Date-Leaf Carbon Particles for Green Enhanced Oil Recovery

Green enhanced oil recovery (GEOR) is an environmentally friendly enhanced oil recovery (EOR) process involving the injection of green fluids to improve macroscopic and microscopic sweep efficiencies while boosting tertiary oil production. Carbon nanomaterials such as graphene, carbon nanotube (CNT), and carbon dots have gained interest for their superior ability to increase oil recovery. These particles have been successfully tested in EOR, although they are expensive and do not extend to GEOR. In addition, the application of carbon particles in the GEOR method is not well understood yet, requiring thorough documentation. The goals of this work are to develop carbon nanoparticles from biomass and explore their role in GEOR. The carbon nanoparticles were prepared from date leaves, which are inexpensive biomass, through pyrolysis and ball-milling methods. The synthesized carbon nanomaterials were characterized using the standard process. Three formulations of functionalized and non-functionalized date-leaf carbon nanoparticle (DLCNP) solutions were chosen for core floods based on phase behavior and interfacial tension (IFT) properties to examine their potential for smart water and green chemical flooding. The carboxylated DLCNP was mixed with distilled water in the first formulation to be tested for smart water flood in the sandstone core. After water flooding, this formulation recovered 9% incremental oil of the oil initially in place. In contrast, non-functionalized DLCNP formulated with (the biodegradable) surfactant alkyl polyglycoside and NaCl produced 18% more tertiary oil than the CNT. This work thus provides new green chemical agents and formulations for EOR applications so that oil can be produced more economically and sustainably.

Introduction
The oil production processes from a reservoir are grouped into three classes: primary, secondary, and tertiary [1]. In the primary stage, oil is produced due to natural drive mechanisms, for example, water, gas cap, solution gas, etc. The secondary process is launched after the weakening of natural energy. Waterflooding and pressure maintenance are common secondary recovery methods. The tertiary oil recovery, known as the enhanced oil recovery (EOR) method, is introduced when the second technique is no longer economically feasible [2,3]. The common EOR methods are chemical, gas, thermal, and others [4][5][6]. The chemical EOR method involves injecting surfactant, polymer, alkaline, and alcohol chemicals to alter interfacial tension (IFT), wettability, phase behavior, and

Carbon Nanoparticle in EOR
The influence of MWCNT on IFT and surface tension was examined at room temperature by Soleimani et al. [18]. The optimum MWCNT concentration was achieved at 0.3 wt%. This solution produced 18.57% incremental oil from a glass bead experiment. The rheological properties of a mixture of an acrylamide polymer and MWCNT were tested in a high-pressure high-temperature (HP-HT) and high salinity environment [19]. Improvements in viscosity and stability in the harsh HP-HT environment were achieved by negative polyelectrolyte and polyampholytic polymers. The dispersion effects of CNT hybrids in foam and emulsion were studied in porous media by Kadhum et al. [20]. It was found that a stable CNT dispersion was obtained using a highly polarized polymer such as Arabic gum and polyvinyl pyrrolidone. An experimental study was conducted to examine the foam stability and viscosity of a surfactant polymer and MWCNT blend [21]. Investigation reveals that MWCNT could improve flow behavior in the foam of porous media. A-Dots or Arab-D dots were applied in a giant Ghawar field in Saudi Arabia to explore EOR potential [22]. A core flood experiment was conducted at 95 • C before the field trial and was followed by a post-flood with 120,000 ppm salinity brine. The average porosity, permeability, and pore volume values of the core plug were 20.3%, 9.89 mD, and 18.74%, respectively. A concentration of 0.001% w/w (10 ppm) of A-Dots solution was injected at a rate of 0.10 cm 3 /min. The solution occupied about 20% of the total pore volume (3.8 cm 3 ). The oil recovery factor reported was 96%. A huff-and-puff method was applied in an Arab-D field trial. The production period was two days and the shut-in time was three days. The distance between the injection and production wells was 1 to 3 km. A total of 5 kg of A-Dot particles were mixed with 255 bbl of injected water. The solution was then injected at a rate 3300 bbl/day. The injection pressure and temperature were 1500 psi and 90 • C, respectively. The overall field trial outcome reported an oil recovery of approximately 82% implying that nano agent concepts are promising in boosting the recovery amount of trapped oil. Table 1 is a brief highlight of some nanomaterials with their EOR potentials. [24]

MWCNTs/ Biopolymer
The effect of CNT in a harsh high-salinity high-temperature (HS-HT) environment in EOR.

High salinity and temperature
Free-radical copolymerization NMR, GPC, FTIR, DSC and TEM.
After several syntheses, the negative polyelectrolyte and polyampholytic polymers served as the best candidate for MWCNTs/polymers in remote environments. [19] Magnetic iron core-carbon shell nanoparticles IFT studies via spinning drop. Adsorption reduction studies. EOR potential.
Achieved an optimum concentration with nano-additives. Nanoparticles reduced surfactant adsorption by 33%.
A final oil recovery of 98% was achieved. [

Nanoparticle Preparation
Carbon-based nanomaterials (CBNs) are emerging as an essential topic in the fields of science and technology. Carbon and its allotropes have been used widely in various applications (such as fiber optics) due to unique aspects such as its excellent physical, chemical, thermal, electrical, and biological properties [28]. Other applications include electrochemical sensors [29,30], electronics [31], drug delivery [32], energy storage [33], solar cells [34], environmental pollutant removal [35], construction materials [36,37], and various materials science applications [38][39][40][41][42][43][44][45]. While these nanomaterials are receiving significant attention, the conventional preparation methods for CBNs are complicated and expensive, thus, limiting their utilization. Consequently, alternative forms of developing CBNs via relatively simple, cost-effective, and sustainable approaches are of great interest. CBN production from biomass could offer an ideal economic and sustainable system. The leaves from trees and other forestry are abundantly available and often go unused. It would be perfect to utilize this biological waste as a cheap material for conversion into value-added carbon products useful in several potential applications.
Ball milling and pyrolysis, among various methods, were adopted for nanomaterial preparation and carbonization respectively. Ball milling is a simple and economical method that allows for the synthesis of nanomaterials on a large scale. It is a top-down technique where any powdered material is mechanically milled into nanoparticles using balls of various stiffnesses. The kinetics of milling depends on the milling energy, type, and size of the balls, milling speed, temperature, and duration of the milling process. Various nanocrystalline/amorphous materials were synthesized using this methodology [46][47][48][49]. On the other hand, pyrolysis is a simple and popular controlled thermochemical treatment technique that is employed to convert waste or any other biomass into valuable products. It is commonly used to prepare biochar, charcoal, and biogas for various commercial applications. Many waste materials, such as rice husk [50], jute sticks [35], date palm [51], wood waste [52], and tree/plant leaves [53,54], were converted into value-added products using this technique.

Materials
The date leaves were collected from the date gardens of the King Fahd University of Petroleum and Minerals (KFUPM) campus. Concentrated H 2 SO 4 and concentrated HNO 3 were obtained from Sigma-Aldrich. Deionized water was used in the experiments. Other materials used in this work are: (1) 99.9% pure sodium chloride (NaCl), (2) distilled water, and (3) chemical dissolvent: naphtha, ethel-methyl ketone, and acetone.

Preparation of the Date-Leaf Carbon Nanoparticles and Carboxylic Acid Functionalization
Date leaves collected from the date gardens on the KFUPM campus served as the source of raw material. After collection, they were dried entirely in the sunlight and then cleaned with distilled water and oven-dried at 110 • C for 24 h to remove any moisture content. The leaves were then shredded into pieces 2 to 3 cm long and pyrolyzed in a tube furnace under N 2 atmosphere at 850 • C for approximately 3 h at heating and cooling rates of 10 • C and 5 • C, respectively. The pyrolytic carbon from the dried date leaves was then powdered using a kitchen blender for approximately 5 min. (This carbon powder is referred to as "ground carbon" in the remainder of the manuscript). Next, the ground carbon was subjected to a high-energy ball-milling technique to synthesize the nanoparticles. The ball-milling machine was operated at a speed of 3000 rpm for approximately 15 h. The particle size reduction was monitored at different intervals of 3 h, 9 h, and 15 h using microscopic analysis. Zirconium balls 600 to 800 µm in size at a ratio of 1:20 by mass of Zr to carbon content were used during the ball-milling process. After completing the ball milling, the surface of the nanocarbon was modified via carboxylic functionalization with sulfuric (H 2 SO 4 ) and nitric acid (HNO 3 ) as per previous studies [35,55]. Typically, 1 g of the carbon particles (obtained after 15 h of ball milling) was added to a mixed acid solution (1 L of concentrated H 2 SO 4 and concentrated HNO 3 ) at a ratio of 3:1 by volume. The mixture was ultrasonicated for 5 h to form the carboxylic group. The sonicated solution was then diluted with deionized water and kept undisturbed for 8 h to settle the carboxylated carbon mass on the bottom of the beaker. The mother liquid segregated at the top was decanted. This procedure was repeated six times. Finally, the carboxylic-functionalized carbon was filtered and dried for 24 h at 60 • C. The preparation process for the functionalized date carbon is shown schematically in Figure 1.
nanoparticles. The ball-milling machine was operated at a speed of 3000 rpm for approximately 15 h. The particle size reduction was monitored at different intervals of 3 h, 9 h, and 15 h using microscopic analysis. Zirconium balls 600 to 800 µm in size at a ratio of 1:20 by mass of Zr to carbon content were used during the ball-milling process. After completing the ball milling, the surface of the nanocarbon was modified via carboxylic functionalization with sulfuric (H2SO4) and nitric acid (HNO3) as per previous studies [35,55]. Typically, 1 g of the carbon particles (obtained after 15 h of ball milling) was added to a mixed acid solution (1 L of concentrated H2SO4 and concentrated HNO3) at a ratio of 3:1 by volume. The mixture was ultrasonicated for 5 h to form the carboxylic group. The sonicated solution was then diluted with deionized water and kept undisturbed for 8 h to settle the carboxylated carbon mass on the bottom of the beaker. The mother liquid segregated at the top was decanted. This procedure was repeated six times. Finally, the carboxylic-functionalized carbon was filtered and dried for 24 h at 60 °C. The preparation process for the functionalized date carbon is shown schematically in Figure 1.

Characterization
The morphologies of the ground carbon, 15 h ball-milled carbon, and carboxylic acidfunctionalized carbon were characterized by field emission scanning electron microscopy (FE-SEM) (Tescan Lyra-3, Kohoutovice, Czech Republic) and transmission electron microscopy (TEM) (JEM-2011; JEOL, Tokyo, Japan). Elemental analysis was carried out using energy-dispersive X-ray spectroscopy (the Lyra-3 attachment to the FE-SEM through the LINK INCA program, Oxford, UK). Surface area, pore size distribution, and structural information were obtained using BET and Raman spectroscopic analysis. A micro-focusing X-ray monochromator XPS (ESCALAB 250Xi XPS Microprobe, Thermo Scientific, Waltham, MA, USA) was used for the X-ray photoelectron spectroscopy (XPS) analysis. All analyses were performed at the Research Institute of KFUPM in Saudi Arabia.

