Several methods of quantitative analysis through UV spectrophotometry allow the computation of concentration of sample solutions. This experiment examines methodological analysiss of the standardization of the soaking up against concentration graph, the absolute method which involves the computation of the specific optical density, and the comparing of the optical density of the sample and standard solution. Sodium aminosalicylate solutions in different concentration are used as the sample with 0.1M Na hydrated oxide ( NaOH ) as the space of the UV spectrum analysis. The concluding consequences are interpreted through the standardization of a graph, demoing a additive relationship between the optical density and the concentration of the solution. The gradient of the graph is calculated as the specific optical density 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 computation with the Beer-Lambert equation. It can besides be calculated in comparing 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 little fluctuation due to the possibility of measurement mistake. Therefore, the application for quantitative analysis of a sample merchandise must be considered in footings of truth for better quality consequence of the merchandises.
This experiment examines the methodological analysiss of quantitative analysis with UV spectrophotometry on an antitubercular drug, Na aminosalicylate. UV spectrophotometry measures the soaking up of visible radiation by the sample, which governed by the Beer-Lambert Law equation [ 1 ] . Absorbance of a sample is accounted by the figure of UV absorbing molecules ; therefore the addition in figure of these absorbing molecules will increase the soaking up value. In other words, higher concentration of a solution will ensue in a higher UV optical density. Under the application of UV spectrophotometry, the soaking up by Na aminosalicylate in several different concentrations are measured, in which the consequences will so be further interpreted through different methodological analysiss in acquisition of the concentrations of any given sample solutions. The samples may necessitate intervention before it can be appropriately measured by the instrument [ 3 ] . This experiment besides requires the choice of a suited wavelength, usually the I»max, obtained through the soaking up of a spectrum of wavelengths from a criterion of the sample [ 3 ] . These methodological analysiss will be discussed relatively based upon their public presentation features, and a tax write-off for a suited method to be used in future analysis can be determined for the production or use of a quality merchandise.
The spectrum of a 1-cm bed of 0.001 % w/v solution of Na aminosalicylate in 0.1M NaOH was analysed over a wavelength of 235 – 325nm with 0.1M NaOH as the space. The consequences obtained were used to cipher an approximative value of specific optical density for the undermentioned experiment.
Solutions of Na 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 space, the optical density of 0.0010 % w/v stock solution with the I»max set at 264nm was checked at a few nm each side of the wavelength. The wavelength with the highest value of optical density was selected as the I»max for the approaching measuring of optical density of the diluted concentrations. The reading was replicated for each solution. The concluding consequences are interpreted into a graph of optical density against concentration with the gradient determined as the specific optical density [ A ( 1 % , 1cm ) ] of Na aminosalicylate.
Based on old consequences, two unknown concentrations of the solution ( labeled “ Unknown 1 ” , “ Unknown 2 ” ) is to be determined utilizing different attack.
( a ) . Calibration graph
The optical density of Unknown 1 is first determined and so diluted to set the optical density to the mid scope of the old graph consequences. The dilution factor was decided as 1 in 4 dilution factor with the concluding volume as 20mL. The optical density of the diluted solution is determined and interpreted through the graph to happen the existent concentration.
( B. ) Absolute method
Optical density of Unknown 2 is measured and the existent concentration is calculated based on the Beer Lambert equation [ A=A ( 1 % , 1cm ) .c.l ] .
