Application of Sol–Gels Modified with Natural Plants Extracts as Stationary Phases in Open-Tubular Capillary Electrochromatography

Ethanol extracts of three widely growing plants were added to silica sol–gel solutions, which were subsequently applied as wall surface modifiers in inner quartz capillaries. Modified capillaries were used for open-tubular capillary electrochromatographic separation of nucleotides and amino groups containing biological compounds (neurotransmitters, amino acids and oligopeptides). The experiments were performed at physiological pH 7.40, and eventual changes of effective mobilities were calculated. Specific compounds characteristic for each plant were tested as sol–gel additives as well, and thus-modified capillaries were used for the separations of the same analytes under identical conditions. The aim of this study was to find out possible interactions between physiological compounds and extracts of freely available plants anchorded in the sol-gel stationary phase in the flowing system. Even though the amount of the modifier in each capillary is very small, basic statistical evaluation showed some not negligible changes in effective mobility of tested analytes. These changes were bigger than ±5% for separations of nucleotides in capillaries with curcuma, Moringa or the mixture of synthetic additives as the sol-gel aditive, and for separations of amino compounds where these changes varying by additive, analyte by analyte.


Introduction
Over the past few decades, numerous stationary phases have been widely used in open-tubular capillary electrochromatography (OT-CEC) separation [1]. Different types of natural or synthetic materials have been tested as the inner-surface capillary modifiers for the separation of a large spectrum of analytes [1,2]. Concurrently, another rapidly growing area of interest is the entrapment of biological materials into silica sol-gel matrices and the application of these matrices as biomaterials in bone tissue regeneration, drug delivery, biosensors, biomedical applications and many other applications [3][4][5][6][7][8][9][10]. The combination of the advantages of OT-CEC and the entrapping possibilities of the sol-gel technique (inner capillary wall modification) allows the possibility of tracking the interactions between incorporated natural substances and biological analytes in a relatively short time [11], which is the subject of our interest. Plant-derived products have been used for healing in many nations and cultures for centuries, and most current drugs originate from natural products [12]. Therefore, we examined the physiological importance and applications of three natural products-Curcuma longa, Moringa oleifera and Hypericum perforatum.