IFT Measurement
The force per unit length at the fluid-fluid and rock-fluid interfaces is referred to as interfacial tension (IFT). IFT between two fluids represents the amount of work required

Characterization
The morphologies of the ground carbon, 15 h ball-milled carbon, and carboxylic acidfunctionalized carbon were characterized by field emission scanning electron microscopy (FE-SEM) (Tescan Lyra-3, Kohoutovice, Czech Republic) and transmission electron microscopy (TEM) (JEM-2011; JEOL, Tokyo, Japan). Elemental analysis was carried out using energydispersive X-ray spectroscopy (the Lyra-3 attachment to the FE-SEM through the LINK INCA program, Oxford, UK). Surface area, pore size distribution, and structural information were obtained using BET and Raman spectroscopic analysis. A micro-focusing X-ray monochromator XPS (ESCALAB 250Xi XPS Microprobe, Thermo Scientific, Waltham, MA, USA) was used for the X-ray photoelectron spectroscopy (XPS) analysis. All analyses were performed at the Research Institute of KFUPM in Saudi Arabia.

IFT Measurement
The force per unit length at the fluid-fluid and rock-fluid interfaces is referred to as interfacial tension (IFT). IFT between two fluids represents the amount of work required to create a new unit of surface area at the interface. It can also be assumed as a measure of the immiscibility of two fluids. IFT values between the Arabian light crude oil and the date-leaf solutions were measured according to standard ASTM D971-99a at different mixing ratios. Five samples of DLCNP were prepared at 200 mg/L to 800 mg/L concentration with deionized distilled water. Arabian light crude oil (ρ @ 25 • C = 0.8286 g/cm 3 ) was obtained from the Saudi Aramco company. The IFT values were then measured via the ASTM D 971-99a method [56]. The step-by-step process is presented here. First, the glass containers for the experiment were cleaned using naphtha, ethel-methyl ketone, and an acetone chemical solvent. After rinsing with tap and distilled water, the glass containers were dried and covered using aluminum foil to keep them safe and clean. Second, naphtha, ethel-methyl ketone, and an acetone chemical dissolvent were then used to clean the Du Noüy ring. A butane gas flame was used as a flaming agent for approximately 5 s until the equipment slightly glowed orange. The ring dimensions are given in Table A1 in Appendix A. Third, at an ambient temperature of 25 • C, the densities of the crude oil and samples were measured to ascertain the density difference between the two liquid phases, Table A2 in Appendix A. Fourth, the heating system was then connected to the tensiometer and set to the temperature mentioned above. Fifth, intermittently during the experiment, the glass containers were cleaned and the tensiometer calibrated to avoid an error. This was achieved by ensuring that the surface tension of the distilled water measured at 25 • C fell within 71 to 73 dyne/cm. Any value outside this range signified an error from the Nanomaterials 2022, 12, 1245 6 of 21 calibration or glass. Sixth, each sample was poured into a clean glass container to a depth of 10 to 15 mm. After waiting for approximately 12 min, the temperature stabilized at 25 • C. Seventh, the ring was immersed in the sample such that its depth was less than 6 mm. Then, the heavy phase was carefully covered with crude oil such that its center was more than 10 mm. Eighth, after aging the sample-oil interface for approximately 30 s, the rupture value was recorded by lowering the platform. Finally, the IFT was then corrected through the software built into the Sigma 702 force tensiometer. The Zuidema and Waters method was used to correct the IFT value [56].

Contact Angle Measurement
The tendency for a solid to prefer one fluid over another is called wettability. Wettability is a function of chemical composition and it has been largely attributed to changes in the pore system (adsorption), which may be due to rock fluid interaction, fluid-fluid interactions, rock mineralogy, and brine chemistry [57]. The surface can be either oil-wet or water-wet, depending on the chemical composition of the fluids. The wettability can be determined through contact angle measurement. The contact angle can be defined as the angle formed by the intersection of the liquid-solid interface and the liquid-vapor interface. A contact angle in the range of 0-70 • indicates water-wet conditions, a contact angle in the range of 70-100 • indicates intermediate-wet conditions and a contact angle in the range of 105-180 • indicates oil-wet conditions [57]. The Drop Shape Analyzer (DSA) 100 is designed to measure contact angle if one fluid is transparent because it measures the drop shape. The 800 mg/L (ppm) DLCNP solution and Arab light crude were selected for measuring the contact angle. The drop was made by DLCNP, and the surrounding phase was Arab Light crude oil. Both phases were dark black, and the image was not captured. Due to this difficulty, the contact angle measurement was not successful. It could be possible if a low concentration such as 100 (mg/L) was taken. However, the 100 mg/L concentration was not selected for the core flood which is vital for EOR performance prediction.

Influence of Pressure and Temperature on IFT
The effect of temperature and pressure on the DLCNP was determined by DSA 100. A pictorial representation of DSA 100 is shown in Figure S1 in the Supplementary Material. First, N-Hexane was used to clean the IFT cells, lines, and cylinder before applying acetone and water. The equipment was then dried with an airflow. Second, the IFT of water at 72.2 mN/m was used to calibrate the IFT cell under ambient conditions. The single-piston syringe pump and/or hand pump of the automated HP were used to fill both cells by transferring the relevant and required fluids. Third, the IFT cell was filled with brine using the hand pump before calibrating the system. The system calibration was performed by measuring the needle size while the software was running. Then, an oil bubble was formed by pumping crude oil through the needle. The injection of brine set the test pressure into the cell, and a thermocouple was used to set the temperature surrounding the cell. Finally, after the desired pressure and temperature were attained for the measurement, the cell was kept for approximately 30 min under stable conditions to allow the two phases to reach equilibrium. At this point, the IFT was measured by actively running the software.

Phase Behavior Study
The phase behavior of a green surfactant system is an important parameter for the oil recovery process. The aims were to determine the middle phase microemulsion and optimum salinity of the green surfactant system. The phase behavior is a simple laboratory technique utilizing the salinity concept. The surfactant concentration was kept constant, NaCl concentration was varied, and the water-oil ratio (WOR) was 1. The volume of oil was 4.5 mL, and the volume of the aqueous phase was 4.5 mL. The aqueous phase consisted of 0.50% APG surfactant and varied (1-7%) NaCl concentrations. The stable phase volume was measured when it reached equilibrium. The calculated volumes are given in Table 2 with an error limit of ±0.01.

Core Flood Experiments
Core flood experiments were performed to determine the behavior of nanofluid inside the reservoir at reservoir pressure and temperature levels. The amount of oil recovered by a given formulation during a core flood can provide a good indication of the possible tertiary recovery potential under real reservoir conditions. Initially, the core samples after cutting and oven-dried (50 • C) for 3 days were measured and placed in the core holder where an overburden pressure (1050 psi) was applied. Thereafter, a vacuum was applied to the core for 24 h to remove any air. The core was saturated with a brine of 2% NaCl concentration and then flooded with 30 • API Arab light crude oil (drainage) until the core reached residual water saturation (S wi ); at this point, the core was saturated with oil. Then imbibition process of the oil-saturated core sample began by flooding with 2% NaCl brine at an injection rate of 0.50 cm 3 /min until it was near residual oil saturation (Sor) and ready for the formulations (nanofluid/surfactants) treatment. Typically, brine flooding was stopped when only a trace of oil was being produced. The next step was to continue injection by the different formulations at 0.50 cm 3 /min. Post flooding was carried out thereafter, to ensure that no oil remains in the tube and cores. The schematics for the core flood experiment setup are presented in Figure S2 in the Supplementary Material. The properties of the core samples used in the experiment are presented in Table 3 whereas the formulation of the three nanoparticle/surfactant combinations is given in Table 4.  from 0.5 µm to 6 µm. The elemental analysis of the ground carbon indicated that C and O existed in significant quantities, while only traces of Si, Ca, K and Mg were present. Note that Cu and Au peaks were caused by the copper substrate and gold coating on the surface of the sample.

3
800 ppm CNT + 0.5% APG + 2% NaCl 1050 50 0.5 30 Figure 2a,b are the FE-SEM images of the ground carbon at two different magnifications. The corresponding elemental analysis is shown in Figure 2c. From Figure  2a,b, it is clear that the particle sizes of the carbon powder were widely distributed, typically ranging from 0.5 µm to 6 µm. The elemental analysis of the ground carbon indicated that C and O existed in significant quantities, while only traces of Si, Ca, K, and Mg were present. Note that Cu and Au peaks were caused by the copper substrate and gold coating on the surface of the sample. The size reduction of the carbon particles was monitored by measuring the grain size via FE-SEM imaging. Figure 3a-f show the FE-SEM images of the ball-milled carbon at three intervals (3,9, and 15 h) on two magnification scales intended to measure the size reduction, while Figure 3g represents the chemical composition of the 15 h ball-milled carbon. Figure 3a,b are the FE-SEM images of the 3hr ball-milled carbon at two magnifications (1 µm and 500 nm). The particle size was reduced significantly by the ball The size reduction of the carbon particles was monitored by measuring the grain size via FE-SEM imaging. Figure 3a-f show the FE-SEM images of the ball-milled carbon at three intervals (3, 9 and 15 h) on two magnification scales intended to measure the size reduction, while Figure 3g represents the chemical composition of the 15 h ball-milled carbon. Figure 3a,b are the FE-SEM images of the 3 h ball-milled carbon at two magnifications (1 µm and 500 nm). The particle size was reduced significantly by the ball milling. A typical particle size measurement on the FE-SEM images indicated particles of the following sizes: 28% > 1 µm, 60% between 1 µm and 500 nm, and 12% between 500 and 300 nm. The average particle size was around 840 nm. Figure 3c,d are the FE-SEM images of the 9 h ball-milled carbon at two magnifications (i.e., 1 µm and 500 nm). It can be observed that the particle size was further reduced by continued ball milling. Here, the particle size was 7% > 1 µm, 30% between 1 µm and 500 nm, 50% between 500 and 300 nm, and 12% between 300 and 100 nm with an average particle size of 570 nm. Similarly, 15 h ball milling, the particle size was significantly further reduced, i.e., particle sizes were 0% > 1 µm, 11% between 1 µm and 500 nm, 26% between 500 and 300 nm, 37% between 300 and 100 nm, and 26% < 100 nm, with an average particle size of 270 nm, Figure 3e,f. Hence, ball milling reduced the particle size of carbon from a few microns to the submicron/nanoscale. The EDS shown in Figure 3g indicates the elemental composition of the nanocarbon. The presence of Zr is attributable to the zirconium balls used for the ball milling. Figure 4a is the TEM image of the nanosized date carbon after functionalization with sulfuric and nitric acid at 200 nm magnification. The particle grain boundaries on the TEM grid are visible in Figure 4a, indicating that the particle size was reduced due to the carboxylic functionalization; most of the particles shrank to the 50 to 150 nm range. The HRTEM image shown in Figure 4b indicates the amorphous nature of the carbon, as no lattice fringes can be observed. This was also confirmed from the selected area electron diffraction (SAED) pattern shown in Figure 4c. The elemental composition of the functionalized nanosized date carbon is shown in Figure 4d, indicating that C and O were the major elements while Si and Zr appeared only in trace amounts. nm, and 12% between 300 and 100 nm with an average particle size of 570 nm. Similarly, 15 h ball milling, the particle size was significantly further reduced, i.e., particle sizes were 0% > 1 µm, 11% between 1 µm and 500 nm, 26% between 500 and 300 nm, 37% between 300 and 100 nm, and 26% < 100 nm, with an average particle size of 270 nm, Figure 3e,f. Hence, ball milling reduced the particle size of carbon from a few microns to the submicron/nanoscale. The EDS shown in Figure 3g indicates the elemental composition of the nanocarbon. The presence of Zr is attributable to the zirconium balls used for the ball milling.  Figure 4a is the TEM image of the nanosized date carbon after functionalization with sulfuric and nitric acid at 200 nm magnification. The particle grain boundaries on the TEM grid are visible in Figure 4a, indicating that the particle size was reduced due to the carboxylic functionalization; most of the particles shrank to the 50 to 150 nm range. The HRTEM image shown in Figure 4b indicates the amorphous nature of the carbon, as no lattice fringes can be observed. This was also confirmed from the selected area electron grid are visible in Figure 4a, indicating that the particle size was reduced due to the carboxylic functionalization; most of the particles shrank to the 50 to 150 nm range. The HRTEM image shown in Figure 4b indicates the amorphous nature of the carbon, as no lattice fringes can be observed. This was also confirmed from the selected area electron diffraction (SAED) pattern shown in Figure 4c. The elemental composition of the functionalized nanosized date carbon is shown in Figure 4d, indicating that C and O were the major elements while Si and Zr appeared only in trace amounts.

