Selective Removal of Nitrophenols from Aqueous Solutions Using Acrylic Acid–Acrylamide Molecularly Imprinted Polymers

Nitrophenol isomers are hazardous aromatic compounds that their toxicity, solubility, acidity, and environmental behavior are largely dictated by the different position of the nitro and hydroxyl group [1]. Of which, PNP is the most environmentally persistent and toxic, commonly entering aquatic systems through the breakdown of pesticides [2], industrial production [3], fossil fuel combustion [4], and biomass burning [5]. Nitrophenols have been detected in surface waters at levels ranged from nanograms to a few micrograms per liter, mostly near industrial and agricultural regions [3]. Toxicity data indicate that PNP is especially harmful to aquatic life [6], and human. These compounds classified by GHS as toxic by inhalation, ingestion, or dermal contact and capable of causing serious eye damage, skin irritation, and organ toxicity with repeated exposure [3]. Moreover, nitrophenols can impair cellular functions, induce oxidative stress, and may act as endocrine disruptors, raising environmental concerns due to their ability to persist and accumulate in ecosystems [7].

 Pollutant removal from aqueous solutions involves techniques such as adsorption, membrane filtration, advanced oxidation processes, biological treatment, and electrochemical methods, each suited to specific contaminants like heavy metals, organics, or pathogens [8, 9]. While methods like adsorption and advanced oxidation processes offer high efficiency, challenges such as cost, and limited selectivity affect their large-scale applicability [10]. Molecular imprinting is a technique used to create synthetic polymers with highly specific recognition sites for a target molecule, known as the template [11]. During the process, functional monomers form a complex with the template molecule, and this complex is polymerized in the presence of a cross-linker to form a rigid matrix. After polymerization, the template is removed, leaving behind cavities that are complementary in size, shape, and functional groups to the original molecule [11]. These cavities enable the polymer, known as a molecularly imprinted polymer (MIP), to selectively rebind the target molecule with high affinity. Compared to conventional adsorbents, MIPs offer significant advantages in terms of selectivity and efficiency [12]. While conventional materials rely on non-specific interactions and typically exhibit broad-range adsorption, MIPs provide highly specific recognition due to their tailor-made binding sites [13]. These characteristics make MIPs particularly useful in applications such as sensors, drug delivery, solid-phase extraction, chromatography, and environmental monitoring, where selective and efficient molecule targeting is essential.

Several studies have demonstrated the effectiveness of molecularly imprinted polymers (MIPs) for the selective removal of nitrophenol compounds from aqueous solutions. A cellulose-based MIP grafted with DMAEMA showed high affinity for PNP, achieving an adsorption capacity of 26.9 mg/g and strong selectivity over structurally similar compounds like MNP and catechol [14]. Similarly, a MIP synthesized with acrylamide for 2,4-dinitrophenol exhibited favorable adsorption isotherms and high selectivity in the presence of phenol derivatives [15]. Another study has investigated enhancing surface area and accessibility, silica-coated MIP nanoparticles for PNP extraction from pharmaceutical samples, displaying improved binding and rapid adsorption [16]. Meanwhile, magnetic MIPs based on Fe₃O₄ functionalized nanoparticles achieved a high adsorption capacity (129 mg/g) within 90 minutes PNP and allowed for magnetic recovery, which simplifies separation processes [17]. Additionally, early work using methacrylamidoantipyrine (MAAP) as a functional monomer demonstrated that π–π interactions and hydrogen bonding could markedly improve binding strength, with adsorption capacities surpassing traditional MAA-based MIPs [18]. Together, these studies illustrate a clear trend toward surface-imprinted, magnetically responsive, and multi-target MIPs, addressing key limitations in selectivity, kinetics, and operational practicality for nitrophenol removal

In this study, we investigate the selective removal of specific nitrophenol isomers from aqueous solution using Acrylic Acid–Acrylamide MIP, for the first time. In addition to the selectivity, we have assessed the thermal stability and extraction efficiency under different pH condition, which are relevant to industrial waste water. Overall, the outcome of this research provides a practical approach to selectively remove nitrophenol form aqueous solution, specifically in industrial and anthropogenically-influenced environments.

