Ternary Mobile Phase Research Articles

Ligand Exchange Chromatography of Nitrophenol Positional Isomers on Co‐CMEDA‐Silica Gel

Nitrophenols are common pollutants detected in industrial effluents because they are used as basic raw materials in various industrial processes. Due to their toxicity to living things they are chemical species that need to be constantly monitored. Hence, the separation of nitrophenol isomers is very important to determine their levels in the environment. Since technical p‐nitrophenol is contaminated with m‐nitrophenol and the other aromatic compounds, the selective separation of p‐nitrophenol from undesired impurities has become one of the important issues of the paracetamol synthesis. In the present work, a new chromatographic method for the separation of nitrophenol isomers on Co(II)‐tris(carboxymethyl)ethylenediamine‐silica gel (Co‐CMEDA‐Silica gel) has been developed using high performance liquid chromatography (HPLC). The chromatographic conditions were optimized by studying the effects of mobile phase composition. The positional isomers of nitrophenol (o‐, m‐, and p‐nitrophenol) could be separated by using ternary mobile phases with water/methanol/acetonitrile in the ratio 5:20:75 under isocratic conditions. The baseline separation of nitrophenol isomers has been achieved successfully with the stepwise gradient elution.

Isolation, Purification and Quantification of Ginsenoside F₅ and F₃ Isomeric Compounds from Crude Extracts of Flower Buds of Panax ginseng.

In this paper, the isolation, purification and quantification of ginsenoside F5 and F3 isomeric compounds from crude extracts of flower buds of Panax ginseng (CEFBPG) was investigated by reversed-phase high-performance liquid chromatography (RP-HPLC) for the first time. The satisfied separation at analytical scale was achieved using a Zorbax Eclipse XDB C-18 column with a ternary mobile phase of acetonitrile–water–phosphoric acid (28:71:1) at a flow rate of 1.0 mL/min within 40 min. UV detection was set at 203 nm. Ginsenoside F5 and F3 was 4.21 mg and 5.13 mg in 1 g flower buds of P. ginseng (FBPG), respectively. The preparation of ginsenoside F5 and F3 at semi-preparative scale was performed by using a Daisogel C-18 column and gradient elution system of acetonitrile–water (32:68 → 28:72) at a flow rate of 10 mL/min with a sample load of 20–30 mg, and yielded ginsenosides in purity of more than 96%. Their structures were characterized by NMR and high resolution electrospray ionization mass spectrometry (HRESIMS). All the method validations showed acceptable limits. The results indicate a new source to obtain ginsenoside F5 and F3, and show that the method developed here appears to be reliable for simultaneously preparing them from CEFBPG.

Planar Electrochromatography Using an Electrospun Polymer Nanofiber Layer

Electrospun polymer nanofiber stationary phases were examined for their application to planar electrochromatography (PEC). Separations were performed on polyacrylonitrile nanofiber ultra-thin-layer chromatography (UTLC) plates in 1-2 min using a ternary mobile phase. The influences of buffer concentration and pH, ratio of organic modifier, and development time on analyte migration distances were studied. Band broadening in this system was studied as a function of distance. The plate height initially decreased and then plateaued with a minimum plate height value as low as 11 μm. Nanofiber alignment considerably increased analyte migration rate, but larger spot sizes were noted when nearly complete fiber alignment was used. The easily tunable stationary phase thickness can be tailored to a given separation, where thinner layers promote faster separations and thicker layers are ideal for more complex mixtures. Compared to UTLC, PEC offers unique selectivity and decreased analysis time (>4 times faster over 15 mm). Results for a two-dimensional separation using UTLC and PEC are also reported. These rapid separations required 11 min using a 40 × 40 mm plate and exhibited a significant increase in separation number (70-77).

