Simultaneous determination of citalopram, paroxetine, fluoxetine, and sertraline by high-temperature liquid chromatography European Journal of Chemistry 9 (3) (2018) 182-188 European Journal of Chemistry View Journal Online View Article Online Simultaneous determination of citalopram, paroxetine, fluoxetine, and sertraline by high-temperature liquid chromatography Sema Akay * and Berkant Kayan Department of Chemistry, Faculty of Arts and Science, Aksaray University, Aksaray, TR 68100, Turkey e-mail: sema.akay7@gmail.com (S.A.), berkantkayan@gmail.com (B.K.) * Corresponding author at: Department of Chemistry, Faculty of Arts and Science, Aksaray University, Aksaray, TR 68100, Turkey. Tel: +90.382.2882166 Fax: +90.382.2882145 e-mail: sema.akay7@gmail.com (S. Akay). 10.5155/eurjchem.9.3.182-188.1738 Received: 25 May 2018 Received in revised form: 27 June 2018 Accepted: 29 June 2018 Published online: 30 September 2018 Printed: 30 September 2018 A lack of serotonin in the brain is associated with depression. Selective serotonin reuptake inhibitors (SSRIs) are widely used to help treat depression and associated symptoms. A method has been developed for the simultaneous determination of SSRIs by high- temperature liquid chromatography (HTLC). Citalopram, paroxetine, fluoxetine, and sertraline compounds, which are widely used as antidepressant active agents, have been chosen as SSRIs. The separation of the SSRIs have been carried out by using four different column types, including XTerra MS C18, Zorbax SB-Phenyl, Alltima C18 and Phenyl Hypersil columns, and their chromatographic performances have been evaluated. The best separation has been obtained on the Zorbax SB-Phenyl column (150 mm × 4.6 mm, 5 μm) among the four different columns studied. The separation temperature and the composition of mobile phase were examined for the optimization of chromatographic separation. Chromatographic separation of SSRIs has been carried out at temperatures ranging from 100 to 200 °C with variable flow rates (0.5-1.5 mL/min). Water:acetonitrile:acetic acid mixtures containing with 10 or 20% acetonitrile and 2% acetic acid have been used as mobile phase. The best separation was observed at volume ratio of 78:20:2 (water:acetonitrile:acetic acid) at elevated temperature on the Zorbax SB-Phenyl column. The wavelength of UV detector was set at 254 nm. All four analytes were eluted within 8 min at 200 °C. At the end of working, it was observed that the retention times of all four analytes decreased with increasing temperature and was stated that the temperature was an effective parameter for chromatographic separation. Furthermore, the relationship between retention factor and separation temperature was examined using Van’t Hoff plots and the results demonstrated with correlation coefficient greater than 0.91 on Zorbax SB Phenyl column. Consequently, the proposed HTLC method for separation and analysis of SSRIs may be used as a green alternative technique. Column Green chemistry Chromatography High-temperature Antidepressant agent Selective serotonin reuptake inhibitor Cite this: Eur. J. Chem. 2018, 9(3), 182-188 Journal website: www.eurjchem.com 1. Introduction Over the last decade, green chromatography techniques have gained increasing attention and acceptance from researchers both academia and industry. High-temperature liquid chromatography is one of these techniques [1-9]. HTLC exists in a temperature beyond room temperature (ca. 40 C) and below supercritical temperatures [10]. This technique deals with liquid chromatography separations performed at temperatures typically within a range from 40 to 200 °C using organic solvent-water mixtures as the mobile phase [3,5,8]. Because of the long retention times in conventional reversed phase-high performance liquid chromatography (RP- HPLC) separations, the solvent consumption is propor- tionately high [11]. HTLC has several advantages when compared with the conventional HPLC. Major advantages of HTLC include increased efficiency, shortened analysis time, and reduction in mobile phase used an organic solvent. Due to the reduction of the dielectric constant of liquids at elevated temperature could be reduce the ratio of organic solvents in the mobile phase, as long as the temperature is raised appropriately [12,13]. Moreover, the accompanying decrease in mobile phase viscosity provides a lowering of column back pressure, allowing faster separations [13]. The decrease of viscosity at increasing temperatures allows the use of higher flow rates, as well [11]. Consequently, the increase in temperature increases diffusivity of the analyte, resulting in improved column efficiency. All of these positive effects have ensured an increased interest in conducting liquid chroma- tography separations at elevated temperature [13,14]. High-temperature LC (HTLC) is a promising eco-friendly technique for conventional RP-HPLC. However, it is not widely applied in analysis of pharmaceuticals due to limited availa- bility of thermally stable stationary phases at elevated temperature [15]. But, in a series of papers, Yang et al. [3,7,16- 18] concluded that the results obtained by a long-term stability study are quite encouraging. With the help of newly developed stationary phases that are thermally more stable and reliable, liquid chromatographic separations may be ABSTRACT RESEARCH ARTICLE KEYWORDS European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2018 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.9.3.182-188.1738 http://dx.doi.org/10.5155/eurjchem.9.3.182-188.1738 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.9.3.182-188.1738&domain=pdf&date_stamp=2018-09-30 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.9.3.182-188.1738 mailto:sema.akay7@gmail.com mailto:berkantkayan@gmail.com mailto:sema.akay7@gmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.9.3.182-188.1738&domain=pdf&date_stamp=2018-09-30� Akay and Kayan / European Journal of Chemistry 9 (3) (2018) 182-188 183 Table 1. Description of columns tested. Column name Length (mm) I.D. (mm) Particle size (μm) Pore size (Å) Base material X-Terra MS C18 100 4.6 3.5 125 Hybrid silica Zorbax-SB C18 150 4.6 5 80 Porous silica Alltima C18 150 4.6 5 100 Spherical silica Phenyl Hypersil 150 4.6 5 120 Porous silica carried out at elevated temperatures without decomposition of the packing materials. Depression is a common, life-disrupting, potentially lethal illness that can affect all ages [19]. A lack of serotonin in the brain is associated with depression [20,21] that is why drugs called selective serotonin reuptake inhibitors (SSRIs) are widely used to help treat depression [19]. The SSRIs are also effectively used in anxiety, obsessive-compulsive disorder, panic disorder and social phobia [22], and they have occasionally been prescribed for pre-menstrual dysphoric disorder and in the treatment of eating disorders, as well [23]. The SSRIs have a different, and generally better tolerated, adverse effect profile when compared to the other antidepressants with approximately equivalent efficacy [19,24,25]. The antidepressants such as citalopram, paroxetine, fluoxetine, fluvoxamine and sertraline belong to the class of SSRIs [19]. Some studies have been published for the simultaneous determination of several compounds of the SSRI by analytical methods. Almost all assays are based either on separation of SSRIs by high-performance liquid chromatography (HPLC) or gas chromatography (GC) [26,27] and detection by various detectors such as ultraviolet [28], diode array detection [29], fluorescence [30], electrochemical detector, mass spectro- metry, nitrogen-phosphorus detector [31,32]. However, no study has been conducted to determine the four compounds of the SSRI simultaneously by high-temperature liquid chromatography. In the present study, was investigated the potential application of the HTLC technique for separation of SSRIs. The SSRIs such as citalopram, paroxetine, fluoxetine, and sert- raline, which are pharmacologically and environmentally prescriptive and selected as model molecules, have been used. The separation temperature and the composition of mobile phase were examined for the optimization of chromatographic separation conditions. Furthermore, the influence of tempe- rature on retention factor was investigated using Van’t Hoff plots. 2. Experimental 2.1. Chemicals and equipments Citalopram hydrobromide (CIT), paroxetine hydrochloride (PRX), fluoxetine hydrochloride (FLU), and sertraline hydrochloride (SRT) were purchased from Sigma Aldrich Chemical (Steinheim, Germany). HPLC grade acetonitrile and acetic acid were acquired from Merck Chemical (Istanbul, Turkey). Ultrapure water (18.2 MΩ×cm) was obtained from a MilliPore Milli-Q-Gradient water purification system (Billerica, MA, USA). XTerra MS C18 column was purchased from Waters Corporation (Milford, USA), and Zorbax SB-Phenyl column was acquired from Agilent Technologies (Santa Clara, CA, USA). Alltima C18 column was obtained from Grace Materials Technologies (Columbia, USA), and Phenyl Hypersil column