XPS Analysis
The functional group's formation on the surface of the carboxylic functionalized carbon was studied using the XPS technique. The XPS survey and deconvoluted C1s and O1s spectrum before (i.e., 15 h ball-milled carbon) and after the acid functionalization carbon are shown in Figure 5. The XPS survey spectrum in Figure 5a,d indicates the existence of C and O as major constituents of the carbon, Si is attributed from the glass substrate/from the carbon sample as a trace amount of Si was observed in EDS analysis of the carbon. While minor traces of Zr are from the ZrO 2 ball used during ball milling. Figure 5b,c depict the deconvoluted C1s and O1s XPS spectrum of 15 h ball-milled carbon. The C1s deconvoluted XPS spectrum indicates the presence of a C-C sp 2 hybridized peak at 284.6 eV, C-C sp 3 peak at 285.6 eV, and O-C=OH at 289.0 eV, respectively, while the O1s deconvoluted XPS spectrum exhibits two peaks at 534.26 eV and 533.38 eV corresponding to -(C=O) and -(C=O)-C bond, respectively [58]. The deconvoluted XPS spectrum of C1s and O1s after carboxylic acid functionalization is shown in Figure 5e,f. The functionalized carbon C1s deconvoluted spectra exhibited a C-C sp 2 peak at 284.6 eV, C-O-C sp 3 peak at 286.6 eV, and O-C=OH peak at 288.5 eV, respectively, while the deconvoluted O1s XPS spectrum of the functionalized carbon indicates two peaks at 532.0 eV and 530.68 eV, respectively, corresponding to -(C=O)-C and O=C bond [58]. It is noted that the peak area of the O-C=OH (Figure 5e) peak in the acid-functionalized carbon is much higher than the ball-milled carbon (Figure 5b). Thus, the XPS technique indicates the formation of carboxylic functional groups after the acid functionalization of the carbon. The functionalized group formation is similar to the study reported earlier [35,55,59]. Table 5 is a quantitative elemental analysis from the XPS survey spectrum before and after carboxylic acid functionalization of the carbon.
respectively, corresponding to −(C=O)-C and O=C bond [58]. It is noted that the peak area of the O-C=OH (Figure 5e) peak in the acid-functionalized carbon is much higher than the ball-milled carbon (Figure 5b). Thus, the XPS technique indicates the formation of carboxylic functional groups after the acid functionalization of the carbon. The functionalized group formation is similar to the study reported earlier [35,55,59]. Table 5 is a quantitative elemental analysis from the XPS survey spectrum before and after carboxylic acid functionalization of the carbon.

BET Analysis
The specific surface area of the carbon was determined using N 2 adsorption-desorption isotherms by the Brunauer-Emmett-Teller (BET) method [60], while the pore size distribution was analyzed using the Barrett-Joyner-Halenda (BJH) method. The N 2 adsorption/desorption isotherms at 77 K for the ground carbon, 15 h ball-milled carbon, and 15 h ball-milled carboxylic-functionalized carbon are depicted in Figure 6. As per the IUPAC classifications, the isotherms for the ground and functionalized carbon appeared to be Type I, indicating that the size distribution of the carbon was microporous. The overlapping adsorption-desorption curves suggest that the micro-porosity of the carbon was narrow and uniform. The 15 h ball-milled carbon belonged to the Type II isotherm, with a relatively high adsorption/desorption capacity. The complete hysteresis loop between 0.4 and 1.0 (P/P o ) indicated mesopores, while the sudden increase in adsorption from 0.9 to 1.0 (P/P o ) demonstrated the presence of macropores [61]. The BET surface area of the ground carbon increased from 254.4 m 2 /g to 331.3 m 2 /g due to the 15 h of ball milling, while the carboxylic functionalization of the 15 h ball-milled carbon significantly decreased the BET surface area from 331.3 to 73.7 m 2 /g. This reduction in BET surface area, nearly five times that of the ball-milled carbon, can be attributed to the oxidation of the carbon with the acid. The decrease in BET surface area due to carboxylic functionalization is consistent with data reported in other studies [35,62].  Figure 7 shows the Raman spectrum of the ground pyrolytic date carbon (pyrolysis at 850 °C) after being ball-milled for 15 h, and subsequently, acid-functionalized, measured at a wavelength of 532 nm. The two intense peaks in the spectrum correspond to graphitization (G-band) and defects (D-band). The G-band and D-band peaks for the ground carbon can be observed at around 1591 cm −1 and 1352 cm −1 , respectively. For the 15 h ball-milled carbon, the G-band and D-band values were also around 1589 cm −1 and 1352 cm −1 . In contrast, for the functionalized carbon, the G-band and D-band values were 1591 cm −1 and 1357 cm −1 . The intensities of the G-and D-bands of the ground and functionalized carbon were almost the same, but significantly lower and broader for the 15 h ball-milled carbon. The ID/IG ratio (ID signifies the intensity of the D band, IG signifies the intensity of the G band) indicates the defect sites (the degrees of disorder) for the ground carbon, ball-milled carbon, and functionalized carbon are 3.78, 2.67, and 2.74, respectively. It indicates the improvement in the ordered carbon with ball milling and functionalization. The BJH pore size distributions of the ground, 15 h ball-milled, and functionalized carbons are shown in Figure S3 in the Supplementary Material. Figure S3a depicts the pore distribution of the ground carbon, with a pore width ranging from 1.9 nm to 230 nm, an average pore width of 2.1 nm, and a total pore volume of 0.13 cm 3 /g. The average pore width and total pore volume increased to 3.67 nm and 0.30 cm 3 /g after 15 h of ball-milling, Figure S3b. However, carboxylic functionalization of the ball-milled carbon reduced the average pore width to 3.164 nm and total pore volume to 0.058 cm 3 /g, as shown in Figure S3c. The physical properties of the date carbon at various stages of processing are tabulated in Table 6.  Figure 7 shows the Raman spectrum of the ground pyrolytic date carbon (pyrolysis at 850 • C) after being ball-milled for 15 h, and subsequently, acid-functionalized, measured at a wavelength of 532 nm. The two intense peaks in the spectrum correspond to graphitization (G-band) and defects (D-band). The G-band and D-band peaks for the ground carbon can be observed at around 1591 cm −1 and 1352 cm −1 , respectively. For the 15 h ball-milled carbon, the G-band and D-band values were also around 1589 cm −1 and 1352 cm −1 . In contrast, for the functionalized carbon, the G-band and D-band values were 1591 cm −1 and 1357 cm −1 . The intensities of the G-and D-bands of the ground and functionalized carbon were almost the same, but significantly lower and broader for the 15 h ball-milled carbon. The I D /I G ratio (I D signifies the intensity of the D band, I G signifies the intensity of the G band) indicates the defect sites (the degrees of disorder) for the ground carbon, ballmilled carbon, and functionalized carbon are 3.78, 2.67 and 2.74, respectively. It indicates the improvement in the ordered carbon with ball milling and functionalization. Nanomaterials 2022, 12, x FOR PEER REVIEW 14 of 23

IFT Measurements and Optimum Concentration at 25 °C
Five samples of DLCNP were prepared at 200 mg/L to 800 mg/L concentration with deionized distilled water. Table A3 in Appendix A presents the IFT values, and Figure 8a is a plot of the IFT values against their respective DLCNP concentrations. At a DLCNP concentration of 0.00 mg/L, the IFT was 23 dyne/cm. This value decreased to about 8.5 dyne/cm when the DLCNP concentration increased to 600 mg/L, above which it plateaued out; the optimum concentration was thus taken to be 600 mg/L. This was also consistent with our earlier study on IFT [27].

IFT Measurements and Optimum Concentration at 25 • C
Five samples of DLCNP were prepared at 200 mg/L to 800 mg/L concentration with deionized distilled water. Table A3 in Appendix A presents the IFT values, and Figure 8a is a plot of the IFT values against their respective DLCNP concentrations. At a DLCNP concentration of 0.00 mg/L, the IFT was 23 dyne/cm. This value decreased to about 8.5 dyne/cm when the DLCNP concentration increased to 600 mg/L, above which it plateaued out; the optimum concentration was thus taken to be 600 mg/L. This was also consistent with our earlier study on IFT [27].

