Spectrophotometric Determination of p-aminobenzoic acid via

Diazotization and Coupling reaction

 

Rana S. Al-Saffar¹, Safaa A. Zakaria¹, Nabeel S. Othman²*

¹Northern Technical University, Mosul Technical Institute, Mosul, Iraq.

²Department of Chemistry, College of Science, University of Mosul, Mosul, Iraq.

 

ABSTRACT:

A simple and accurate spectrophotometric method to assay p-aminobenzoic acid (PABA) has been suggested. The method based on the diazotization of PABA via reaction with nitrite ions which liberated in the medium through the reaction of sodium nitrite with hydrochloric acid, then coupling the dizotised-PABA(D-PABA)with 1-naphthylamine -7-sulphonic acid (1-NASA) to form an intense colored pink azo dye ,it is water-soluble , very stable and has a maximum absorbance at 525 nm. The absorbance was increase with the increasing of PABA amounts in the solution, and the linearity from 2.5 to70 µg in 10 ml (0.25to7 µg.ml^-1).The molar absorptivity and Sandell ҆s sensitivity values have been calculated they equal to 3.6057x10⁴ L.mol^-1 cm ^-1 and 0.0037993 µg. cm^-2 respectively. The two values above demonstrated a good sensitivity of the suggested method. The method has been used in estimated PABA resulted from analysis of folic acid in tablet.

 

 

INTRODUCTION:

4-Aminobenzoic acid or also p-aminobenzoic acid, the simple symbol used by authors is PABA. PABA is an crystalline substance with white to gray color, it can be soluble in water with slight manner , in alcoholic medium it is freely soluble, highly soluble in solution of alkaline medium. It has the molecular formula C₇H₇NO₂ and the following structure in Scheme 1 ^1,2.

 

M.wt.=137.1 g/mol.

Scheme 1:The structure of PABA.

 

Common medical uses of PABA, especially the salt potassium acid in fibrous skin infections medications, as well as to determine the levels of urinary sodium, potassium or nitrogen, and the most important medical use in preparations the protect from sunlight^3,4. PABA is involved in preparing a number of antibiotics^5,6.

 

The most important industrial uses of PABA is in the study of air pollution due to pesticides used against pests, their use nowadays is very limited ⁷. Also used in preparing different azo dyes after diazotisation and coupling with another agent. Through the investigation in the literature about the techniques or analytical methods used in estimating PABA, these methods, include the using of the hyphenated technique LC- MS/MS ^8,9 and also HPLC ^10,11. The spectrophotometric methods remain desirable for many researchers because the devices are available, price of the device is cheap compared to other techniques and does not require continuous maintenance ,also the availability of reagents to estimate the various pharmaceutical compounds^12-15, it is used in the estimation of more than one component in the same pharmaceutical preparation^16,17, sensitivity to the method can also be developed using the derivative spectroscopy^18,19. The important characterize of PABA is that, the contains of amino group as a function group, which can enter a number of reactions, including: diazotisation and coupling with coupling agents such as phloroglucinol ²⁰ and 2,4,6-tri-hydroxy benzoic acid ²¹ ,oxidative coupling reactions promethazine in presence of hypochlorite ²², xylenol using sodium periodate as oxidant²³,charge transfer complex using 2, 3 dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)²⁴, indirect methods such as: reaction of PABA with iron(III) in acidic medium, then reaction of resulting iron(II) with 1,10-phenathroline²⁵ and the reaction of PABA with hypochlorite in acidic medium and the subsequent measurement of the residual chlorine by using the well-known reaction with o-toluidine²⁶. The aim of the current research is to suggest a simple and accurate spectral method for estimating PABA and researching the possibility of estimating PABA in its pharmaceutical preparation and indirect estimating of folic acid.

 

EXPERIMENTAL:

Apparatus:

Spectral measurements were made by using the UV-Vis Hach´s DR6000 spectrophotometer with two quartz cells(1 cm optical path). HR – 200AND sensitive scale balance to perform the weighing operation.

 

Material and solutions:

1-Naphthylamine-7-sulphonic acid (1-NASA) solution ,0.333%:

 0.5 g of the 1-NASA reagent was dissolve in 120 ml of distilled water with heating to a boil, then filter, cool and add 30 ml of glacial acetic acid. The reagent prepared daily and kept in a dark bottle ²⁷.

 

Other solution:

50 µg.ml^-1 PABA,0.3 % sodium nitrite and 0.5% sulphamic acid were prepared by dissolving 0.0050, 0.3 and 0.5 g in 100 ml distilled water in a calibrated flasks respectively. Also 1M hydrochloric acid by diluting the concentrated acid with distilled water.

 

Pharmaceutical formulation:

New care capsules solution 100 µg. ml^-1:

The contents of 5 capsules were weighted and mixed well (each capsule contains 20 mg PABA), then the weight equivalent to 0.0100g as pure PABA is dissolved in small volume (2 ml) ethanol and 40 ml distilled water was added ,then heating until all sample dissolved, and the volume is completed to 100 ml in a volumetric flask to prepare a 100 ppm PABA solution.

 

Folic acid solution:

Ten tablet of folic acid each one contain 5 mg are crushed, then the powder is dissolved in 50 ml of dilute NaOH solution (0.1 standard) and withdraw 1 ml of the above solution and add a mixture consisted from 75 ml water + 18 ml dilute hydrochloric acid (0.1 N) +1 ml of gelatin solution and complete the volume to 100 ml. Withdraw 75 ml of the final solution to a conical flask and add 0.5 g of zinc powder and leave it for 15 minutes with shaking then filter the solution and neglect the first 10 ml of the filtrate. The rest of the filtrate is collected and kept in a dark bottle ²⁸, the final solution concentration is 3.1 µg PABA .ml^-1 .

 

Standard and calibration curve:

The standard curve of the suggested method is prepared after the optimum conditions have been established in practice to estimate the PABA as follows: Increased volumes of PABA solution are added so that it contains 2.5 to70 µg to a series of volumetric flasks of 10 ml, then add 1 ml of 1M hydrochloric acid solution and 0.3 ml of 0.3% sodium nitrite solution, and leave the solutions for 3 minutes with shake from time to time, after that add 1 ml of 0.5% sulfamic acid solution and leave the solutions for 4 minutes with stirring to get rid of excess nitrite ions , then add 1.5 ml of 0.33% reagent solution 1-NASA. It is followed by the addition of 3 ml of ethanol, then the volumes are complemented with distilled water up to the marks limit. The absorbance of the solutions is measured against the blank solution at the wavelength of 525 nm, and the standard curve shown in Figure (1) is in agreement with the Beer's law in the concentration range 2.5 to 70 µg / 10 ml. 0. 25-7.0 µg. ml^-1), and there is a negative deviation at concentrations above 50µg/10^-1 mL, the molar absorptivity value of the resulting dye is 3.6057x10⁴ L.mol^-1 cm ^-1, while the value of Sandell's indication is 0.0037993 µg. cm-².

 

Figure 1.The calibration cure to PABA estimation.

 

The value of the detection limit (LOD) and the value of the quantitative limit (LOQ) were calculated²⁹ by measuring the absorption of 10 blank solutions against distilled water at the wavelength of 525 nm and according to the working method, then the mean and standard deviation (ɕB) are found. Then the below relationships have been used in determination of LOD and LOQ

LOD = 3ɕB /S = 0.0480 µg. 10 ml^-1

LOQ = 10ɕB /S = 0.1603 µg. 10 ml^-1

 

RESULTS AND DISCUSSION:

The effect of different conditions affecting the absorbance of the resulting azo dye was studied by using 0.5 ml of PABA solution (50 µg. ml-1) in a final volume of 10 ml

 

Principle of the procedure:

1-Forming of diazotised-PABA(D-PABA) : via reaction of PABA with HNO₂ which is formed in solution by adding NaNO₂ and the acid.

 

2-Removing the excess of NO₂ ^- before adding the coupling agent 1-NASA the an reacted HNO₂ must be removed by adding sulphamic acid.

 

HNO₂ + H₂NSO₃H    N₂↑ + H₂SO₄ + H₂O

3-Forming azo dye and appearing the color: the pink azo dye formed immediately after adding 1-NASA reagent.

 

Selection of the coupling agent:

A number of organic compounds that can be used as coupling agents with D-PABA have been tested, the results are shown in Table 1 indicated that 1-NASA gave best results (high absorbance and high color contrast), therefore it consider as the optimum coupling reagent.

 

Table 1:The selection of coupling agent.

 

The type and quantity of the acid used in the diazotisation:

The effect of acid type's has been studied and the results in Table2 shows that HCl gave high absorbance and it recommended to added 1 ml to give sufficient acidy to produce the azo dye and according to the high absorbance and excellent R² (Table 3),so that it was the optimum acid.

 

Table 2:The optimum acid used in diazotisation.

 

Table 3:The optimum amount of HCl.

