Startseite Separation of sulphonamides on a C12-diol mixed-mode HPLC column and investigation of their retention mechanism
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Separation of sulphonamides on a C12-diol mixed-mode HPLC column and investigation of their retention mechanism

  • Hayriye Aral EMAIL logo und Yekun Altun
Veröffentlicht/Copyright: 12. August 2025
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Open Chemistry
Aus der Zeitschrift Open Chemistry Band 23 Heft 1

Abstract

Sulphonamides exhibit broad-spectrum antibacterial effects. However, their extensive use poses health risks, such as antimicrobial resistance. Therefore, advanced and accurate analytical techniques are needed to monitor the presence of sulphonamide residues. In this study, a mixture of eight sulphonamides was separated on a C12-diol mixed-mode column containing both a hydrophobic C12 chain and a hydrophilic diol group. The influence of various experimental parameters, including pH, buffer concentration, mobile phase composition, temperature, and flow rate, on the separation efficiency of the column was investigated. All eight sulphonamides were separated within 20 min via gradient elution with an acetonitrile/phosphate buffer (pH 6.5, 40 mM KH2PO4/K2HPO4) solution at 15°C and a detection wavelength of 250 nm. Examination of the retention times of the analytes at different pH values ranging from 3 to 7 revealed that the sulphonamides were significantly affected by the acidity of the medium, thus enabling a detailed discussion of the mixed-mode separation mechanism. The interaction mechanism between the eight sulphonamides and the C12-diol mixed-mode column was explored using quantitative structure–retention relationship parameters such as log D, log S, and pK a. Although the interaction of the sulphonamides with the C12-diol mixed-mode column partly showed the classic reverse-phase feature, polar interactions played a major role, and the mixed-mode interaction mechanism was dominated by polar and apolar interactions. The results obtained were compared with a commercial ODS-C18 column in terms of both separation performance and retention behaviour.

Keywords: mixed-mode; sulphonamides; retention mechanism; log D ; log S ; quantitative structure–retention relationship

1 Introduction

Sulphonamides, a class of synthetic antimicrobial agents, have been extensively used in human and veterinary medicine because of their broad-spectrum antibacterial effects [1]. They are frequently used in livestock farming to treat and prevent bacterial infections and enhance production efficiency. However, the increased use of sulphonamides has raised concerns about the accumulation of drug residues in food products such as eggs, meat, and milk, which could pose a risk to consumers and potentially contribute to the growing problem of antimicrobial resistance [2]. Regulatory bodies worldwide, including the European Union and the US Food and Drug Administration, have imposed strict maximum residue limits for sulphonamides in foodstuffs to ensure public health safety [3]. These actions have driven the need for more advanced and accurate analytical techniques to monitor the presence of these residues [2,4,5].

High-performance liquid chromatography (HPLC) has long been preferred for the detection and quantification of sulphonamide residues owing to its high efficiency and sensitivity [6–11]. However, conventional HPLC methods such as reversed-phase chromatography often struggle with the complex physicochemical properties of sulphonamides, which contain both hydrophobic and ionic functional groups. Traditional methods require steps such as ion-pairing or sample derivatisation, increasing the risk of sample contamination and complicating the analytical process. These limitations have encouraged researchers to explore more flexible techniques that can handle the dual nature of sulphonamides [1].

In recent years, mixed-mode HPLC has gained attention as an alternative to traditional HPLC methods, offering greater flexibility and selectivity for separating challenging analytes such as sulphonamides [12,13]. Mixed-mode HPLC integrates multiple retention mechanisms, often combining reverse-phase, normal-phase, and ion-exchange interactions within a single stationary phase [14–20]. This dual-mode approach allows for more precise control over the separation process, rendering it especially useful for analysing compounds such as sulphonamides, which vary widely in terms of polarity and ionisation state [21]. Researchers have found that adjusting parameters such as the mobile phase composition and pH in mixed-mode systems leads to improvements in resolution and peak shape for sulphonamides, allowing for more reliable quantification, even in complex food samples [22,23]. Methylimidazolium-modified silica microparticles, a new type of mixed-mode stationary phase intended for use in the hydrophilic interaction liquid chromatography (HILIC)/anion-exchange regime, have been proposed and synthesised, enabling the separation of six sulphonamides after 10 and 12 min [24]. Novel amide-modified silica stationary phases have also been developed and evaluated for the isocratic separation of seven sulphonamides using sequential injection chromatography. The seven sulphonamides could be successfully separated within 20 min. In this system, a mobile phase composed of acetonitrile (ACN) and water with a flow rate of 0.45 mL/min was employed. The findings suggest a mixed-mode retention mechanism for sulphonamides on stationary phases, involving multiple interactions such as dipole–dipole and π–π interactions and hydrogen bonding [25].

The separation mechanism in mixed-mode HPLC can be further understood and optimised using quantitative structure–retention relationships (QSRRs) and various physicochemical parameters [14]. The key parameters are logarithmic parameters such as log P, log D, and log S, which represent the partition coefficient, distribution coefficient, and solubility of the analytes, respectively. These parameters provide valuable insights into the hydrophobic and ionic interactions governing analyte retention in the stationary phase. Specifically, log P offers an understanding of the hydrophobicity of the analytes, while log D reflects the pH-dependent ionisation state of the molecules. Additionally, other quantitative parameters such as p (polarity), k (partitioning coefficient), and a (activity coefficient) were utilised to better predict and control the separation behaviour of complex compounds [26–28]. By applying these parameters, chromatographers can fine-tune the separation process, enhance the selectivity of the method, and improve the resolution of analytes in mixed-mode HPLC. Such an approach is crucial for analysing compounds with diverse functional groups and ionisation states, rendering it especially relevant for complex food matrices in which multiple sulphonamides may coexist.

