Sweeping of alprenolol enantiomers with an organic solvent and sulfated β‐cyclodextrin in capillary electrophoresis

Sweeping, an on‐line sample concentration technique in CE, is the picking and accumulation of analytes by the pseudostationary phase or complexing additive. In the presence of an electric field, the analytes concentrated at the additive front that initially penetrated the sample zone. Here, we describe the sweeping of cationic alprenolol enantiomers using sulfated β‐CD and organic solvent. The separation solution contained the anionic additive while ACN was in the sample solution. With fused silica capillaries, positive polarity, and solutions buffered at pH 3, the direction of the enantiomers' effective electrophoretic mobility was the same as the electrophoretic mobility (or electrophoretic mobility without additive). When the amount of ACN in the sample was increased (i.e. 60%), the interaction between the analytes and additive became negligible. This caused the sweeping boundary to shift from the electrophoretically moving β‐CD front to the zone between the sample and separation solution. The equation that described the narrowing of injected sample zone was derived. The performance of sweeping with 60% ACN in the sample was then studied under different operating conditions (e.g. type of injection, injection time, and CD concentration). The low interaction between enantiomers and additive gave only moderate increases in sensitivity (approximately tenfold), but was improved when field enhancement was used during electrokinetic injection. With a conductivity difference (separation/sample solution) of 70 and a short injection time of 30 s at 20 kV, peak improvements of >100‐fold was easily achieved.     

In-capillary preconcentration of pirimicarb and carbendazim with a monolithic polymeric sorbent prior to separation by CZE

CZE was assayed for the separation of carbamate pesticides susceptible to protonation (Pirimicarb, Carbendazim). Different electrophoretic media with high organic contents were explored, adequate separation and resolution being achieved when a BGE based on ACN with acetic acid in the presence of SDS as an ionic additive was used. With a view to increasing the sensitivity of the method, an in-capillary SPE step prior to the electrophoretic separation was developed. We employed a monolithic polymer formed in situ within the capillary as a medium for analyte retention. The synthesized monolithic bed exhibited high porosity and allowed samples to be loaded at flow rates of about 65 microL/min by applying a pressure of 12 bar. A 5-cm length of monolithic sorbent was used to preconcentrate the target analytes from aqueous samples. The analytes retained were eluted from the polymeric phase directly in the separation capillary with the same electrophoretic medium used for their further separation by CZE. For a 15-min preconcentration time, the in-line SPE-CZE approach proposed here permitted the determination of these pesticides in drinking water at a concentration level of 0.1 microg/L, as demanded by current EU legislation.     

Nonaqueous CE using contactless conductivity detection and ionic liquids as BGEs in ACN

N,N'-Alkylmethylimidazolium cations have been separated in NACE when one of the N,N'-dialkylimidazolium salts (ionic liquids (ILs)) was used as an electrolyte additive to the organic solvent separation medium. The separated species were 1-methyl-, 1-ethyl-, 1-butyl-, 1-octyl-, 1-decyl-3-methylimidazolium and N-butyl-3-methylpyridinium cations and BGE composed of 1-ethyl-3-methylimidazolium ethylsulfate or 1-butyl-3-methylimidazolium trifluoroacetate [BMIm][FAcO] (A6; B2) diluted in ACN. It was demonstrated that contactless conductivity detection (CCD) may be applied to monitoring the separation process in nonaqueous separation media, allowing to use the UV light-absorbing imidazolium-based electrolyte additives. There could be marked three concentration regions of added ILs; at first ionic strength of BGE below 1-2 mM, and then the actual electrophoretic mobility of analytes rises from 0. At concentrations above 1-2 mM, the added IL facilitated separation. In concentration region of 1-20 mM, the actual electrophoretic mobility of analyzed imidazolium cations was increasing with decrease in separation medium ionic strength. At higher concentrations of BGE (above 30 mM), the conductivity of the separation media became too high for this detector. Some organic dyes were also successfully separated and detected by contactless conductivity detector in a 20 mM A6 separation electrolyte in ACN.     

