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Sodium p-Aminosalicylate is an parallel of para-aminobenzoic acid that inhibits or prevents the folic acid synthesis in bacteriums known as Mycobacterium TB and it is used as an anti-tuberculosis agent by forestalling the generation of the tubercle B. [ 3 ] It is besides used to handle Crohn ‘s disease today. [ 6 ] Quantitative analysis of Na p-Aminosalicylate utilizing the UV or seeable spectrometry determines the optical density of the bonds within the molecules in sodium p-Aminosalicylate. The concentration of sodium p-Aminosalicylate can be determined by three methods. [ 6 ] The first method is utilizing standardization graph by mensurating the optical density of the unknown concentration of solution for a scope of criterions and plotting a Beer-Lambert graph. [ 7 ] The 2nd method is known as absolute method where the computation is carried out by mensurating the optical density of the unknown with known specific optical density. [ 7 ] The 3rd method is comparative method where computations can be carried out by mensurating optical density of a individual criterion and optical density of the unknown. [ 7 ]

A I»max values was selected from the standard graph of Na p-aminosalicylate 0.0010 % w/v, the I»max was used for determine the “ unknown 1 and 2 ” and the specific optical density was determined utilizing the incline of the gradient. The 0.1M Na hydrated oxide was used as clean agent. Dilution was done on “ unknown 1 ” due to the high optical density value. For the comparative method, the I»max used to find “ unknown 2 ” was obtained from the standardization graph provided. The concentration of “ unknown 2 ” was determined by utilizing absolute method where computation was carried out after the specific optical density was obtained from the standard graph and by comparative method where computation was carried after the optical density of “ unknown 2 ” had measured.

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From the consequence, the concentration of “ unknown 1 ” was 0.0026 % w/v by utilizing the standard graph and “ unknown 2 ” was 0.0006 % w/v for both comparative and absolute method. The consequences obtained in this analysis were good and precise. This concluded the UV molecular spectrometry has wide soaking up sets and used chiefly for quantitative analysis intents in pharmaceutical.

1. Introduction:

Sodium p-Aminosalicylate besides known as sodium 4-amino-2-hydroxybenzoate or 4-Amino-2-hydroxybenzoic acid is a white to blanch xanthous crystalline pulverization. [ 1 ] The chemical expression is C7H7NO3Na [ 2 ] It is practically odourless and freely soluble in H2O, meagerly soluble in intoxicant and practically indissoluble in quintessence. It dissolves in H2O give a clear solution which is colourless. [ 1 ] The pH of the solution of Sodium p-Aminosalicylate in the scope between 7.0 to 7.5. [ 1 ] The Na salt of salicylate is prepared from Na phenolate under warming and force per unit area with gas C dioxide. [ 3 ] It contains a hydroxyl and a carboxyl group and these groups are reacted with an acid or an intoxicant. [ 3 ] Ester produced as end merchandise in the reaction of carboxyl group with intoxicants. [ 3 ]

Para-Aminosalicylic acid is an parallel of para-aminobenzoic acid that inhibits or prevents the folic acid synthesis in Mycobacterium TB and it is known as bacteriostatic agents where it inhibits growing of the tubercle B. [ 3 ] Para-Aminosalicylic acid and its Na salt signifier known as Na p-Aminosalicylate are bacteriostatic agent where it acts against inhibit the growing of mycobacterium and it used in the intervention of TB when administered orally. [ 3 ] Aminosalicylic acids are active ingredients in pharmaceutical sector including it acts as anti-infectives agent, where it acts against colds, grippe and some sorts of viral infections. [ 3 ] 5-aminosalicylic acid is an active metabolite of sulfasalazine in compound known as mesalamine, it used to handle redness disease occurred in the rectum and lower colon, mild to chair ulcerative inflammatory bowel disease, and proctitis. [ 4 ]

Quantitative analysis utilizing the UV or seeable spectrometry is simple and cost- effectivity. [ 5 ] It produces rapid consequence therefore sensitive to absorbance which about 1- 10ppm. [ 5 ] It is about cosmopolitan and prone to interventions in debasement merchandises, metabolites, excipients and endogenous stuffs. The measuring of light soaking up by molecules in a solution is governed by the Beer-Lambert jurisprudence, logI0/It= A= Iµbc. The Pharmacopoeial methods rely on simple analysis by ultraviolet spectrophotometry to find active ingredients in preparations. All of the pharmaceutical merchandises, concentrations and sums are expressed in gms ( g ) or mgs ( milligram ) instead than moles and therefore the Beer-Lambert jurisprudence is expressed as A = A ( 1 % , 1cm ) .c.l. The path-length is normally 1cm because the measuring is made in a 1cm cell. [ 5 ]