The continued development of mixed-mode HPLC as a tool for the detection and quantification of sulphonamides highlights its potential to meet the stringent regulatory requirements set by food safety authorities. Given its ability to accommodate a variety of analytes in complex matrices and maintain high sensitivity, this chromatographic approach has proven essential for the routine monitoring of sulphonamide residues in foods such as eggs, where contamination risks are particularly high [1,2,29]. Further optimisation of these methods is likely to enhance their detection capabilities, ensuring that food safety standards are met worldwide. However, almost all studies on sulphonamides in the literature were conducted with a traditional C-18 column. Since mixed-mode HPLC is still a developing field, mixed-mode columns are very few in the market. Therefore, there is a need to study the separation performances and properties of existing columns in detail. In particular, understanding the mixed-mode interaction mechanism is very important for the development of more suitable columns. In particular, examining the interaction mechanism using QSRR parameters rather than speculative interpretations will provide more accurate information.

This study aimed to separate a mixture of eight sulphonamides, a class of compounds exhibiting both polar and non-polar characteristics as well as high sensitivity to environmental acidity, using a mixed-mode column featuring a C12-diol structure. The eight sulphonamides selected in this study include compounds used in both human and veterinary medicine. Although these analytes may not co-occur in a single real-world sample, their diverse physicochemical profiles were intentionally selected to enable a broad evaluation of the retention and separation behaviour of the C12-diol mixed-mode stationary phase. Through separation experiments, we intend to investigate the intricacies of the mixed-mode separation mechanism by employing the QSRR parameters log D, log S, and pK a. The results obtained were compared with a commercially available ODS-C18 column, focusing on both separation performance and retention behaviour. This comparison was made to assess how the C12-diol-mixed-mode column performs relative to a widely used and established column, ODS-C18, in terms of efficiency, selectivity, and retention times under optimised conditions.

2 Experimental

2.1 Materials

An AcclaimTM mixed-mode column HILIC-1 (250–4.6 mm, 5 µm, 100 Å) was purchased from Thermo Fisher (Waltham, Massachusetts, USA). HPLC-grade ACN, methanol (MeOH), ethanol, and isopropanol were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used for HPLC. All sulphonamides (sulphadiazine [SDZ], sulphamethoxazole [SMX], sulphamerazine [SMR], sulphathiazole [STZ], sulphamethazine [SMZ], sulphamethoxypyridazine [SMOP], sulphadimethoxine [SDM], and sulphaquinoxaline [SQX]; Figure 1) used in this study were obtained from Sigma-Aldrich. Deionised water was purified using a Millipore Milli-Q Water System (Merck, USA). Log D, log S, and pK a values were calculated using ChemAxon Chemicalize software. ACE C18 column (250–4.6 mm, 5 µm, 100 Å) was purchased from Avantor (USA).

Figure 1 Structures of the eight sulphonamides separated in this study.
Figure 1

Structures of the eight sulphonamides separated in this study.

2.2 HPLC conditions

Chromatographic analyses were conducted using an Agilent 1260 HPLC system equipped with a quaternary pump, degasser, autosampler, diode-array detector (DAD), and thermostated column compartment. Standard stock solutions of each sulphonamide (1 mg/mL) were prepared in ACN and stored at 4°C, and working solutions were diluted with deionised water to a final concentration of 0.1 mg/mL. Detection was performed at 250 nm. Buffer solutions were prepared using ammonium formate/formic acid for pH 3–4, ammonium acetate/acetic acid for pH 4–6, and potassium dihydrogen phosphate/potassium monohydrogen phosphate for pH 6–8. For optimal separation, a 40 mM KH₂PO₄/K₂HPO₄ buffer at pH 6.5 was used, prepared by dissolving the appropriate salts in deionised water, adjusting the pH with dilute phosphoric acid or KOH, and filtering through a 0.45 µm membrane. For gradient elution (Figure 2a), the mobile phase consisted of ACN and buffer, starting at 10% ACN and 90% buffer, increasing linearly to 40% ACN and 60% buffer from 0 to 14 min, then held constant at 40% ACN and 60% buffer from 14 to 20 min; the flow rate was gradually increased from 1.0 to 1.5 mL/min between 0 and 14 min and maintained at 1.5 mL/min from 14 to 20 min; the column temperature was maintained at 15°C and the injection volume was 10 µL (analyte concentration: 100 ppm). For isocratic elution (Figure 2b), the mobile phase was a fixed composition of 10% ACN and 90% buffer, with a constant flow rate of 1.5 mL/min, a column temperature of 15°C, an injection volume of 10 µL (100 ppm), and detection at 250 nm. The dead time (t₀) of the column was determined by adding a small amount of methanol to the mobile phase and measuring it at each temperature, resulting in values ranging from 2.77 to 2.97 min, which were used for all retention factor (k′) calculations. The sulphonamides analysed were sulphadiazine (SDZ), sulphamethoxazole (SMX), sulphamerazine (SMR), sulphathiazole (STZ), sulphamethazine (SMZ), sulphamethoxypyridazine (SMOP), sulphadimethoxine (SDM), and sulphaquinoxaline (SQX); their log D and log S values were calculated using ChemAxon Chemicalize software. Deviations from the chromatographic conditions described in this section are detailed in the corresponding figures.

Figure 2 Optimum (a) gradient and (b) isocratic conditions for the separation of the eight sulphonamides. Conditions: (a) ACN/buffer (pH 6.5, 40 mM KH2PO4/K2HPO4, 10/90 (v/v)); 0–14 min, 40/60; 14–20 min, flow rate: 1 mL/min; 0–14 min, 1–1.5 mL/min; 14–20 min; temperature: 15°C; injection volume: 10 mL/100 ppm; and detection wavelength: 250 nm. (b) ACN/buffer (pH 6.5, 40 mM KH2PO4/K2HPO4, 10/90 (v/v)), flow rate: 1.0 mL/min, temperature: 15°C, injection volume: 10 µL/100 ppm, detection wavelength: 250 nm. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX).
Figure 2

Optimum (a) gradient and (b) isocratic conditions for the separation of the eight sulphonamides. Conditions: (a) ACN/buffer (pH 6.5, 40 mM KH2PO4/K2HPO4, 10/90 (v/v)); 0–14 min, 40/60; 14–20 min, flow rate: 1 mL/min; 0–14 min, 1–1.5 mL/min; 14–20 min; temperature: 15°C; injection volume: 10 mL/100 ppm; and detection wavelength: 250 nm. (b) ACN/buffer (pH 6.5, 40 mM KH2PO4/K2HPO4, 10/90 (v/v)), flow rate: 1.0 mL/min, temperature: 15°C, injection volume: 10 µL/100 ppm, detection wavelength: 250 nm. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX).

