Several methods of quantitative analysis through UV spectrophotometry allow the calculation of concentration of sample solutions. This experiment examines methodologies of the calibration of the absorption against concentration graph, the absolute method which involves the calculation of the specific absorbance, and the comparison of the absorbance of the sample and standard solution. Sodium aminosalicylate solutions in different concentration are used as the sample with 0.1M sodium hydroxide (NaOH) as the blank of the UV spectrum analysis. The final results are interpreted through the calibration of a graph, showing a linear relationship between the absorbance and the concentration of the solution. The gradient of the graph is calculated as the specific absorbance value, which is further used in absolute analysis. An unknown concentration of the sample solution can be determined either based upon the graph or through calculation with the Beer-Lambert equation. It can also be calculated in comparison with a standard solution of known concentration, known as comparative method. Absolute and comparative analyses show a close reading of the concentration of the sample with slight variation due to the possibility of measurement error. Therefore, the application for quantitative analysis of a sample product must be considered in terms of accuracy for better quality effect of the products.
Introduction:
This experiment examines the methodologies of quantitative analysis with UV spectrophotometry on an antitubercular drug, sodium aminosalicylate. UV spectrophotometry measures the absorption of light by the sample, which governed by the Beer-Lambert Law equation [1]. Absorbance of a sample is accounted by the number of UV absorbing molecules; thus the increase in number of these absorbing molecules will increase the absorption value. In other words, higher concentration of a solution will result in a higher UV absorbance. Under the application of UV spectrophotometry, the absorption by sodium aminosalicylate in several different concentrations are measured, in which the results will then be further interpreted through different methodologies in acquisition of the concentrations of any given sample solutions. The samples may require treatment before it can be suitably measured by the instrument [3]. This experiment also requires the selection of a suitable wavelength, normally the λmax, obtained through the absorption of a spectrum of wavelengths from a standard of the sample [3]. These methodologies will be discussed comparatively based upon their performance characteristics, and a deduction for a suitable method to be used in future analysis can be determined for the production or usage of a quality product.
Experimental:
The spectrum of a 1-cm layer of 0.001% w/v solution of sodium aminosalicylate in 0.1M NaOH was analysed over a wavelength of 235 - 325nm with 0.1M NaOH as the blank. The results obtained were used to calculate an approximate value of specific absorbance for the following experiment.
Solutions of sodium aminosalicylate at 50mL volume with concentrations of 0.0002, 0.0004, 0.0006, 0.0008%w/v was prepared from a 0.0010%w/w stock solution. With 0.1M of NaOH as the blank, the absorbance of 0.0010%w/v stock solution with the λmax set at 264nm was checked at a few nm each side of the wavelength. The wavelength with the highest value of absorbance was selected as the λmax for the upcoming measurement of absorbance of the diluted concentrations. The reading was replicated for each solution. The final results are interpreted into a graph of absorbance against concentration with the gradient determined as the specific absorbance [A (1%, 1cm)] of sodium aminosalicylate.
Based on previous results, two unknown concentrations of the solution (labeled "Unknown 1", "Unknown 2") is to be determined using different approach.
(a). Calibration graph
The absorbance of Unknown 1 is first determined and then diluted to adjust the absorbance to the mid range of the previous graph results. The dilution factor was decided as 1 in 4 dilution factor with the final volume as 20mL. The absorbance of the diluted solution is determined and interpreted through the graph to find the actual concentration.
(b.) Absolute method
Absorbance of Unknown 2 is measured and the actual concentration is calculated based on the Beer Lambert equation [A=A (1%, 1cm).c.l].