The FT-IR spectra of poly (AA-co-AAm) and its MIP are shown in Figures 6 and 7, respectively. The Figures showed a broad absorption band at (3000-3700) cm^-1, which corresponds to OH vibrations and NH stretching bands, indicating the presence of hydroxyl and amide groups [21, 22].

The frequency Peak at (1670-1675) cm^-1 is attributed to the carbonyl group C=O of the acrylamide functional group, which may shift slightly during polymerization [21, 22], while peak at (1630-1650) cm^-1 was associated with the C=O stretching of the carboxylate groups of acrylic acid [21, 22]. Additionally, the peak at (2938-2949) cm^-1 was attributed to C-H stretching vibrations of the main chain of polyme [22, 23].

For MIP of copolymer, peak at 1724 cm^-1 was attributed to stretching carbonyl of carboxylic acid groups in acrylic acid units, while the peak at 1670 cm^-1 corresponds to the carbonyl stretching of the carbetamide groups in acrylamide units. These data are in good agreement with common carbonyl stretching (C=O) peaks that are generally observed around 1724 cm^-1 and 1670 cm^-1 [24, 25].

Peaks around 2924 cm^-1 are due to stretching vibrations C-H in the main chain of polymer [24]. Also, a wide band between (3000-3700) cm^-1 corresponds to vibrations OH and NH stretching, indicating the presence of hydroxyl groups of acrylic acid and amine groups of acrylamide [21]. The important peaks in IR spectrum are listed in Table 1 match to those previously published [21-25].

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Figure 6. FT-IR spectrum of copolymer

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Figure 7. FT-IR spectrum of MIP

Table 1. The important IR bands of copolymer (Co.) and its MIP

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Figure 8. TGA Thermogram of MIP

Table 2. Evaluation of the rebinding efficiency (%E) for the nitrophenol isomers

Figure 8 is shown the TGA Thermogram of MIP for copolymer. Its, shows a 4.2% initial weight loss between 25℃ and 224.1℃. The decomposition proceeds in three stages: 5.6% weight loss between 224.1℃ and 327℃, followed by a 25.2% loss from 327℃ to 509.3℃. The char content is relatively high 60%, and the maximum decomposition rate is the lowest among the three 2.9%/min, indicating enhanced thermal stability, likely due to the synergistic effect of the copolymer composition.

Figures 9 shows the relative percentage of the remained nitrophenol isomers as a function to the volume of methanol used to rinse the template (50 ml portion of methanol each time using an ultrasonic water bath for 30 min). The reduction percentage of the initial concentration in of nitrophenol present in the template was tracked until the no more analyte was detected.

It was noted that the both of ONP and MNP needed 400 and 500 ml of methanol respectively to be completely removed from the copolymer template, unlike PNP which was completely removed using 150 ml. This behavior likely associated with the more intramolecular interaction (hydrogen bonding) of the ONP compared to PNP [26, 27], increasing its binding to the active sites, delaying its removal from the template. Thus, less time, sonication, and volume of methanol are needed to remove PNP from the template compares to the other studied isomers (ortho and meta).

Table 2 summarizes the results obtained in this study. The rebinding efficiency of the three prepared MIPs with the copolymer studied was evaluated. The evaluation study was performed used spectrophotometry.

The results summarized in the above table indicate that the cross-linker, MBA, plays a crucial role in the binding of template molecules. The presence of the functional group (-CO-NH-CH₂-NH-CO-) in the cross-linker likely contributes significantly to the enhancement of binding efficiency, therefore %E of MIP is higher than %E of NIP [20].

Copolymer-based MIPs can be attributed to the specific nature and strength of interactions formed between the template molecule MNP and the functional monomers during the imprinting process.

