As can be seen from the structure of quinine (fig.1), quinine is a polycyclic aromatic compound and as such, quinine can be estimated by fluorescence spectroscopy at levels as low as 0.1μg/cm3
Changes in the system pH, if it has an effect on the charge status of the chromaphore, may influence pH. A change in the pH of the solution may alter the shape of the excitation spectrum of the fluorescent compound. The presence of anions, such as chloride, bromide, iodine and nitrate may affect fluorescence. Quinine sulphate is highly fluorescent in 0.1M H2SO4, but becomes non-fluorescent in 0.1M HCl.
Concentration of the analyte has an effect on fluorescence intensity. The power of fluorescence emission, F, is proportional to the radiant power of the excitation beam that is absorbed by the system. That is,
Equation 1
Where P0 is the power of the beam incident upon the solution and P is its power after traversing a length, b, of the medium. The constant K1 depends upon the quantum efficiency of the fluorescence process. In order to relate F to the concentration, c, of the fluorescing species, we write Beer’s Law in the form
Equation 2
Where e is the molar absorptivity of the fluorescing molecules and ebc is the absorbance, A. By substitution of equation 2 into equation 1 we get
This equation containing an exponential term can be expanded as a Maclaurin series. Ultimately the following is derived:
At constant P0, F = Kc
Thus, a plot of the fluorescence power of a solution versus concentration of the emitting species should be linear at low concentrations. When c becomes great enough so that the absorbance is larger than about 0.05, linearity is lost; F then lies below an extrapolation of the straight-line plot.
Two other factors, also responsible for negative departures from linearity at high concentration, are self-quenching and self-absorption. Self-quenching is the result of collisions between excited molecules. Radiationless transfer of energy occurs. Self-quenching can be expected to increase with concentration because of the greater probability of collisions occurring.
Self-absorption decrease fluorescence efficiency, which is defined as the ratio of the number of photons emitted as fluorescence to the number absorbed. For strong fluorescence, such as quinine, at high concentrations the incident intensity Po falls very rapidly across the sample and strong fluorescence occurs only from the front layer. The desired situation is for even excitation along the path of the beam, hence solutions must be adequately diluted and high intensity exciting sources employed
Atomic Fluorescence Spectrophotometer
The atomic fluorescent spectrophotometer works by employing double beam optics as shown in fig.2, in order to compensate for fluctuations in the power of the source. The sample beam first passes through an excitation filter or a monochromator, which transmits radiation to excite fluorescence but exclude radiation of the wavelength of the fluorescence emission. Fluorescence from the sample is propagated in all directions but is most conveniently observed at right angles to the excitation beam; at other angles, large errors in the measurement of intensity may be caused as a result of the increase scattering from the solution and the cell walls. The emitting radiation reaches a photo transducer after passing through a 2nd filter or monochromator that isolates the fluorescence for measurement.
The reference beam passes through an attenuator that reduces its power to approximately that of the fluorescence radiation. Signals from the reference are then fed into a difference amplifier whose output is displayed by a meter.
Fig.2
Components Of A Fluorescence Spectrophotometer
The Standard Addition Technique
Standard addition is an alternate calibration technique to external standardisation. It is applicable to all types of analytical methods not just fluorescence spectroscopy. Standard addition is used instead of external standardisation if is possible that the sample solution contains interference. The basic idea is to insure that the interference present in the sample solution is also present in each of the standard solutions.
When standard solutions are prepared a stock solution of known concentration of which dilute aquiots are produced to known volumes to give the concentration range that is required. The stock aliquiots are added to a fixed amount of the sample solution, and then made to volume. The volume of the sample must be constant.
The detector response of all the standard solutions including the 0 mg cm3 standard solutions are measured. A graph is then plotted of the variation in detector response with concentration of analyte added to the solution. The graph will not pass through the origin because the sample solution contains the analyte. The graph is then extrapolated so that it cuts the negative x-axis. The point at which the graph cuts the negative x-axis give the concentration due to the analyte in the diluted sample solution.
