Quantitative Analysis Of The Sodium P Aminosalicylate Biology Essay

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Sodium p-Aminosalicylate is an analogue of para-aminobenzoic acid that inhibits or prevents the folic acid synthesis in bacteria known as Mycobacterium tuberculosis and it is used as an anti-tuberculosis agent by preventing the multiplication of the tubercle bacillus. [3]It is also used to treat Crohn's disease nowadays. [6]Quantitative analysis of sodium p-Aminosalicylate using the ultraviolet or visible spectroscopy determines the absorbance 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 using calibration graph by measuring the absorbance of the unknown concentration of solution for a range of standards and plotting a Beer-Lambert graph. [7]The second method is known as absolute method where the calculation is carried out by measuring the absorbance of the unknown with known specific absorbance. [7]The third method is comparative method where calculations can be carried out by measuring absorbance of a single standard and absorbance of the unknown. [7]

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A λmax values was selected from the standard graph of sodium p-aminosalicylate 0.0010%w/v, the λmax was used for determine the "unknown 1 and 2" and the specific absorbance was determined using the slope of the gradient. The 0.1M sodium hydroxide was used as blank agent. Dilution was done on "unknown 1" due to the high absorbance value. For the comparative method, the λmax used to determine "unknown 2" was obtained from the calibration graph provided. The concentration of "unknown 2" was determined by using absolute method where calculation was carried out after the specific absorbance was obtained from the standard graph and by comparative method where calculation was carried after the absorbance of "unknown 2" had measured.

From the result, the concentration of "unknown 1" was 0.0026%w/v by using the standard graph and "unknown 2" was 0.0006%w/v for both comparative and absolute method. The results obtained in this analysis were good and precise. This concluded the ultraviolet molecular spectroscopy has broad absorption bands and used mainly for quantitative analysis purposes in pharmaceutical.

1. Introduction:

Sodium p-Aminosalicylate also known as sodium 4-amino-2-hydroxybenzoate or 4-Amino-2-hydroxybenzoic acid is a white to pale yellow crystalline powder. [1] The chemical formula is C7H7NO3Na [2] It is practically odorless and freely soluble in water, sparingly soluble in alcohol and practically insoluble in ether. It dissolves in water give a clear solution which is colourless. [1] The pH of the solution of Sodium p-Aminosalicylate in the range between 7.0 to 7.5. [1] The sodium salt of salicylate is prepared from sodium phenolate under heating and pressure with gas carbon dioxide. [3] It contains a hydroxyl and a carboxyl group and these groups are reacted with an acid or an alcohol. [3]Ester produced as end product in the reaction of carboxyl group with alcohols. [3]

Para-Aminosalicylic acid is an analogue of para-aminobenzoic acid that inhibits or prevents the folic acid synthesis in Mycobacterium tuberculosis and it is known as bacteriostatic agents where it inhibits growth of the tubercle bacillus. [3] Para-Aminosalicylic acid and its sodium salt form known as sodium p-Aminosalicylate are bacteriostatic agent where it acts against inhibit the growth of mycobacteria and it used in the treatment of tuberculosis when administered orally. [3] Aminosalicylic acids are active ingredients in pharmaceutical sector including it acts as anti-infectives agent, where it acts against colds, flu and some kinds of viral infections. [3] 5-aminosalicylic acid is an active metabolite of sulfasalazine in compound known as mesalamine, it used to treat inflammation disease occurred in the rectum and lower colon, mild to moderate ulcerative colitis, and proctitis. [4]

Quantitative analysis using the ultraviolet or visible spectroscopy is simple and cost- effectiveness. [5] It produces rapid result hence sensitive to absorbance which approximately 1- 10ppm. [5] It is almost universal and prone to interferences in degradation products, metabolites, excipients and endogenous materials. The measurement of light absorption by molecules in a solution is governed by the Beer-Lambert law, logI0/It= A= εbc. The Pharmacopoeial methods rely on simple analysis by ultraviolet spectrophotometry to determine active ingredients in formulations. All of the pharmaceutical products, concentrations and amounts are expressed in grams (g) or milligrams (mg) rather than moles and thus the Beer-Lambert law is expressed as A = A (1%, 1cm).c.l. The path-length is usually 1cm because the measurement is made in a 1cm cell. [5]