Analyte zone sharpening in pressurized capillary electrochromatography based on electrophoretic migration under a heterogeneous field

Significant peak width reductions, or peak height enhancements, of angiotensins were observed when a high voltage was applied to hydrophilic interaction pressurized capillary electrochromatography using gradient elution with mobile phases containing perchloric acid. The investigation using a contactless conductivity detector revealed that perchloric acid was adsorbed on the surface of the stationary phase, when the acetonitrile content in the mobile phase was high, and released from the stationary phase by increasing the water content during a gradient procedure. The released perchloric acid formed a highly concentrated zone moving from the column inlet to the outlet. The electrochromatographic behavior of the analytes, primarily electrophoretic migration, was changed in this zone. As a consequence of the significant variation in migration velocity of the analytes, the sample band width was reduced similar fashion to on‐capillary concentration in capillary electrophoresis. Using this result, the reduction of band width and enhancement in separation efficiency was demonstrated in reversed‐phase pressurized electrochromatography, in which the conductivity of the mobile phase was significantly altered using a step gradient. The resolution between benzoic acid and 1‐naphthalene sulfonic acid was successfully improved from 2.7 to 4.3 by using the band width reduction method based on field‐amplified stacking.     

Separation of negatively charged nonsteroidal anti-inflammatory drugs by reversed-phase capillary electrochromatography

Reversed-phase capillary electrochromatography in a 5-microm C18 fully packed capillary was employed to optimize the separation of negatively charged nonsteroidal anti-inflammatory drugs. The effect of the physico-chemical parameters and different analysis modes on the separation of 2-arylpropionic acids was studied and evaluated. The mobile phase composition, buffer type, concentration and pH differently influenced the peak efficiency and resolution, selectively modulating the analytes interaction with the stationary phase. The use of zwitterionic MES or acetate mobile phases strongly modulated the analytes migration order and peak efficiency. The optimum experimental conditions were found in MES buffer, pH 5.0, containing the 75% acetonitrile-methanol (1:1). All the analytes were baseline separated in a mixture in less than 13 min with peak efficiencies in the range of 78,500-84,200 N/m. Under these conditions the analytes were negatively charged and their effective electrophoretic mobilities played a role in the separation. The analysis of different pharmaceutical preparations containing anti-inflammatory drugs, e.g. drops and tablets, is also presented after a very simple sample pretreatment.     

Monolithic column with double mixed-modes of hydrophilic interaction/cation-exchange and reverse-phase/cation-exchange stationary phase for pressurized capillary electrochromatography

A monolithic capillary column with double mixed-modes of hydrophilic interaction/cation-exchange and RP/cation-exchange stationary phase was prepared by in situ thermal polymerization and then hydrolyzed with hydrochloric acid. The polymerization solution containing glycidyl methacrylate (GMA), 3-sulfopropyl methacrylate potassium salt (SPMA), and ethylene dimethacrylate (EDMA) in a binary porogenic solvent consisting of dimethylformamide (DMF) and 1,4-butanediol was polymerized in a fused-silica capillary pretreated with 3-(trimetoxysilyl) propyl methacrylate. The epoxy groups on the surface were hydrolyzed to diol groups with hydrochloric acid to enhance the polarity of the stationary phase. By simply altering the ACN content in the mobile phase, two mixed-mode mechanisms could be achieved on the same monolithic column in different mobile phase condition. Hydrophilic interaction (or hydrophilic interaction/cation-exchange) was observed at high ACN content, as well as RP (or RP/cation-exchange) was observed at low ACN content. The monolithic column provided good selectivity and high efficiency for separation of neutral polar analytes and basic compounds. Phenols, anilines, alkaloids, nucleic acid bases, and narcotic pharmaceuticals have been successfully separated. Effects of salt concentration and ACN content on the separation have also been investigated. High column efficiencies of up to 352 000 plates/meter were obtained by the separation of narcotic pharmaceuticals.     