3 Results and discussion

The chromatographic analysis of the eight sulphonamides was performed using a mixed-mode column. The analytes were SDZ, SMX, SMR, STZ, SMZ, SMOP, SDM, and SQX (Figure 1). The influence of the mobile phase composition, pH, buffer concentration, flow rate, and temperature on the chromatographic behaviour of the sulphonamides was investigated. The optimal separation conditions were determined using a isocratic elution program with an ACN/buffer (pH 6.5, 40 mM KH2PO4/K2HPO4, 10/90 (v/v)) solution, flow rate of 1 mL/min, detection wavelength of 250 nm, and a gradient elution program with an ACN/buffer (pH 6.5, 40 mM KH2PO4/K2HPO4, 10/90 (v/v)): 0–14 min, 40/60; 14–20 min, flow rate: 1 mL/min; 0–14 min, 1–1.5 mL/min; 14–20 min; temperature: 15°C; injection volume: 10 µL/100 ppm; and detection wavelength: 250 nm. Under these conditions, the eight sulphonamides were successfully separated within 22 min (Figure 2). These results are better than those obtained from other stationary phases with mixed-mode structures [23,30]. The calculated chromatographic parameters indicate that the separation performance under the optimized conditions was satisfactory. The selectivity (α) values for all consecutive sulphonamide pairs were greater than 1.0, confirming that each analyte could be discriminated from its neighbour. Furthermore, the resolution (Rs) values were above the baseline separation threshold of 1.5 for most analyte pairs, except for a slight overlap between peaks 4 and 5, which still remained within an acceptable range (Rs = 1.30). These findings demonstrate that the mixed-mode C12-diol stationary phase provided efficient retention and separation of the eight sulphonamides with minimal peak overlap. Detailed values are presented in Table S1.

The QSRR parameters (log D, log S, and pK a) of the analytes were calculated (Table 1) to explore the interaction mechanism of sulphonamides with the C12-diol mixed-mode column. By examining the mixed-mode properties of the column together with these quantitative parameters, we can obtain a deeper understanding of the retention and elution behaviours of sulphonamides.

Table 1

Calculated log D 1, log S 1, and pK a values of the sulphonamides used in this study2

HPLC elution order Analyte Log D Log S pK a 1
1 SDZ 0.28 −2.38 6.99
2 SMX 0.24 −1.47 5.86
3 SMR 0.41 −2.54 6.99
4 STZ 0.36 −1.83 5.73
5 SMZ 0.54 −2.77 6.99
6 SMOP 0.33 −2.75 6.84
7 SDM 1.14 −3.44 6.91
8 SQX 1.40 −2.74 6.79

1Calculated at an optimum pH of 6.5. 2All values were calculated using Chemaxon Chemicalize software.

A comparison of the elution order of the sulphonamides with their log D and log S values listed in Table 1 under the optimized chromatographic conditions indicates a mixed-mode interaction between the sorbent and analytes. In contrast to the classical reverse-phase mechanism, in which the retention time decreases with increasing polarity owing to enhanced aqueous solubility, the observed elution order did not strictly follow this trend. For instance, SMX, which had the lowest log D (0.24) and highest log S (−1.47) among the sulphonamides, was the second analyte to elute from the column. Similarly, STZ, which had the second-highest aqueous solubility (log S = −1.83) and fourth-highest hydrophilicity (log D = 0.36), was the fourth analyte to elute from the column. These observations suggest that polarity plays a more significant role than aqueous solubility in the separation and retention behaviour of the analytes. Furthermore, SMOP, as the third most-polar compound with a log D of 0.33, was the sixth to elute from the column owing to its low aqueous solubility (log S = −2.77), indicating the dominance of aqueous solubility in this case. A similar comparison can be made for the two most hydrophobic compounds, SDM and SQX, which eluted last at all pH values, as shown in Figures 2 and 3. Although these results are consistent with the reverse-phase mechanism, the log D and log S values present an interesting discrepancy. SDM has low aqueous solubility (log S = −3.44), whereas SQX has high hydrophobicity (log D = 1.40). Despite having a lower log D value, SQX was eluted later than SDM. This finding aligns with the reverse-phase mechanism in terms of log D but contradicts the trend observed for the log S values, suggesting that in addition to non-polar interactions, polar interactions, such as hydrogen-bonding, ion–dipole, and ion–ion interactions, play a role in the interaction between the stationary phase and sulphonamides. Hydrogen bonding dominates for some compounds (high log S and aqueous solubility), whereas high polarity (low log D) determines the polar interactions for others. Similarly, for non-polar interactions, high hydrophobicity (high log D) dominates for some compounds, whereas low log S (low aqueous solubility) dominates for others.

Figure 3 Effect of pH on the retention time (R.T.) of sulphonamides on the C12-diol mixed-mode column. Conditions: ACN/buffer (20 mM formate, acetate, or phosphate buffers, 10/90 (v/v)), flow rate: 1 mL/min, temperature: 25°C, injection volume: 10 µL/100 ppm, and detection wavelength: 250 nm. Analytes: (1) SDZ: sulphadiazine, (2) SMZ: sulphamethazine, (3) SDM: sulphadimethoxine, (4) SMX: sulphamethoxazole, (5) SMOP: sulphamethoxypyridazine, (6) SMR: sulphamerazine, (7) SQX: sulphaquinoxaline, and (8) STZ: sulphathiazole.
Figure 3

Effect of pH on the retention time (R.T.) of sulphonamides on the C12-diol mixed-mode column. Conditions: ACN/buffer (20 mM formate, acetate, or phosphate buffers, 10/90 (v/v)), flow rate: 1 mL/min, temperature: 25°C, injection volume: 10 µL/100 ppm, and detection wavelength: 250 nm. Analytes: (1) SDZ: sulphadiazine, (2) SMZ: sulphamethazine, (3) SDM: sulphadimethoxine, (4) SMX: sulphamethoxazole, (5) SMOP: sulphamethoxypyridazine, (6) SMR: sulphamerazine, (7) SQX: sulphaquinoxaline, and (8) STZ: sulphathiazole.