Wettability Measurement and Influence of Temperature and Pressure on IFT
As previously highlighted in Section 3.5, the wettability via contact angle measurement was not successful. This was attributed to the fact that since both phases were dark black, distinguishing between the drop and the surrounding phase from the image was not successful. Moreover, the DSA 100 is designed to measure contact angle if one fluid is transparent because it measures the drop shape. Furthermore, at higher concentrations, the carbon materials appeared dark, hence, measurement was unsuccessful.
The impact of temperature and pressure on IFT was observed by DSA 100 as highlighted in Figure 8. IFT was measured at temperatures ranging from 25 • C to 81 • C. It was observed that IFT increased from 14 dyne/cm to 17 dyne/cm as the temperature increased from 25 • C to 81 • C, Figure 8b and Table A4 in Appendix A. Thus, a 56-degree rise in temperature only increased IFT by 3 dyne/cm. This means that temperature had only a minimal influence on the IFT values of DLCNP solutions. Furthermore, the IFT values of the DLCNP solutions were measured with pressures varying from 3000 psi to 5000 psi. There was a 2 dyne/cm change in IFT (from 28 dyne/cm to 30 dyne/cm), Figure 8c and Table A5 in Appendix A; thus, a minimum influence of pressure on IFT from the 3000 psi to 5000 psi pressure range. The study by Soleimani et al. [23], presented in Table 1, also observed an increase in surface tension measurement (since theoretically, surface tension has an indirect relationship with IFT), between CNT and air (because IFT measurement between CNT and the Dubai crude oil sample used was unsuccessful due to a very dark cloudy phase), with a maximum value of 33.46 dyne/cm at 0.3 wt% CNT. The authors conclude that CNT synthesized via chemical vapor deposition technique can lower IFT, hence, an improvement in oil recovery can be achieved. Though the authors did not measure the effect of temperature and pressure [23], perhaps, it may be attributed to the minimal impact the CNT had as we did observe in our DLCNP as seen in Figure 8b,c.
Five samples of DLCNP were prepared at 200 mg/L to 800 mg/L concentration with deionized distilled water. Table A3 in Appendix A presents the IFT values, and Figure 8a is a plot of the IFT values against their respective DLCNP concentrations. At a DLCNP concentration of 0.00 mg/L, the IFT was 23 dyne/cm. This value decreased to about 8.5 dyne/cm when the DLCNP concentration increased to 600 mg/L, above which it plateaued out; the optimum concentration was thus taken to be 600 mg/L. This was also consistent with our earlier study on IFT [27].

Wettability Measurement and Influence of Temperature and Pressure on IFT
As previously highlighted in Section 3.5, the wettability via contact angle measurement was not successful. This was attributed to the fact that since both phases were dark black, distinguishing between the drop and the surrounding phase from the image was not successful. Moreover, the DSA 100 is designed to measure contact angle if one fluid is transparent because it measures the drop shape. Furthermore, at higher concentrations, the carbon materials appeared dark, hence, measurement was

Optimum Salinity of Green Surfactant
The optimum salinity of the APG and NaCl system was measured through Healy and Reed correlation. The calculated data are given in Table 7. The solubilization parameters of oil and brine in the micro-emulsion phases were calculated using Equations (1) and (2). Then the parameters were plotted against salinity. The optimum salinity was obtained by the point of intersection between the oil and water curves. Figure 9 describes the optimum salinity of the APG-NaCl system where an optimum salinity of about 2.75% was observed.
Solubilization parameter of oil, of oil in microemulsion phase Vol. of surfactant in microemulsion phase (1) Solubilization parameter of water, of water in microemulsion phase Vol. of surfactant in microemulsion phase (2)  Figure 9. Optimum salinity of the APG-NaCl system.

Core Flooding
In this study, three core flood experiments were conducted to test the EOR capability of functionalized and non-functionalized DLCNP. The first experiment was carried out to see how functionalized DLCNP worked in smart flooding and increased the tertiary oil. The goal of the second core flood test was to observe how a mixture of non-functionalized DLCNP and non-ionic green surfactant boosted residual oil in the EOR process. The last formulation was conducted to compare the tertiary oil recovery (TOR) of the DLCNP with that of commercial CNT. Po and Pw Salinity (%, NaCl)

Po -Oil Solubilization
Pw -Water Solubilization Figure 9. Optimum salinity of the APG-NaCl system.

Core Flooding
In this study, three core flood experiments were conducted to test the EOR capability of functionalized and non-functionalized DLCNP. The first experiment was carried out to see how functionalized DLCNP worked in smart flooding and increased the tertiary oil. The goal of the second core flood test was to observe how a mixture of non-functionalized DLCNP and non-ionic green surfactant boosted residual oil in the EOR process. The last formulation was conducted to compare the tertiary oil recovery (TOR) of the DLCNP with that of commercial CNT.

Functionalized DLCNP in Smart Water Flooding
The impact of DLCNP and distilled water on oil recovery was studied via a core flood experiment. In the formulation reported here, 800 mg/L (ppm) of DLCNP was mixed with distilled water. After water flooding, the secondary oil recovery was 40% of the oil initially in place (OIIP). About three pore volumes of nanofluid were then injected into the core, and incremental oil recovery was 9%; thus, total oil recovery was 49% of OIIP. Figure 10 shows the complete recovery calculated after brine and DLCNP mixture flooding.

DLCNP in Green Surfactant Flooding
The influence of non-functionalized DLCNP and non-ionic green surfactants mixture together with NaCl on oil recovery was studied in this experiment. The non-ionic surfactant APG 264 of 0.5% and non-functionalized DLCNP of 800 mg/L (ppm) were mixed with 2% NaCl. Results of this formulation are presented in Figure S4 in the Supplementary Material. In the water flooding stage, secondary oil recovery was 44% of Oil Initially in Place (OIIP). In surfactant and carbon nanomaterial flooding, there was 33% oil production after 4.6 PVs of the fluid injection and 12.33% oil production after 1.3 PVs of brine injection. Total tertiary oil recovery was 45%.

Non-Functionalized DLCNP with CNT
A batch of (non-functionalized) DLCNP was prepared and compared with (nonfunctionalized) CNT. The core flood experiment was described in detail earlier [8][9][10][11]. The non-ionic surfactant APG 264 of 0.5% and CNT of 800 mg/L (ppm) were mixed with 2% NaCl. Results of this formulation are presented in Figure S5 in the Supplementary Material. The secondary oil recovery was 50% of Oil Initially in Place (OIIP). In surfactant flooding, there was 23% oil production after 3.3 PVs of nanofluid injection and 4% oil production after 0.6 PVs of brine injection. Total tertiary oil recovery was 27% [63]. A summary of the three formulation results is presented in Table 8, whereas Figures S5 and S6 represent the oil recovery observed. The efficacy of using green surfactant and cosurfactant systems for EOR was studied previously by [12]. The surfactant contained 0.5% of APG 264 and 0.5% butanol as co-surfactant. The oil recovery due to waterflood was

DLCNP in Green Surfactant Flooding
The influence of non-functionalized DLCNP and non-ionic green surfactants mixture together with NaCl on oil recovery was studied in this experiment. The non-ionic surfactant APG 264 of 0.5% and non-functionalized DLCNP of 800 mg/L (ppm) were mixed with 2% NaCl. Results of this formulation are presented in Figure S4 in the Supplementary Material. In the water flooding stage, secondary oil recovery was 44% of Oil Initially in Place (OIIP). In surfactant and carbon nanomaterial flooding, there was 33% oil production after 4.6 PVs of the fluid injection and 12.33% oil production after 1.3 PVs of brine injection. Total tertiary oil recovery was 45%.

Non-Functionalized DLCNP with CNT
A batch of (non-functionalized) DLCNP was prepared and compared with (nonfunctionalized) CNT. The core flood experiment was described in detail earlier [8][9][10][11]. The non-ionic surfactant APG 264 of 0.5% and CNT of 800 mg/L (ppm) were mixed with 2% NaCl. Results of this formulation are presented in Figure S5 in the Supplementary Material. The secondary oil recovery was 50% of Oil Initially in Place (OIIP). In surfactant flooding, there was 23% oil production after 3.3 PVs of nanofluid injection and 4% oil production after 0.6 PVs of brine injection. Total tertiary oil recovery was 27% [63]. A summary of the three formulation results is presented in Table 8, whereas Figures S5 and S6 represent the oil recovery observed. The efficacy of using green surfactant and co-surfactant systems for EOR was studied previously by [12]. The surfactant contained 0.5% of APG 264 and 0.5% butanol as co-surfactant. The oil recovery due to waterflood was about 41% and the tertiary oil recovery of this formulation amounted to 41%. This is a considerable amount of additional oil recovery, implying a total oil recovery of approximately 82% of OIIP. The tertiary oil recovery of APG and DLCNP (formulation II) is 45% which is about 4% more than the APG and butanol system studied by [12]. This, therefore, shows that the carbon particle might be an eco-friendly option for boosting oil production.

Conclusions
This study experimentally investigated the potential application of carbon nanomaterial for green EOR studies. Based on this research, the main findings of this paper are:

1.
A carbon nanoparticle was prepared from a cheap source via a direct method (i.e., pyrolysis at 850 • C and ball milling for 15 h). The surface of the nanocarbon was carboxylic acidfunctionalized using acid treatment. The prepared carbons were characterized using FE-SEM, TEM, EDS, XPS, Raman spectroscopy, and BET.

2.
The particle size of the carboxylic acid-functionalized carbon ranged from 50 to 159 nm.

3.
The IFT values between the Arabian light crude oil and different concentrations of aqueous DLCNP were measured. The DLCNP reduced the IFT by 41% from 14.46 to 8.56 dyne/cm at 25 • C.

4.
Only a minimal influence of pressure and temperature on the IFT between the DLCNP solutions and Arabian light crude oil was observed.

5.
Core flood experiments with Berea sandstone confirmed that a concentration of 800 ppm carboxylated DLCNP mixed with distilled water could recover 9% of the residual oil and 49% of the OIIP. 6.
An 800 ppm sample of non-functionalized DLCNP was blended with 0.5 wt% nonionic green surfactant APG and 2 wt% NaCl brine; this produced 45% tertiary oil and 89% OIIP recovery. This formulation performed better than the commercially available CNT.
The date tree is available all over the Kingdom of Saudi Arabia and in the middle east. The nanoparticle developed from the leaf and utilized to boost oil recovery will add value to the national economy.

Recommendations
It is recommended that the wettability study via contact angle measurement between carbon particle solution and crude oil system be investigated further. Additionally, since both fluids are black, it would be better to develop a new and effective measuring technique. Additionally, the narrow-down salinity scanning technique should be conducted to get a better understanding of the optimum salinity.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nano12081245/s1, Figure S1: Drop shape analyzer (DSA) 100, Figure S2: Core flooding experimental setup, Figure S3: BJH pore size distribution of the ground, ball-milled (15 h), and functionalized date carbon, Figure S4: Total oil recovery measured for the green surfactant and nanomaterial formulation, Figure S5: Total oil recovery measured for the three formulations tested, Figure S6: Comparison of the DLCNP with carbon nanotubes.  Acknowledgments: Corresponding authors would like to thank the College of Petroleum and Geosciences (CPG) at KFUPM for providing funding and Inter-disciplinary Research Center for Hydrogen and Energy Storage for giving research support.