Fig.3
Example of a Standard Addition Graph
Equation 3
Equation 3 shows that the concentration of the analyte in the sample is equal to the concentration due to the analyte in the diluted sample multiplied by the dilution factor.
Justification Of Method
Fluorescence spectroscopy is a suitable method of analysis for the analyte quinine because, for applicable compounds, fluorescence gives high sensitivity (in the low parts per million) and high specificity. High Sensitivity results from the difference in wavelength between the exciting and fluorescence radiation. This results in a signal contrasted with essentially zero background; it is always easier to measure a small signal directly than a difference between two large signals. (As is done in absorption spectrophotometry.) High specificity results from dependence on two spectra, the excitation and emission spectra, and the possibility of measuring the lifetimes of the fluorescent state. Fluorescence spectroscopy has assumed a major role in analysis, particularly in the determination of trace contaminants in our environment, industries and bodies.
Some fluorescence instruments, such as dispersive fluorescence instruments appear to be more complex and potentially more expensive to purchase and maintain then instrumentation for other methods of analysis. However, non-dispersive instruments are more simple and low-cost.
Experimentation
For the experiment, we were supplied with the following reagents;
-
1 mg cm-3 Standard solution of quinine sulphate in 0.05 mol dm-³ sulphuric acid.
- 0.05 mol dm³ sulphuric acid
- Unknown urine sample containing approx 100 μg cm³
The unknown urine sample, containing approx 100 μg cm³ quinine, was accurately diluted by pipetting 5cm3 into a volumetric flask and then made to 100cm3 by pouring the sulphuric acid into the flask. As sulphuric acid is corrosive, safe laboratory practice was ensured. Accuracy of the measurement was ensured by the use of a Pasteur pipette to make the solution up to 100 cm3. A range of standard addition solutions were then prepared containing the same volume of diluted urine and varying concentrations of added standard quinine sulphate. This was achieved by pipetting 5 cm3 of the diluted urine solution into each of the six 100 cm3 volumetric flasks. 0;5;10;15;20 and 25 cm3 volumes of the standard solution of quinine sulphate were then pipetted into each flask in turn, and made to the mark with sulphuric acid. Accuracy was again ensured, by using a Pasteur pipette to level the meniscus of the solution to 100 cm3. Each flask was labelled with its corresponding volume of standard solution of quinine sulphate. The fluorescence intensity of each solution was then measured in the fluorescence spectrophotometer. The excitation wavelength was set at 350 nm and the emission wavelength at 460 nm. The spectrophotometer was set to zero with 0.05 mol dm-3 sulphuric acid. In order to ensure maximum accuracy, the outside of the cuvette was wiped before each measurement with a tissue to allow optimum fluorescence readings. The cuvette was rinsed and dried thoroughly during in between readings to prevent any error occurring. The spectrophotometer was set to maximum sensitivity after setting to zero, with the solution containing 25 cm3 of added quinine sulphate, the highest concentration of added quinine sulphate. The fluorescence intensity was then measured and recorded for each solution.
Results
The results obtained from the experiment were as follows:
Fig. 4
A table to show the variation in fluorescence intensity against the volume of standard solution of quinine added.
Excitation wavelength = 350 nm
Emission wavelength = 460 nm
The data in fig. 4 are shown graphically in fig. 5.
The graph data in fig. 5 was extrapolated and it was found that the line of best fit crossed the x-axis at 24.22. This is the concentration due to the analyte in the diluted sample. The dilution factor is 5 because 5 cm³ of the sample urine was diluted to 100 cm³ using 0.05 mol dm³ sulphuric acid.
The concentration of analyte in sample is then found by using equation 3.
μg cm³
The concentration of the quinine in the unknown urine sample was found to be 121 μg cm³
Discussion
The measurements taken during the experimentation may have contained random errors or systematic errors. Repeated results would have improved accuracy for random errors. Possible systematic errors were:
- Personal error.
- Instrumental error.
- Unsuitable method.
- Interference in sample.
- Sample history.