Fast Separation Method Development for Supercritical Fluid Chromatography Using an Autoblending Protocol

Supercritical fluid chromatography (SFC) is a powerful separation technique particularly in the area of enantioseparations. With rapid analysis speed, wide polarity compatibility, higher column efficiency and lower cost of the mobile phase, SFC is regarded as a better choice than high-performance liquid chromatography for drug discovery. In the development of separation method, the choice of modifier and/or additive is the key point of optimum separation. However, such screening of SFC is typically time-consuming. In this study, an autoblending protocol was introduced to speed up the modifier and/or additive screening process, which was performed on a separate programmable gradient proportioning system. The protocol prepares mobile phases on the fly to speed up the screening of modifiers and/or additives and reduces the waste of solutions. Furthermore, by switching mobile phase in the same run, separation of different types of compounds could also be achieved. This system was successfully applied to screen modifier–additive combinations of three alkaloids and three polyphenols by switching to two mobile phase conditions, as well as by a ternary additive mobile phase on an SFC system. The proposed protocol allows fast separation method development of SFC, which was proved to be rapid, simple, and reproducible.

High-performance liquid chromatography assay with programmed flow elution for cisatracurium in human plasma: Application to pharmacokinetics in infants and children

A high-performance liquid chromatography (HPLC) assay with fluorescence detection (FLD) for quantification of cisatracurium in human plasma was developed and fully validated. Liquid-liquid extraction was employed for sample preparation. The separation was carried out on a C18 column with ternary mobile phase composed of 30mmolL(-1) phosphate buffer (pH 3.0), acetonitrile and methanol (60:35:5, v/v/v). Verapamil was used as the internal standard. The isocratic elution with programmed flow rate was employed by setting at 0.8mLmin(-1) from 0 to 3.5min, 0.5mLmin(-1) from 3.5 to 6min, and 1.0mLmin(-1) from 6 to 10min. The fluorescence detection was performed at 236nm for excitation and 324nm for emission. The assay was linear from 50 to 2800ngmL(-1), with a detection limit of 12ngmL(-1). The correlation coefficient (r) for linear regression was 0.9997. The intra-day coefficients of variation (CVs) were less than 2.0%, and the inter-day CVs were less than 4.0%. The mean recoveries were in the range of 92.1-100.4%. The total HPLC run time was less than 10min. The developed HPLC method was fast, simple, sensitive, accurate and suitable for studying the pharmacokinetics of cisatracurium in infants and children after intravenous administration.

HPLC–ED of low-molecular weight brominated phenols and tetrabromobisphenol A using pretreated carbon fiber microelectrode

Electrochemically pretreated carbon fiber microelectrode was used to develop a simple, fast and sensitive HPLC-ECD method for the determination of brominated phenols. In addition to simple mono-, di- and tri-bromophenols (4-bromophenol, 2,4-dibromophenol, 2,6-dibromophenol, 2,4,6-tri-bromophenol) the possibility of electrochemical detection of 3,3',5,5'-tetrabromobisphenol A in oxidation mode is reported for the first time. The isocratic separation was achieved within 14 min using ternary mobile phase consisting of 50mM-phosphate buffer (pH 3.5), acetonitrile and methanol (35/15/50, v/v), and detection potential of E=+1450 mV (vs. Ag/AgCl). The carbon fiber microelectrode permitted to use high anodic potentials (up to +1800 mV vs. Ag/AgCl), the optimum analytical response was achieved at +1450 mV vs. Ag/AgCl. The limits of detection (LOD) for the studied analytes were within the range of 1.8-56.6 ng mL(-1). The developed method was applied to determination of brominated phenols in spiked water samples. Furthermore, after simple extraction with methyl tert-butyl ether, it was possible to quantify tetrabromobisphenol A (TBBA) in a piece of CRT monitor plastic casing. The found amount of TBBA was 10.22 mg kg(-1) (±0.43).

How Ternary Mobile Phases Allow Tuning of Analyte Retention in Hydrophilic Interaction Liquid Chromatography