was purchased from Thermo Fisher Scientific (Waltham, USA). A Perkin-Elmer Flexar LC system (Waltham, USA) with an ISCO 260-D syringe pump (Teledyne ISCO, Lincoln, NE, USA), Teknosem column heating unit (Istanbul, Turkey), a Peltier cooling unit (Teknosem, Istanbul, Turkey), Flexar LC injector with a 10 µL loop, a back pressure regulator and the Flexar UV detector was used. 2.2. Chromatographic conditions An ISCO 260-D syringe pump and a Perkin-Elmer Flexar LC system were combined and converted into a high-temperature liquid chromatograph. The detailed procedures are described in our previous study [18]. Chromatographic separations were achieved on the determined four commercial columns at different temperatures (100, 150, 175, and 200 °C). Water: acetonitrile:acetic acid mixtures containing with 10 or 20% acetonitrile and 2% acetic acid have been used as mobile phase in separations. Acetic acid was used to control the pH of the mobile phase. Analyte concentrations in test mixture were ranging 20-40 mg/L, and they were freshly prepared using ultra-pure water as the solvent. Chromatographic separations were carried out at flow rates ranging from 0.5 to 1.5 mL/min. The injection volume was 10 μL and the wavelength of detector was set at 254 nm. Data acquisition and analysis were performed with the Perkin-Elmer Chromera chromatography data system. The obtained chromatograms were investigated by taking into account the qualitative separation of solutes. 3. Results and discussion 3.1. Column selection and chromatographic separations The performance of the chromatographic system is determined by the separation carried out in the column, namely the selection and use of the appropriate column filling material. Most of columns used in reversed-phase liquid chromatography contain silica-based phases such as C8, C18. Silica-based RP columns have been widely employed in terms of the popularity and good efficiency. In this study were chosen silica-based different column types such as XTerra MS C18, Zorbax SB-Phenyl, Alltima C18 and Phenyl Hypersil, because literature search shows that these stationary phases are relatively stable at high temperatures [3,7,9]. The description of all four columns is given in Table 1. 3.1.1. Zorbax SB-phenyl column The separation of SSRIs was first investigated on the Zorbax SB-Phenyl column. Chromatographic separations on this column were carried out at temperatures ranging from 100 to 200 °C with ranging 1.0-1.3 mL/min flow rates. Firstly, water-organic solvent mixtures (water:acetonitrile:acetic acid, 93:5:2, v:v:v) have been used as mobile phase in separation. Despite the use of high temperatures, good separation was not achieved and the retention times were relatively long. Separations of the same test mixture were also performed using a stronger mobile phase with 20% acetonitrile and the obtained chromatograms are shown in Figure 1. To limit the length of this paper, only chromatograms obtained at 150 and 175 °C are used. As shown in Figure 1, that all four analytes are well separated and no extra peaks are observed. The retention times of all four analytes decreased at elevated temperatures with adding organic solvent. In addition, it can clearly be seen in Table 2. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.3.182-188.1738 184 Akay and Kayan / European Journal of Chemistry 9 (3) (2018) 182-188 Table 2. HTLC data obtained on four different columns depending on the amount of acetonitrile in the mobile phase with the temperature. Temperature (ᵒC) Flow rate (mL/min) Compounds Retention time Retention factor Tailing factor Peak width Zorbax SB Phenyl (20 % Acetonitrile) 100 1.0 Citalopram 12.16 36 0.94 1.39 Paroxetine 13.48 49 0.93 1.35 Fluoxetine 17.84 67 0.84 1.58 Sertraline 21.60 75 0.79 1.67 150 1.0 Citalopram 7.42 29 0.99 1.30 Paroxetine 9.01 36 0.95 1.24 Fluoxetine 12.04 48 0.93 1.49 Sertraline 14.61 58 0.89 1.56 175 1.2 Citalopram 4.58 21 1.02 1.17 Paroxetine 6.15 24 0.98 1.11 Fluoxetine 8.62 33 0.97 1.40 Sertraline 9.96 39 0.95 1.45 200 1.3 Citalopram 3.01 17 1.05 1.03 Paroxetine 4.12 22 1.02 1.07 Fluoxetine 6.95 27 0.99 1.37 Sertraline 7.87 32 0.97 1.39 XTerra MS C18 (10 % Acetonitrile) 100 0.5 Citalopram 16.34 64 0.80 2.15 Paroxetine 29.57 99 0.69 2.36 Sertraline 52.12 157 0.51 2.67 Fluoxetine 52.58 159 0.52 2.69 150 0.8 Citalopram 6.47 25 0.93 1.89 Paroxetine 10.12 40 0.89 2.22 Sertraline 15.81 61 0.91 2.28 Fluoxetine 15.96 64 0.89 2.29 175 0.9 Citalopram 4.10 15 0.97 1.81 Paroxetine 5.87 23 0.92 2.14 Sertraline 9.65 38 0.95 2.17 Fluoxetine 