1M HCl, Solution added (ml)	Absorbance /µg PABA in 10 ml				R2
	10	20	30	50
0.2	0.165	0.381	0.512	0.786	0.970978
0.5	0.256	0.522	0.745	0.943	0.969874
1	0.398	0.634	0.912	1.196	0.987593
1.2	0.385	0.587	0.897	1.023	0.948979
1.5	0.297	0.466	0.811	0.978	0.960685

 

The amount of sodium nitrite and time of reaction:

The presence of the nitrite ion in the solution which is prepared from the addition of sodium nitrite and hydrochloric acid is essential for the formation of the diazonium salt , also a another point is the reaction time. Therefore different amounts of sodium nitrite (were added and left for different time periods from 1 to 5 minutes before adding sulphamic acid. The results illustrated in Table 4 show that the 0.3 ml of sodium nitrite with a reaction time of 3 minutes gave the best results of high absorbance and color stability, therefore 0.3 ml and 3 minutes as standing time are recommended in subsequent experiments.

 

Table 4 :The effect of NaNO₂ amount and time on absorbance.

Amount of NaNO2, (0.3%) soln.	Absorbance / Standing time( min.)
	Immediately*	1	3	5
0.1	0.338	0.390	0.416	0.378
0.2	0.351	0.391	0.486	0.422
0.3	0.395	0.432	0.624	0.472
0.4	0.345	0.395	0.583	0.432

*There is no standing time immediately adding sulphamic acid.

 

The effect of sulphamic acid and time on absorbance:

The excess of nitrite ions is not desirable in diazotisation and coupling reactions for its entry into unwanted side reactions and therefore was eliminated by adding sulfamic acid. The results in Table 5 show that adding 1ml of sulfamic acid with a 4 minutes as a reaction time is sufficient to break down the nitrite ions, and nitrogen gas bubbles can be observed in the reaction medium the volumetric flasks treated with sulfamic acid gave the highest absorption of the azo dye and the blank gave the lowest absorption at the wavelength of determination.

 

The 5:The effect of sulphamic acid and time in destroying the excess of nitrite ions.

*There is no standing time after adding acid, immediately adding 1-NASA solution sulphamic.

 

The optimum amount of coupling reagent:

The last step is to produce the pink azo dye by adding the coupling agent 1-NASA Table 6 contains the results of adding different quantities of the 1-NASA solution(0.4%) to the range of concentration from 10 to 50 µg PABA/10 ml and we conclude that 1.5 ml is sufficient to give the highest absorbance of the range of the PABA concentrations used in this experiment and the highest value of the determination coefficient(R²) .

 

Table 6: The optimum amount of 1- NASA reagent.

1-NASA (0.4% ) solution	Absorbance of different amounts of PABA (µg) in10 ml				R2
	10	20	40	50
0.5	0.188	0.284	0.486	0.874	0.945698
1	0.286	0.476	0.705	0.983	0.985791
1.2	0.375	0.567	0.811	1.102	0.986139
1.5	0.474	0.684	0.976	1.211	0.99602
2	0.382	0.561	0.788	1.156	0.970899
3	0.275	0.478	0.622	1.035	0.944794

 

Reaction medium:

Alkaline solution(1M NaOH) was added in different amounts , and it was found that a decrease in the absorbance and turbidity occurred by adding large volumes of NaOH. Also, volumes of hydrochloric acid (1M HCl) were added, the resulting azo dye has been destroyed and there is no color contrast, a recommendation to keep the additions of all reagents(the components of reaction mentioned before, which gave solution at pH=4.1) without adding a base or acid.

 

Stability of the pink azo dye:

A number of attempts were made to increase the stability of the color, including the use of a buffer solution, and the best results were obtained by adding of 3 ml ethanol to the reaction before dilution with distilled water (Table 7).

 

Table 7. The stability of the azo dye in presence of 3 ml ethanol.

Time (min .)	Absorbance/ µg of PABA/ 10 ml
	10 µg	30 µg	50 µg
After dilution	0.401	0.897	1.282
5	0.422	0.903	1.291
10	0.421	0.903	1.291
15	0.420	0.903	1.291
20	0.421	0.903	1.291
25	0.421	0.903	1.291
30	0.421	0.903	1.289
35	0.421	0.902	1.289
40	0.419	0.902	1.289
45	0.419	0. 901	1.289
50	0.419	0.901	1.288
55	0.419	0.900	1.288
60 (1hr)	0.418	0.900	1.288

The optimum conditions to formed an stable pink azo dye are illustrated in Table8.

 

Table 8: The optimum conditions of suggested method

The variable	The optimum
Acid used ,volume	HCl,1.0 ml
Volume of NaNO2,standing time	0.3 ml,3 minutes
Volume of sulphamic acid, standing time	1.0 ml, 4 minutes
Volume of reagent (1-NASA)	1.5 ml
Stability ,volume of ethanol	60 minutes,3.0 ml

 

Absorption spectrum:

When PABA reacted with 1-NASA reagent according to what is mentioned in the procedure of the suggested method, the absorption spectrum has high absorbance at maximum wavelength 525 nm and the blank solution gave little absorbance at the same wavelength (Fig. 2).