The chromatographic behaviour of the sulphonamides on the C12-diol column was attributed to a mixed-mode interaction mechanism. In addition to the non-polar interactions provided by the hydrophobic chain, the diol group contributes to retention through hydrogen-bonding and ion–dipole interactions.

3.1 Effect of pH on the separation and retention of the sulphonamides

To investigate the influence of pH on the separation and retention behaviour of the sulphonamides, we selected an initial mobile phase composition of an ACN/buffer (pH 3, 20 mM H₃PO₄/KH₂PO₄, 10/90 (v/v)) for pH optimisation. After determining the optimal pH, we optimised the ACN concentration at three different pH values (pH 3, 5, and 6.5).

Prior knowledge of the polarities of the analytes and how their polarity is influenced by pH changes is crucial to understanding the effect of pH variation on the retention of sulphonamides. Figure 4 illustrates the variation in the log D values of the studied sulphonamides as a function of pH. As depicted in the figure, the hydrophobicity of the analytes remained relatively stable within the pH range of 3–5. However, below pH 3 and above pH 5, particularly above pH 6, the polarity and water solubility of all analytes increased (lower log D and higher log S values), as shown in Figure 5a–c. With increasing resolution, detention times also decreased. Retention times appear to be higher at pH 3.0, while shorter at pH 5.0 and 6.0.

Figure 4 Log D values of the eight sulphonamides as a function of pH. Analytes: SDZ: sulphadiazine, SMZ: sulphamethazine, SDM: sulphadimethoxine, SMX: sulphamethoxazole, SMOP: sulphamethoxypyridazine, SMR: sulphamerazine, SQX: sulphaquinoxaline, and STZ: sulphathiazole.
Figure 4

Log D values of the eight sulphonamides as a function of pH. Analytes: SDZ: sulphadiazine, SMZ: sulphamethazine, SDM: sulphadimethoxine, SMX: sulphamethoxazole, SMOP: sulphamethoxypyridazine, SMR: sulphamerazine, SQX: sulphaquinoxaline, and STZ: sulphathiazole.

Figure 5 Effect of ACN content (ACN%) in the aqueous buffer on the retention time (R.T.) of sulphonamides on the C12-diol mixed-mode column at (a) pH 6.5 (20 mM KH2PO4/K2HPO4), (b) pH 5.0 (20 mM CH3COOH/CH3COONH4), (c) pH 3.0 (20 mM H3PO4/KH2PO4), (d) U-shape curve for ACN% in water versus ln k′. Other conditions: Flow rate: 1 mL/min, temperature: 25°C, injection volume: 10 mL/100 ppm, and detection wavelength: 250 nm. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX).
Figure 5

Effect of ACN content (ACN%) in the aqueous buffer on the retention time (R.T.) of sulphonamides on the C12-diol mixed-mode column at (a) pH 6.5 (20 mM KH2PO4/K2HPO4), (b) pH 5.0 (20 mM CH3COOH/CH3COONH4), (c) pH 3.0 (20 mM H3PO4/KH2PO4), (d) U-shape curve for ACN% in water versus ln k′. Other conditions: Flow rate: 1 mL/min, temperature: 25°C, injection volume: 10 mL/100 ppm, and detection wavelength: 250 nm. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX).

Although no substantial change in the retention times of the analytes was observed within the pH range of 3–5, a decrease in retention times was noted at pH values above 5 or 6. As indicated in Table 1, the pK a values of the analytes fall within the range of 5–7. The increase in water solubility and corresponding decrease in retention times above pH 5–6 were attributed to the transition of the analytes to their anionic forms.

However, the log D and log S values of all analytes were not affected to the same extent above pH 5. While SMX and STZ exhibited intermediate log D and log S values compared with the other analytes (SMX fifth order and STZ sixth order), at pH 7, SMX displayed the lowest log D value, whereas STZ ranked fourth. This differential behaviour of the analytes in terms of the effect of pH on their hydrophobicity is not reflected in the graph of retention time versus pH. According to the classical one-dimensional interaction mechanism of reversed-phase chromatography, as the polarity increases, the water solubility of the analytes also increases, leading to a decrease in the retention time; thus, more polar analytes elute earlier than apolar ones. However, the behaviour observed in the present case deviates from this expectation. Generally, the retention times of the analytes decreased with increasing hydrophilicity above pH 6, but the retention times of SMX and STZ did not show a parallel decrease in log D. This optimal separation occurs within the pH range of 6–7, even though the log D values that are quite similar within this range are particularly intriguing. This result unequivocally demonstrates that as analytes transition to their anionic form, polar interactions, such as ion–dipole interactions, intensify and play a significant role alongside apolar interactions. This mixed-mode interaction mechanism is responsible for the optimal separation achieved at pH 6.5.

3.2 Dual-mode retention behaviour of sulphonamides under varying ACN ratios and solvent systems

Binary and ternary mixtures of water, ACN, and MeOH were used to determine the optimal mobile phase composition. ACN/water binary mixtures were more effective for separation than mixtures containing MeOH. As is well-known, methanol exhibits higher polarity than ACN due to its capacity to form hydrogen bonds. Consequently, water/methanol mixtures possess significantly greater polarity compared to water/ACN mixtures. While sulphonamides contain polar functional groups, their overall apolar character dominates, leading to predominantly reverse-phase chromatographic behaviour. This implies that both polar and apolar interactions are more pronounced in water/ACN mixtures than in water/methanol mixtures. Therefore, it is anticipated that sulphonamides will be more effectively separated in the less polar water/ACN mobile phase compared to the more polar water/methanol mobile phase. Therefore, different ACN/water mixtures were tested to determine the optimal mobile phase ratio.

To identify the optimal conditions and better understand the effect of mobile phase composition on the retention behaviour of the analytes, we performed mobile phase optimisation at three pH values (6.5, 5.0, and 3.0). The effect of the ACN ratio on the separation and retention behaviours of the sulphonamides at each pH value is presented in Figure 5a–c. Owing to the high hydrophobicity of all the analytes at pH 3.0 and 5.0, their retention times were also high. Because using an ACN ratio below 18% significantly increased the analysis time under the experimental conditions employed in this study, ACN optimisation was conducted in the range of 18–25%. However, at pH 6.5, the analysis time decreased because of the increased water solubility of the analytes. Thus, the optimum ACN composition at pH 6.5 was 10%.

As seen in all three plots in Figure 5, the retention times of all analytes decreased logarithmically with increasing ACN ratio at all three pH values. This effect was particularly pronounced at pH 6.5, where the increased hydrophilicity of the analytes enhanced their sensitivity to ACN. The effect of ACN on analyte retention was more significant and sharper at this pH than at the other pH values; specifically, the logarithmic relationship in this graph was more distinct. Although the hydrophilicity of the analytes increased at higher pH values and the interaction mechanism was mixed mode, the behaviour of these analytes towards ACN was consistent with a reversed-phase mechanism.

To elucidate the retention mechanism of sulphonamides on a C12-mixed-mode column, the retention behaviour of all analytes was investigated under both reversed-phase and HILIC conditions. Isocratic elution was carried out using a mobile phase gradient of 10–90% ACN in water. The retention factor (k′) of each analyte was calculated, and a plot of log k′ versus % ACN was constructed (Figure 5d). The plot exhibited a characteristic U-shape, indicating a transition from reversed-phase to HILIC retention. At lower ACN concentrations (10–20%), strong retention was observed due to hydrophobic interactions with the C12 stationary phase. As the ACN proportion increased, retention decreased, consistent with the reduced hydrophobic character of the mobile phase. However, at very high ACN concentrations (>80%), a slight increase in retention was observed, suggesting a contribution from HILIC interactions. These results demonstrate that the retention behaviour of sulphonamides on this mixed-mode column is governed by a combination of reversed-phase and HILIC mechanisms, with the relative contribution of each mechanism depending on the composition of the mobile phase.

3.3 Effect of buffer concentration on the separation and retention of the sulphonamides

To investigate the influence of the buffer concentration on the separation and retention of the sulphonamides, a series of KH₂PO₄/K₂HPO₄ buffer solutions (10, 20, 30, 35, and 40 mM) were employed under the optimised conditions of pH 6.5, 10% ACN, 25°C, and 1 mL/min flow rate. Although a buffer concentration of 10 mM resulted in unsatisfactory separation, no significant differences were observed at higher concentrations, with 40 mM yielding the best separation performance (Figure 6a).

Figure 6 (a) Effect of buffer concentration on the retention time (R.T.) of sulphonamides on the C12-diol column. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX). (b) Effect of temperature on the retention time (R.T.) of the sulphonamides on the C12-diol column. (a) retention times versus temperature and (b) ln k′ versus 1/T (Van’t Hoff equation). Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX). The retention factors (k′) were calculated using dead time values measured at each temperature, ranging from 2.77 to 2.97 min. (c) Effect of flow rate on the retention time (R.T.) of sulphonamides on the C12-diol column. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX).
Figure 6

(a) Effect of buffer concentration on the retention time (R.T.) of sulphonamides on the C12-diol column. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX). (b) Effect of temperature on the retention time (R.T.) of the sulphonamides on the C12-diol column. (a) retention times versus temperature and (b) ln k′ versus 1/T (Van’t Hoff equation). Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX). The retention factors (k′) were calculated using dead time values measured at each temperature, ranging from 2.77 to 2.97 min. (c) Effect of flow rate on the retention time (R.T.) of sulphonamides on the C12-diol column. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX).

Increasing the buffer concentration did not significantly alter the retention times of the analytes. However, a slight increase in retention time was observed for SDM and SQX with increasing buffer concentration. Due to their higher hydrophobicity (higher log D values), SDM and SQX were more affected by the increase in buffer concentration. An elevated buffer concentration enhances the ionic character and polarity of the mobile phase, which decreases the solubility of these hydrophobic analytes in the mobile phase, resulting in prolonged retention times.

Given that the optimal separation of analytes 5 and 6 (SMZ and SMOP, respectively) was achieved at a buffer concentration of 40 mM, this concentration was considered optimal and used for subsequent flow rate and temperature optimisations.

3.4 Effect of temperature on the separation and retention of the sulphonamides

Temperature plays a critical role as a key parameter in chromatographic separation processes. The separation performance can be directly influenced by temperature, depending on the type of method used, the chemical properties of the analytes, and the characteristics of the column. In liquid chromatography techniques such as HPLC, temperature variations typically alter the interactions of the analytes with the stationary phase, resulting in significant effects on separation efficiency and resolution [31].

In this study, the separation of various sulphonamides was investigated under different temperature conditions using a C12-DIOL mixed-mode column, which exhibits a hybrid interaction mechanism. To determine the optimal temperature for separation, we repeated the experiments at temperatures of 10, 15, 20, 25, 30, and 40°C (Figures S34–39) under the previously optimised conditions of pH 6.5, 10% ACN, 40 mM buffer concentration, and 1 mL/min flow rate. The optimal temperature was identified as 15°C, as this temperature provided the best baseline separation between SMZ and SMOP. As shown in Figure 6b, the retention times of the analytes decreased with increasing temperature. This decrease was not strictly linear but exhibited a logarithmic relationship.

The Van’t Hoff equation provides a useful framework for examining how temperature affects the chromatographic retention behaviour. Although no chemical reaction occurs during separation, plotting the natural logarithm of the retention factor (ln k′, where k′ reflects analyte retention time relative to dead time in the range of 2.77–2.97 min for the corresponding temperatures) versus the reciprocal of temperature (1/T, in K⁻¹) allows for the extraction of thermodynamic parameters such as enthalpy (ΔH°) and entropy (ΔS°), as shown in Table 2. This analysis offers mechanistic insights into the analyte–stationary phase interactions under different thermal conditions.