(c.) Comparative method
With λmax selected at 300nm, the absorbance of 0.0010% w/v stock solution and Unknown 2 is both measured and calculated with the given relationship,
Results:
From the spectrum of a 1-cm layer of 0.001%w/v solution of sodium aminosalicylate in 0.1M NaOH, results are shown as follow:
λmax / nm
Absorbance
264
0.63
300
0.43
Using the λmax of 264nm,
A = A (1%, 1cm).c.l
0.63 = A (1%, 1cm) 0.0001 1
A (1%, 1cm) = 630nm
Using the λmax of 300nm,
A = A (1%, 1cm).c.l
0.43 = A (1%, 1cm) 0.0001 1
A (1%, 1cm) = 430nm
Concentration of solution / %w/v
First absorbance reading
Second absorbance reading
Average absorbance reading
0.0002
0.127
0.130
0.129
0.0004
0.251
0.253
0.252
0.0006
0.377
0.372
0.375
0.0008
0.482
0.481
0.482
0.0010
0.624
0.625
0.625
Gradient of the graph = nm
From the graph, using points (0.52, 0.00082) to calculate molar absorptivity:
0.00082 g in 100mL = 0.00082 100 1000 g L-1 = 0.0082 g L-1
0.0082 211.15 = 3.883 10-5 mol L-1
Beer-Lambert Law equation for molar absorbtivity:
A = ε c l;
0.52 = ε 3.883 10-5 1
ε = 13392 L mol-1 cm-1
(a). Calibration graph
Solution
First reading
Second reading
Average reading
Unknown 1 before dilution
1.195
1.197
1.196
With considerations from the previous readings, a 1 in 4 dilution factor is selected for the second absorbance with the final solution volume being 20mL
Solution
First reading
Second reading
Average reading
Unknown 1 after dilution
0.319
0.323
0.323
Based on the graph from previous results, unknown 1 has an estimated concentration of 0.00052%w/v after dilution. The actual concentration can then be calculated through the equation, C1V1 = C2V2.
C1= actual concentration, C2=0.00052, V1=5mL, V2=20mL
C1 = = 2.08 %w/v
(b). Absolute method
Solution
First reading
Second reading
Average reading
Unknown 2
0.543
0.549
0.546
Using equation A=A (1%, 1cm).c.l ;
A = 0.546, A (1%, 1cm) = 607.14, l = 1
c = 8.99 %w/v
(c). Comparative method
Solution
First reading
Second reading
Average reading
0.0010 %w/v solution
0.366
0.370
0.368
Unknown 2
0.336
0.339
0.338
; A2 = 0.338, AStd = 0.368
Therefore, C2 = %w/v
Discussion:
Beer-Lambert law shows the relationship of the concentration of the sample solution and its absorption through the equation, A = ε c l; A defined as absorption of the sample, ε as the molar absorption coefficient, l as the path length of the cell in centimeters (usually 1cm), and c as the concentration of the sample solution in mol L-1. As pharmaceutical products are mostly expressed in grams or milligrams, therefore, Beer-Lambert law equation is also expressed as A=A (1%, 1cm).c.l ; with A (1%, 1cm) as the specific absorbance and the concentration is expressed in grams per 100mL [1].
The sample solution is calibrated initially to determine the relationship between the concentration and the absorption of the sample solution in different consecutive concentrations. The plotted graph shows a linear relationship which is supported by the Beer-Lambert law equation, whereby the intercept is theoretically zero, provided that the concentration of the sample solution is below a specific concentration value [2]. The graph provides a method for acquiring an unknown concentration of the sample based upon the absorption value of the sample as to the chemical analysis of "Unknown 1". As "Unknown 1" initially showed an absorbance out of range from the graph plotted, the solution was diluted to obtain an absorbance within the mid range value of the graph. The dilution factor was considered into the final calculation for the actual concentration of "Unknown 1". However, the accuracy of this application can be questioned through the external calibration such as preparing the sample, measurement of the sample, and calibration of the instrument [3].
The absolute method which was used to analyse "Unknown" 2 involves a physical constant value, determined based upon fundamental experimental conditions. In this experiment, the specific absorbance of the sample solution is calculated as the constant from the gradient of the calibrated graph. With the value substituted into the Beer-Lambert equation and absorbance measurement of the sample, the concentration can be calculated. This method shows more precision in the final results and is comparatively more accurate than the calibration method due to the factor that it omits the error in preparation of the sample. There may be error in precalibration of the measuring instrument for this method, but the results may still be accurate provided that the error is accounted in the final results [3].
The comparative method, also known as the relative method, determines the unknown concentration of the sample through comparison with the standard of the sample [4].This analysis method shows the same possibility of errors that may occur in the absolute measurement. It may be slightly inaccurate than the absolute measurement as it requires the measurement of two samples rather than just "Unknown 2". However, pre-calibration error can be omitted as well if the final results consider that particular error.
Based upon the final results, there is still a slight difference in the concentration of "Unknown 2" between the absolute method and comparative method. This could be due to the measurement error in either of the methods.
Conclusion:
There are various methods in quantitative analysis of a chemical substance through UV spectrophotometry. All aspects of the methods for accuracy and precision must be considered when selecting methods for analysis.