Insights into cyclodextrin interactions during sample stacking using capillary isotachophoresis with on-line microcoil NMR detection

On-line capillary isotachophoresis (cITP)-NMR experiments were used to probe the interactions of the pharmaceutical compounds S-alprenolol, S-atenolol, R-propranolol, R-salbutamol and S-terbutaline with beta-cyclodextrin (beta-CD) during cITP concentration. In cITP, ionic analytes are concentrated and separated on the basis of their electrophoretic mobility. Because neutral molecules have an electrophoretic mobility of zero, they are normally not concentrated or separated in electrophoretic experiments like cITP. Most of the analytes studied were concentrated by cITP sample stacking by a factor of around 300. For analytes that formed a strong inclusion complex, beta-CD co-concentrated during cITP sample stacking. However, once the focusing process was complete, a discrete diffusional boundary formed between the cITP-focused analyte band and the leading and trailing electrolyte, which restricted diffusion into and out of the analyte band.     

Use of large-volume sample stacking in on-line solid-phase extraction-capillary electrophoresis for improved sensitivity

We present a new system for the sensitive analysis of cephalosporins by CE using both on-line SPE and large-volume sample stacking (LVSS). Sample volumes of 250 muL were loaded onto the SPE microcolumn which was then desorbed with 426 nL of ACN. The SPE elution plug was injected into the CE system via an in-line valve interface filling approximately 60% of the volume of the separation capillary. Subsequently, LVSS was performed by applying a voltage of -5 kV, which resulted in the simultaneous removal of the elution solvent and the preconcentration of the analytes in a narrow zone. This way the amount of analyte loaded into the capillary could be considerably increased without serious loss of CE separation efficiency. LODs for cefoperazone and ceftiofur were in the ng/L range which represents an improvement of a factor of 8450 and 11 450 when compared with direct CE injection. The cephalosporin test compounds presented a good linear response (corrected peak area) between 0.5 and 10 mug/L with correlation coefficients higher than 0.995. The final method is compared with previously reported LVSS-CE and SPE-CE systems for the analysis of cephalosporins.     

Discontinuous electrolyte systems for improved detection of biologically active amines and acids by capillary electrophoresis with laser-induced native fluorescence detection

On-line concentration and separation of biologically active amines and acids by capillary electrophoresis (CE) in conjunction with laser-induced fluorescence using an Nd:YAG laser at 266 nm under discontinuous conditions is presented. The suitable conditions for simultaneous analysis of amines and acids were: samples were prepared in a solution (pH* 3.1) consisting of 10 mM citric acid, 89% acetonitrile (ACN), and water; a capillary was filled with 1.5 M Tris-borate (TB) buffer (pH 10.0); and the anodic vial contained PTG10 buffer (pH* 9.0) that consists of 50 mM propanoic acid, Tris, 10% glycerol, and water. After injecting a large-volume sample, amines and acids were separately stacked at the front (cathodic side) and back (anodic side) of the acidic sample zone, mainly because of changes in their electrophoretic mobilities as a result of changes in pH, viscosity, and electric field when high voltage was applied. When the sample was injected at 15 kV for 360 s, the concentration limits of detection (LODs) for 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) were 0.27 and 0.31 nM, respectively, which are about 400- and 800-fold sensitivity improvements when compared to those injected at 1 kV for 10 s. For the analysis of amines, samples were prepared in 100 mM citric acid (pH* 1.8) containing 89% ACN and both the capillary and anodic vial were filled with 400 mM PTG20 (propanoic acid, Tris, 20% glycerol, and water) at pH* 4.5. Using a large injection volume (15 kV for 360 s), we achieved concentration LODs of 17 pM and 0.3 nM for tryptamine and epinephrine, which are about 5200- and 14,000-fold sensitivity improvements, respectively, in comparison with those injected at 1 kV for 10 s. The features of simplicity (no sample pretreatment), rapidity (12 min), and sensitivity for identification of amines and acids of interest in urine samples show diagnostic potential of the two approaches developed in this study.     