( c. ) Comparative method
With I»max selected at 300nm, the optical density of 0.0010 % w/v stock solution and Unknown 2 is both mensural and calculated with the given relationship,
From the spectrum of a 1-cm bed of 0.001 % w/v solution of Na aminosalicylate in 0.1M NaOH, consequences are shown as follow:
I»max / nanometer
Using the I»max of 264nm,
A = A ( 1 % , 1cm ) .c.l
0.63 = A ( 1 % , 1cm ) 0.0001 1
A ( 1 % , 1cm ) = 630nm
Using the I»max 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 optical density reading
Second optical density reading
Average optical density reading
Gradient of the graph = nanometer
From the graph, utilizing points ( 0.52, 0.00082 ) to cipher molar absorption factor:
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 = Iµ degree Celsius cubic decimeter ;
0.52 = Iµ 3.883 10-5 1
Iµ = 13392 L mol-1 cm-1
( a ) . Calibration graph
Unknown 1 before dilution
With considerations from the old readings, a 1 in 4 dilution factor is selected for the 2nd optical density with the concluding solution volume being 20mL
Unknown 1 after dilution
Based on the graph from old consequences, unknown 1 has an estimated concentration of 0.00052 % w/v after dilution. The existent concentration can so be calculated through the equation, C1V1 = C2V2.
C1= existent concentration, C2=0.00052, V1=5mL, V2=20mL
C1 = = 2.08 % w/v
( B ) . Absolute method
Using equation A=A ( 1 % , 1cm ) .c.l ;
A = 0.546, A ( 1 % , 1cm ) = 607.14, cubic decimeter = 1
degree Celsiuss = 8.99 % w/v
( degree Celsius ) . Comparative method
0.0010 % w/v solution
; A2 = 0.338, AStd = 0.368
Therefore, C2 = % w/v
Beer-Lambert jurisprudence shows the relationship of the concentration of the sample solution and its soaking up through the equation, A = Iµ c cubic decimeter ; A defined as soaking up of the sample, Iµ as the molar soaking up coefficient, cubic decimeter as the way length of the cell in centimetres ( normally 1cm ) , and degree Celsius as the concentration of the sample solution in mol L-1. As pharmaceutical merchandises are largely expressed in gms or mgs, hence, Beer-Lambert jurisprudence equation is besides expressed as A=A ( 1 % , 1cm ) .c.l ; with A ( 1 % , 1cm ) as the specific optical density and the concentration is expressed in gms per 100mL [ 1 ] .
The sample solution is calibrated ab initio to find the relationship between the concentration and the soaking up of the sample solution in different back-to-back concentrations. The aforethought graph shows a additive relationship which is supported by the Beer-Lambert jurisprudence 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 geting an unknown concentration of the sample based upon the soaking up value of the sample as to the chemical analysis of “ Unknown 1 ” . As “ Unknown 1 ” ab initio showed an optical density out of scope from the graph plotted, the solution was diluted to obtain an optical density within the mid scope value of the graph. The dilution factor was considered into the concluding computation for the existent concentration of “ Unknown 1 ” . However, the truth of this application can be questioned through the external standardization such as fixing the sample, measuring of the sample, and standardization of the instrument [ 3 ] .
The absolute method which was used to analyze “ Unknown ” 2 involves a physical changeless value, determined based upon cardinal experimental conditions. In this experiment, the specific optical density of the sample solution is calculated as the invariable from the gradient of the graduated graph. With the value substituted into the Beer-Lambert equation and optical density measuring of the sample, the concentration can be calculated. This method shows more preciseness in the concluding consequences and is relatively more accurate than the standardization method due to the factor that it omits the mistake in readying of the sample. There may be error in precalibration of the measurement instrument for this method, but the consequences may still be accurate provided that the mistake is accounted in the concluding consequences [ 3 ] .
The comparative method, besides known as the comparative method, determines the unknown concentration of the sample through comparing with the criterion of the sample [ 4 ] .This analysis method shows the same possibility of mistakes that may happen in the absolute measuring. It may be somewhat inaccurate than the absolute measuring as it requires the measuring of two samples instead than merely “ Unknown 2 ” . However, pre-calibration mistake can be omitted every bit good if the concluding consequences consider that peculiar mistake.
Based upon the concluding consequences, there is still a little difference in the concentration of “ Unknown 2 ” between the absolute method and comparative method. This could be due to the measuring mistake in either of the methods.
There are assorted methods in quantitative analysis of a chemical substance through UV spectrophotometry. All facets of the methods for truth and preciseness must be considered when choosing methods for analysis.