9.73 40 0.92 2.18 200 1.0 Citalopram 2.48 9 1.00 1.76 Paroxetine 3.90 11 0.98 2.05 Sertraline 6.01 27 0.99 2.11 Fluoxetine 6.04 27 0.99 2.12 Alltima C18 (20 % Acetonitrile) 100 1.0 Citalopram 12.58 40 0.80 2.24 Paroxetine 15.94 59 0.75 2.47 Fluoxetine 24.01 97 0.71 2.56 Sertraline 26.29 101 0.73 2.59 150 1.1 Citalopram 7.02 27 0.89 2.13 Paroxetine 9.88 38 0.84 2.33 Fluoxetine 18.72 71 0.81 2.49 Sertraline 18.93 72 0.80 2.51 175 1.1 Citalopram 5.21 20 0.92 1.94 Paroxetine 7.85 31 0.87 2.19 Fluoxetine 13.87 54 0.92 2.42 Sertraline 14.02 56 0.91 2.43 200 1.2 Citalopram 3.98 14 0.97 1.85 Paroxetine 5.66 23 0.90 2.07 Fluoxetine 9.70 38 0.99 2.31 Sertraline 9.81 39 0.97 2.31 Phenyl Hypersil (10 % Acetonitrile) 100 1.0 Citalopram 72.10 251 0.84 6.02 Paroxetine 95.21 339 0.78 7.10 Fluoxetine - - - - Sertraline - - - - 150 1.3 Citalopram 49.18 193 0.96 4.88 Paroxetine 70.03 278 0.90 5.92 Fluoxetine 94.60 357 0.89 7.21 Sertraline - - - - 175 1.5 Citalopram 33.37 134 1.00 4.35 Paroxetine 51.76 203 0.98 5.47 Fluoxetine 76.89 295 0.96 6.54 Sertraline - - - - 200 1.5 Citalopram 18.95 72 1.03 3.92 Paroxetine 32.35 135 1.00 5.09 Fluoxetine 58.11 248 0.98 6.29 Sertraline - - - - 3.1.2. Separations on XTerra MS C18 The separation of SSRIs was investigated on the XTerra MS C18 column at temperatures ranging from 100 to 200 °C. The separations were obtained with a mobile phase consisting of 10% acetonitrile (water:acetonitrile:acetic acid, 88:10:2, v:v:v) at flow rates ranging from 0.5 to 1.0 mL/min. During the experiments it was stated that the separation with increasing amount of organic solvent performed better, and the elution accelerated with increasing temperature. Figure 2 has shown the chromatograms obtained on this column at different temperatures and varied flow rates. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.3.182-188.1738 Akay and Kayan / European Journal of Chemistry 9 (3) (2018) 182-188 185 (a) (b) Figure 1. Separation of the SSRIs with HTLC on Zorbax SB Phenyl column at (a) 150 ᵒC and (b) 175 ᵒC. Peak identification: 1, citalopram; 2, paroxetine; 3, fluoxetine and 4, sertraline. (a) (b) Figure 2. Separation of the SSRIs with HTLC on XTerra MS C18 column at (a) 150 ᵒC and (b) 175 ᵒC. Peak identification: 1, citalopram; 2, paroxetine; 3, sertraline and 4, fluoxetine. As investigated is Figure 2, no extra peak were observed but, fluoxetine and sertraline were co-elueted in separation, and then citalopram and paroxetine were also co-elueted at 200°C. Thus, good separation was not obtained. It was concluded that this may be due to the fact that XTerra MS C18 column has different properties such as the column length and particle size than the other columns studied. However, it was stated that the retention times of all four analytes decreased with increasing temperature and then the shortest retention time among all columns was achieved on the XTerra MS C18 column at 200 °C with a time of less than 7 minutes. 3.1.3. Separations on Alltima C18 The separation of SSRIs was performed on the Alltima C18 column at temperatures ranging from 100 to 200 °C with ranging 1.0-1.2 mL/min flow rates. The separations were obtained with a mobile phase consisting of 20% acetonitrile (water:acetonitrile:acetic acid, 78:20:2, v:v:v). Some of the chromatograms obtained by Alltima C18 column is shown in Figure 3. The retention times of all four analytes decreased at elevated temperature, but fluoxetine and sertraline were co- elueted as in XTerra column, and so good separation was not achived on this column, as well. In order to make a better separation, it was stated that the composition of mobile phase should be changed, that is, the ratio of the organic solvent should be increased. However, it was concluded that even if the ratio of organic solvent is increased, citalopram and paroxetine peaks will be co-eluted with increasing temperature, so that an effective separation cannot be made. 3.1.4. Separations on phenyl hypersil The separation of SSRIs was finally investigated on the Phenyl Hypersil column (Figure 4). The separations were obtained with a mobile phase consisting of 10% acetonitrile (water:acetonitrile:acetic acid, 88:10:2, v:v:v) at temperatures ranging from 100 to 200 °C with ranging 1.0-1.5 mL/min flow rates. As shown in Figure 4, all three analytes were eluted within 95 min in 150 ᵒC, and sertraline peak was not detected in the seperations. It was stated that the reason is due to the structure of the stationary phase and its interaction with the analytes. Despite the use of high temperatures and high flow rates, good separations were not achieved and the retention times were relatively long on the Phenyl Hypersil column. In addition, the solvent consumption was proportionately high due to the long retention times in separations. Thus, good separation was not obtained on this column. 