An attractive yet hardly explored feature of hydrophilic interaction liquid chromatography (HILIC) is the tuning of analyte retention through the addition of an alcohol to the water (W)-acetonitrile (ACN) mobile phase (MP). When retention times increase sharply between 10/90 and 5/95 (v/v) W/ACN, intermediate retention values are stepwise accessible with a ternary MP of 5/90/5 (v/v/v) W/ACN/alcohol by switching from methanol to ethanol to isopropyl alcohol. We investigate the physicochemical basis of this retention tuning by molecular dynamics simulations using a model of a 9 nm silica pore between two solvent reservoirs. Our simulations show that alcohol molecules insert themselves neatly into the retentive W-rich layer at the silica surface, without disrupting the layer's structure or altering its essential properties. With the decreasing tendency of an alcohol (methanol > ethanol > isopropyl alcohol) to move toward the silica surface, the contrast between the W-rich layer and the bulk MP sharpens as the latter becomes more organic, while the W density near the silica surface remains high. Analyte retention increases with the ratio between the W mole fraction in the diffuse part of the W-rich layer and that in the bulk MP. We predict that tuning of HILIC retention is possible over a wide range through the choice of the third solvent in a W/ACN-based ternary MP, whereby the largest retention values can be expected from W-immiscible solvents that fully remain in the bulk MP.

MSn, LC‐MS‐TOF and LC‐PDA studies for identification of new degradation impurities of bupropion

Three new degradation impurities of bupropion were characterized through high performance liquid chromatography coupled to photodiode array detection and to time-of-flight mass spectrometry. Bupropion was subjected to the ICH prescribed stress conditions. It degraded to seven impurities (I-VII) in alkaline hydrolytic conditions which were optimally resolved on an XTerra C18 column (250 × 4.6 mm, 5 µm) with a ternary mobile phase comprising ammonium formate (20 mm, pH 4.0), methanol and acetonitrile (75:10:15, v/v). The degradation impurities (III-V and VII) were characterized on the basis of mass fragmentation pattern of drug, accurate mass spectral and photodiode array data of the drug and degradation impurities. Compound V was found to be a known degradation impurity [1-hydroxy-1-(3-chlorophenyl)propan-2-one], whereas III, IV and VII were characterized as 2-hydroxy-2-(3'-chlorophenyl)-3,5,5-trimethylmorpholine, (2,4,4-trimethyl-1,3-oxazolidin-2-yl)(3-chlorophenyl)-methanone and 2-(3'-chlorophenyl)-3,5,5-trimethylmorphol-2-ene, respectively. Compound III was a known metabolite of the drug. This additional information on the degradation impurities can help in the development of a new stability-indicating assay method to monitor the stability of the drug product during its shelf-life as well as in development of a drug product with increased shelf-life.

SIMULTANEOUS DETERMINATION OF FOUR FLAVONOID AGLYCONE ANALOGS IN THE INFLORESCENCE OF JUGLANS REGIA L. BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-DIODE ARRAY DETECTION

A reversed-phase high-performance liquid chromatography coupled with a diode array detection method and a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method were first developed and validated for the simultaneous determination of four flavonoid aglycone analogs in the inflorescence of Juglans regia L. grown in different areas of Gansu province, China, surmounting the challenges rising from the complexity of the matrices owing to different origins of the plant. The separation of the four compounds was accomplished through a ternary mobile phase, with good specificity and satisfactory resolution. The chromatographic peaks of the four investigated compounds were identified by comparing their retention time and mass spectrometric profile with the corresponding reference compounds. The method showed a good linearity (r2 > 0.9998), high precision (RSD <5%), and recoveries (ranged from 96.6–102.7%) were acceptable. The limits of detection (LOD) and the limits of quantification (LOQ) are ranged from 0.26–0.58 µg/ml and from 0.86–1.90 µg/ml, respectively. The results demonstrated that this analytical approach is ideal for the determination of such bioactive compounds as flavonoid aglycones, and that it constructed a basis for the comprehensive evaluation of the quality of Juglans regia L.

QUANTIFICATION OF PROTODIOSCIN AND PROTOTRIBESTIN IN FRUITS OF TRIBULUS TERRESTRIS L. COLLECTED FROM DIFFERENT PHYTO-GEOGRAPHICAL ZONES OF INDIA

Fruits of Tribulus terrestris L. are known for its medicinal and dietary application in the traditional medicine of India and other parts of the World. Protodioscin and Prototribestin are steroidal saponins notably found in the T. terrestris. The aim of the present study is to quantify protodioscin and prototribestin in fruits of T. terrestris collected from different phytogeographical zones of India to check the chemotaxonomic variation, if any, through HPTLC using ternary mobile phase of n butanol:glacial acetic acid:water (80:06:20, v/v/v) on precoated silica gel 60 F254 aluminum plates. Analytical studies showed considerable variations of Protodioscin and Prototribestin content in T. terrestris fruits, and it was observed that maximum percentage of Protodioscin (0.317%) found in samples from Western Ghat of India and maximum percentage of Prototribestin (0.636%) found in sample growing in the Arid zone of India.