Conflicts of Interest:
The authors declare no conflict of interest.


s2fieldsofstudy: Chemistry Biology Physics

text:
De novo creation of a naked eye–detectable fluorescent molecule based on quantum chemical computation and machine learning

Designing fluorescent molecules requires considering multiple interrelated molecular properties, as opposed to properties that straightforwardly correlated with molecular structure, such as light absorption of molecules. In this study, we have used a de novo molecule generator (DNMG) coupled with quantum chemical computation (QC) to develop fluorescent molecules, which are garnering significant attention in various disciplines. Using massive parallel computation (1024 cores, 5 days), the DNMG has produced 3643 candidate molecules. We have selected an unreported molecule and seven reported molecules and synthesized them. Photoluminescence spectrum measurements demonstrated that the DNMG can successfully design fluorescent molecules with 75% accuracy (n = 6/8) and create an unreported molecule that emits fluorescence detectable by the naked eye.

Correlation with number of aromatic rings
To see the correlations of absorption wavelength to S1 states/emission wavelength from S1 states and its oscillator strengths (OSs) with the number of aromatic rings, the correlation graphs with them are shown in Figure S1. We used the tools of RDkit (59) to count aromatic rings. Figure S1. Correlation graphs of fluorescence/absorption wavelength and their intensities with aromatic rings. Upper two are correlation graphs of number of aromatic rings with S0 absorption wavelength, its Oscillator strength. Bottom two are correlation graphs with S1 emission wavelength, its oscillator strength (bottom two).

Correlation with conjugate length
To see the correlations of absorption wavelength to S1 states/emission wavelength from S1 states and its oscillator strengths (OSs) with conjugate length, the correlation graphs with them are shown in Figure S1. We have count conjugate length whose unity is defined as single-doublesingle bond sequence. Figure S2. Correlation graphs of fluorescence/absorption wavelength and their intensities with conjugate rings. Upper two are correlation graphs of number of conjugate lengths with S0 absorption wavelength, its Oscillator strength. Bottom two are correlation graph with S1 emission wavelength, its oscillator strength.

Prediction of absorption and emission by machine learning model
For investigation of the relationship between molecular features and the properties of absorption and fluorescence, we developed prediction models based on random forest using the Mordred descriptors. We employed an implementation of the random forest regression model in scikit-learn library. The number of trees was set to 100, and other settings were used as default. The prediction performance was evaluated using 5-fold cross validation. The average of correlations of the prediction results for each property is shown in Table S1. The features with high feature importance in each prediction model are shown in Figure S3. Table S1. Pearson's correlation coefficients (R values) of wavelengths and their intensities of absorption to S1 states and fluorescence at the B3LYP/3-21G* level from S1 states with the predicted values by the trained random forest models.

Selected molecules
From the viewpoint of detectability, we have selected 87 molecules in accordance with the condition mentioned in the main text. In Table S2, selected 87 molecules in SMILES string are summarized with their properties computed at the B3LYP/3-21G* level. A-G are molecules or its tautomers that are found in Scifinder and synthesized for the experimental validation. The unreported molecule we synthesize in this study is PC. I-IV are expected to emit near-infrared light.

General methods
ATR-FTIR spectra were obtained using a Thermo-Nicolet 760X FTIR spectrophotometer equipped with a SMART-iTX ATR accessory. 1 H-NMR spectra were obtained using a JEOL JNM-ECA400 spectrometer operating at 400 MHz and using tetramethylsilane (TMS) as an internal standard. Proton decoupled 13 C-NMR spectra were obtained using a JEOL JNM-ECA400 spectrometer operating at 101 MHz and using TMS as an internal standard. Data was processed using Delta version 5.0.5.1. 1 H NMR chemical shifts (δ) are reported in ppm relative to TMS in DMSO-d6 (δ = 0.00). 13 C NMR chemical shifts (δ) are reported in ppm relative to the solvent reported. Coupling constants (J) are expressed in Hertz (Hz), shift multiplicities are reported as singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), multiplet (m) and broad singlet (bs). High resolution ESI-MS mass spectra were measured using a Thermo Scientific Q-Exactive Plus instrument in methanol with 0.1% formic acid.

Photoluminescence (PL) spectra of PC under N 2
PL spectra under air and N2 (after N2 bubble for 20 min) are measured at room temperature. Since there is no difference between them, phosphorescence should not be involved in PL of PC. Figure S12. PL spectra of PC under air and N 2 .

Materials
The materials of A-G (Table S2) were obtained from Tokyo Chemical Industry Co., Ltd. through custom synthesis. Spectroscopic grade solvents (DCM or THF) were obtained from Fujifilm Wako Pure Chemical Corporation.

Characterization of A-G
ATR-FTIR spectra were obtained using a Thermo-Nicolet 760X FTIR spectrophotometer equipped with a SMART-iTX ATR accessory. 1 H-NMR spectra were obtained using a JEOL JNM-ECA400 or JNM-ECZ400S spectrometer operating at 400 MHz and using tetramethylsilane (TMS) as an internal standard. Proton decoupled 13 C-NMR spectra were obtained using a JEOL JNM-ECA400 or JNM-ECZ400S spectrometer operating at 101 MHz and using TMS as an internal standard. 19 F-NMR spectra were obtained using a JEOL JNM-ECZ400S spectrometer operating at 376 MHz. Data was processed using Delta version 5.0.5.1. 1 H NMR chemical shifts (δ) are reported in ppm relative to TMS in DMSO-d6 (δ = 0.00). 13 C NMR chemical shifts (δ) are reported in ppm relative to the solvent reported. Coupling constants (J) are expressed in Hertz (Hz), shift multiplicities are reported as singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), multiplet (m) and broad singlet (bs). High resolution ESI-MS mass spectra were measured using a Thermo Scientific Q-Exactive Plus instrument in methanol with 0.1% formic acid or methanol with 10% tetrahydrofuran.

Quantum yields of PC and A-G
Quantum yields ( ) in solid and solution states of PC and A-G molecules are summarized in Table  S5. Absolute fluorescence quantum yields were determined with a Hamamatsu Photonics C-9920-02 calibrated integrating sphere system.


ANALYTICAL METHODS FOR THE QUANTIFICATION OF RITONAVIR IN PHARMACEUTICALS, A COMPARATIVE EVALUATION

. Ritonavir is an antiretroviral medication used to treat AIDS (Acquired Immune Deficiency Syndrome)/HIV (Human Immunodeficiency Virus). This article describes the development and validation of spectrophotometric and liquid chromatographic techniques for ritonavir quantification in pharmaceutical formulations. Liquid chromatographic analyzes were carried out using a C18 column and a mobile phase consisted of 20 mM KH 2 PO 4 (pH = 3.0) and acetonitrile (45:55, v/v), with a flow rate of 1.2 mL min -1 and UV detection at 235 nm. For the spectrophotometric analyses, ethanol was employed as a solvent, The UV spectra of the standard and sample solution were scanned between 200 and 800 nm, and λ max was determined as 235 nm. Analytical techniques have been validated in accordance with the processes outlined in the ICH (International Council for Harmonization) recommendations. The results showed that the analytical procedures were linear, accurate, precise, and robust. The recovery values of the methods are within the standard norms (98  102%). Following that, a statistical comparison of analytical techniques was carried out, and the findings revealed no significant difference. Consequently, the developed analytical techniques might be used quality control analysis of ritonavir in pharmaceuticals.

INTRODUCTION
As of March 07, 2022, WHO had documented 5,998,301 deaths and 445,096,612 cases of Covid-19 worldwide [1]. There is presently no antiviral or therapeutic treatment available to treat Covid-19. Covid-19 illness is uncontrolled and has become a severe worldwide concern due to its high fatality rates.
Infected people have been given off-label treatments [2][3][4]. A large variety of antivirals have been investigated in in vitro research as well as approved clinical trials. The majority of them are protease inhibitors from a biological perspective [5][6][7][8][9]. Protease is a virus-growing enzyme. Protease inhibitors can prevent these enzyme activities. These inhibitors bind selectively to viral proteases and block the proteolytic cleavage of protein precursors required to produce infectious viral particles. As a result, viral replication is prevented. Ritonavir, indinavir, amprenavir, nelfinavir, saquinavir atazanavir drugs are in this class. They are used in highly active antiretroviral therapy. Among these drugs, ritonavir is used for the treatment of HIV/AIDS. It is often used alone or in combination with other anti-retroviral drug [10]. The chemical properties of ritonavir have been presented in Table 1.
The ritonavir monograph is officially available in both the United States Pharmacopoeia and the British Pharmacopoeia describing the high-pressure liquid chromatography (HPLC) technique for the quantification of ritonavir in bulk powder [11,12].
According to a literature review, various spectrophotometric methods for determination of ritonavir in pharmaceutical formulations have been developed [1315]. In the literature, several HPLC methods for determination of ritonavir have been published [1618]. Some of these are for the simultaneous detection of antiretrovirals in human plasma by reversed phase high performance liquid chromatography methods [1922], two high performance liquid chromatography methods [23] and one high performance thin layer chromatography method [24] for determining ritonavir in pharmaceutical formulations were published in the literature.
However, some of these techniques were quite complex and had limitations such as sample preparation, gradient elution, and long run time. These procedures also require the use of expensive equipment, specialized reagents, and substantial volumes of organic solvents.
The aim of this study is to develop analytical methods that are simple, quick, inexpensive, and well-validated for the quantification of ritonavir in pharmaceutical formulations using LC chromatographic and UV spectrophotometric techniques. In addition, these methods to be developed will be less time-consuming and cheaper than other published methods. The validation of the developed analytical methods was carried out according to the procedures described in ICH guidelines Q2(R1) for the validation of analytical methods [25,26]. Furthermore, the applicability and reliability and of these methods have been assessed by concentrating on routine quality control analyses. The results obtained from analytical methods were compared statistically using the least-squares method. This is the first study to statistically compare two different analytical methods developed for the quantification of ritonavir in pharmaceutical preparations.
However, no studies on a comparative comparison of spectrophotometric and liquid chromatographic techniques for ritonavir quantification have been published. The purpose of this research is to develop analytical methods that are simple, quick, cheap, and well-validated for the quantification of ritonavir in pharmaceutical formulations using LC chromatographic and UV spectrophotometric techniques. In addition, these methods to be developed will be less time consuming and cheaper than other published methods. This is the first study to statistically compare two different analytical methods developed for the quantification of ritonavir in pharmaceutical preparations.

Instruments
An Agilent 1260 HPLC system consisting of Chemstation software, and UV-Vis detector was used during method development and validation in chromatographic analysis. Shimadzu UV-1800 spectrophotometer consisting of UV-Probe software and a double beam using 1.0 cm quartz cells was used during spectrophotometric analysis.

Chemicals
Milli-Q water purification system (Millipore) was used to produce HPLC grade water. Pure ritonavir and Norvir (100 mg) were supplied from Abbvie Pharmaceuticals. (Istanbul, Turkey). All solvents used in the study were of HPLC grade and were obtained from Merck (Germany).