3.2. Influence of temperature on retention A good strategy for greening LC methods is to minimize the consumption of organic solvents is to speed up the analysis by elevating the mobile phase temperature [15]. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.3.182-188.1738 186 Akay and Kayan / European Journal of Chemistry 9 (3) (2018) 182-188 (a) (b) Figure 3. Separation of the SSRIs with HTLC on Alltima C18 column at (a) 150 ᵒC and (b) 175 ᵒC. Peak identification: 1, citalopram; 2, paroxetine; 3, fluoxetine and 4, sertraline. (a) (b) Figure 4. Separation of the SSRIs with HTLC on Phenyl Hypersil column at (a) 150 ᵒC and (b) 175 ᵒC. Peak identification: 1, citalopram; 2, paroxetine; 3, fluoxetine. Figure 5. Influence of temperature on retention times of SSRI in XTerra MS C18 column. Peak identification: 1, citalopram; 2, paroxetine; 3, sertraline and 4, fluoxetine. Because temperature is a strong variable that affects selectivity and efficiency. Elevated temperature can signi- ficantly reduce the retention times of analytes (Figure 5). In addition, Table 2 summarizes chromatographic data such as retention times (min), retention factors, tailing factors, and peak width (min) for HTLC separations at working tempe- ratures. Along with the increased flow rate, the peak widths of the compounds were reduced as well as the retention times. Besides the mobile phase additives such as acetic acid, the high temperature has also improved the peak symmetries of the compounds. The relationship between separation temperature and retention factor was examined using van’t Hoff plots. The result demonstrated linear correlation between ln(k′) and 1/T for separations on Zorbax SB Phenyl column with correlation coefficient greater than 0.91 (Figure 6). 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.3.182-188.1738 Akay and Kayan / European Journal of Chemistry 9 (3) (2018) 182-188 187 R² = 0.9109 R² = 0.942 R² = 0.9524 R² = 0.9215 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1 4.3 4.5 0.0020 0.0021 0.0022 0.0023 0.0024 0.0025 0.0026 0.0027 0.0028 ln k 1/T (K⁻¹) Citalopram Paroxetine Fluoxetine Sertraline Figure 6. Van’t Hoff Plots for separations on Zorbax SB Phenyl column. (a) (b) Figure 7. Influence of composition of mobile phase on HTLC separations in Zorbax SB Phenyl column. (a) 10 % Acetonitrile at 150 ᵒC (b) 20 % Acetonitrile at 150 ᵒC. Peak identification: 1, citalopram; 2, paroxetine; 3, fluoxetine and 4, sertraline. In addition, the standard enthalpy (ΔHo) values for SSRIs were calculated from the slope of the Van’t Hoff plots and were obtained the values for all solutes are negative. The standard enthalpy values calculated for citalopram, paroxetine, fluoxetine and sertraline are ΔHCITᵒ= -10.81 kJ/mol; ΔHPRXᵒ= - 12.18 kJ/mol; ΔHFLUᵒ= -13.30 kJ/mol; ΔHSRTᵒ= -12.60 kJ/mol, respectively. It was stated that solute transfer is an exothermic process, and it is energetically more suitable for the solutes to remain in the stationary phase. Due to the viscosity of the mobile phase is significantly decreased with increasing temperatures, the obtained low back-pressure allows to be employed much higher flow rates [3]. Thus, the increased flow rate greatly decreases the retention time, and shortens the exposure of the analyte to high temperatures. In addition, analyte degradation is not observed, and stationary phases are not much damaged, as well. Therefore, the advantages of studying at elevated temperatures such as reduced solvent consumption, shorten analysis time, and efficient separation allow HTLC to be used as a green tool for routine analysis. 3.3. Influence of composition of mobile phase on separation The composition of mobile phase is another important parameter in separation. Most of the HTLC studies [1,13,18,33] used of water-organic solvent mixtures as mobile phases. This is because the elution strength of pure heated water is not strong enough for analysis most of the non-polar analytes in commercially available reversed-phase columns [33]. Considering that water acts as an organic solvent during the separations carried out at high temperatures [3,7,34,35], the ratio of modifier used in the mobile phase was kept constant at maximum 20% (Figure 7). In addition, it is believed that a high ratio of organic solvent is disadvantageous both in terms of chemical consumption and environment. As shown in Figure 7, these significant improves in separation efficiency with modifier in mobile phase can clearly be seen as an added benefit to performing the separations at elevated temperatures. Consequently, at elevated temperature the use of acetonitrile-water mixture as a mobile phase can be an efficient method to separate SSRIs with the benefit of consuming little acetonitrile than conventional separation methods. 