Results and Discussion
Several factors need to be considered at the beginning of this section. First, we were observing interactions in flowing system. Secondly, electroseparations of these analytes are usually performed at lower (about 2.5) or higher (about 9.0) pH values of the running buffer. The electroosmotic flow is suppressed to minimum at low pH values, which provides a longer time for interaction, and increases with the increasing pH. Thus, the time for possible interaction shortens accordingly to the buffer pH set for analyses. Thirdly, the electroseparations of physiological compounds (substances) are seldom used in neutral buffer, because it is more difficult to separate them from each other, if an additive (i.e., sodium dodecyl sulphate) is not included in the buffer.
To approximately mimic physiological conditions, the separations were performed in 0.05 mol/L phosphate buffer at pH 7.40. Importantly, analysis of a group of analytes had to be carried out as fast as possible, ideally within one day-a requirement that was not always achieved because of the long duration of the analyses, particularly during nucleotide separations. Thus, migration times could shorten from day to day. This phenomenon could be caused by changes in the inner capillary surface due to chemical and/or structural modifications or wiping or leaking of an additive during separations, each of these phenomena differing by the number of repeated separations. However, unless otherwise stated, the separations of the final complete mixtures were performed at least three times in one day. The other reason for the shortening of the migration time, especially for the curcuma-modified capillary, is that curcumin is unstable at neutral and basic pH values and undergoes degradation to ferulic acid and feruloylmethane [12]. Most curcumin is rapidly degraded within 30 min when placed in phosphate buffer systems at pH 7.2 [12]. It is also worth mentioning that the process of sol-gel condensation could be accelerated by the use of a basic compound, e.g., aminopropyl triethoxysilane, but this was not applied to maintain the maximum access of the separated analytes to the components of the extract dispersed in a sol-gel layer.
The electrochromatograms obtained during the separations of amino group compounds are shown in Figure 1. Extreme changes in the separation of these substances were not observed, except for better resolution of glutathione (7) and HIAA (8), especially in capillaries modified with curcuma extract (line B, resolution R 7,8 = 2.50) and a mixture of plant extracts (line E, resolution R 7,8 = 3.62). The separation in the last-mentioned capillary modified with a sol-gel containing a mixture of plant extracts gave the longest migration time. The analytes migrated in the same order in all nine capillaries, which is why the only numerically described electrochromatogram is the first one on the top of Figure 1 (line A) of the pure sol-gel modifier. The resolution of glutathione (7) and HIAA (8) in all other capillaries varied by the interval R 7,8 = 1.59-2.30.
The percentage changes in the effective mobilities of amino group analytes compared to 100% mobility in a capillary modified with pure sol-gel are graphically presented in Figure 2. Interestingly, angiotensin showed two peaks in the electrochromatograms, regardless of the type of capillary installed for separation. Thus, the two peaks of angiotensin in Figures 1 and 2 are marked as A 1 and A 2 in both the electrochromatogram and graphical representation, respectively. Figure 2 also shows that the two positively charged analytes, i.e., acetylcholin chloride (AChCl) (1) and epinephrine (2), migrate through the capillaries almost untouched by the inner-wall modifiers, in contrast with the negatively charged anions migrating behind electroosmotic flow (EOF), which seemed to be more affected by the sol-gel modifier.
Similarly, the electropherograms of separations of nucleotides in modified capillaries are summarized in Figure 3. In capillary modified with sol-gel containing St. John's wort extract, CTP and UTP did not elute from the capillary in the required time of 60 min (Line D). Furthermore, none of the modified capillaries contributed to better resolution; this was especially true of CTP (10) and UTP (11), which usually migrated in one peak or only partially divided.  The percentage changes in the effective mobilities of amino group analytes to 100% mobility in a capillary modified with pure sol-gel are graphically pr Figure 2. Interestingly, angiotensin showed two peaks in the electrochromato gardless of the type of capillary installed for separation. Thus, the two peaks of sin in Figures 1 and 2 are marked as A1 and A2 in both the electrochromato Similarly, the electropherograms of separations of nucleotides in modified capillaries are summarized in Figure 3. In capillary modified with sol-gel containing St. John's wort extract, CTP and UTP did not elute from the capillary in the required time of 60 min (Line D). Furthermore, none of the modified capillaries contributed to better resolution; this was especially true of CTP (10) and UTP (11), which usually migrated in one peak or only partially divided. Relative effective mobility changes of amino group compounds in capillaries with modified sol-gels compared to 100% of the capillary with pure sol-gel without additive (line A). A t-test p < 0.05 is marked by the symbol above colored bars. The symbol indicates that the values of the effective mobility changes were greater than ±5% of the standard deviation of Gaussian normal distribution, compared to the pure sol-gel capillary.
The changes in effective mobilities are graphically represented in Figure 4, where the mobilities of nucleotides calculated in the pure sol-gel capillary are taken as 100% again. A t-test revealed that the most significant changes of effective mobility of individual compounds, marked with , were registered for capillaries modified with curcuma extract (line B), Moringa extract (line C) and a mixture of synthetic modifiers (line I). The same three capillaries showed changes greater than 5% (more than the statistical standard deviation) in the effective mobility of nucleotides (marked as ) compared to the pure sol-gel capillary with no additives. As in the separation of amino group compounds, the order of nucleotides migrating to the detector remained the same, which is why the complete numerical description is only for the first capillary (line A) modified with pure sol-gel with no additive. Other specifically numbered electrochromatograms described nucleotides migrating broadly (line B and C) or undetectably (line D). The biggest differences in resolution were noted for substances GTP (7), CDP (8) and ATP (9), as follows: the resolution between GTP (7) and CDP  The changes in effective mobilities are graphically represented in Figure mobilities of nucleotides calculated in the pure sol-gel capillary are taken as A t-test revealed that the most significant changes of effective mobility of ind pounds, marked with , were registered for capillaries modified with cur (line B), Moringa extract (line C) and a mixture of synthetic modifiers (line three capillaries showed changes greater than 5% (more than the statistical resolution were noted for substances GTP (7), CDP (8) and ATP (9), as follows: the resolution between GTP (7) and CDP (8)   Let's summarize the overall positive results. It could be assumed that the effect of the plant extract anchored in the sol-gel was greater than the effect of the plant-dominant substance alone. Even though the amount of the dominant substance in an extract is much lower than when used as a single synthetic equivalent, and lowers again because of its dilution in the sol-gel solution and creating only thin layer on the wall, these trace amounts still seem to have not negligible effect on the separation. Notable changes of the effective mobility might be probably caused by the multiple interactions with the many extract components. Interactions between the modifier and an analyte took place, particularly for amino group-possessing compounds, but these varied from capillary to capillary, analyte to analyte. The sol-gel OT-CEC approach can be used to separate amino compounds at neutral conditions that are not possible with unmodified capillaries and can be tailored by various additives. In addition, we chose this method mainly as a way to monitor the additive-analyte interaction, not primarily to improve the separation. Let's summarize the overall positive results. It could be assumed that the effect of the plant extract anchored in the sol-gel was greater than the effect of the plant-dominant substance alone. Even though the amount of the dominant substance in an extract is much lower than when used as a single synthetic equivalent, and lowers again because of its dilution in the sol-gel solution and creating only thin layer on the wall, these trace amounts still seem to have not negligible effect on the separation. Notable changes of the effective mobility might be probably caused by the multiple interactions with the many extract components. Interactions between the modifier and an analyte took place, particularly for amino group-possessing compounds, but these varied from capillary to capillary, analyte to analyte. The sol-gel OT-CEC approach can be used to separate amino compounds at neutral conditions that are not possible with unmodified capillaries and can be tailored by various additives. In addition, we chose this method mainly as a way to monitor the additive-analyte interaction, not primarily to improve the separation.
The most significant changes in nucleotides separations were noted for capillaries modified with curcuma, Moringa and mixture of synthetic additives.
Several factors may play a role in all these changes mentioned above: the possible character and quantity of non-covalent interactions between the analyte and an additive (e.g., host-guest interaction, metal-ligand coordination, hydrogen bonds, π-π stacking), and the higher number of potential reactants (components) in a natural modifier. Another aspect, better or worse, of the separations in modified capillaries, is the peak broadening, whether a natural or synthetic additive was added to the sol-gel. It might be brought about by narrowing of the capillary inner surface due to the layer of the sol-gel and eventual interactions with the modifier. The advantages of the stationary phase thus prepared for OT-CEC include easy preparation of the plant ethanol extract and the sol-gel solution, easy modification process and the fact, that the modified sol-gels do not interfere with PDA detection.
Of course, there are some undesirable facts. Firstly, the possibility of unstable modification caused by either leaking of the extract or the mechanical loss of the modifier.
Secondly, investigation of interactions of the test analytes with each of many substances present in the extract would be time consuming. In this article, we focused on the question/answer of whether the addition of the whole extract or a dominant compound causes any noticeable change at all or not.
Thirdly, a natural or synthetic additives fastened into the sol-gel modifier may lose a degree of freedom, thus losing their ability to form spatial interactions.
Because this methodology of monitoring possible interactions is rather unique, it is difficult to compare the obtained results with other similar publications.

Conclusions
The aim of this work was to determine the capability of biological compounds to interact with plant extracts using OT-CEC as an experimental method to approximately mimic interactions in biological systems. While some aspects of the study require improvemente.g., more stable sol-gel modification, longer interaction times, higher concentration of either natural or synthetic additives etc.-partial interactions were demonstrated and calculated under near-physiological conditions. The interaction of the whole herb extract with the compounds of this model system can consist even in low doses of all active substances present in it together. Synergistic interactions between the components of individual herbs or mixtures of herbs are an important part of their therapeutic efficacy. And since the number of possible plant extracts of enormous number of plants all around the world is almost unlimited and offers a vast number of combinations, we can fine-tune the proportions of the basic sol-gel reaction components and various additives following the required purpose of its final applications.

Electrochromatographic Analyses
Electrochromatographic analyses were performed on the Beckmann Coulter P/ACE 5500 (Fullerton, CA, USA) apparatus. Detection with a photodiode array (PDA) detector was tuned to the 254 nm wavelength for nucleotides and 214 nm for amino group- The dried plant parts were immersed in pure ethanol (EtOH) to yield the following final concentrations: curcuma (5.257 mg/mL), St. John's wort (67.666 mg/mL) and Moringa (109.2 mg/mL). The dried plants were left to leach in ethanol for at least 2 months at room temperature in ground glass beakers covered with aluminum foil to prevent light access. The liquid fraction (extract) was then aspirated with a syringe and filtered to a vial from which the appropriate amount was used for the sol-gel modification procedure. The filtered extracts of the natural plants were optically clear and had vibrant colors: bright yellow for curcuma, blood red for St. John's wort and grass green for Moringa. The three synthetic additives, curcumin, isoquercetin and hypericin, were dissolved in ethanol to yield a concentration of 1 mg/mL. The mentioned process is simply illustrated in the Scheme 1.