Within the experiment, personal errors were likely to be minimal as procedures were carried out carefully. Personal error can generally be reduced by reading instruments carefully, and double-checking measurements. Instrumental error may have occurred though the use of inaccurate glassware such as pipettes; this could be reduced using glassware of improved tolerance; and incorrect calibration of spectrophotometer. Errors from an unsuitable method were unlikely to occur as it had been carried out before and was shown to be reliable.
Interference in the sample may have occurred as a result of chloride ions being present due to salt being excreted in the urine. The presence of anions such as chloride in the sample may have suppressed the presence of quinine. If the solution under investigation contains, beside the fluorescing molecules, a solute that absorbs either the exciting or the fluorescing radiation, the measured fluorescent power is reduced. To minimise this effect, the sample can be diluted to reduce interfering absorbance provided that the concentration of the analyte is not reduced to an intolerably low level. The method of standard additions, as was used, works to minimise this effect since the absorbance for the interfering component remains constant.
The presence of paramagnetic species such as, dissolved oxygen and other heavy atoms, strongly affects the rate of intersystem crossing which in turn alters the quantum efficiency for fluorescence. To improve the accuracy of this experiment, paramagnetic species could have been separated and excluded from the sample solution.
Errors relating to sample history may have occurred if the sample had been contained in plastic of rubber laboratory equipment resulting in the leech of fluorescent contaminants. This error can be reduced by careful storage of the sample in suitable containers. Another source of error could arise from the graph as the points taken were scattered and the best-fit line could have been drawn inaccurately.
As previously stated the analysis of quinine in urine is important in forensic science as quinine is frequently used as an adulterant in illicit heroin samples. Its presence can therefore be tested for in order to determine the presence of heroin in the body. However if the test was carried out errors could occur in the results if the individual being tested had previously been taking anti-malarial medication. Therefore further tests would need to indicate the presence of heroin or its metabolites such as morphine.
Conclusion
The concentration of quinine in the unknown urine sample was found to be 121 μg cm³. It can be concluded from this experiment that the method of standard addition was an accurate method to use as an approximation of 100 μg cm³ was known before the experiment was carried out, therefore, the value obtained is likely to be close to the approximate concentration contained in the unknown sample.
Sources of error may have included personal and instrumental errors as a result of inaccurate glassware, inaccurate measurements and incorrect calibration of the spectrophotometer.
The aims of the ecperioment as outlined at the beginning of the report were met successfully and were as follows;
- To determine the variation in fluorescence with quinine sulphate concentration of standard addition solutions.
- To determine the quinine sulphate concentration of an unknown urine sample.
- To assess the data and judge whether other components of the urine interfere.
- To adhere to all safety regulations required when working in a laboratory.
- To carry out the experiment to a high level of accuracy.
- To employ standard addition techniques.
The first aim was achieved through the preparation of the six standard addition solutions and the measurement of the fluorescence of each. The results were tabulated and shown graphically in figs. 4 and 5 respectively.
The second aim was met by extrapolating the graph of variation in fluorescence with added quinine sulphate concentration shown in fig. 5, to determine the quinine sulphate concentration of the diluted urine solution and hence the unknown urine sample. This was calculated to be 121 μg cm³.
The third aim was met in the discussion where the data represented graphically was assessed as being linear. Other components of the urine such as chloride ion present due to salt being excreted in the urine were judged to possibly interfere with the fluorescence of the sample.
The forth and fifth aims were met through safe lab practice and careful procedural techniques.
The sixth aim was achieved by the method employed in the experiment.
References
Charles E. White and Robert J. Argauer, Fluorescence Analysis – A practical Approach, Marcel Dekker Inc., New York 1970.
D. Betteridge and H.E. Hallam, Modern Analytical Methods, The Chemical Society, 1972.
Hobart H. Willard, Lynne L. Merritt Jr, John A. Dean, Frank A. Settle Jr, Instrumental Methods Of Analysis, 7th Edition, Wadsworth Publishing Company, 1988.
John Daintith (Ed), A Dictionary Of Chemistry, 4th Edition, Oxford University Press, 2000