4. Conclusion Chromatographic analysis of citalopram, paroxetine, fluoxetine, and sertraline were carried out on four commercial columns using HTLC. Due to their better stability at higher temperatures, these columns were chosen in this work. The best separations were achieved on Zorbax SB-Phenyl column. No extra peaks were observed in columns, namely analyte degradation is not observed and also these stationary phases 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.3.182-188.1738 188 Akay and Kayan / European Journal of Chemistry 9 (3) (2018) 182-188 are stable under high-temperature liquid chromatography conditions. Although HTLC separation of SSRIs samples were carried out in this work, industrial application of the HTLC methods developed in this study will require further validation studies. The separation temperature is the most important parameter for the optimization of chromatographic separation conditions in this study. In that, the relationship between retention factor and separation temperature was examined using van’t Hoff. It was observed that the retention times of all four solutes decreased with increasing tempe- rature. Besides, the ratio of organic solvent in mobile phase is considerably reduced with increasing temperature. Thus, the results obtained in this study indicated that using acetonitrile– water mixtures at elevated temperatures as an alternative to the conventional RP-HPLC mobile phases can be reduce the ratio of acetonitrile used to achieve effective separation. On the other hand, it has been noted in the study that the mobile phase composition, i.e. the acetonitrile ratio, is the second effective parameter for separation. The best separation was observed at volume ratio of 20% acetonitrile at elevated temperature with the Zorbax SB-Phenyl column. Namely, the power of elution of the pure water was increased with acetonitrile in mobile phase. It was stated that the separation with increasing amount of organic solvent performed better. Therefore, the retention time significantly decreased with increasing temperature combined with a stronger modifier. It was stated the advantages of studying at elevated tempe- ratures such as shorten analysis time and efficient separation allow HTLC usage. Consequently, the data of study demonst- rated that HTLC may be used as a green alternative technique for separation and analysis of SSRIs. Acknowledgments The authors gratefully thank the Scientific Research Project Unit (BAP) of Aksaray University (2016/038) for the financial support of this work. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Author contributions: All authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. ORCID Sema Akay http://orcid.org/0000-0001-8887-6653 Berkant Kayan http://orcid.org/0000-0002-9892-5284 References [1]. Greibrokk, T.; Andersen, T. J. Chromatogr. A 2003, 1000, 743-755. [2]. Teutenberg, T.; Goetze, H. J.; Tuerk, J.; Ploeger, J.; Kiffmeyer, T. K.; Schmidt, K. G.; Kohorst, W.; Rohe, T.; Jansen, H. D.; Weber, H. J. Chromatogr. A 2006, 1114, 89-96. [3]. Yang, Y. J. Sep. 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Fundamental and Applied Studies on Chromatographic Separation of Cold Drugs and Skincare Creams and Extraction of Salvia Miltiorrhiza Using Subcritical Water, The Scholarship ECU, 2014. Copyright © 2018 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.3.182-188.1738 http://orcid.org/0000-0001-8887-6653 http://orcid.org/0000-0002-9892-5284 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Chemicals and equipments 2.2. Chromatographic conditions 3. Results and discussion 3.1. Column selection and chromatographic separations 3.1.1. Zorbax SB-phenyl column 3.1.2. Separations on XTerra MS C18 3.1.3. Separations on Alltima C18 3.1.4. Separations on phenyl hypersil 3.2. Influence of temperature on retention 3.3. Influence of composition of mobile phase on separation 4. Conclusion Acknowledgments ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: