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Most pharmaceutical products absorb UV radiation with some of them, which are coloured compounds absorb radiation in the visible range. The absorption of UV radiation occurs through the excitation of electrons within the ground state to a higher energy state. Only most easily excited electrons show absorption in the UV-visible region. Thus, choice of reagent becomes significant in order to get a reliable result for the quantitative analysis. In this case, sodium aminosalicylate is chosen as the absorbing species. In addition, sodium aminosalicylate is bacteriostatic against Mycobacterium tuberculosis by inhibiting its growth and multiplication and hence used as an antitubercular drug and also is often used to treat Crohn's disease by reducing inflammation. Three approaches are used to determine the concentration of both unknown solutions. They are calibration graph method, absolute method and comparative method. All of them are based on the absorbance readings recorded at the Î»max chosen. Transmittance is the ratio of the transmitted intensity to the incident intensity. Absorbance is defined as the common logarithm of the reciprocal of transmittance of a pure sample. Furthermore, UV spectroscopy is commonly used for quantitative analysis for BP assays and limit tests in terms of its reliability, easy in data handling, availability of the equipment and its broad absorption bands.
A set of standard solutions was prepared in order to construct a calibration graph for the determination of the unknown analyte concentration. 50mL volumes of 0.0002, 0.0004, 0.0006 and 0.0008%w/v of sodium aminosalicylate solutions in 0.1M NaOH were prepared from the 0.0010%w/v stock solution. For example, 10mL of stock solution was pipetted out and mixed with 40mL of 0.1M NaOH in order to make 50mL of 0.0002%w/v solution in a round-bottomed flask.
Absorbance were measured using a single beam spectrophotometer. Firstly, blanking the cell was carried out between both cells with 0.1M NaOH. Any cell difference should be recorded and subtracted from any later readings of the sample.
The absorbance of each of the five standard solutions were recorded and replicate readings were obtained for each solution. This was done by emptying, refilling and replacing the cuvette between readings. All the data gathered was used to plot a graph of absorbance against concentration. The slope of the graph was the specific absorbance, A(1%,1cm). Molar absorptivity could be calculated from the data obtained.
Initial absorbance of unknown 1 was measured at 263nm. Dilution of 1 in 4 was carried out in unknown 1 in order to get the absorbance which fell in the mid range of the graph plotted. The diluted solution was made up to 100mL with 25mL unknown 1 and 75mL 0.1M NaOH. Absorbance was measured again. Read-off from the graph gave the diluted concentration. Actual concentration can be found by multiplying back the dilution factor.
There were two ways used to find out the concentration of unknown 2, which were absolute method and comparative method. For the absolute method, the absorbance of unknown 2 was measured and its concentration was determined using Beer-Lambert law. In this case, the specific absorbance was obtained previously from the slope of the calibration graph. For the comparative method, the Î»max was changed to 300nm and measured only the absorbance of a single standard (0.0010%w/v) and unknown 2. The concentration of unknown 2 was calculated using a relationship tabulated below.
A spectrum of a 0.001%w/v solution of sodium aminosalicylate in 0.1M NaOH was obtained over the wavelength range 235 to 325nm. From this spectrum, two Î»max and their corresponding absorbance were recorded. The specific absorbance was calculated using Beer- Lambert law:
A = A(1%,1cm).c.l
Table 1 Absorbance at Î»max (Reference and estimation)
Selection of suitable Î»max:
From the above, Î»max at 264nm gave the highest absorbance. Absorbance a few nm each side of 264nm were recorded in order to select the most appropriate Î»max. Î»max at 263nm was selected.
Table 2 Absorbance at different wavelengths
Graph 1 Selection of Î»max
Preparation of standard solutions:
A set of 50mL calibration solutions with 0.0002, 0.0004, 0.0006 and 0.0008%w/v in 0.1M NaOH was prepared from 0.0010%w/v stock solution. For example,
C1V1 = C2V2
0.0010 x V1 = 0.0002 x 50
V1 = 10mL
Volume of stock
Volume of NaOH
Final diluted volume
Table 3 Serial dilutions
Absorbance of standard solutions:
Difference between cells: -0.02
Table 4 Absorbance of standard solutions
Graph 2 Calibration graph (Beer-Lambert Law)
Molar absorptivity of sodium aminosalicylate in 0.1M NaOH at 263nm:
Mr of sodium aminosalicylate = 211.15
Concentration of sodium aminosalicylate = 0.0002 x 10 ÷ 211.15 = 9.472 x 10-6 mol dm-3
Absorbance of 0.0002%w/v sodium aminosalicylate at 263nm is 0.122.
A = ÎµMl
0.122 = Îµ x 9.472 x 10-6 x 1
Îµ = 12880.15 dm3mol-1cm-1
Determination of concentrations of unknown 1 & 2:
For unknown 1,
Initial absorbance at 263nm = 1.180
25mL of unknown 1 was taken and made up to 100mL solution with 0.1M NaOH.
Dilution factor: 1 in 4.
Absorbance (diluted) at 263nm = 0.302
According to the calibration graph drawn, the concentration of unknown 1 is 5.20 x 10-4 %w/v.