The wavelength scope 200-700nm is leting the radiation passed through a solution of a compound. The soaking up of UV or seeable radiation occurs through the excitement of negatrons within the molecular construction to a higher energy province. [ 5 ] The short wavelength & lt ; 150nm can do damaging to populating beings because the strongest bond in organic molecules break. [ 5 ] Longer wavelength & gt ; 200nm is excited the weaker bonds molecules normally used in analysis method. [ 5 ] The maximal wavelength or abbreviated I»max gives maximal sensitiveness and it has little alteration in optical density with alteration of wavelength where little mistakes in I» scene does non impact truth of the optical density reading. Most of the compounds have more than one I»max value and the highest is selected to minimise the interventions. [ 5 ]

The concentration of sodium p-Aminosalicylate can be determined by three methods. [ 7 ] The first method is utilizing standardization graph. The computation can be carried out by mensurating the optical density for a scope of criterions and plotting a Beer-Lambert graph. [ 7 ] The concentration of unknown solution can be obtained from the graph after measured of the optical density of the unknown solution. This is good method if the unknown concentration may change well and one-dimensionality is established. [ 7 ] The incline gives specific optical density. The 2nd method is known as absolute method where the computations is carried out by mensurating the optical density if the unknown under defined conditions and utilizing a known specific optical density. It is British Pharmacopeia preferred method and equation used is concentration ( % w/v ) = A/A ( 1 % , 1cm ) in 1cm cell. [ 7 ] The 3rd method is comparative method where computations can be carried out by mensurating optical density of a individual criterion and optical density of the unknown. It is reasonably rapid but requires a pure criterion. [ 7 ] The equation used is C2/0.001= A2/Astd. It is best if the criterion and sample concentrations are closed. [ 7 ] It is United Stated Pharmacopeia ( USP ) preferred method. [ 7 ]

2. Experimental:

2.1. Materials:

An ultraviolet molecular spectrophotometer was used in this experiment. 0.1M Na hydrated oxide solution was used as clean solution and as dilution agent for the sodium-p-aminosalicylate. Stock solution was 0.0010 % w/v sodium-p-aminosalicylate. The setup used were 10mL, 20mL, 50mL pipettes, beakers and 50mL volumetric flasks ; cuvette was used to make full the solutions and placed into the spectrophotometer. [ 6 ]

2.2. Methods:

Absorption spectrum of 0.0010 % w/v Sodium Aminosalicylate in 0.1M Na hydroxide solution.

The spectrum of a 1-cm bed of a 0.0010 % w/v solution of Na aminosalicylate in 0.1M Na hydrated oxide solution over the wavelength scope 235 to 325nm was scanned and examined. The baseline was obtained before entering the spectrum utilizing 0.1M sodium hydroxide solution in both cells. The wavelengths or I»max and the optical density values at these wavelengths of the two upper limits were measured from the spectrum. The approximative specific optical density was calculated at each of these upper limits utilizing the Beer-Lambert jurisprudence equation ;

A = A ( 1 % , 1cm ) .c.l

Where, A= optical density

A ( 1 % , 1cm ) = specific optical density of a 1cm bed of a 1 % w/v solution

c= concentration ( % w/v )

l= way length ( centimeter )

Beer- Lambert jurisprudence, specific optical density and molar absorption factor

50mL volumes of 0.0002 % w/v, 0.0004 % w/v, 0.0006 % w/v, and 0.0008 % w/v solutions of aminosalicylate in 0.1M Na hydrated oxide were prepared from the 0.0010 % w/v stock solution. A I»max values was selected and the undermentioned processs were carried out utilizing a individual beam spectrophotometer.

The wavelength was set to the selected value, the 0.1M Na hydrated oxide as the dissolver was placed in the both cells and the optical density reading was set to zero on the instrument. The 0.0010 % w/v stock solution was used, the optical density a few nm each side of the selected wavelength was checked. The optical density to zero was set at each wavelength utilizing the space. A I»max values was selected from the tabular array of optical density values and the spectrophotometer was set to this wavelength. The wavelength in the spectrophotometer remained set until the subdivision completed. The optical densities of a 1-cm bed of each of the five solutions of Na aminosalicylate in difference concentrations were recorded. The replicate readings for each solution were obtained by empty, refill and replace the cuvette between readings. A graph of optical density against concentration was plotted and the specific optical density ( A ( 1 % , 1cm ) ) was determined utilizing the gradient of the graph. The British Pharmacopoeia ( BP ) defined the molar absorption factor ( Iµ ) as the optical density of a 1-cm bed of a 1M solution. The molar absorption factor of Na aminosalicylate in 0.1M Na hydrated oxide was calculated at the selected upper limit.