Standard solutions
An appropriate amount of ritonavir was dissolved in ethanol and a stock standard solution (500 µg mL -1 ) was prepared. Six working standard solutions in the concentration range of 1060 µg mL -1 were prepared by serial dilutions of the stock standard solution with ethanol. These standard solutions were then filtered using 0.22 micron syringe filters.

Sample solutions
A total of 10 tablets were weighed and ground into fine powder by grinding in a mortar. The powder equivalent to 25 mg of ritonavir was weighed accurately, taken into a 50 mL of volumetric flask. 20 mL of ethanol was added to dissolve ritonavir, the volume was diluted to 50 mL with ethanol when dissolution was complete. Finally, ethanol was added up to the marked line, and shaked homogenously for 15 min. This supernatant was filtered through membrane filter (0.22 µm pore size). 1 mL of this solution was taken and transferred to a 10 mL of volumetric flask. The volume was made up with ethanol and a ritonavir solution (50 µg mL -1 ) was obtained.  It is soluble in methanol, ethanol, and isopropanol, but practically insoluble in water Chemical structure

Determination of λmax
Using ethanol, the spectrophotometer was first calibrated to zero. Standard ritonavir solution at a concentration of 30 µg mL -1 was scanned between 200 and 800 nm wavelengths in a spectrophotometer device to determine the wavelength at which it absorbed the maximum.

Calibration curves
The standard solutions (20 μL) were injected into the liquid chromatographic system. It was determined that the peak areas obtained with the concentrations of the standard solutions were proportional. A calibration curve was created by plotting the concentration against the peak area for the chromatographic method, Slope, intercept and corelation coefficient were calculated from this calibration curve. These standard solutions were scanned in the wavelength range of 200 -800 nm in a spectrophotometer and λmax was determined as 235 nm. The absorption values of each standard solution at a wavelength of 235 nm were determined. It was determined that the corresponding absorbance values were proportional to the concentrations of the standard solutions. A calibration curve has been developed by plotting the concentration against the absorbance for the spectrphotometric method, Slope, intercept and corelation coefficient were calculated from this calibration curve.

Assay of ritonavir in tablets
Three sample solutions prepared were injected into the HPLC system (20 µL). It was analyzed using the developed method, the peak areas were determined, and the ritonavir contents in the tablet were calculated.

Analysis method conditions
An Agilent brand 1260 model HPLC apparatus with a UV-Vis detector was used for chromatographic analyses. The mobile phase was a 20 mM KH2PO4 solution (pH 3.0, with ortophosphoric acid) at a flow rate of 1.2 mL min -1 . The mobile phase was filtered by passing through a 0.22 µm membrane filter and degassed in an ultrasonic bath before use. The column was ODS (4.6 mm x 250 mm, 3.0 µm particle size) and stored at 25 °C. Ritonavir detection was performed at 235 nm with a UV detector. The run time was 15 minutes under these conditions. All absorbance measurements were taken with a dual-beam spectrophotometer at 235 nm.

Analytical method validation
Analytical techniques were validated according to the procedures specified in the ICH guideline Q2(R1) [25,26]. All studies such as accuracy, linearity, sensitivity, precision, robustness, specificity, system suitability were investigated as validation parameters. Six working standard solutions (10 -60 g mL -1 ) were prepared by serial dilutions of the stock solution with ethanol. These standard solutions were then filtered using 0.22 micron syringe filters. Then, 20 µl of each standard solution was injected into the liquid chromatographic system three times for validation of the chromatographic method. Ritonavir's peak areas and retention times were determined and recorded. The calibration curve was created by graphing the peak area against the concentration, and its linearity was assessed using regression analysis. The correlation coefficient, slope, and intercept were all calculated. Each analysis was carried out in triplicate. The absorbances of standard solutions at a wavelength of 235 nm were measured on a spectrophotometer. Analyzes were performed in triplicate.
The standard addition method was used to determine the methods's accuracy. A known amount of standard was added to the previously tested sample solution at three different levels (80, 100, and 120 %). The developed methods were used to examine all of the samples. The RSD and recovery for each concentration were determined. Absorbance values was measured directly for UV spectrophotometric technique by comparing the estimated and observed concentrations, the recovery percent was computed.
The precision of the analytical methods was evaluated by intra-day repeatability and inter-day repeatability. Standard solutions at the concentrations of 40, 50, and 60 µg mL -1 were examined three times in the same day in intraday precision studies and standard solutions of the same concentration were evaluated three times on three consecutive days for inter-day precision experiments. The results of precision studies were presented as a percentage of RSD.
The sensitivity of analytical techniques was evaluated using the limits of detection (LOD) and the limits of quantitation (LOQ). To calculate the LOD and LOQ from the calibration curve, we utilized the following formulas.
where, σ indicates the standard deviation of the calibration curve's intercept and S indicates the calibration curve's slope. Peak purity was determined by comparing the chromatograms at the peak's beginning, pinnacle, and end for specificity in the HPLC method.
The analytical techniques' robustness was evaluated by looking at the effects of tiny but purposeful of parameter modifications on results. The same sample was examined under varied circumstances, such as variations in flow rate of the mobile phase (0.1 mL min -1 ) and detector wavelengths (2 nm), to test the robustness of the chromatographic method, and the influence has been detected on the system suitability characteristics. Standard solution (30 µg mL -1 ) was injected six times for system suitability testing. The peak area, theoretical plate number, tailing factor, capacity factor, and asymmetry factor were all determined. The standard solution was kept at room temperature for up to two days and refrigerated (4 o C) for up to five days for stability experiments. The developed methods were then used to analyze these solutions.

Comparative analysis
Analytical techniques were found to be adequate for determining the quantity of ritonavir in pharmaceutical formulations after validation. Statistically, the recovery percentages of analytical techniques used on commercial medications were compared. For comparison, the F-and t-tests were utilized.

RESULTS AND DISCUSSION
The validation of analytical methods was carried out in accordance with the methods outlined in the ICH guidelines.

HPLC method
Using the peak shape and analysis time, the chemical composition of the mobile phase and flow rate were determined. Many mobile phases were tried. Finally, it was established that 20 mM KH2PO4 (pH: 3) was the optimal mobile phase for short run duration and symmetrical peak point. Figure 1 depicts the overlap chromatograms of the standard solutions, whereas Figure 2 depicts the chromatographic method's linearity plot. Ritonavir had a retention time of 10.00 min. The developed methods have been validated following ICH guidelines for accuracy, linearity, precision, specificity, system suitability, robustness, and sensitivity (LOD and LOQ). Since the peak purity index of ritonavir was determined as 0.9998, the method was determined to be specific. According to linear regression analysis, peak area and analyte concentration show a very good correlation The calibration curve had a correlation coefficient (r 2 ) of 0.9998±0.0006 and was linear over the concentration range of 10 to 60 μg mL -1 . Linear regression equation was y = 5.0509x1.3133. The detection and quantification limits were determined as 0.90 µg mL -1 and 2.70 µg mL -1 , respectively, and these values indicated the sensitivity of the method.
The results and statistical evaluations presented in Table 1 show the high accuracy and precision of the method. Low relative standard deviation (RSD) values were obtained from the recovery experiments and showed good accuracy. The RSD for intraday and interday repeatability studies as recommended in ICH guidelines. Values were determined as < 2. The RSD values for intraday repeatability and interday repeatability ranged from 0.0940 to 0.1515 and 0.1840 to 0.2615, respectively. This showed that the developed liquid chromatographic method is precise. Regression and validation parameters for analytical methods are presented in Table 2. Minor modifications made intentionally in the experimental conditions did not make a statistically significant difference on the system suitability parameters. RSD values for each method parameter were calculated and determined to be < 2.
System suitability tests demonstrated the suitability of the proposed liquid chromatographic method for routine quality control analysis. The capacity factor was determined as 9.85. The tailing factor was set at 1.155, which indicates strong peak symmetry. The theoretical plate number was determined as 6542, this value indicates high level of column efficiency. The results of the system suitability tests show that the devised technique fits these requirements within acceptable limits. Standard solutions are stable at room temperature, according to solution stability studies.

UV-Vis spectrophotometric method
The spectrophotometric technique was validated in accordance with ICH standards. Standard solutions were scanned in the spectrophotometer in the wavelength range of 200800 nm. It was observed that ritonavir had maximum absorption at 260 nm wavelength. The overlapping spectrum of the standard solutions is presented in Figure 3. A graph of absorbance values against concentration values of standard solutions was created. The linearity graph of the standard solutions is presented in Figure 4. Standard solutions demonstrated linear relationship in the range of 10 to 60 µg mL -1 . Correlation coefficient (r 2 ) was 0.9995±0.0009. The regression equation was found to be y = 0.0122x-0.0043. In the recovery studies, the standard addition technique was used for the three concentrations and acceptable RSD, ranging from 99.20% to 100.98%. Detection and quantification limits were determined as 1.00 µg mL -1 and 3.00 µg mL -1 , respectively.  The spectrophotometric approach devised allowed for the quick measurement of ritonavir in pharmaceutical formulations. Ethanol was chosen as the solvent due to its excellent solubility and stability. This solvent also has good spectrum characteristics. Low RSD results imply that the procedure is precise. It was observed that the method was highly accurate and the average recovery values approached 100%. In addition, the small values of LOD and LOQ show that the method is quite sensitive.

Analysis of pharmaceutical formulations by analytical methods developed
Analytical methodologies established were used to commercial pharmaceutical formulations. Table 3 shows the findings of the examination of ritonavir-containing pills. The study yielded values that were extremely close to the levels stated on the labels.

Statistical analysis
The F-test and the t-test were used to compare analytical methods statistically. The statistical tests revealed that the experimental data obtained from the analytical methods were not statistically different. It was determined that the calculated F and t values were lower than the table values of the analytical methods with a 95% confidence interval. The analytical methods presented in this study proved to be amenable to the accurate measurement of ritonavir in pharmaceutical formulations. Table 4 displays data on the statistical comparison outcomes of analytical methodologies.
In this investigation, two independent analytical techniques were devised to determine the amount of ritonavir in pharmaceuticals: chromatographic and spectrophotometric methods. The conditions of the developed analytical methods have been optimized and validated. Several analytical methods have been reported in the literature to determine the amount of ritonavir in pharmaceutical products. Some of these techniques are quite complex, requiring long analysis times, expensive apparatus, special reagents, and large amounts of organic solvents. In addition, the spectrofluorimetric and spectrophotometric methods that have been published in the literature require complex and time-consuming sample preparation. No research has yet been conducted in which two different analytical methods are created and compared statistically in any of these studies. Therefore, our study is the first study in this direction. The characteristics of the previously reported HPLC methods and the proposed method are presented in Table 5. Table 5. Characteristics of previously reported HPLC methods and the proposed method.