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Figure 9. Removal (%) of nitrophenol isomers from the Co. templates

Copolymerization plays important role in the efficiency of rebinding. The use of copolymers in molecular imprinting can diminish the specificity of interactions between the functional monomer and the template molecule. The incorporation of multiple monomer types may dilute these specific interactions, resulting in reduced formation of well-defined binding sites. Additionally, the structural heterogeneity introduced through copolymerization can disrupt the optimal spatial arrangement necessary for high-affinity binding, ultimately leading to decreased binding efficiency for MNP [28].

In contrast, cross-linked copolymer-based MIPs often exhibit reduced binding efficiency due to increased compositional complexity, which can disrupt optimal binding site formation and weaken template recognition [28, 29].

*The initial concentration (C_i): It represents a concentration of 15 mg L^-1, which are nitrophenol solutions that were previously prepared with chloroform. Polymers prepared with chloroform solvent dissolve to a high degree because it is a solvent with weak hydrogen bonding [30, 31].

*The final concentration (C_f): It is the concentration of the filtrate measured in milligrams per liter of the original solution after 24 hours of placing the sample in it.

*Moles of substrate bound per gram of polymer (S_b): It is calculated from the following two relationships [30]:

$S_b=C_i-C_f$     (1)

$S_b, g=S_b / W_p$     (2)

*The partition coefficient (K_p): is the partition coefficient of substrate on the MIP divided by the substrate in solution [30]:

$K_p=S_b / C_f$     (3)

*The imprinting factor (I.F.): The value is determined by the ratio of the partition coefficient of a substrate on the molecularly imprinted polymer (K_{p MIP}) to that on the non-imprinted polymer (K_{p NIP}), both prepared using the same monomer formulation (Eq. (4)) [20, 30]:

$I.F. =K_{p, M I P} / K_{P, N I P} K_p=S_b / C_f$     (4)

*Gibbs free energy of interaction (ΔG): The free energy of substrate binding by MIP is determined using the equation [30]:

$\Delta G=-R T \operatorname{Ln} K_p$     (5)

Tables 3-5 are shown the results of selectivity study by 0.2 grams of MIP and NIP samples were placed in 10 ml of each of 15 mg L^-1 nitrophenol, phenol and m-cresol solutions in a shaker and left for 24 hrs. with mixing for certain periods at a speed of 50 rpm. Then the selectivity was measured after filtering the solutions, and the same was true for the rest of the samples [20].

Table 3. Selectivity study of MIP_ONP and NIP_ONP towards phenol and m-cresol

Table 4. Selectivity study of MIP_MNP and NIP_MNP towards phenol and m-cresol

Table 5. Selectivity study of MIP_PNP and NIP_PNP towards phenol and m-cresol

The selectivity of MIP and NIP towards various templates was evaluated using mixed target compounds, ONP/ phenol, ONP/ m-cresol, MNP/ phenol, MNP/ m-cresol, PNP/ phenol, PNP/ m-cresol for the nine templates respectively.

The effect of molecular imprinting on selectivity was determined using the following relation [32]:

$K_d=\frac{\left(C_i-C_f\right)}{W_p} \times V$     (6)

where, K_dis the distribution coefficient, C_i and C_f are the initial and final concentrations of the analyte, W_p is the weight of the MIP, and V is the volume.

The selectivity coefficient (K) of the template in the presence of structurally similar molecules can be calculated as:

$k=\frac{\mathrm{Kd}(\text { nitrophenol })}{\mathrm{Kd}(\text { phenol or } m-\text { cresol })}$     (7)

The relative selectivity coefficient (k') is defined as the ratio of the selectivity coefficients of MIP and NIP [20]:

$K^{\prime}=K_{M I P}-K_{N I P}$     (8)

Nitrophenols are ubiquitous environmental pollutant. To evaluate the efficiency of extracting the three studied nitrophenol pollutants from the aqueous solution. 45 ml of the three prepared aqueous nitrophenol solutions were taken and placed in a separating funnel. 5 ml of chloroform solution was added and shaken for ten minutes. The solutions were then left to separate the organic and aqueous layers. Then the chloroform layer was removed and the second and third extractions were repeated in the same way for all three solutions. The concertation of the aqueous nitrophenol solution was measured after each extraction.

The extraction process was repeated for acidic solutions at a pH =2 and for basic solutions at a pH =10, as indicated in Table 6 and Table 7. The point of choosing these pH values is because pKa value of the ortho, meta, and para nitrophenol are 7.23, 8.35, and 7.15, respectively [33-35]. Therefore, checking the effect of extraction at pH fewer and higher than the pKa will help in explaining the effect of protonation on the extraction efficiency.

Since the volume of the organic layer is not equal to the volume of the aqueous layer, the value of D, the distribution ratio was defined as the ratio of the concentration of a compound in the organic phase to that in the aqueous phase, was calculated from the first extraction efficiency for each acidity function from the following mathematical relationship as follows [36]:

$D=\frac{\text { Corg. }}{\text { Caq. }}$     (9)

where, C_org. is the concentration of the remaining substance in the aqueous layer after the first extraction, C_aq. is the concentration of the original substance in the aqueous layer [36].

Then it is possible to know the volume of the organic layer, the chloroform layer, required for any volume of the aqueous layer contaminated with nitrophenols, with high efficiency, using the following mathematical relationship [36]:

$\% E=\frac{100 D}{D+\left(\frac{\text { Vaq. }}{\text { Vorg. }}\right)}$     (10)

where %E it is the percentage of extraction efficiency, D distribution ratio, V_org. The size of organic layer, V_aq. The size of aqueous layer [36].

Table 6 presents the neutral isomers of nitrophenol, which exhibit lower degrees of ionization compared to the acidic isomers listed in Table 7. The ionized forms demonstrate reduced solubility in organic solvents, leading to decreased distribution ratios and extraction efficiencies. In contrast, at pH 2 an acidic aqueous environment all isomers predominantly exist in their neutral forms. These neutral species have limited solubility in aqueous media but exhibit enhanced solubility in organic solvents such as chloroform. Consequently, higher distribution ratios and extraction efficiencies are anticipated due to favorable partitioning into the organic phase.

Table 6. Concentrations of nitrophenol isomers before and after three extraction cycles at pH= 7

At pH = (7 and 2), all nitrophenol isomers are predominantly in their protonated (neutral) forms. This is due to the fact that their pKa values are above 7, indicating that at this acidic pH, the compounds remain largely undissociated. Consequently, their solubility in aqueous solutions is low, and they are more readily extracted into organic solvents like chloroform [37].

Table 7. Concentrations of nitrophenol isomers before and after three extraction cycles at pH= 2

At pH = 10, all three isomers are predominantly deprotonated, existing as phenoxide anions (Table 8). This deprotonation significantly alters their solubility characteristics. Phenoxide ions are highly soluble in aqueous solutions but have poor solubility in organic solvents like chloroform. As a result, the distribution of these compounds between the aqueous and organic phases is less favorable for extraction at this basic pH [37].

Table 8. Concentrations of nitrophenol isomers before and after three extraction cycles at pH= 10

As presented in Table 8, all isomers are predominantly ionized. Extraction at pH higher than the pKa (nitrophenols are deprotonated) shows a fewer efficiency, as expected. In addition, the ionized nature of these isomers leads to poor solubility in organic solvents, thereby resulting in low distribution ratios and reduced extraction efficiencies.  Thus, it is highly recommended using acidic medium when extracting nitrophenols from aqueous solutions.

Analysis of the data presented in the Tables 6 and 7 indicates that a decrease in the pH corresponds to an increase in the D values. Specifically, for MNP, the D value at pH of 2 was 4.6, representing a more than sevenfold increase compared to the value of 0.6 observed at a neutral pH of 7. Whereas, at higher pH value, the extraction efficiency significantly declined (Figure 8).