoted by 1-T, where T represents transmittance. In line with this, if no light is absorbed, then transmittance is zero.
In conducting the experiment, several assumptions were made. In the first place, the cell was assumed to have no influence on the light from the source. This possibly could have reduced the intensity of light as well as transmittance results. Secondly, the sample used was assumed to absorb some light. However, in case, the solute was blank, and then transmittance would be zero.
Raw Data and Calculations
The first objective was to observe the light photons as absorbed through a sample. The table below indicates this.
Table 1
| Time,t,in seconds | Photons | Intensity,in Photons per second |
| 5 | 188 | 37.612p/s |
| 10 | 383 | 37.586p/s |
| 15 | 578 | 37.557p/s |
| 20 | 740 | 37.544p/s |
| 25 | 942 | 37.515p/s |
| 30 | 1230 | 37.511p/s |
The data was collected through observation of the photons across the blank as well as over a sample. The total number of photons was 1020 through the blank and the intensity was 37.541 photons per second. On the other hand, a total of 1020 photons were passed through a sample and the intensity was 23.752.
The transmittance was given by the formula; I/Io
23.752 photons per second/37.541 photons per second. The result had no units and was 0.632695.
The percentage transmittance was further calculated as follows:
(Number of photons per second through the sample/Number of photons per second through the blank) multiplied by 100. This gave a result of 63.3%. Which indicates that 63.3% of the light was transmitted through the sample?
It is also possible to calculate the percentage of light that was absorbed by the sample and this was determined as follows:
1-(Number of photons per second through the sample/Number of photons per second through the blank) multiplied by 100.
In short, (1-T)*100 which was equal to 100%-63.3%. This implied that 36.7% of light was not transmitted but was absorbed.
Using the formula earlier on outlined in the report, absorbance is given by the negative log of T.
Absorbance, denoted by A equals to –Log T.
Therefore, A=-Log (0.633)
A=0.198596
Absorbance was also calculated at various wavelengths and at different periods and was as follows:
Table 2
| Time, t | Io=300 | I | T=I/Io | wavelength | A=-Log(T) |
| 5.2 | 300 | 281.7 | 0.939 | 380 | 0.027334 |
| 5.9 | 300 | 275.08 | 0.916933 | 385 | 0.037662 |
| 5.9 | 300 | 272.13 | 0.9071 | 390 | 0.042345 |
| 5.9 | 300 | 267.33 | 0.8911 | 395 | 0.050074 |
| 5.9 | 300 | 261.43 | 0.871433 | 400 | 0.059766 |
| 5.9 | 300 | 234.242 | 0.780807 | 420 | 0.107456 |
| 5.9 | 300 | 192.096 | 0.64032 | 440 | 0.193603 |
| 5.9 | 300 | 150.4688 | 0.501563 | 460 | 0.299675 |
| 5.9 | 300 | 57.29 | 0.190967 | 520 | 0.719042 |
| 5.9 | 300 | 42.15 | 0.1405 | 560 | 0.852324 |
| 5.9 | 300 | 51.29 | 0.170967 | 600 | 0.767089 |
| 5.9 | 300 | 98.548 | 0.328493 | 640 | 0.483473 |
| 5.9 | 300 | 183 | 0.61 | 680 | 0.21467 |
| 5.9 | 300 | 252.068 | 0.840227 | 720 | 0.075604 |
| 5.9 | 300 | 274.46 | 0.914867 | 740 | 0.038642 |
| 5.9 | 300 | 295.245 | 0.98415 | 780 | 0.006939 |
After the analysis as shown above, an absorbance spectrum was constructed and appeared as shown below:
An Absorbance spectrum for a Known Sample
The following data was used to form an absorbance spectrum of an unknown sample.