Combining poly (methacrylic acid‐co‐ethylene glycol dimethacrylate) monolith microextraction and octadecyl phosphonic acid‐modified zirconia‐coated CEC with field‐enhanced sample injection for analysis of antidepressants in human plasma and urine

A method based on poly (methacrylic acid‐co‐ethylene glycol dimethacrylate) monolith microextraction and octadecylphosphonic acid‐modified zirconia‐coated CEC followed by field‐enhanced sample injection preconcentration technique was proposed for sensitive CE‐UV analysis of six antidepressants (doxepin, clozapine, imipramine, paroxetine, fluoxetine and chlorimipramine) in human plasma and urine. A poly(methacrylic acid‐co‐ethylene glycol dimethacrylate) monolithic capillary column was introduced for the extraction of antidepressants from urine and plasma samples. The hydrophobic main chains and acidic pendant groups of the monolithic column make it a superior material for extraction of basic analytes from aqueous matrix. After extraction, the desorption solvent, which normally provided an excellent medium to ensure direct compatibility for field‐enhanced sample injection in CE, was analyzed by CE directly. By the use of alkylphosphonate‐modified zirconia‐coated CEC for separation of the basic compounds of antidepressants, high separation efficiency and resolution were achieved because that both hydrophobic interaction between analytes and alkylphosphonate‐modified zirconia coat and electrophoretic effect work on the separation of antidepressants. The best separation was achieved using a buffer composed of 0.3 M ammonium acetate (adjusted to pH 4.5 with 1 M acetic acid) and 35% ACN v/v, with a temperature and voltage of 20°C and 20 kV, respectively. By applying both preconcentration procedures, LODs of 11.4–51.5 and 3.7–17.0 μg/L were achieved for the six antidepressants in human plasma and urine, respectively. Excellent method of reproducibility was found over a linear range of 50–5000 μg/L in plasma and urine sample.     

Investigations on the behaviour of acidic, basic and neutral compounds in capillary electrochromatography on a mixed-mode stationary phase

This work describes the separation of acidic, basic and neutral organic compounds as well as inorganic anions in a single run by capillary electrochromatography employing a stationary phase which exhibits both strong anion-exchange and reversed-phase chromatographic characteristics. The positive surface charge of this stationary phase provided a substantial anodic electroosmotic flow. The analytes were separated by a mixed-mode mechanism which comprised chromatographic interactions (hydrophobic interactions, ion-exchange) as well as electrophoretic migration. The influence of ion-exchange and hydrophobic interactions on the retention/migration of the analytes could be manipulated by varying the concentration of a competing ion and/or the amount of organic modifier present in the background electrolyte. Additionally the effects of pH changes on both the chromatographic interactions as well as the electrophoretic migration of the analytes were investigated.     

Improving sensitivity by large-volume sample stacking combined with sweeping without polarity switching by capillary electrophoresis coupled to photodiode array ultraviolet detection

An easy, simple, and highly efficient on-line preconcentration method for polyphenolic compounds in CE was developed. It combined two on-line concentration techniques, large-volume sample stacking (LVSS) and sweeping. The analytes preconcentration technique was carried out by pressure injection of large-volume sample followed by the EOF as a pump pushing the bulk of low-conductivity sample matrix out of the outlet of the capillary without the electrode polarity switching technique using five polyphenols as the model analytes. Identification and quantification of the analytes were performed by photodiode array UV (PDA) detection. The optimal BGE used for separation and preconcentration was a solution composed of 10 mM borate-90 mM sodium cholate (SC)-40% v/v ethylene glycol, without pH adjustment, the applied voltage was 27.5 kV. Under optimal preconcentration conditions (sample injection 99 s at 0.5 psi), the enhancement in the detection sensitivities of the peak height and peak area of the analytes using the on-line concentration technique was in the range of 18-26- and 23-44-fold comparing with the conventional injection mode (3 s). The detection limits for (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (C), (-)-epigallocatechin gallate (EGCG), and (-)-epicatechin gallate (ECG) were 4.3, 2.4, 2.2, 2.0, and 1.6 ng/mL, respectively. The five analytes were baseline-separated under the optimum conditions and the experimental results showed that preconcentration was well achieved.     