 

Fig. 2:Absorption spectrum of 30 μg/10 ml PABA against blank (A), the azo dye measured against D.W. (B) and blank measured against D.W.(C).

 

The nature of the pink azo dye:

For the purpose of knowing the correlation ratio of diazonium salt corresponding to PABA with a reagent 1-NASA. The continuous variation method (Job's method) and mole ratio methods ³⁰ have been applied according to the following working steps: A number of solutions containing different volumes (0.1-0.5) ml of 3.647 x 10^-4 M PABA and (0.5-0.1) ml of the 3.647 x10^-4 M reagent 1-NASA were prepared, with the addition of the other solutions under optimum conditions, then the absorbance of these solutions is measured against their blank solutions at the wavelength of 525 nm. Fig. (3 and 4) shows the ratio of the association of the PABA with the 1-NASA reagent.

 

Fig. 3:The continuous variation curve.

 

Fig.4: The mole ratio method curve.

 

The scheme2 below included the coupling of D-PABA with 1-NASA reagent on para position to amino group on the reagent ³¹ ,the suggested chemical structure of pink azo dye is agrees with the results above from Fig. 3 and 4.

 

 

Scheme 2: The pink azo dye structure.

 

Application of the methods:

The method has been applied to estimation PABA in capsule(Newcare,20 mg PABA/ each one) and PABA resulting from analysis of folic acid. Accurate and precise results were obtained and the error in RE% or RSD% were within the permissible limit(Table 9 ).

 

Table 9:The results of application part.

Formulation	PABA taken , µg	Recovery* %	RE%	RSD %
Newcare 20 mg PABA / capsule Asia Pharma, Ind.-Syria	30	97.8	-2.2	± 0.5
	50	98.1	-1.9	±1.2
Folic acid SAFA CO. Diala-Iraq	0.31	98.2	-1.8	± 0.2
	0.62	97.7	-2.3	± 0.4

*Average of five determinations.

 

The results in Fig.5 and from the equation of linearity , folic acid as contented in the tablet can be determined by using the below relationships :

 

10 tablet contained 50 mg folic acid dissolved in 50 ml

50 mg/50 ml =1mg /ml = 1000 µg/ml =1000 ppm

1ml diluted to 100 ml

 

The final concentration of 1ml folic acid = 10 µg folic acid /ml = 10 ppm folic acid

From the linearity relationships (y =0.079X + 0.2421) in Fig4:

 

When y = 0 ….X = 0.2421 / 0.079 = 3.064 µg PABA in 1ml folic acid

 

µg folic acid/ ml = µg PABA X [M .wt. of folic acid / M wt. of PABA]

 

µg folic acid/ ml = 3.064 x[ 441.4 / 137.1] = 9.864 µg folic acid/ ml (ppm)

 

The recovery% = [µg folic acid/ ml (Practically)] / [µg folic acid/ ml (Theoretically, calculated above )] = [ 9.864/10] x 100 = 98.64 %

 

 

Fig.5:The standard addition method in determination PABA resulted from folic acid tablet.

 

Comparison of the method:

Some of the analytical parameters were selected and compared with the same for methods in literature. The results are illustrated in Table 10.

 

Table 10: Comparison of main analytical parameters.

Parameter	Present method	Method 1(24)	Method 2 (25)
Reaction types	Diazotisation and coupling	Charge transfer complex	Oxidation-reduction
The main reagent	1-naphthylamine -7-sulphonic acid	DDQ	1,10-phenathroline
Maximum wavelength, nm	525	474	510
Linearity (μg/ml )	0.25-7	5-90	0.2- 3.2
The sensitivity, ε (L.mol-1cm-1)	3.647924x104	1.06 x103	4.93×104
Stability of product(minutes)	60	……..	45
RSD%	Not more than 1.2	Not more than 0.17	Not more than 0.507
R2	0.997	0.994	0.9923
Temperature ,°C	Room temp.	60	80 for 40 min.
Applications	PABA in capsule and PABA result from folic acid analysis	PABA in tablets	PABA in capsule

 

Comparative results in Table 10 indicated that the present method was characterized by good analytical specifications, as it does not need heating ,the estimation is carried out at room temperature ,a good value of molar absorptivity value , high stability of the azo dye and it has a wider applications.

 

CONCLUSION:

The proposed method was simple, accurate, and precise , in addition to good sensitivity through molar absorption value and the possibility of determination of PABA in capsule and PABA result from folic acid tablet. The future work is the indirect estimation of folic acid in its pharmaceutical preparations.

 

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Accepted on 05.01.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(10):5313-5318.