Table 3
| Time,t | Wavelength | Io | I | T=I/Io | A=-Log T |
| 6.4 | 380 | 300 | 288.125 | 0.960417 | 0.01754 |
| 6.4 | 385 | 300 | 289.06 | 0.963533 | 0.016133 |
| 6.5 | 390 | 300 | 286 | 0.953333 | 0.020755 |
| 6.4 | 395 | 300 | 284.03 | 0.946767 | 0.023757 |
| 6.3 | 420 | 300 | 267.968 | 0.893227 | 0.049038 |
| 6.3 | 460 | 300 | 233.968 | 0.779893 | 0.107965 |
| 6.3 | 500 | 300 | 193.174 | 0.643913 | 0.191173 |
| 6.3 | 540 | 300 | 158.9705 | 0.529902 | 0.275805 |
| 6.3 | 580 | 300 | 136.7188 | 0.455729 | 0.341293 |
| 6.3 | 620 | 300 | 99.0769 | 0.330256 | 0.481149 |
| 6.3 | 660 | 300 | 54.838 | 0.182793 | 0.73804 |
| 6.3 | 700 | 300 | 84.4117 | 0.281372 | 0.550719 |
| 6.3 | 740 | 300 | 229.666 | 0.765553 | 0.116025 |
| 6.3 | 780 | 300 | 297.903 | 0.99301 | 0.003046 |
An Absorbance Spectrum for the Unknown Sample
It is important to note that the absorbance is plotted against the wavelengths in the two diagrams provided above.
Table 4
Concentration of the Unknown Sample
The following data was used to calculate the concentration of the unknown sample.
| Conc. | Time,t | Io | I | T=I/Io | A=-Log T |
| Blank | 6.2 | 660.674 | |||
| 20 | 6.2 | 381.656 | 0.577677 | 0.238315 | |
| 40 | 6.2 | 220.062 | 0.333087 | 0.477442 | |
| 60 | 6.2 | 130.225 | 0.197109 | 0.705293 | |
| 80 | 6.2 | 73.625 | 0.111439 | 0.952962 | |
|
|
In order to determine the concentration of the unknown sample, it was important to use Beer’s Law that predicts a relationship between absorbance and concentration.
According to this law, absorbance equals to the sum of the slope of the calibration curve/line and the intercept. On the other hand, concentration of the unknown sample can be calculated using the following formula:
Concentration of Unknown sample= (Absorbance of the Unknown sample-Intercept of the calibration line)/ the slope of the calibration line.
Using the results from the above experiment, it is clear that the calibration line cuts through the origin. In short, the equation of the line is A=0.01 C (Where A represents Absorbance while C represents Concentration.
In such an equation, the absorbance intercept is Zero while the slope can be calculated as follows:
Slope of the calibration line= Displacement of the Absorbance axis divided by displacement in the concentration axis.
By taking any two points from table 4; for instance (0.238, 20) and (0.477, 40), the slope equals to 0.01.
Discussions
From the experiment conducted, it was found out that, absorbance increases with increases in the wavelength but with more increments in the wavelength, the absorbance ultimately declines. The absorbance spectrum for the known sample attested to this. In this case, the curve rose from 0.027334 to the highest absorbance value of 0.8523 at a wavelength of 560λ and then declined to an absorbance value of 0.006939 at a wavelength of 780 λ.
These results showed that the absorbance of light photons increases with increment in wavelengths but eventually declines with further increments in the wavelengths.
Similar results were obtained when light photons were passed over an unknown sample. However, the figures were significantly different from those obtained from a known sample. In this case, the absorbance rose from a minimum value of 0.01754 at a wavelength of 380 λ to a maximum of 0.73804 at a wavelength of 660 λ, and then declined to an absorbance of 0.003046 at a wavelength of 780 λ.
In order to determine the concentration of the unknown sample, a graph of the absorbance and the concentration of known samples was plotted, which showed a linear relationship between the two variables. In this case, the absorbance increased with increase in concentration of the samples.
The unknown sample had an absorbance of 0.638501 which lies on the line of best fit as shown above. The equation A (Absorbance) =0.01C (Concentration) was used to determine the concentration of the unknown sample. Using the above formula, the concentration of the unknown sample was 63.85 µ Moles per litre.
Conclusion
From the experiment above, it was fair to suggest that spectrometry is valuable in determining the concentration of unknown solutions. Indeed, the steps outlined were closely followed and in fact, good results, as expected, were obtained. More accurate results could be obtained by ensuring that experimenters accurately record their observations. An accurate observation ensures that the results are less erroneous thereby being consistent with the theories from the secondary sources as well as previous experiments conducted.
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