Selective spectrophotometric determination of some primary amines using 2,4-dinitrofluorobenzene reagent

2.1 Apparatus

All absorption measurements were made on a Shimadzu UV-210A double-beam spectrophotometer supplied with a digital printer DP80Z and matched 1-cm optical silica cells. Heating of solutions was carried out on a water bath of frost instruments, LTD. The reading of pH was made on a PW 9420 pH meter supplied with an electrode type CE 10–12 pH. Weighing was carried out on a balance type of Mettler H 54 AR.

2.2 Reagents

Analytical grade chemicals, acetonitrile and distilled water were used. Standard solutions of primary aliphatic amines (250 μg/ml) in acetonitrile were prepared and aromatic amines (100 μg/ml) were dissolved in a small amount of absolute ethanol and diluted with distilled water. These amines are supplied by FLUKA & BDH companies. 0.01 M of DNFB (Sigma Co.) was prepared in acetonitrile for the determination of aliphatic amines or by dissolving in a small amount of absolute ethanol and diluting with distilled water for the determination of aromatic amines. 0.1% of sodium dodecyl sulphate (SDS) and Triton-x 100, 0.001 M of sodium hydroxide, 0.01 M of sodium bicarbonate and sodium acetate solutions were prepared in distilled water.

2.3 General procedures

3.1 Effect of solvents

In the preliminary investigation work, it was found that DNFB reagent reacted with primary, secondary, tertiary aliphatic and aromatic amines and amides in the alcoholic medium of methanol, acetonitrile, acetone, ethanol, in addition to water. It was observed that most of these amines reacted with DNFB to produce various colours except the acetonitrile when used as a solvent for aliphatic amines and DNFB with diluting the same solvent lead to the production of yellow coloured products with aliphatic amines (in the presence of a base in the case of 1,6-diaminohexane) and gave maximum absorption at wavelengths that ranged between 355 and 357 nm for given amines (Fig. 1 and Table 2 for 1,6-diaminohexane as example), whereas other amines were either unreacted or gave low response. On the other hand, it was found that DNFB reacted with primary aromatic amines and produce yellow coloured products when water is used as the solvent for both DNFB and aromatic amine in the presence of a base and diluting with water gave a maximum absorption at wavelengths that ranged between 355 and 377 nm for given amines (Fig. 2 and Table 2 for benzidine as example). Therefore; these systems of solvents are recommended for the determination of aliphatic and aromatic amines.

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Figure 1

Absorption spectra of 15 μg/5 ml 1,6-diaminohexane with DNFB against reagent blank (a) and blank against acetonitrile (b) plotted under the general procedure conditions.

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Figure 2

Absorption spectra of 400 μg/25 ml benzidine with DNFB against reagent blank (a) and blank against distilled water (b) plotted under the general procedure conditions.

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3.2 Effect of pH and buffer solutions

The effect of pH on the absorption of the products was studied using different pH values of HCl or NaOH that ranged from 2 to 12. As shown in Fig. 3, it was found that the products formed with maximum absorption when the final pH of the solutions was 8.40 for 1,6-diaminohexane and 6.02 for benzidine as examples from the studied amines after the addition of NaOH, and a decrease in absorbances was observed through the addition of HCl, which may be attributed to the liberation of hydrogen fluoride. Therefore, different buffers with the above optimum pH were prepared to examine the sensitivity. It was found that they have no effect on the colour intensity.

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Figure 3

Effect of pH on the absorbance of DNFB product with (a) 10 μg/ml benzidine, (b) 2 μg/ml 1,6-diaminohexane.

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3.3 Effect of base

Various bases, such as NaOH, KOH, Na₂CO₃, NaHCO₃, NH₄OH and CH₃CO₂Na were examined in order to obtain high sensitivity. It was found that these bases reduced the intensity of the aliphatic amine-DNFB products except of 1,6-diaminohexane which shows increasing colour intensity in the presence of NaOH. For the aromatic amines, it was found that NaHCO₃ gave high intensity with benzidine and p-phenylenediamine and CH₃CO₂Na for m-aminophenol. The optimum amounts of these bases are cited in Table 4.

3.4 Effect of reaction time and temperature

The reaction time was determined by following the colour development at room temperature and in thermostatically controlled water-bath at different temperatures. The absorbance was measured at 5 min intervals against the reagent blank treated similarly. It was observed that the absorbance reached a maximum after a time that ranged between 30 and 60 min at room temperature (cyclohexylamine at 40 °C) and remains constant between 30 and 60 min for aliphatic amines, and between 40 and 60 min at a temperature that ranged between 50 °C and 70 °C and remains constant between 45 and 80 min for aromatic amines (Table 4).

3.5 Effect of DNFB concentration

The effect of changing the DNFB concentration on the absorbance of a solution containing a fixed amount of the primary amine was studied. It is evident that the absorbance increases with increasing DNFB concentration and reached a maximum at optimum concentrations cited in Table 4.