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The wavelength range 200-700nm is allowing the radiation passed through a solution of a compound. The absorption of ultraviolet or visible radiation occurs through the excitation of electrons within the molecular structure to a higher energy state. [5] The short wavelength <150nm can cause damaging to living organisms because the strongest bond in organic molecules break. [5] Longer wavelength >200nm is excited the weaker bonds molecules usually used in analysis method. [5] The maximum wavelength or abbreviated λmax gives maximum sensitivity and it has small change in absorbance with change of wavelength where small errors in λ setting does not affect accuracy of the absorbance reading. Most of the compounds have more than one λmax value and the highest is selected to minimize the interferences. [5]

The concentration of sodium p-Aminosalicylate can be determined by three methods. [7] The first method is using calibration graph. The calculation can be carried out by measuring the absorbance for a range of standards and plotting a Beer-Lambert graph. [7]The concentration of unknown solution can be obtained from the graph after measured of the absorbance of the unknown solution. This is good method if the unknown concentration may vary considerably and linearity is established. [7] The slope gives specific absorbance. The second method is known as absolute method where the calculations is carried out by measuring the absorbance if the unknown under defined conditions and using a known specific absorbance. It is British Pharmacopeia preferred method and equation used is concentration (%w/v) = A/A (1%, 1cm) in 1cm cell. [7]The third method is comparative method where calculations can be carried out by measuring absorbance of a single standard and absorbance of the unknown. It is fairly rapid but requires a pure standard. [7]The equation used is C2/0.001= A2/Astd. It is best if the standard 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 sodium hydroxide solution was used as blank solution and as dilution agent for the sodium-p-aminosalicylate. Stock solution was 0.0010%w/v sodium-p-aminosalicylate. The apparatus used were 10mL, 20mL, 50mL pipettes, beakers and 50mL volumetric flasks; cuvette was used to fill the solutions and placed into the spectrophotometer. [6]

2.2. Methods:

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

The spectrum of a 1-cm layer of a 0.0010%w/v solution of sodium aminosalicylate in 0.1M sodium hydroxide solution over the wavelength range 235 to 325nm was scanned and examined. The baseline was obtained before recording the spectrum using 0.1M sodium hydroxide solution in both cells. The wavelengths or λmax and the absorbance values at these wavelengths of the two maxima were measured from the spectrum. The approximate specific absorbance was calculated at each of these maxima using the Beer-Lambert law equation;

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

Where, A= absorbance

A (1%, 1cm) = specific absorbance of a 1cm layer of a 1%w/v solution

c= concentration (%w/v)

l= path length (cm)

Beer- Lambert law, specific absorbance and molar absorptivity

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 sodium hydroxide were prepared from the 0.0010%w/v stock solution. A λmax values was selected and the following procedures were carried out using a single beam spectrophotometer.

The wavelength was set to the selected value, the 0.1M sodium hydroxide as the solvent was placed in the both cells and the absorbance reading was set to zero on the instrument. The 0.0010%w/v stock solution was used, the absorbance a few nm each side of the selected wavelength was checked. The absorbance to zero was set at each wavelength using the blank. A λmax values was selected from the table of absorbance values and the spectrophotometer was set to this wavelength. The wavelength in the spectrophotometer remained set until the section completed. The absorbances of a 1-cm layer of each of the five solutions of sodium 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 absorbance against concentration was plotted and the specific absorbance (A (1%, 1cm)) was determined using the gradient of the graph. The British Pharmacopoeia (BP) defined the molar absorptivity (ε) as the absorbance of a 1-cm layer of a 1M solution. The molar absorptivity of sodium aminosalicylate in 0.1M sodium hydroxide was calculated at the selected maximum.

Determination of concentrations

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Ultraviolet- visible spectroscopy was frequently used as a method of qualitative analysis for the BP assays and limit tests. The concentration of two sodium-p-aminosalicylate solutions, "Unknown 1" and "Unknown 2" with unknown concentration were determined using following method.

Calibration graph was used to determine the absorbance of a 1-cm layer of "Unknown 1". The "Unknown 1" solution was diluted so that its absorbance was in the mid range of the graph of absorbance against concentration plotted. The graph of absorbance against concentration was used to determine the concentration of the diluted solution and the concentration of "Unknown 1" was calculated.