CONCLUSION
Chemicals that are detrimental to the environment and human health are avoided as much as feasible in the spectrophotometric and chromatographic procedures described in this study. The gadgets employed in the new procedures are no more expensive than those used in published investigations. Both analytical approaches are cost-effective. The results show that these methods are appropriate for estimating the quantity of ritonavir in pharmaceutical formulations. The devised spectrophotometric approach has no complicated processes. It is less expensive and requires less time. These are the benefits of the spectrophotometric method we created above the chromatographic approach we just devised. When statistically contrasted, the chromatographic approach is more accurate and exact than the spectrophotometric method. Excipients in pharmaceutical formulations had no effect on the results of either technique of analysis. Finally, because the proposed procedures are simple, specific, quick, accurate, and precise, they may be successfully used for ritonavir measurement in pharmaceutical formulations.


In Situ Construction of Bronze/Anatase TiO2 Homogeneous Heterojunctions and Their Photocatalytic Performances

Photocatalytic degradation is one of the most promising emerging technologies for environmental pollution control. However, the preparation of efficient, low-cost photocatalysts still faces many challenges. TiO2 is a widely available and inexpensive photocatalyst material, but improving its catalytic degradation performance has posed a significant challenge due to its shortcomings, such as the easy recombination of its photogenerated electron–hole pairs and its difficulty in absorbing visible light. The construction of homogeneous heterojunctions is an effective means to enhance the photocatalytic performances of photocatalysts. In this study, a TiO2(B)/TiO2(A) homogeneous heterojunction composite photocatalyst (with B and A denoting bronze and anatase phases, respectively) was successfully constructed in situ. Although the construction of homogeneous heterojunctions did not improve the light absorption performance of the material, its photocatalytic degradation performance was substantially enhanced. This was due to the suppression of the recombination of photogenerated electron–hole pairs and the enhancement of the carrier mobility. The photocatalytic ability of the TiO2(B)/TiO2(A) homogeneous heterojunction composite photocatalyst was up to three times higher than that of raw TiO2 (pure anatase TiO2).

Introduction
Environmental pollution is one of the biggest crises facing humans, and many environmental treatment technologies have been developed to control pollution [1,2]. Photocatalytic degradation has become one of the most promising emerging environmental pollution control technologies due to its mild reaction conditions, lack of secondary pollution generation, and environmental friendliness [3][4][5][6]. The key to the practical large-scale application of photocatalysis is the preparation of efficient, low-cost photocatalysts. However, most currently available photocatalysts fail to achieve a balance between a high efficiency and a low cost [7,8]. The modification of inexpensive, widely available photocatalysts to enhance their photocatalytic performances has become an important research direction for photocatalytic degradation technology [9][10][11]. TiO 2 is a non-toxic, non-hazardous, and low-cost photocatalyst with stable performance, but it exhibits poor photocatalytic performance, which must be enhanced to meet the needs of practical applications [12][13][14][15][16]. The low performance of TiO 2 photocatalysts is attributed to the easy recombination of photogenerated electron-hole pairs and the difficulty of visible light absorption. The construction of a homogeneous heterojunction is an effective method for suppressing the recombination of photogenerated electron-hole pairs and enhancing the carrier mobility, thereby improving the photocatalytic performance of photocatalysts [17][18][19][20][21][22]. As a rare phase in TiO 2 , TiO 2 (B) (bronze TiO 2 ) has a good electrical conductivity, a loose, porous structure, and it can be constructed with TiO 2 in the anatase phase (denoted as TiO 2 (A)), to form a homogeneous heterojunction crystal lattice with very small mismatches. As a result, the TiO 2 (B)/TiO 2 (A) homogeneous heterojunction will exhibit good photocatalytic degradation performance [23][24][25][26]. In this study, TiO 2 (B)/TiO 2 (A) homogeneous heterojunctions were constructed in situ by a simple hydrothermal method, utilizing TiO 2 (A) as the raw material with ion exchange and calcination. The prepared TiO 2 (B)/TiO 2 (A) homogeneous heterojunction material exhibited an excellent photocatalytic degradation performance, which was up to three times higher than that of raw TiO 2 . At present, the research on TiO 2 homogeneous heterojunctions mostly focuses on anatase/rutile TiO 2 , while much less research focuses on bronze/anatase TiO 2 . This study will provide new ideas and beneficial attempts for the research of TiO 2 homogeneous heterojunctions.

Materials
The rhodamine B (RhB, C 28 H 31 CIN 2 O 3 , Purity: 95%) was purchased from china Beijing Solarbio Technology, and both the sodium hydroxide (NaOH, Purity: 96%) and nano titanium dioxide (TiO 2 , Purity: 99.8%) were purchased from china Shanghai Aladdin Industrial Company. All reagents used in the experiments were of analytical grade and were used without further purification.

Preparation of Samples
A 100 mL 10 M NaOH aqueous solution was prepared, and 1 g raw TiO 2 was added. The mixture was stirred well, poured into a 150 mL hydrothermal reactor, heated to 220 • C in a blast drying oven, and held for 8 h. The reaction product was taken out and rinsed with a large amount of deionized water to near neutrality, after which it was dried at 60 • C for 10 h to obtain an intermediate product, which was labeled DNaT-220. The DNaT-220 was then soaked in 1000 mL 0.1 M dilute hydrochloric acid for 72 h, rinsed with a large amount of deionized water to near neutrality, dried at 60 • C for 10 h, and finally calcined in a muffle furnace at 500 • C for 2 h with a heating rate of 5 • C/min to obtain the final product, which was labeled DT-220. The intermediate products , and DNaT-160 as well as the final products DT-200, DT-190, DT-180, and DT-160 were prepared in the same way by changing only the temperature of the hydrothermal reaction. The number after the hyphen indicates the reaction temperature in • C.

Analysis and Testing
The phases of the samples were tested and analyzed using X-ray powder diffraction (XRD, D8 Advance, Bruker, Bremen, Germany) operated with a Cu target, wide-angle diffraction, a scanning range of 10-70 • , a scanning rate of 10 • /min, a test wavelength of 1.5406 Å, a tube voltage of 40 kV, and a tube current of 40 mA. The morphologies and structures of the samples were observed and analyzed using a field-emission scanning electron microscope (FE-SEM, Gemini SEM 300, Zeiss, Oberkochen, Germany) operated at a working voltage of 3 kV. The morphologies and structures of the samples were also observed and analyzed using a field-emission transmission electron microscope (Talos F200X, FEI, Hillsboro, OR, USA) operated at a working voltage of 200 kV, with a maximum magnification of 1.1 million times and a limit resolution of 0.12 nm. The photocatalytic degradation performance for RhB and the ultraviolet-visible (UV-vis) absorption spectra of the samples were tested by UV-vis spectrophotometry (UV-3600, Shimadzu, Japan) in the wavelength range of 200-2500 nm. The photocurrents and impedances of the samples were tested using an electrochemical workstation (CHI-760E, Chinstruments, Shanghai, China).
To test the photocatalytic degradation performance, 2 L of 20 mg/L RhB solution was prepared. In each experiment, 100 mL of RhB solution was added to 20 mg of the sample, and the mixture was sonicated for 30 min. Then, the mixture was placed under the light source of a solar simulator (Solar-500Q, Newbit, Beijing, China) with an intensity of 600 W/m 2 (at the liquid surface), and the photocatalytic degradation experiment was performed for 60 min. Every 10 min, 5 mL of liquid was taken from the mixture and placed in a centrifuge for centrifugation at 10,000 rpm for 10 min. After this, the supernatant was removed, and the absorbance (A n ) was measured by UV-vis spectrophotometry. The solution concentration (C n ) was proportional to A n , and thus, C n /C 0 = A n /A 0 , where A 0 is the absorbance of the 20 mg/L RhB solution. Figure 1a shows the XRD patterns of the raw TiO 2 and the intermediate products DNaT-220, DNaT-200, DNaT-190, DNaT-180, and DNaT-160. The raw TiO 2 diffraction peaks matched well with the standard PDF card JCPDS#21-1272, which corresponds to the most common anatase TiO 2 phase. The XRD patterns of the DNaT-220 and DNaT-200 had identical peak positions and shapes, coinciding with the PDF card JCPDS#31-1329, which indicated that both samples contained the same Na 2 Ti 3 O 7 phase. The peaks of the samples with lower hydrothermal reaction temperatures, DNaT-190, DNaT-180, and DNaT-160, changed significantly, which mainly manifested as increases in the half-peak widths of the spectral lines. All the samples contained Na 2 Ti 3 O 7 . However, the positions of the strongest and the second strongest peaks (corresponding to the TiO 2 in the anatase phase) were close to those of the peaks of Na 2 Ti 3 O 7 , which were relatively strong. Therefore, the peaks of the anatase TiO 2 phase (corresponding to the incompletely reacted raw TiO 2 ) were not clearly observed.    Figure 1b shows the XRD patterns of the raw TiO 2 and the final products-DT-220, DT-200, DT-190, DT-180, and DT-160. The peak positions and shapes of the DT-220 and DT-200 spectra were identical, and their standard PDF card number was JCPDS#35-0088, which corresponded to the bronze TiO 2 phase. When the hydrothermal reaction temperature dropped to 190 • C, the diffraction peaks of the bronze TiO 2 phase (corresponding to the product DT-190) were significantly weakened, whereas significant diffraction peaks of the anatase TiO 2 phase were observed. As the hydrothermal reaction temperature continued to be reduced, only the diffraction peaks completely consistent with those of raw TiO 2 were observed in the XRD patterns of the products DT-180 and DT-160. The TiO 2 ·xH 2 O produced by the generated Na 2 Ti 3 O 7 via ion exchange was converted to TiO 2 (B) after calcination at 500 • C.

