Most pharmaceutical merchandises absorb UV radiation with some of them, which are colored compounds absorb radiation in the seeable scope. The soaking up of UV radiation occurs through the excitement of negatrons within the land province to a higher energy province. Merely most easy aroused negatrons show soaking up in the UV-visible part. [ 3 ] Thus, pick of reagent becomes important in order to acquire a dependable consequence for the quantitative analysis. In this instance, Na aminosalicylate is chosen as the absorbing species. In add-on, Na aminosalicylate is bacteriostatic against Mycobacterium TB by suppressing its growing and generation and therefore used as an antitubercular drug and besides is frequently used to handle Crohn ‘s disease by cut downing redness. [ 4 ] Three attacks are used to find the concentration of both unknown solutions. They are calibration graph method, absolute method and comparative method. All of them are based on the optical density readings recorded at the I»max chosen. Transmittance is the ratio of the familial strength to the incident strength. [ 2 ] Absorbance is defined as the common logarithm of the reciprocal of transmission of a pure sample. [ 2 ] Furthermore, UV spectrometry is normally used for quantitative analysis for BP checks and bound trials in footings of its dependability, easy in informations handling, handiness of the equipment and its wide soaking up sets.
A set of standard solutions was prepared in order to build a standardization graph for the finding of the unknown analyte concentration. 50mL volumes of 0.0002, 0.0004, 0.0006 and 0.0008 % w/v of Na aminosalicylate solutions in 0.1M NaOH were prepared from the 0.0010 % w/v stock solution. For illustration, 10mL of stock solution was pipetted out and assorted with 40mL of 0.1M NaOH in order to do 50mL of 0.0002 % w/v solution in a round-bottomed flask.
Optical density were measured utilizing a individual beam spectrophotometer. First, blanking the cell was carried out between both cells with 0.1M NaOH. Any cell difference should be recorded and subtracted from any ulterior readings of the sample.
The optical density of each of the five standard solutions were recorded and replicate readings were obtained for each solution. This was done by emptying, replenishing and replacing the cuvette between readings. All the information gathered was used to plot a graph of optical density against concentration. The incline of the graph was the specific optical density, A ( 1 % ,1cm ) . Molar absorption factor could be calculated from the informations obtained.
Initial optical density of unknown 1 was measured at 263nm. Dilution of 1 in 4 was carried out in unknown 1 in order to acquire the optical density which fell in the mid scope of the graph plotted. The diluted solution was made up to 100mL with 25mL unknown 1 and 75mL 0.1M NaOH. Absorbance was measured once more. 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 happen out the concentration of unknown 2, which were absolute method and comparative method. For the absolute method, the optical density of unknown 2 was measured and its concentration was determined utilizing Beer-Lambert jurisprudence. In this instance, the specific optical density was obtained antecedently from the incline of the standardization graph. For the comparative method, the I»max was changed to 300nm and measured merely the optical density of a individual criterion ( 0.0010 % w/v ) and unknown 2. The concentration of unknown 2 was calculated utilizing a relationship tabulated below.
A spectrum of a 0.001 % w/v solution of Na aminosalicylate in 0.1M NaOH was obtained over the wavelength scope 235 to 325nm. From this spectrum, two I»max and their corresponding optical density were recorded. The specific optical density was calculated utilizing Beer- Lambert jurisprudence:
A = A ( 1 % ,1cm ) .c.l
I»max ( nanometer )
A ( 1 % ,1cm )
Table 1 Optical density at I»max ( Reference and appraisal )
Choice of suited I»max:
From the above, I»max at 264nm gave the highest optical density. Absorbance a few nm each side of 264nm were recorded in order to choose the most appropriate I»max. I»max at 263nm was selected.
Wavelength, I» ( nanometer )
Table 2 Optical density at different wavelengths
Graph 1 Selection of I»max
Preparation of standard solutions:
A set of 50mL standardization 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 illustration,
C1V1 = C2V2
0.0010 ten V1 = 0.0002 x 50
V1 = 10mL
( % w/v )
Volume of stock
( milliliter )
Volume of NaOH
( milliliter )
Final diluted volume
( milliliter )
Table 3 Consecutive dilutions
Optical density of standard solutions:
Difference between cells: -0.02
Concentration ( % w/v )
Table 4 Absorbance of standard solutions
Graph 2 Calibration graph ( Beer-Lambert Law )
Molar absorption factor of Na aminosalicylate in 0.1M NaOH at 263nm:
Mr of Na aminosalicylate = 211.15
Concentration of Na aminosalicylate = 0.0002 ten 10 ? 211.15 = 9.472 ten 10-6 mol dm-3
Optical density of 0.0002 % w/v Na aminosalicylate at 263nm is 0.122.
A = IµMl
0.122 = Iµ x 9.472 x 10-6 ten 1
Iµ = 12880.15 dm3mol-1cm-1
Determination of concentrations of unknown 1 & A ; 2:
For unknown 1,
Initial optical density 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
Harmonizing to the standardization graph drawn, the concentration of unknown 1 is 5.20 ten 10-4 % w/v.