Table 2

ΔH and ΔS values obtained from the Van’t Hoff equation (Figure 6b)

HPLC elution order Analyte ΔH (kJ/mol K) ΔS (J/mol K)
1 SDZ −10.60 −34.00
2 SMX −12.77 −39.44
3 SMR −9.09 −24.75
4 STZ −12.17 −33.92
5 SMZ −7.59 −13.83
6 SMOP −10.45 −25.73
7 SDM −16.30 −38.24
8 SQX −16.22 −39.99

Note: The thermodynamic parameters ΔH° and ΔS° reported here are not standard molar enthalpy or entropy values, but derived interaction-related descriptors from the Van’t Hoff plot, reflecting the analyte–stationary phase behaviour under given chromatographic conditions.

In this study, Van’t Hoff plots (Figure 6b) were used to assess the retention characteristics of eight sulphonamides. The resulting lines were nearly linear (R 2 > 0.97), indicating that thermodynamic modelling is appropriate despite minor curvature, which may arise from mixed-mode retention contributions. The varying slopes and intercepts observed for different analytes reflect distinct interaction profiles with the stationary phase, likely involving both hydrophobic and hydrophilic effects.

The negative ΔH° values observed in Table 2 indicate that the binding events are exothermic. As a result, with increasing temperature, the analytes’ tendency to be retained on the column decreases, and k′ becomes smaller. The negative ΔS° values suggest that the system becomes more ordered during the interaction. The negative ΔS° values for analytes 2, 7, and 8 are higher compared to those for analytes 5 and 6, which may imply that analytes 2, 7, and 8 form more structurally defined and thermodynamically consistent interactions with the stationary phase.

Ultimately, this study provides important insights into the separation behaviour of sulphonamides in interaction with the C12 mixed-mode column under varying temperatures. The findings show that temperature plays a significant role in the interaction of sulphonamides with the column, with different analytes exhibiting distinct behaviours towards the temperature changes. These results will be valuable in analysing sulphonamides using different columns.

3.5 Effect of flow rates on the separation and retention of sulphonamides

To optimise the flow rate, we conducted a series of experiments at flow rates of 0.75, 1.0, 1.25, 1.50, and 1.75 mL/min under the following optimised conditions: pH 6.5, 10% ACN, 40 mM buffer, and 15°C (Figures S40–44). As shown in Figure 6c, an increase in the flow rate resulted in a logarithmic decrease in the analyte retention time. Although the analysis time increases as the flow rate decreases, the separation selectivity between SMZ and SMOP does not change significantly. For this reason, a flow rate of 1.5 ml/min, which provides a shorter analysis time, was accepted as the optimum flow rate.

3.6 Comparison of the C12-mixed-mode column with ODS C18

To better evaluate the separation performance and retention behaviour of the C12-mixed-mode column, the results were compared to those obtained with a commercially available ODS-C18 column (Figure 7). For a fair comparison, eight sulphonamides were initially injected into the ODS-C18 column under the optimised conditions established for the C12-mixed-mode column, as outlined in Figure 2. Under these conditions, seven sulphonamides were successfully separated in 26 min (Figure 7b). While the seven sulphonamide compounds exhibited excellent peak sharpness and high selectivity, the separation of SMR and STZ was not achieved under these conditions. Since these conditions were optimised for the C12-mixed-mode column, the focus was on comparing the analysis time and retention behaviour rather than the separation performance. Except for SDZ (1), all analytes exhibited longer retention times on the ODS-C18 column. As previously mentioned, it is expected that sulphonamides, with their highly apolar characteristics and predominantly reverse-phase behaviour, would be retained longer on the ODS-C18. At pH 6.5, two key differences between the two columns were observed: the ODS-C18 provided sharper peaks, while the C12-diol-mixed-mode column displayed better selectivity by successfully separating eight analytes.

Figure 7 Comparison of (a) C12-diol-mixed mode and (b) ODS C18 for the separation of eight sulphonamides under the optimised conditions given for C12-diol-mixed mode column in Figure 2 (pH 6.5).
Figure 7

Comparison of (a) C12-diol-mixed mode and (b) ODS C18 for the separation of eight sulphonamides under the optimised conditions given for C12-diol-mixed mode column in Figure 2 (pH 6.5).

To further enhance the reliability and fairness of the separation performance comparison, the conditions for the ODS-C18 column were also optimised. Various parameters (such as the mobile phase ratio and buffer concentration) across pH 3–7.5 were tested to identify the optimal separation conditions. At pH 5.0, with an ACN/water ratio of 20/80, the separation of eight analytes was achieved in 25 min (Figure 8). Under these conditions, SDM (7) and SQX (8) were separated nearly baseline in the 22–24-min range. When the analysis time was reduced to 20 min, compounds 7 and 8, as well as 1 and 4, began to overlap.

Figure 8 Optimised separation of the eight sulphonamides on an ODS C18 column. ACN/buffer (pH 5.0, 20 mM CH3COOH/CH3COONH4, 20/80 (v/v)), flow rate: 1.0 mL/min, temperature: 25°C, injection volume: 5 µL/100 ppm, and detection wavelength: 260 nm. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX).
Figure 8

Optimised separation of the eight sulphonamides on an ODS C18 column. ACN/buffer (pH 5.0, 20 mM CH3COOH/CH3COONH4, 20/80 (v/v)), flow rate: 1.0 mL/min, temperature: 25°C, injection volume: 5 µL/100 ppm, and detection wavelength: 260 nm. Analytes: (1) sulphadiazine (SDZ), (2) sulphamethoxazole (SMX), (3) sulphamerazine (SMR), (4) sulphathiazole (STZ), (5) sulphamethazine (SMZ), (6) sulphamethoxypyridazine (SMOP), (7) sulphadimethoxine (SDM), and (8) sulphaquinoxaline (SQX).

Referring to the data in Figure 4 and Table 1, the log D order of the analytes at pH 6.5 is 2 < 1 < 6 < 4 < 3 < 5 < 7 < 8, and at pH 5.0 it is 1 < 6 < 3 < 5 < 2 < 4 < 7 < 8. This order reflects an increase in hydrophobicity from left to right. The elution order, however, does not follow this trend at either pH. The ODS-C18 is a stationary phase that interacts through one-dimensional hydrophobic interactions. Therefore, the elution order was expected to correlate with hydrophobicity. However, the results did not align with this expectation. This discrepancy is likely due to the polar interactions of ACN, which have not been fully accounted for. Although ACN is generally considered an apolar solvent in a water/ACN mixture, its dipole moment is significantly higher than that of water, making it a more polar solvent. Consequently, in addition to hydrophobic interactions with non-polar groups, it is likely that dipole–dipole and ion–dipole interactions occur between the sulphonamide compounds (which contain a polar sulphonamide group, specifically an amide NH) and ACN. While the analytes interact hydrophobically with ACN, the simultaneous polar interactions stabilise the environment. In essence, while analytes interact hydrophilically with water, they can interact both hydrophilically and hydrophobically with ACN. This dual interaction likely explains the discrepancy between the log D order and the elution order.