Determination of concentrations

Ultraviolet- seeable spectrometry was often used as a method of qualitative analysis for the BP checks and bound trials. The concentration of two sodium-p-aminosalicylate solutions, “ Unknown 1 ” and “ Unknown 2 ” with unknown concentration were determined utilizing following method.

Calibration graph was used to find the optical density of a 1-cm bed of “ Unknown 1 ” . The “ Unknown 1 ” solution was diluted so that its optical density was in the mid scope of the graph of optical density against concentration plotted. The graph of optical density against concentration was used to find the concentration of the diluted solution and the concentration of “ Unknown 1 ” was calculated.

Absolute method was used to find the optical density of a 1-cm bed of “ Unknown 2 ” . The optical density of “ Unknown 2 ” was recorded. The concentration of “ Unknown 2 ” was calculated utilizing the specific optical density ( A ( 1 % , 1cm ) ) value determined antecedently.

Comparative method was used to compare the optical density of a 1-cm bed of the 0.0010 % w/v solution and a 1-cm bed of “ Unknown 2 ” . The wavelength was set to the second of the I»max value from the supplied spectrum. The optical density of the 1-cm bed of 0.0010 % w/v solution and a 1-cm bed of the “ Unknown 2 ” were recorded. The concentration of the “ Unknown 2 ” , C2, was calculated utilizing the relationship.

C2/0.001 = A2/Astd

3. Consequences:

The measuring of the wavelength or I»max and the optical density values of the two upper limit in the supplied spectrum were shown below ( table 1 ) ; the approximate specific optical density at each upper limit wavelength was shown in table 1.

Maxima on the supplied spectrum

Wavelength ( nanometer )

Absorbance value ( A )

Specific optical density ( A ( 1 % ,1cm ) )

First I»max

264

0.630

630

Second I»max

300

0.425

425

Table 1: The wavelength figure, optical density value and specific optical density of the two upper limit on the supplied spectrum.

The volume required for the dilutions of the sodium-p- aminosalicylate harmonizing to the concentration stated from 0.0010 % w/v stock solution by utilizing 0.1M Na hydrated oxide was shown in table 2. Calculation of the volume required shown in portion 1 in auxiliary inquiry. The expression for the dilution was C1V1= C2V2. All the optical density of the 0.0010 % w/v sodium-p- aminosalicylate from wavelength scope 260nm to 265nm was shown in table 3. The maximal wavelength used for finding of the dilutions of the sodium-p- aminosalicylate was 265nm due to the highest optical density in 0.0010 % w/v sodium-p- aminosalicylate. All the optical density of the dilutions sodium-p- aminosalicylate was determined by the figure of wavelength 265nm. The standardization graph of concentration ( % w/v ) against optical density ( I» ) was plotted harmonizing to the tablet 4. From the standardization graph 1, there was a additive secret plan obtained. The specific optical density A ( 1 % , 1cm ) was obtained through the expression Y = maxwell +c, where Y =absorbance, m =specific optical density, ten =concentration and degree Celsius =intercept. The gradient of the secret plan where represent the specific optical density was 595nm. ( Refer to the standardization graph 1 following page )

Concentration of 50mL sodium-p- aminosalicylate ( % w/v ) diluted with 0.1M NaOH solution

Volume of 0.0010 % w/v stock solution required ( milliliter )

0.0002

10

0.0004

20

0.0006

30

0.0008

40

0.0010

50

Table 2: The volume of 0.0010 % w/v stock solution required fixing the difference concentration of 50mL sodium-p- aminosalicylate ( % w/v ) diluted with 0.1M NaOH solution.

Wavelength

Optical density of 0.0010 % w/v sodium-p- aminosalicylate

1streading

Optical density of 0.0010 % w/v sodium-p- aminosalicylate

2ndreading

Mean

260

0.546

0.544

0.545

261

0.559

0.558

0.559

262

0.572

0.574

0.573

263

0.587

0.587

0.587

264

0.596

0.594

0.595

265

0.599

0.599

0.599

Table 3: The optical density of 0.0010 % w/v sodium-p- aminosalicylate with 0.1M NaOH solution as space.

Concentration of sodium-p- aminosalicylate ( % w/v )

Optical density

1streading

Optical density

2ndreading

Mean

0.0002

0.111

0.109

0.110

0.0004

0.231

0.226

0.229

0.0006

0.349

0.347

0.348

0.0008

0.468

0.469

0.469

Table 4: The optical density of difference concentration sodium-p- aminosalicylate with 0.1M NaOH solution as space.