Determination of mercaptopurine and its four metabolites by large-volume sample stacking with polarity switching in capillary electrophoresis

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.     

On-line stacking techniques for the nonaqueous capillary electrophoretic determination of acrylamide in processed food

In the present study, field amplified sample stacking (FASS) techniques in the nonaqueous capillary electrophoresis method (NACE) were introduced for the on-line concentration of the acrylamide to improve acrylamide detection at 210 nm by diode-array detection. Acetonitrile (ACN) as a nonaqueous solvent permits acrylamide to be protonated through the change of its acid-base chemistry, allowing capillary electrophoretic separation of this compound. Choosing 30 mmol L(-1) HClO(4), 20 mmol L(-1) NaClO(4), 218 mmol L(-1) CH(3)COOH in ACN as the separation electrolyte and employing sample stacking methods, the LOD value of acrylamide was decreased to 2.6 ng mL(-1) with electrokinetic injection and 4.4 ng mL(-1) with hydrodynamic injection. Optimized stacking conditions were applied to the determination of acrylamide in several foodstuffs. The method is simple, rapid, inexpensive, and widely applicable for the determination of acrylamide in food samples.     

磺化β-环糊精的合成及其在毛细管电泳手性拆分中的应用

 采用直接磺化法合成了 3种不同取代度的磺化β-环糊精 ,并对合成产物进行了表征。将这 3种负电性的β-环糊精衍生物应用于毛细管电泳手性化合物的拆分研究中 ,考察了不同 p H值和不同电极性条件下负电性磺化β-环糊精对手性化合物的立体选择作用。     

Simultaneous determination of mitotane,its metabolite,and five steroid hormones in urine samples by capillary electrophoresis using β-CD2SDS1 complexes as hydrophobic compounds solubilizers

Mitotane is a cytotoxic drug used in the treatment of inoperable adrenocortical carcinoma, it inhibits steroidogenesis as well, and therefore monitoring the level of steroid hormones in patients treated with mitotane is a crucial point of therapy. Hence, we have developed a simple, fast, and efficient electrophoretic method combined with reverse polarity sweeping as online preconcentration technique and dispersive liquid–liquid microextraction for the simultaneous determination of mitotane, its main metabolite DDA, and five steroid hormones (progesterone, testosterone, epitestosterone, cortisol, and corticosterone) in urine samples. In addition, a new sample matrix consisting of β-CD₂SDS₁ complexes for a high hydrophobic compounds solubilization was developed. Approach based on the application of β-cyclodextrin and SDS complex of a ratio 2:1 allowed for hydrodynamic injection into the capillary of a solution containing both mitotane and other analytes. The detection limits of the analytes for the reverse polarity sweeping-dispersive liquid–liquid microextraction method were found to be in the range of 1.5–3 ng/mL, which were approximately 1000 times lower than in the conventional hydrodynamic injection (5 s, 0.5 psi) without any preconcentration procedure. All analytes were completely resolved in less than 13 min by uncoated silica capillary with an inner diameter of 75 μm (ID) × 60 cm. Electrophoretic separation was performed in reverse polarity with a voltage of –25 kV with a background electrolyte (BGE) consisting of 100 mM SDS, 25% ACN, 25 mM phosphate buffer (pH 2.5), and 7 mM β-cyclodextrin.     