Results and Discussion
The above phase analysis revealed that the entire phase-transformation process under the present experimental conditions was as follows: At hydrothermal reaction temperatures of 220 • C and 200 • C, the raw TiO 2 was completely converted by hydrothermal reactions to Na 2 Ti 3 O 7 , which then led entirely to the product TiO 2 (B) after ion exchange and hightemperature calcination. At a hydrothermal reaction temperature of 190 • C, the raw TiO 2 was partly converted via a hydrothermal reaction to Na 2 Ti 3 O 7 , and then ion exchange and high-temperature calcination led partly to the product TiO 2 (B) and partly to the incompletely reacted anatase TiO 2 phase. With a further decrease in the hydrothermal reaction temperature, the anatase phase TiO 2 was converted to Na 2 Ti 3 O 7 at a slower rate, the proportion of Na 2 Ti 3 O 7 converted by the reaction further decreased, and the amount of the remaining unreacted anatase TiO 2 phase further increased accordingly. Through controlled hydrothermal reactions, the structure of the raw TiO 2 could be easily modulated between anatase and bronze phases, providing a method for homogeneous heterojunction formation in situ, as well as electrical property adjustment. Combined with the XRD analysis results, we concluded that the band-like material in the two sets of SEM images was Na 2 Ti 3 O 7 (Figure 2a,b), which retained this morphology after ion exchange and calcination (Figure 2e,f) but was converted to TiO 2 (B). These band-like Na 2 Ti 3 O 7 and TiO 2 (B) were also observed in samples DNaT-220 and DT-220, respectively. When the hydrothermal reaction temperature dropped to 190 • C, a three-dimensional network started to appear in the DNaT-190 and DT-190 samples and many fine particles were dispersed on the network; these were TiO 2 (A) crystals that had not been converted. As the hydrothermal reaction temperature was further reduced, the conversion rate of the raw TiO 2 to Na 2 Ti 3 O 7 decreased. When the hydrothermal reaction temperature decreased to 180 • C, band-like Na 2 Ti 3 O 7 could no longer be generated by the reaction. Morphologically, the product consisted of Na 2 Ti 3 O 7 with a network structure and granular TiO 2 (A) dispersed in the network structure that had not completely reacted, as shown in Figure 2c Figure 4a shows the photocatalytic degradation curves of the raw TiO 2 , DT-200, DT-190, DT-180, and DT-160, respectively (Each sample was tested three times and the average value was taken). The photocatalytic degradation performances of the two-phase composite products, DT-190, DT-180, and DT-160, composed of TiO 2 (B) and TiO 2 (A) were significantly higher than those of the raw TiO 2 and pure TiO 2 (B). The DT-180 significantly outperformed the DT-190 and DT-160, and the photocatalytic degradation performance of the DT-180 heterojunction composite was nearly three times higher than that of raw TiO 2 . In addition, we can see that the samples had a small amount of adsorption on RhB during 30 min of ultrasound. Figure 4b quantitatively compares the catalytic efficiencies of the above samples, according to the kinetic calculation results. In terms of the Langmuir-Hinshelwood model, degradation can be considered as a first-order reaction at low RhB concentration. Therefore, the fitting calculation of degrading kinetics can be simplified to be linear, and this reaction satisfies the apparent first-order reaction rate equation: ln(C/C 0 ) = −k 1 t, where k is the apparent rate constant of the first-order reaction, and ln(C/C 0 ) is a function of irradiation time t. The calculated k values of no catalyst, Raw TiO 2 , DT-200, DT-190, DT-180 and DT-160 are 0 min −1 , 0.004 min −1 , 0.006 min −1 , 0.013 min −1 , 0.020 min −1 and 0.014 min −1 , respectively. These results suggest that the ratio of TiO 2 (B) to TiO 2 (A) in the product was related to the photocatalytic performance of the TiO 2 (B)/TiO 2 (A) homogeneous heterojunction composite photocatalyst, and that the composite exhibited the best photocatalytic performance when the optimal ratio was approached. In addition, the recyclability of DT-180 in RhB degradation reaction under UV-Vis irradiation was investigated, as shown in Figure 4c. After three successive cycles, the DT-180 sample retained nearly consistent photocatalytic efficiency, indicating that the TiO 2 (B)/TiO 2 (A) homogeneous heterojunction composite is stable during the degrading process.  Figure 5a shows the UV-vis absorption spectra of the raw TiO 2 , DT-200, and DT-180. Compared with those of raw TiO 2 and pure TiO 2 (B), the light absorption performance of the TiO 2 (B)/TiO 2 (A) homogeneous heterojunction composite photocatalyst in the UV region was not improved, but rather decreased due to the successful construction of homogeneous heterojunctions. The light absorption performance in the visible region was not significantly improved, with only a very slight red-shift. Figure 5b shows the Tauc plot of the raw TiO 2 , DT-200, and DT-180. The band gap energy of the semiconductors can be obtained from the x-axis intercept of the extended line. From the graph, the band gap energy values for the above samples were determined to be 3.33 ev, 3.18 ev, and 3.24 ev, respectively, which suggests that the band gap energy of TiO 2 (A) was reduced after introducing TiO 2 (B) to TiO 2 (A) to form the homogeneous heterojunction composite.  To investigate the origins of the improved photocatalytic performance, the light responses and electrical properties were measured. Figure 6a shows the transient photocurrent responses of the raw TiO 2 , DT-200 (representative of TiO 2 in the bronze phase), and DT-180. The photocurrent of DT-200 was higher than that of the raw TiO 2 in the anatase phase by a factor of approximately 1.5, and that of DT-180 was significantly higher than that of DT-200, by a factor of more than 1.5. Usually, a higher photocurrent indicates a weaker recombination of photogenerated electrons and holes. Thus, the recombination of photogenerated electron-hole pairs was significantly suppressed when the structure of TiO 2 was transformed from the anatase phase into the bronze phase. This suggests that TiO 2 (B) exhibited a slower recombination rate of photogenerated carriers than TiO 2 (A). In addition, the formation of TiO 2 (B)/TiO 2 (A) homogeneous heterojunctions further boosted the photogenerated charge separation because of the small built-in potential [25,27], leading to the substantially increased photocurrent shown in Figure 6a. Figure 6b shows the electrochemical impedance plots of the raw TiO 2 , DT-200, and DT-180. Generally, a larger arc radius indicates a higher impedance of the sample. Therefore, it was concluded that the impedance of DT-200 was lower than that of the raw TiO 2 , while that of DT-180 was much lower than those of both the raw TiO 2 and DT-200. This indicated that the conductivity and carrier mobility were improved by the transformation of TiO 2 (A) to TiO 2 (B) and were further enhanced by the formation of TiO 2 (B)/TiO 2 (A) homogeneous heterojunctions. Overall, the construction of TiO 2 (B)/TiO 2 (A) homogeneous heterojunctions not only benefitted the charge separation in the photocatalytic process, but also improved the electrical characteristics. The synergistic effects generated excellent photocatalytic activities, showing great potential for low-cost and high-efficiency photodegradation catalysts in environmental applications.

Conclusions
In this study, a novel photocatalyst consisting of TiO 2 (B)/TiO 2 (A) homogeneous heterojunctions was successfully prepared through facile hydrothermal processes. The structural characterizations revealed that a transformation occurred in the structure from the anatase phase to the bronze crystal phase, during which a series of TiO 2 (B)/TiO 2 (A) homogeneous heterojunctions formed. No significant improvement in the light absorption was observed, except for a very small red-shift in the absorption spectra. However, a substantial increase in photocurrent was detected for TiO 2 (B) in the transient light response measurement compared to that of TiO 2 (A). Additionally, further growth of the photocurrent for the homogeneous heterojunction was clearly observed, which was likely due to the small built-in field at the heterojunction interface. Moreover, the electrochemical impedance test revealed that the TiO 2 (B)/TiO 2 (A) homogeneous heterojunction possessed a substantially higher conductivity than that of TiO 2 (A), which also benefited the photocatalytic activities. The results of this study also indicated that the photocatalytic degradation performance of the TiO 2 (B)/TiO 2 (A) homogeneous heterojunction composite photocatalyst can be further enhanced by improving its light absorption capacity. This work provides a new structural design concept and a simple preparation method for the development of low-cost and high-efficiency composite photocatalysts.


s2fieldsofstudy: Chemistry Biology

text:
5-Methyl-cytosine stabilizes DNA but hinders DNA hybridization revealed by magnetic tweezers and simulations

Abstract 5-Methyl-cytosine (5mC) is one of the most important DNA modifications and plays versatile biological roles. It is well known that 5mC stabilizes DNA duplexes. However, it remains unclear how 5mC affects the kinetics of DNA melting and hybridization. Here, we studied the kinetics of unzipping and rezipping using a 502-bp DNA hairpin by single-molecule magnetic tweezers. Under constant loading rates, 5mC increases the unzipping force but counterintuitively decreases the rezipping force at various salt and temperature conditions. Under constant forces, the non-methylated DNA hops between metastable states during unzipping and rezipping, which implies low energy barriers. Surprisingly, the 5mC DNA can’t rezip after fully unzipping unless much lower forces are applied, where it rezips stochastically in a one-step manner, which implies 5mC kinetically hinders DNA hybridization and high energy barriers in DNA hybridization. All-atom molecular dynamics simulations reveal that the 5mC kinetically hinders DNA hybridization due to steric effects rather than electrostatic effects caused by the additional methyl groups of cytosines. Considering the possible high speed of DNA unzipping and zipping during replication and transcription, our findings provide new insights into the biological roles of 5mC.

Section1. Preparation of DNA for MT experiments.
As shown in Figure 1 in the main text, we prepared the DNA hairpin sample by following steps.
(ii) We digested the four dsDNA segments to generate the 4-nt sticky ends using BsaI-HF (New England Biolabs).
(iii) We ligated the four dsDNA segments to generate a 1424-bp combined dsDNA using T4 DNA ligase (New England Biolabs).
(iv) We added biotin groups to 3' ends of the dsDNA using Terminal transferase (New England Biolabs) and biotin-11-dUTP (Roche).
(v) We anchored the thiol-labelled end of dsDNA to the sulfo-SMCC (Thermo Fisher) coated glass surface.
Then, we tethered a streptavidin-coated superparamagnetic microbead Dynabeads) to the other end of dsDNA through the biotin-streptavidin bond. We exerted a stretching force of ~40 pN on the dsDNA using magnetic tweezers.
(vi) We peeled off the untethered ssDNA strand in the dsDNA at 100 mM NaOH and left the tethered ssDNA stretched at ~40 pN.
(vii) Because the two hairpin stems contained complementary sequences, the long DNA hairpin zipped at 8 pN when we changed the buffer into 1 mM Tris-HCl pH7.5 and 150 mM NaCl.
For the other DNA sample used in Figure S9, S10 and S12B, we amplified the head-to-head two 502-bp dsDNA segments (40701-41202 bp of λ-DNA) as the new hairpin stem through PCR using 2_hairpinF2-BsaI/2_hairpinR2_BsaI and 2_hairpinF3_BsaI/2_hairpinR3_BsaI as primers, respectively. We prepared this DNA sample by above steps except changed the sequence of the long hairpin.
We synthesized all the oligos with the following sequences (Sangon Biotech).  Figure S1. Home-built MT and flow cell. We built the MT using an inverted microscope equipped with an oil objective, on the top of which a flow cell was placed. We controlled the temperature with an objective heater.
To assemble one flow cell, we stuck together two cover slides with doubles-side taps and draw the inlet and outlet with silicone rubber.      Figure S13. The effects of 5mC on the unzipping and rezipping forces obtained at 150 mM NaCl using MD simulations. We stretched the 2-bp DNA hairpin through a force-cycle containing a force-increasing scan through a set of constant forces where the hairpin unzipped, followed by a force-decreasing scan through the same set of constant forces where the hairpin rezipped. At each constant force, we held the DNA for 150 ns during which we calculated the average in the extension of DNA in the last 50 ns. The force-cycle yielded an unzipping force-extension curve (filled symbols) and a rezipping force-extension curve (empty squares symbols).
We determined the unzipping force to be the lowest force where an abrupt increase in extension occurred in the unzipping force-extension curve (upward arrow). Similarly, we determined the rezipping force to be the highest force where an abrupt decrease in extension occurred in the rezipping force-extension curve (downward arrow).