The unknown concentrations of the sodium-p- aminosalicylate ( % w/v ) were determined and the optical density for “ Unknown 1 ” was & gt ; 1 and hence it is excessively high to find the concentration harmonizing to standardization graph 1. ( Refer table 5 ) The highest optical density obtained from the graph 1 was about 0.7 and the optical density of the unknown concentration sodium-p- aminosalicylate was 1.189. Dilution of the “ unknown 1 ” sodium-p- aminosalicylate was done to accomplish a new optical density value which in the scope of the standardization graph 1 plotted. ( Refer table 5 ) The entire volume of dilution unknown concentration sodium-p- aminosalicylate by utilizing 0.1M Na hydrated oxide was 20mL and 5mL of “ unknown 1 ” concentration sodium-p- aminosalicylate was calculated. ( Refer portion A ) . The optical densities of the “ unknown 1 ” concentration of sodium-p- aminosalicylate was in the mid-range of the standardization graph 1 ( Refer table 6 )

Unknown concentration of sodium-p- aminosalicylate ( % w/v )

Optical density

1streading

Optical density

2ndreading

Mean

Unknown 1

1.189

1.195

1.192

Unknown 2

0.336

0.339

0.338

Table 5: The optical density of unknown concentration of sodium-p- aminosalicylate with 0.1M NaOH solution as space.

Unknown concentration of sodium-p- aminosalicylate ( % w/v )

Optical density

1streading

Optical density

2ndreading

Mean

Unknown 1 ( diluted )

0.366

0.370

0.368

Unknown 2 ( undiluted )

0.336

0.339

0.338

Table 6: The optical density of “ unknown 1 ” sodium-p- aminosalicylate after dilution and “ unknown 2 ” with 0.1M NaOH solution as space.

The wavelength used was 300nm which was the 2nd upper limit of I»max measured in the supplied spectrum for the optical density of “ unknown 2 ” sodium-p- aminosalicylate. ( Refer to postpone 7 )

Concentration of sodium-p- aminosalicylate ( % w/v )

Optical density

1streading

Optical density

2ndreading

Mean

0.0010

0.643

0.641

0.642

Unknown 2

0.336

0.339

0.338

Table 7: The optical density of “ unknown 2 ” and 0.0010 % w/v sodium-p- aminosalicylate with 0.1M NaOH solution as space by utilizing wavelength 300nm.

The finding of the two unknown concentrations of sodium-p- aminosalicylate was shown below.

Part A:

From the standardization graph ( graph 1 ) , the optical density value of “ Unknown 1 ” was 0.0006 % w/v. The entire volume after dilution was 20mL and the volume of the “ Unknown 1 ” sodium-p- aminosalicylate was 5mL. By utilizing equation, C1V1= C2V2

C1V1= C2V2

( 0.00065 % w/v ) ( 20mL ) = ( X ) ( 5mL )

X= ( 0.00065 % w/v ) ( 20mL ) / ( 5mL )

= 0.0026 % w/v

Part B:

The concentration of “ Unknown 2 ” was determined utilizing absolute method where Beer- Lambert jurisprudence was applied, A = A ( 1 % , 1cm ) .c.l ; A= 0.338, optical density of “ Unknown 2 ” ; A ( 1 % , 1cm ) = specific absorbance= 595 nanometer ; hundred =concentration of “ Unknown 2 ” and l= 1cm.

A = A ( 1 % , 1cm ) .c.l

0.338= 595 ten C x 1

C= 0.338/300

= 0.0006 % w/v

Part C: Another method to find the concentration of “ unknown 2 ” was utilizing comparative method where a relationship was applied, C2/0.001= A2/Astd. C2= concentration of “ unknown 2 ” ‘ A2= optical density value for “ unknown 2 ” and Astd= optical density of the standard 0.0010 % w/v sodium-p- aminosalicylate.

C2/0.001= A2/Astd.

C2= ( 0.001 x 0.338 ) /0.599

= 0.0006 % w/v

The molar absorption factor of Na aminosalicylate in 0.1M NaOH at the selected upper limit:

Mr of Na aminosalicylate = 176.074

The concentration of 0.0002 % w/v Na aminosalicylate converted to mol/L. The value obtained is 1.136 ten 10-6mol/L.