In conclusion, comparing the two columns reveals that the ODS-C18 column provides much sharper peaks, whereas the C12-DIOL-mixed-mode column offers better selectivity, successfully separating eight analytes in 20 min. Although some literature reports suggest that HILIC and polar-modified mixed-mode columns may cause a slight decrease in peak sharpness compared to conventional C18 phases due to secondary polar interactions, this effect was not observed under the reversed-phase dominant conditions used in this study (as shown in Figure 5d). It is also worth noting that the lower efficiency observed for the C12-diol column compared to the ODS-C18 column (as shown in Figure 7) is not due to any defect or sorbent degradation. Instead, it can be explained by the lower carbon load and the presence of terminal hydroxyl groups on the C12-diol stationary phase, which may lead to slightly broader peaks even under reversed-phase conditions.

Research in the mixed-mode field aims to develop columns with exceptional efficiency as well as selectivity. Thus, exploring the separation and interaction mechanisms of mixed-mode columns is crucial for the future development of more efficient and durable columns. The findings of this study will contribute significantly to these advancements.

4 Conclusions

The increasing complexity of analytical samples, as well as the desire for efficient separation using a single chromatographic column, has driven the development of mixed-mode chromatography. Although this field has experienced significant growth, the commercial availability of suitable stationary phases remains limited. This study investigated the separation performance and underlying mechanisms of a commercially available C12-diol mixed-mode stationary phase using sulphonamides as model analytes. These analytes, which possess both hydrophobic and hydrophilic functionalities and exhibit pH-dependent behaviour, provide a suitable platform for investigating intricate interactions within mixed-mode systems. The separation of the eight sulphonamides was achieved using both isocratic and gradient elution within 20 min, and the retention behaviour was correlated with the QSRR parameters log P, log D, and pK a. The effects of pH, mobile phase composition, buffer concentration, flow rate, and temperature on the separation and retention of the analytes were studied. The results indicate that the stationary phase operates via a hybrid mechanism that combines the elements of both the HILIC and reversed-phase modes. The use of a mixed-mode column for the HPLC separation of sulphonamides offers a substantial methodological advancement for analytical research. By integrating reversed-phase and secondary polar interactions – including weak electrostatic effects – this approach enables highly selective and precise separations. Given the wide polarity range and chemical diversity of sulphonamides, this method demonstrates broad applicability, extending beyond these compounds to other analytes with similar physicochemical properties. Moreover, it provides a robust and reliable analytical framework for regulatory compliance and quality control in pharmaceutical, food safety, and environmental monitoring. This study underscores the versatility of mixed-mode chromatography, contributing to the development of innovative separation strategies and enhancing the analytical capabilities of modern chromatography techniques.

The results were compared with a commercial ODS-C18 column. While better peak sharpness was achieved with the ODS-C18 column, the C12-DIOL mixed-mode column demonstrated better selectivity. It is generally observed that HILIC and mixed-mode columns, which contain polar groups, exhibit a slight decrease in peak sharpness compared to conventional C18 columns due to the multiple and complex polar interactions. All studies in the mixed-mode field aim to develop columns that not only offer excellent selectivity but also high efficiency. Therefore, the investigation and understanding of mixed-mode separation and interaction mechanisms are crucial for the future development of better, more efficient, and more durable columns. The findings from this study will make a significant contribution to this endeavour. We acknowledge that alternative strategies could offer further insights and hope our study provides a useful foundation for such future efforts.

Acknowledgments

This study constitutes part of Yekun Altun’s master’s thesis. The authors thank the University of Batman for providing laboratory facilities and support.

  1. Funding information: Authors state no funding involved.

  2. Author contributions: Yekun Altun is a master’s student who contributed to the experimental part of this study, which constitutes a part of his master’s thesis. Hayriye Aral contributed to both the conceptualisation and the experimental execution of the study.

  3. Conflict of interest: There are no conflicts of interest to declare.

  4. Data availability statement: The data that support the findings of this study are available in the supplementary material of this article.

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Received: 2025-05-11
Revised: 2025-06-22
Accepted: 2025-06-26
Published Online: 2025-08-12

© 2025 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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  43. Stability studies of titanium–carboxylate complexes: A multi-method computational approach
  44. Efficient adsorption performance of an alginate-based dental material for uranium(vi) removal
  45. Synthesis and characterization of the Co(ii), Ni(ii), and Cu(ii) complexes with a 1,2,4-triazine derivative ligand
  46. Evaluation of the impact of music on antioxidant mechanisms and survival in salt-stressed goldfish
  47. Optimization and validation of UPLC method for dapagliflozin and candesartan cilexetil in an on-demand formulation: Analytical quality by design approach
  48. Biomass-based cellulose hydroxyapatite nanocomposites for the efficient sequestration of dyes: Kinetics, response surface methodology optimization, and reusability
  49. Multifunctional nitrogen and boron co-doped carbon dots: A fluorescent probe for Hg2+ and biothiol detection with bioimaging and antifungal applications
  50. Separation of sulphonamides on a C12-diol mixed-mode HPLC column and investigation of their retention mechanism
  51. Characterization and antioxidant activity of pectin from lemon peels
  52. Fast PFAS determination in honey by direct probe electrospray ionization tandem mass spectrometry: A health risk assessment insight
  53. Correlation study between GC–MS analysis of cigarette aroma compounds and sensory evaluation
  54. Synthesis, biological evaluation, and molecular docking studies of substituted chromone-2-carboxamide derivatives as anti-breast cancer agents
  55. The influence of feed space velocity and pressure on the cold flow properties of diesel fuel
  56. Acid etching behavior and mechanism in acid solution of iron components in basalt fibers
  57. Protective effect of green synthesized nanoceria on retinal oxidative stress and inflammation in streptozotocin-induced diabetic rat
  58. Evaluation of the antianxiety activity of green zinc nanoparticles mediated by Boswellia thurifera in albino mice by following the plus maze and light and dark exploration tests
  59. Yeast as an efficient and eco-friendly bifunctional porogen for biomass-derived nitrogen-doped carbon catalysts in the oxygen reduction reaction
  60. Novel descriptors for the prediction of molecular properties
  61. Special Issue on Advancing Sustainable Chemistry for a Greener Future
  62. One-pot fabrication of highly porous morphology of ferric oxide-ferric oxychloride/poly-O-chloroaniline nanocomposite seeded on poly-1H pyrrole: Photocathode for green hydrogen generation from natural and artificial seawater
  63. High-efficiency photocathode for green hydrogen generation from sanitation water using bismuthyl chloride/poly-o-chlorobenzeneamine nanocomposite
  64. Special Issue on Phytochemicals, Biological and Toxicological Analysis of Plants
  65. Comparative analysis of fruit quality parameters and volatile compounds in commercially grown citrus cultivars
  66. Total phenolic, flavonoid, flavonol, and tannin contents as well as antioxidant and antiparasitic activities of aqueous methanol extract of Alhagi graecorum plant used in traditional medicine: Collected in Riyadh, Saudi Arabia
  67. Study on the pharmacological effects and active compounds of Apocynum venetum L.
  68. Chemical profile of Senna italica and Senna velutina seed and their pharmacological properties
  69. Essential oils from Brazilian plants: A literature analysis of anti-inflammatory and antimalarial properties and in silico validation
  70. Toxicological effects of green tea catechin extract on rat liver: Delineating safe and harmful doses
https://doi.org/10.1515/chem-2025-0185
Schlagwörter für diesen Artikel
mixed-mode; sulphonamides; retention mechanism; log D; log S; quantitative structure–retention relationship
Creative Commons
BY 4.0

Artikel in diesem Heft

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  39. Resveratrol-derived MDM2 inhibitors: Synthesis, characterization, and biological evaluation against MDM2 and HCT-116 cells
  40. Phytochemical constituents, in vitro antibacterial activity, and computational studies of Sudanese Musa acuminate Colla fruit peel hydro-ethanol extract
  41. Chemical composition of essential oils reviewed from the height of Cajuput (Melaleuca leucadendron) plantations in Buru Island and Seram Island, Maluku, Indonesia
  42. Phytochemical analysis and antioxidant activity of Azadirachta indica A. Juss from the Republic of Chad: in vitro and in silico studies
  43. Stability studies of titanium–carboxylate complexes: A multi-method computational approach
  44. Efficient adsorption performance of an alginate-based dental material for uranium(vi) removal
  45. Synthesis and characterization of the Co(ii), Ni(ii), and Cu(ii) complexes with a 1,2,4-triazine derivative ligand
  46. Evaluation of the impact of music on antioxidant mechanisms and survival in salt-stressed goldfish
  47. Optimization and validation of UPLC method for dapagliflozin and candesartan cilexetil in an on-demand formulation: Analytical quality by design approach
  48. Biomass-based cellulose hydroxyapatite nanocomposites for the efficient sequestration of dyes: Kinetics, response surface methodology optimization, and reusability
  49. Multifunctional nitrogen and boron co-doped carbon dots: A fluorescent probe for Hg2+ and biothiol detection with bioimaging and antifungal applications
  50. Separation of sulphonamides on a C12-diol mixed-mode HPLC column and investigation of their retention mechanism
  51. Characterization and antioxidant activity of pectin from lemon peels
  52. Fast PFAS determination in honey by direct probe electrospray ionization tandem mass spectrometry: A health risk assessment insight
  53. Correlation study between GC–MS analysis of cigarette aroma compounds and sensory evaluation
  54. Synthesis, biological evaluation, and molecular docking studies of substituted chromone-2-carboxamide derivatives as anti-breast cancer agents
  55. The influence of feed space velocity and pressure on the cold flow properties of diesel fuel
  56. Acid etching behavior and mechanism in acid solution of iron components in basalt fibers
  57. Protective effect of green synthesized nanoceria on retinal oxidative stress and inflammation in streptozotocin-induced diabetic rat
  58. Evaluation of the antianxiety activity of green zinc nanoparticles mediated by Boswellia thurifera in albino mice by following the plus maze and light and dark exploration tests
  59. Yeast as an efficient and eco-friendly bifunctional porogen for biomass-derived nitrogen-doped carbon catalysts in the oxygen reduction reaction
  60. Novel descriptors for the prediction of molecular properties
  61. Special Issue on Advancing Sustainable Chemistry for a Greener Future
  62. One-pot fabrication of highly porous morphology of ferric oxide-ferric oxychloride/poly-O-chloroaniline nanocomposite seeded on poly-1H pyrrole: Photocathode for green hydrogen generation from natural and artificial seawater
  63. High-efficiency photocathode for green hydrogen generation from sanitation water using bismuthyl chloride/poly-o-chlorobenzeneamine nanocomposite
  64. Special Issue on Phytochemicals, Biological and Toxicological Analysis of Plants
  65. Comparative analysis of fruit quality parameters and volatile compounds in commercially grown citrus cultivars
  66. Total phenolic, flavonoid, flavonol, and tannin contents as well as antioxidant and antiparasitic activities of aqueous methanol extract of Alhagi graecorum plant used in traditional medicine: Collected in Riyadh, Saudi Arabia
  67. Study on the pharmacological effects and active compounds of Apocynum venetum L.
  68. Chemical profile of Senna italica and Senna velutina seed and their pharmacological properties
  69. Essential oils from Brazilian plants: A literature analysis of anti-inflammatory and antimalarial properties and in silico validation
  70. Toxicological effects of green tea catechin extract on rat liver: Delineating safe and harmful doses
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Heruntergeladen am 30.9.2025 von https://www.degruyterbrill.com/document/doi/10.1515/chem-2025-0185/html
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