Electrochromatographic Analyses
Electrochromatographic analyses were performed on the Beckmann Coulter P/ACE 5500 (Fullerton, CA, USA) apparatus. Detection with a photodiode array (PDA) detector was tuned to the 254 nm wavelength for nucleotides and 214 nm for amino group-containing physiological compounds, with a bandwidth of 10 nm. Alternatively, two cartridges with slightly different widths of detection windows were used: 100 µm × 200 µm or 100 µm × 800 µm. Fused silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) with a total length of 27 cm and an effective length of 20.5 cm (75 µm I.D. and 375 µm O.D.) were used for all separations. The injection was performed with an applied pressure of 3.4 kPa.

Preparation of the Sol-Gel Additives
The dried plant parts were immersed in pure ethanol (EtOH) to yield the following final concentrations: curcuma (5.257 mg/mL), St. John's wort (67.666 mg/mL) and Moringa (109.2 mg/mL). The dried plants were left to leach in ethanol for at least 2 months at room temperature in ground glass beakers covered with aluminum foil to prevent light access. The liquid fraction (extract) was then aspirated with a syringe and filtered to a vial from which the appropriate amount was used for the sol-gel modification procedure. The filtered extracts of the natural plants were optically clear and had vibrant colors: bright yellow for curcuma, blood red for St. John's wort and grass green for Moringa. The three synthetic additives, curcumin, isoquercetin and hypericin, were dissolved in ethanol to yield a concentration of 1 mg/mL. The mentioned process is simply illustrated in the Scheme 1.

Modification of the Inner Capillary Surface
First, the fused silica capillary was prewashed in the following steps, each for 10 min: Milli-Q water, 1 mol/L NaOH, Milli-Q water, 1 mol/L HCl, Milli-Q water, ethanol, followed by an air flush. The standard basic solution of a sol-gel contained the following: 50 µL Milli-Q H 2 O, 100 µL EtOH, 50 µL TEOS, 50 µL of an additive and 20 µL 0.1 mol/L HCl. All ingredients were mixed, vortexed and left to react for 20 min. After a transparent clear solution was obtained, the capillary was washed with the solution using suction as follows: one end of the capillary was inserted into home-made tapering plastic tubing connected to an injection syringe, and the other end was immersed in an Eppendorf vial containing the sol-gel solution. After the solution was vacuum-aspirated at the syringe end for 30 min, both ends of the capillary were sealed with parafilm and left at room temperature overnight. To prepare a capillary modified with pure sol-gel without any additive, 50 µL of EtOH was added to the sol-gel solution instead of the additive. (mixture 1). Similarly, the mixture of synthetic plant-specific additives contained 16.6 µL of each characteristic compound dissolved in EtOH (1 mg/mL) to give 50 µL of additive again (mixture 2). On the next day, the rest of the unreacted inner-wall modifier was removed from the capillary with a syringe. The capillary was then flushed with air and nitrogen (5 min each, pressure 200 kPa) and left open at room temperature until experiments had been performed (usually 4 weeks).

Conditioning (Stabilization) of Capillaries and Electrochromatographic Separation
After modification and rest at room temperature, the capillaries were prepared for the separation experiments. Each capillary was first washed with water for 5 min and then washed with the mobile phase (buffer) for 10 min. All experiments were performed in 0.05 mol/L disodium hydrogenphosphate solution at a pH of 7.40, adjusted with 1 mol/L hydrochloric acid.
The basic concentration of the samples was 0.1 mg/mL for compounds with amino groups and 0.5 mg/mL for nucleotides. The separations were performed in a step-by-step process: initially, 20 µL of pure distilled water was used in a microvial with 2.5 µL of the first analyte, and the electrochromatographic separation was left to run. Next, another 2.5 µL of the other analyte was added to the first analyte in a microvial, and the separation proceeded again. Thus, the mixture of analytes was separated sequentially to verify the migration time.

Analytes
Simple compounds important for physiological processes were chosen as the first test set.

Mathematical Calculations
The effective mobilities of individual analytes were calculated according to the equation (µ eff is effective mobility, µ app is apparent mobility and µ EOF is mobility of electroosmotic flow), while µ = l.L t.V (cm 2 /min.V) (l is effective capillary length to the detector, L is total capillary length, t is migration time and V is applied voltage).
Resolution of two consecutive peaks was calculated according to the equation t is migration time of an analyte and w is baseline peak width. 4.6. Statistics t-test was set and calculated as programmed in the MS Excel, Function t-test, (i.e., field 1, field 2, 2 sides, type 3), the standard deviation was calculated in MS Excel program as well.
Author Contributions: Experimental design, data measurement, manuscript preparation, J.S.; overall supervision, final graphical design, manuscript supervisor, I.M. All authors have read and agreed to the published version of the manuscript.