Absolute method was used to determine the absorbance of a 1-cm layer of "Unknown 2". The absorbance of "Unknown 2" was recorded. The concentration of "Unknown 2" was calculated using the specific absorbance (A (1%, 1cm)) value determined previously.

Comparative method was used to compare the absorbance of a 1-cm layer of the 0.0010%w/v solution and a 1-cm layer of "Unknown 2". The wavelength was set to the second of the λmax value from the supplied spectrum. The absorbance of the 1-cm layer of 0.0010%w/v solution and a 1-cm layer of the "Unknown 2" were recorded. The concentration of the "Unknown 2", C2, was calculated using the relationship.

C2/0.001 = A2/Astd

3. Results:

The measurement of the wavelength or λmax and the absorbance values of the two maxima in the supplied spectrum were shown below (table 1); the approximate specific absorbance at each maxima wavelength was shown in table 1.

Maxima on the supplied spectrum

Wavelength (nm)

Absorbance value (A)

Specific absorbance (A(1%,1cm))

First λmax

264

0.630

630

Second λmax

300

0.425

425

Table 1: The wavelength number, absorbance value and specific absorbance of the two maxima on the supplied spectrum.

The volume required for the dilutions of the sodium-p- aminosalicylate according to the concentration stated from 0.0010%w/v stock solution by using 0.1M sodium hydroxide was shown in table 2. Calculation of the volume required shown in part 1 in supplementary question. The formulae for the dilution was C1V1= C2V2. All the absorbance of the 0.0010%w/v sodium-p- aminosalicylate from wavelength range 260nm to 265nm was shown in table 3. The maximum wavelength used for determination of the dilutions of the sodium-p- aminosalicylate was 265nm due to the highest absorbance in 0.0010%w/v sodium-p- aminosalicylate. All the absorbance of the dilutions sodium-p- aminosalicylate was determined by the number of wavelength 265nm. The calibration graph of concentration (%w/v) against absorbance (λ) was plotted according to the tablet 4. From the calibration graph 1, there was a linear plot obtained. The specific absorbance A (1%, 1cm) was obtained through the formulae y = mx +c, where y =absorbance, m =specific absorbance, x =concentration and c =intercept. The gradient of the plot where represent the specific absorbance was 595nm. (Refer to the calibration graph 1 next page)

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

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

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 preparing the difference concentration of 50mL sodium-p- aminosalicylate (%w/v) diluted with 0.1M NaOH solution.

Wavelength

Absorbance of 0.0010%w/v sodium-p- aminosalicylate

1streading

Absorbance 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 absorbance of 0.0010%w/v sodium-p- aminosalicylate with 0.1M NaOH solution as blank.

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

Absorbance

1streading

Absorbance

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 absorbance of difference concentration sodium-p- aminosalicylate with 0.1M NaOH solution as blank.

The unknown concentrations of the sodium-p- aminosalicylate (%w/v) were determined and the absorbance for "Unknown 1" was >1 and hence it is too high to determine the concentration according to calibration graph 1. (Refer table 5) The highest absorbance obtained from the graph 1 was approximately 0.7 and the absorbance of the unknown concentration sodium-p- aminosalicylate was 1.189. Dilution of the "unknown 1" sodium-p- aminosalicylate was done to achieve a new absorbance value which in the range of the calibration graph 1 plotted. (Refer table 5) The total volume of dilution unknown concentration sodium-p- aminosalicylate by using 0.1M sodium hydroxide was 20mL and 5mL of "unknown 1" concentration sodium-p- aminosalicylate was calculated. (Refer part A). The absorbances of the "unknown 1" concentration of sodium-p- aminosalicylate was in the mid-range of the calibration graph 1(Refer table 6)

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

Absorbance

1streading

Absorbance

2ndreading

Mean

Unknown 1

1.189

1.195

1.192

Unknown 2

0.336

0.339

0.338

Table 5: The absorbance of unknown concentration of sodium-p- aminosalicylate with 0.1M NaOH solution as blank.

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

Absorbance

1streading

Absorbance

2ndreading

Mean

Unknown 1 (diluted)

0.366

0.370

0.368

Unknown 2 (undiluted)

0.336

0.339

0.338

Table 6: The absorbance of "unknown 1" sodium-p- aminosalicylate after dilution and "unknown 2" with 0.1M NaOH solution as blank.