Capillary electrophoretic enantioselective determination of zopiclone and its impurities

A capillary electrophoretic enantioselective method with UV detection was developed and validated for the simultaneous quantification of zopiclone enantiomers and its impurities, zopiclone-N-oxide enantiomers, and 2-amino-5-chloropyridine, in tablets. The analytes were extracted from the tablets using ACN and were separated in an uncoated fused-silica capillary (50 μm, 42 cm effective length, 50 cm total length) using 80 mM sodium phosphate buffer pH 2.5 and 5 mM carboxymethyl-β-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A voltage of 27 kV was applied and the capillary temperature was maintained at 25°C. All enantiomers were analyzed within 8 min and linear calibration curves over the concentration range of 0.4-0.8 mg mL?1 for each zopiclone enantiomer, 0.8-1.6 μg mL?1 for 2-amino-5-chloropyridine and 0.4-0.8 μg mL?1 for each zopiclone-N-oxide enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of zopiclone under stress conditions. This application demonstrated the importance of a stability-indicating assay method for this drug.     

Large-volume stacking in capillary electrophoresis using a methanol run buffer

Highly sensitive nonaqueous capillary electrophoresis of weakly acidic organic compounds has been performed using methanol as the run buffer solvent. Methanol provided appropriate suppression of the electroosmotic flow and an increase in the electrophoretic mobilities of anionic solutes compared to water. These two effects allowed large-volume stacking using the electroosmotic flow pump (LVSEP) to be achieved for larger anions using a bare fused-silica capillary under an electric field of reverse polarity, whereas only fast-moving small anions were previously known to be suitable for LVSEP in aqueous media. A field-enhanced sample injection of an additional amount of analytes during the solvent plug removal further enhanced the limits of detection to below the nanomolar range with conventional UV absorption detection. Under optimum conditions, excellent linear responses and reproducibility in the migration times together with the corrected peak areas for ten analytes were obtained in the concentration range of 10-100 nM.     

Nonaqueous capillary electrophoresis with laser-induced fluorescence detection: a case study of comparison with aqueous media

A novel method based on separation by nonaqueous capillary electrophoresis (NACE) combined with laser-induced fluorescence (LIF) detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were chosen as test analytes for their subtle fluorescence properties. In aqueous systems, capillary zone electrophoresis (CZE) was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer. The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response, and the enhancement seems to be comparable for micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). As another alternative, NACE separation was advantageous when performing the analysis under the optimum separation condition of 20 mM sodium tetraborate, 20 mM sodium dodecyl sulfate (SDS), 0.1% (v/v) glacial acetic acid, 20% (v/v) acetonitrile (ACN) in methanol medium after derivatization in ACN/dimethyl sulfoxide (DMSO) (3:2, v/v) mixed aprotic solvents containing 20 mM ammonium acetate. Compared with derivatization and separation in aqueous media, NACE-LIF procedure was proved to be superior, providing high sensitivity and short migration time. Under respective optimum conditions, the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures. In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.     

Capillary electrochromatography of biologically relevant flavonoids

Flavonoids were separated utilizing CEC technique. Baseline separation of biologically relevant flavonoids was obtained using a 100 microm ID fused-silica capillary filled with 3 microm Silica-C18 material and an optimized mobile phase comprising of 20 mM Tris-HCl (pH 6.5), ACN and water at a ratio of 10/40/50 v/v/v. Separations were carried out at 25 kV and a column temperature of 25 degrees C. The influence of relevant parameters for the CEC separation, such as buffer concentration, pH, separation voltage, and ACN concentration, was investigated and optimized. Dependencies of the electroendoosmotic flow (EOF) on these parameters and effects on the resolution of the analytes were studied. During analyses the solvents used for dissolving the samples turned out to have significant effects on the separation of flavonoids. The optimized system was then successfully used for the separation of the flavonoids epicatechin, myricetin, quercetin, naringenin, and hesperetin. CEC turned out to be a useful complementary tool for the economic analysis of flavonoids in addition to common HPLC, muHPLC, and CE methodologies. This method can be used for real applications in phytomics.