A= Iµbc

Iµ= A/ B /c

= 0.110/ 1.136 ten 10-6mol/L

= 96840.7 ( within the typical values 1,000-100,000 )

4. Discussion:

This experiment reveals the qualitative analysis to find the “ unknown 1 ” and “ unknown 2 ” of sodium-p- aminosalicylate. The concentration of the “ unknown 1 ” obtained is 0.00065 % w/v and it was straight obtained from the Beer-Lambert graph plotted after measurement of the optical density of a scope of criterions. The concentration obtained from the standard graph was non the exact concentration of “ unknown 1 ” because there was a dilution done for the ground of the optical density values of the “ unknown 1 ” was excessively high. The “ unknown 1 ” was diluted to 20mL by utilizing sodium hydroxide solution. The precisely concentration of “ unknown 1 ” was 0.0026 % w/v. This method requires a pure criterion and really clip consuming.

The concentration of “ unknown 2 ” was determined by two methods. There were absolute method and comparative method. The standard Beer- Lambert graph plotted after mensurating if the optical density of a scope of criterions sodium-p- aminosalicylate 0.0010 % w/v gives a incline of 595 nanometer as specific optical density and the equation used is concentration ( % w/v ) = A/A ( 1 % , 1cm ) in 1cm cell. The computation of concentration “ unknown 2 ” was determined by direct substituted the values obtained into the equation. This method was known as absolute method. This was the preferred method for British Pharmacopeia for analysis of the active ingredient in a drug. The concentration for “ unknown 2 ” was 0.0006 % w/v.

The comparative method was the preferred method for the United States Pharmacopeia. [ 7 ] It is utile to find the sample if it is reacted or extracted before the optical density is measured. [ 7 ] The computation was carried out by mensurating optical density of a standard optical density and optical density of the “ unknown 2 ” . Then the optical density values obtained were substituted in the equation C2/0.001= A2/Astd. The concentration of “ unknown 2 ” was = 0.0006 % w/v.

The concentrations of “ unknown 2 ” obtained from the absolute method and comparative method were same. This reveals the experiment was done in correct and proper stairss. The experiment was successfully done. The ultraviolet spectrophotometer was accurately calibrated in the experiment. The standard Beer-Lambert graph, optical density against concentration was plotted exactly. The good accomplishment to run the spectrophotometer was applied. The type of cuvette used to make full the solution for the optical density measuring was appropriate for these wavelengths. The choice on the standard wavelength selected was close to the maximal wavelength, I»max on the standardization graph provided. The incline or gradient on the standard graph was accurately calculated. The same cuvette was used for the measuring of both standard sodium-p- aminosalicylate 0.0010 % w/v and “ unknown 1 and 2 ” for sodium-p- aminosalicylate 0.0010 % w/v to accomplish maximal quantitative truth. All the solutions used in the experiment were free from any atoms, this was important to avoid dispersing where evident addition of optical density in the solution and produced good and precise consequence.

5. Decisions:

This analysis was concluded that the UV molecular spectrometry has wide soaking up sets and used chiefly for quantitative analysis intents. Simple analysis by ultraviolet spectrophotometry is used to find active ingredients in pharmaceutical preparations Pharmacopoeial methods for designation cheques. The standard optical density, A ( 1 % , 1cm ) is used for the analysis and this to a great extent depends on the UV spectrophotometer being accurately calibrated. The consequence obtained in this analysis was good and precise.

6. Auxiliary inquiries:

Part 1

Determine the volume of stock solution required for each concentration of sodium-p- aminosalicylate

Calculations:

Formulas used: C1V1= C2V2

Concentration of stock solution: 0.0010 % w/v

a ) To fix 50mL 0.0002 % w/v sodium-p- aminosalicylate ;

C1V1= C2V2

( 50mL ) ( 0.0002 % w/v ) = ( X ) ( 0.0010 % w/v )

X= ( 50mL ) ( 0.0002 % w/v ) / ( 0.0010 % w/v )

= 10mL

B ) To fix 50mL 0.0004 % w/v sodium-p- aminosalicylate ;

C1V1= C2V2

( 50mL ) ( 0.0004 % w/v ) = ( X ) ( 0.0010 % w/v )

X= ( 50mL ) ( 0.0004 % w/v ) / ( 0.0010 % w/v )

= 20mL

degree Celsius ) To fix 50mL 0.0004 % w/v sodium-p- aminosalicylate ;

C1V1= C2V2

( 50mL ) ( 0.0006 % w/v ) = ( X ) ( 0.0010 % w/v )

X= ( 50mL ) ( 0.0006 % w/v ) / ( 0.0010 % w/v )

= 30mL

vitamin D ) To fix 50mL 0.0008 % w/v sodium-p- aminosalicylate ;

C1V1= C2V2

( 50mL ) ( 0.0008 % w/v ) = ( X ) ( 0.0010 % w/v )

X= ( 50mL ) ( 0.0008 % w/v ) / ( 0.0010 % w/v )

= 40mL

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