The wavelength used was 300nm which was the second maxima of λmax measured in the supplied spectrum for the absorbance of "unknown 2" sodium-p- aminosalicylate. (Refer to table 7)

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

Absorbance

1streading

Absorbance

2ndreading

Mean

0.0010

0.643

0.641

0.642

Unknown 2

0.336

0.339

0.338

Table 7: The absorbance of "unknown 2" and 0.0010%w/v sodium-p- aminosalicylate with 0.1M NaOH solution as blank by using wavelength 300nm.

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

PART A:

From the calibration graph (graph 1), the absorbance value of "Unknown 1" was 0.0006%w/v. The total volume after dilution was 20mL and the volume of the "Unknown 1" sodium-p- aminosalicylate was 5mL. By using 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 using absolute method where Beer- Lambert law was applied, A = A (1%, 1cm).c.l; A= 0.338, absorbance of "Unknown 2"; A (1%, 1cm) = specific absorbance= 595 nm; c =concentration of "Unknown 2" and l= 1cm.

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

0.338= 595 x C x 1

C= 0.338/300

= 0.0006%w/v

PART C: Another method to determine the concentration of "unknown 2" was using comparative method where a relationship was applied, C2/0.001= A2/Astd. C2= concentration of "unknown 2"' A2= absorbance value for "unknown 2" and Astd= absorbance 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 absorptivity of sodium aminosalicylate in 0.1M NaOH at the selected maximum:

Mr of sodium aminosalicylate = 176.074

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

A= εbc

ε= A/ b /c

= 0.110/ 1.136 x 10-6mol/L

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

4. Discussion:

This experiment reveals the qualitative analysis to determine 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 directly obtained from the Beer-Lambert graph plotted after measuring of the absorbance of a range of standards. The concentration obtained from the standard graph was not the exact concentration of "unknown 1" because there was a dilution done for the reason of the absorbance values of the "unknown 1" was too high. The "unknown 1" was diluted to 20mL by using sodium hydroxide solution. The exactly concentration of "unknown 1" was 0.0026%w/v. This method requires a pure standard and very time 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 measuring if the absorbance of a range of standards sodium-p- aminosalicylate 0.0010%w/v gives a slope of 595 nm as specific absorbance and the equation used is concentration (%w/v) = A/A (1%, 1cm) in 1cm cell. The calculation 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 preferable 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 preferable method for the United States Pharmacopeia. [7] It is useful to determine the sample if it is reacted or extracted before the absorbance is measured. [7]The calculation was carried out by measuring absorbance of a standard absorbance and absorbance of the "unknown 2". Then the absorbance 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 steps. The experiment was successfully done. The ultraviolet spectrophotometer was accurately calibrated in the experiment. The standard Beer-Lambert graph, absorbance against concentration was plotted precisely. The good skill to operate the spectrophotometer was applied. The type of cuvette used to fill the solution for the absorbance measurement was appropriate for these wavelengths. The selection on the standard wavelength selected was close to the maximum wavelength, λmax on the calibration graph provided. The slope or gradient on the standard graph was accurately calculated. The same cuvette was used for the measurement of both standard sodium-p- aminosalicylate 0.0010%w/v and "unknown 1 and 2" for sodium-p- aminosalicylate 0.0010%w/v to achieve maximum quantitative accuracy. All the solutions used in the experiment were free from any particles, this was crucial to avoid scattering where apparent increase of absorbance in the solution and produced good and precise result.

5. Conclusions:

This analysis was concluded that the ultraviolet molecular spectroscopy has broad absorption bands and used mainly for quantitative analysis purposes. Simple analysis by ultraviolet spectrophotometry is used to determine active ingredients in pharmaceutical formulations Pharmacopoeial methods for identification checks. The standard absorbance, A (1%, 1cm) is used for the analysis and this heavily depends on the ultraviolet spectrophotometer being accurately calibrated. The result obtained in this analysis was good and precise.

6. Supplementary questions:

PART 1

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

Calculations:

Formulae used: C1V1= C2V2

Concentration of stock solution: 0.0010% w/v

a) To prepared 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 prepared 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

c) To prepared 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

d) To prepared 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