Actual concentration of unknown 1 = 5.20 ten 10-4 ten 4 = 2.08 ten 10-3 % w/v
For unknown 2,
Optical density at 263nm = 0.508
A ( 1 % ,1cm ) = incline of standardization graph
= 0.56 – 0.02
0.00096 – 0.00004
A = A ( 1 % ,1cm ) chlorine
0.508 = 586.96 ten degree Celsiuss x 1
degree Celsiuss = 8.65 ten 10-4 % w/v
Difference between cell = -0.009
Optical density of 0.0010 % w/v stock solution at 300nm = 0.371
Optical density of unknown 2 at 300nm = 0.322
C2 = A2
C2 = 0.322
C2 = 8.68 ten 10-4 % w/v
UV has a short wavelength, therefore it has high energy which is adequate to alter the electronic construction of a molecule. It may temporarily impact the bond construction. In this instance, Na aminosalicylate is chosen as the absorbing species due to the presence of OH and NH2 groups in the benzine pealing which leads to higher junction and higher molar absorption factor. This increases the opportunity of electron passage. [ 3 ]
Normally, the optical density of benzine is recorded at 255nm. [ 1 ] However, the extremely conjugated system lowers the energy spread for negatron passage and hence increasing the wavelength of the absorber. Therefore, the approximative I»max of Na aminosalicylate are 264nm and 300nm harmonizing to the spectrum provided. I»max is set to 263nm for the optical density measuring of the standard solutions and unknown 1. This is because the optical density at 263nm is the highest, that is 0.607 for the 0.0010 % w/v stock solution.
The usage of I»max will give maximal sensitiveness for the optical density readings. A little alteration around the I»max will merely give a somewhat alteration in optical density. In other words, the truth of the optical density readings will non be affected excessively much if there is an mistake in wavelength scene. Other than that, most reagent will hold more than one I»max value, hence, the wavelength with highest optical density recorded is frequently chosen in order to understate interventions from other substances and it will be more specific. Furthermore, taking optical density at I»max will understate the mistakes from drosss as drosss may hold different I»max. [ 3 ]
Single beam spectrophotometer is well-suited for quantitative analysis because all the measurings are made at a individual wavelength. When utilizing the individual beam instrument, it is necessary to take the readings of the clean solution and the sample solution consecutively, in order to guarantee that the strength from the light beginning is changeless. [ 2 ] Other than that, blanking the cells are of import before the start of optical density measurings as there may be a little difference between the two cuvettes used.
Beer-Lambert Law states that the optical density depends on the entire figure of absorbing molecules in the light way through the cell. [ 1 ] This besides means that the soaking up is affected by both the analyte concentration ( degree Celsius ) and the way length ( cubic decimeter ) . The jurisprudence is farther simplified into two similar equations, the latter one is normally used in British Pharmacopeia ( BP ) :
A = IµMl
A = A ( 1 % ,1cm ) chlorine
Both equations are merely applied to thin solutions which the optical density is less than 1. Radiation visible radiation will barely to acquire through concentrated solution which optical density is greater than 1 and consequences in inaccurate readings. Premises are made for the Beer-Lambert jurisprudence: the usage of monochromatic visible radiation ( radiation consisting of merely individual wavelength or in practical the visible radiation with narrow wave set ) and the homogeneousness of the absorber. [ 2 ]
Harmonizing to the standardization graph drawn, optical density is straight relative to the concentration ( additive relationship ) . This proves that the Beer-Lambert jurisprudence is obeyed. However, some divergences may originate from the experiment which consequences in non-linearity, peculiarly at high concentrations. The job does non originate in this experiment as low concentrations are used.
Three different quantitative methods are used to look into the concentrations of both unknown solutions. First, the standardization graph methodological analysis. This involves the optical density measuring over a scope of standard solutions and plotting a graph. The unknown concentration can be obtained from the read-off of the graph based on the optical density measured. It is really time-consuming and it requires a pure criterion which stays chemically stable over a period of clip. [ 3 ] Extras are done for optical density measuring to cut down mistakes. Beer-Lambert jurisprudence must be obeyed for all the samples tested.
Following is the absolute methodological analysis. This lone measures the unknown optical density under defined conditions and by utilizing a known specific optical density. [ 3 ] It merely requires a high quality instrument for the optical density measuring and therefore it is easier to be used. It does non necessitate criterions, therefore it is less clip devouring. This is the preferable method in British Pharmacopeia ( BP ) .
The last 1 is comparative methodological analysis. This involves the optical density measuring of a individual criterion and the unknown. Therefore, it needs a pure criterion to forestall any inaccuracies. Since it is based on the relationship between the criterion and the unknown, therefore it is best if the concentration of the criterion and the unknown solution are near. It is more preferable in United States Pharmacopeia ( USP ) . [ 3 ]
Overall, those three methods must obey the one-dimensionality as proposed in Beer-Lambert jurisprudence. The concentrations for both unknown 1 and unknown 2 are 2.08 ten 10-3 % w/v and 8.67 ten 10-4 % w/v severally. The common factors of mistake are the wavelength, spectral declaration and stray visible radiation. Absolute method in comparing with comparative method, is more dependable due to less measurings made. For the comparative method, any divergence or impure criterion will do inaccuracy in concentration finding. For absolute method, the factors of mistake can be reduced to minimum by utilizing the UV spectrophotometer that fulfils the BP cheques.
UV-visible spectrometry is a simple, rapid, cosmopolitan and cost-efficient method in quantitative analysis. The analysis should be done at maximal wavelength so that the optical density will be high and changeless around the chosen wavelength. Sodium aminosalicylate is a good UV absorber. Beer- Lambert jurisprudence must be obeyed in every quantitative method used. The concentrations for both unknown 1 and unknown 2 are 2.08 ten 10-3 % w/v and 8.67 ten 10-4 % w/v severally.