Actual concentration of unknown 1 = 5.20 x 10-4 x 4 = 2.08 x 10-3 %w/v
For unknown 2,
Absorbance at 263nm = 0.508
A(1%,1cm) = slope of calibration graph
= 0.56 - 0.02
0.00096 - 0.00004
A = A(1%,1cm)cl
0.508 = 586.96 x c x 1
c = 8.65 x 10-4 %w/v
Difference between cell = -0.009
Absorbance of 0.0010%w/v stock solution at 300nm = 0.371
Absorbance of unknown 2 at 300nm = 0.322
C2 = A2
C2 = 0.322
C2 = 8.68 x 10-4 %w/v
UV has a short wavelength, therefore it has high energy which is enough to change the electronic structure of a molecule. It may temporarily affect the bond structure. In this case, sodium aminosalicylate is chosen as the absorbing species due to the presence of OH and NH2 groups in the benzene ring which leads to higher conjugation and higher molar absorptivity. This increases the chance of electron transition.
Normally, the absorbance of benzene is recorded at 255nm. However, the highly conjugated system lowers the energy gap for electron transition and hence increasing the wavelength of the absorber. Therefore, the approximate Î»max of sodium aminosalicylate are 264nm and 300nm according to the spectrum provided. Î»max is set to 263nm for the absorbance measurement of the standard solutions and unknown 1. This is because the absorbance at 263nm is the highest, that is 0.607 for the 0.0010%w/v stock solution.
The use of Î»max will give maximum sensitivity for the absorbance readings. A small change around the Î»max will only give a slightly change in absorbance. In other words, the accuracy of the absorbance readings will not be affected too much if there is an error in wavelength setting. Other than that, most reagent will have more than one Î»max value, therefore, the wavelength with highest absorbance recorded is often chosen in order to minimise interferences from other substances and it will be more specific. Furthermore, taking absorbance at Î»max will minimise the errors from impurities as impurities may have different Î»max.
Single beam spectrophotometer is well-suited for quantitative analysis because all the measurements are made at a single wavelength. When using the single beam instrument, it is necessary to take the readings of the blank solution and the sample solution consecutively, in order to ensure that the intensity from the light source is constant. Other than that, blanking the cells are important before the start of absorbance measurements as there may be a slight difference between the two cuvettes used.
Beer-Lambert Law states that the absorbance depends on the total number of absorbing molecules in the light path through the cell. This also means that the absorption is affected by both the analyte concentration(c) and the path length(l). The law is further simplified into two similar equations, the latter one is commonly used in British Pharmacopeia(BP):
A = ÎµMl
A = A(1%,1cm)cl
Both equations are only applied to dilute solutions which the absorbance is less than 1. Radiation light will hardly to get through concentrated solution which absorbance is greater than 1 and results in inaccurate readings. Assumptions are made for the Beer-Lambert law: the use of monochromatic light (radiation consisting of only single wavelength or in practical the light with narrow wave band) and the homogeneity of the absorber.
According to the calibration graph drawn, absorbance is directly proportional to the concentration (linear relationship). This proves that the Beer-Lambert law is obeyed. However, some deviations may arise from the experiment which results in non-linearity, particularly at high concentrations. The problem does not arise in this experiment as low concentrations are used.
Three different quantitative methods are used to investigate the concentrations of both unknown solutions. Firstly, the calibration graph methodology. This involves the absorbance measurement over a range of standard solutions and plotting a graph. The unknown concentration can be obtained from the read-off of the graph based on the absorbance measured. It is very time-consuming and it requires a pure standard which stays chemically stable over a period of time. Duplicates are done for absorbance measurement to reduce errors. Beer-Lambert law must be obeyed for all the samples tested.
Next is the absolute methodology. This only measures the unknown absorbance under defined conditions and by using a known specific absorbance. It only requires a high quality instrument for the absorbance measurement and thus it is easier to be used. It does not need standards, therefore it is less time consuming. This is the preferred method in British Pharmacopeia(BP).
The last one is comparative methodology. This involves the absorbance measurement of a single standard and the unknown. Thus, it needs a pure standard to prevent any inaccuracies. Since it is based on the relationship between the standard and the unknown, therefore it is best if the concentration of the standard and the unknown solution are close. It is more preferred in United States Pharmacopeia(USP).
Overall, those three methods must obey the linearity as proposed in Beer-Lambert law. The concentrations for both unknown 1 and unknown 2 are 2.08 x 10-3%w/v and 8.67 x 10-4%w/v respectively. The common factors of error are the wavelength, spectral resolution and stray light. Absolute method in comparison with comparative method, is more reliable due to less measurements made. For the comparative method, any deviation or impure standard will cause inaccuracy in concentration determination. For absolute method, the factors of error can be reduced to minimum by using the UV spectrophotometer that fulfils the BP checks.
UV-visible spectroscopy is a simple, rapid, universal and cost-effective method in quantitative analysis. The analysis should be done at maximum wavelength so that the absorbance will be high and constant around the chosen wavelength. Sodium aminosalicylate is a good UV absorber. Beer- Lambert law must be obeyed in every quantitative method used. The concentrations for both unknown 1 and unknown 2 are 2.08 x 10-3%w/v and 8.67 x 10-4%w/v respectively.