3.6 Effect of surfactants

Effect of various surfactants including cetyltrimethylammonium bromide (CTAB), cetylperydinum chloride (CPC), sodium dodecyl sulphate (SDS), tween-80 and triton x-100 was tested. It was found that these surfactants caused a decrease in the absorbances of both aliphatic and aromatic amines except for benzidine-DNFB product which shows increasing intensity in the presence of 1.8 mL of tween-80 which is recommended in the procedure (Table 4).

3.7 Effect of order of addition

In order to obtain the high colour intensity the order of addition of reagents for aliphatic and aromatic amines should be carried out as shown in Table 3, otherwise a loss in colour intensity was observed.

However; the optimum reaction conditions for developing the colour intensity of the studied primary amine-DNFB products are summarized in Table 4.

3.8 Analytical parameters

Under the experimental conditions described in Table 4, standard calibration graphs for the studied primary aliphatic and aromatic amines with DNFB were constructed by plotting absorbance versus concentration (Fig. 4a and b). The correlation coefficients ranged from 0.9980 to 0.9999, indicating good linearity. Beer’s law is obeyed in the ranges as cited in Table 5, and the molar absorptivity values indicate the high sensitivity of the proposed method.

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Figure 4a

Calibration graphs for aliphatic amines.

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Figure 4b

Calibration graphs for aromatic amines.

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3.9 Precision and accuracy

Six replicate measurements are performed at three different concentrations within the Beer’s law limits of each amine. The relative standard deviation and recovery % results cited in Table 6 indicate the high precision and accuracy of the proposed method.

3.10 Interferences

The interference from various organic nitrogen compounds including secondary, tertiary amines and amides on the determination of 5 μg/ml of 1,5-diaminopentane (as example for primary aliphatic amines) and 12 μg/ml of m-aminophenol (as example for primary aromatic amines) was examined. It was found that these compounds did not affect the accuracy of the determination of the above compounds indicating the method is selective for the determination of compounds containing the primary amino group. An error of 5.0% in the absorbance readings was considered tolerable. The results are summarized in Table 7.

3.11 Stoichiometric relationship

The molar ratio of the product formed between the studied amines and the reagent used was investigated applying the molar ratio method (Hargis, 1988) using equimolar solutions of the amine and reagent. The results indicated that the product was formed in the ratio of 1:1 (Fig. 5, typical examples have been plotted). This finding supports that the interaction of the studied amino compounds and the reagent used takes place at only one site which was the more sterically free terminal basic primary amino group. However; these results are proved by using Job (Fig. 6) and slope ratio methods (Fig. 7) (Hargis, 1988).

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Figure 5

Mole ratio plots of DNFB products with samples of primary aliphatic and aromatic amines at their corresponding λ_max and under their optimum conditions.

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Figure 6

Job plots for the studied aliphatic and aromatic primary amines.

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Figure 7

Slope ratio plots for DNFB product with (a) 1,5-diaminopentane, (b) cyclohexylamine, (c) m-aminophenol and (d) p-phenylenediamine.

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The apparent stability constant was estimated by comparing the absorbance of a solution containing stoichiometric amounts of the amine and DNFB (As) to one containing an excessive amount of DNFB reagent (Am). The average conditional stability constants of the products are calculated by the following equation: $$\begin{array}{cc}\hfill & K_{\text{c}}=1-\alpha /{\alpha }^2\text{C}\hfill \\ \hfill & \alpha =A_{\text{m}}-A_{\text{s}}/A_{\text{m}}\hfill \end{array}$$ where K_c is the association constant (l mol⁻¹), α is the degree of dissociation and C the concentration of the product which is equal to the concentration of amine. The results shown in Table 8 indicate that the products are relatively stable.

3.12 Reaction mechanism

The reaction of DNFB with amines that own a free primary amine group results in the formation of coloured products in basic medium (Georgion et al., 1991; Correa et al., 2000). This reaction was first introduced by Sanger (Sanger, 1945) as means for the determination of the DNA sequence. Based on the mole ratio method and supported by Job and slope ratio methods, it was found that the primary amine interacted with the DNFB in the ratio of 1:1. This result indicates that the reaction between the amine and the reagent used takes place at only one site which was the more sterically free terminal amino group. The reaction is a typical nucleophilic substitution and proceeds through an intermediate product as cited in Scheme 1.

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Scheme 1

Probable reaction mechanism of primary amines with DNFB reagent.

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However; it was observed that blank gave a faint yellow colour with maximum absorption at 250 nm. This may be attributed to the formation of 2,4-dinitrophenolate ion through its basic hydrolysis (Correa et al., 2000) as cited in Scheme 2.

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Scheme 2

Basic hydrolysis of DNFB.

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