Novel Data on the Effect of Tetrahydrofuran as an Organic Co-Modifier in RP-HPLC

The aim of the study was to characterise the unique chromatographic properties of tetrahydrofuran (THF) based on hydrogen-bonding interactions with weakly acidic compounds (pK a = 7.4–12.48) including steroids with phenolic hydroxyl groups, their substituted derivatives and heterocyclic amides having different polar functional groups (log P = 1.15–4.78). The results suggested that the organic modifier does not simply affect retention by changing the hydrophobicity of eluent, but rather specifically modifies the nature of the analyte–stationary phase interaction. In the water/isopropanol (IPA)/THF eluent mixture THF forms a hydrogen-bonded complex with the phenolic steroid compounds. The apparent formation of the THF–analyte complex depended on the proportion of components in the ternary mobile phase employed (from 70:30:0 to 70:0:30 v/v/v). The weakly acidic model compounds showed an increasing retention time with increasing THF concentration. This effect of THF was found to be a solvent-specific interaction, which was only observed in the presence of IPA. The systematic modification of the phase ratio of organic modifiers exerts a great influence on retention time and changes the separation processes over a wide range. In the case of other protic solvents (methanol, ethanol) we could not observe this selective chromatographic behaviour. From the point of view of chromatographic practice, the use of THF–IPA co-modifiers may increase the selectivity and provide excellent possibilities for separation of weakly acidic compounds including the large family of phenolic compounds.

ANALYSIS OF SULFONAMIDE RESIDUES IN REAL HONEY SAMPLES USING LIQUID CHROMATOGRAPHY WITH FLUORESCENCE AND TANDEM MASS SPECTROMETRY DETECTION

This paper presents new reversed phase liquid chromatographic methods (HPLC-FLD and LC-MS/MS) for the quantification of sulfonamides in spiked and incurred honey samples. The sample preparation was optimized using Oasis HLB (hydrophilic–lipophilic balance) solid-phase extraction (SPE) cartridge. Elutions of sulfonamides were carried out under acidic, neutral, and basic conditions using methanol. Recoveries under acid condition were in the range from 66.8–90%, which were approximately 10% higher than those obtained under other conditions. The sample clean-up was also tested using Strata-XL cartridges. The HPLC-FLD separation was performed using a Varian C18 column and a ternary (methanol-acetonitrile-phosphate buffer, pH 5) mobile phase resulting good selectivity for the determination. The robustness of the ternary gradient method was evaluated by computer simulation (DryLab). LC-MS/MS separation was carried out on a Kinetex XB core-shell type HPLC column that enabled a low limit of detection (0.01–0.5 µg/kg) and faster separation (6 min). The developed methods were validated in accordance with the European Union Commission Decision 2002/657/EC and were applied successfully for more than four hundred honey samples (under a national monitoring program). The concentrations of sulfadimethoxine, sulfachloropyridazine, and trimethoprim residues in samples were found in a concentration range from 0.03 up to 686 µg/kg.

Ternary isocratic mobile phase optimization utilizing resolution Design Space based on retention time and peak width modeling

An optimization procedure of ternary isocratic mobile phase composition in the HPLC method using a statistical prediction model and visualization technique is described. In this report, two prediction models were first evaluated to obtain reliable prediction results. The retention time prediction model was constructed by modification from past respectable knowledge of retention modeling against ternary solvent strength changes. An excellent correlation between observed and predicted retention time was given in various kinds of pharmaceutical compounds by the multiple regression modeling of solvent strength parameters. The peak width of half height prediction model employed polynomial fitting of the retention time, because a linear relationship between the peak width of half height and the retention time was not obtained even after taking into account the contribution of the extra-column effect based on a moment method. Accurate prediction results were able to be obtained by such model, showing mostly over 0.99 value of correlation coefficient between observed and predicted peak width of half height. Then, a procedure to visualize a resolution Design Space was tried as the secondary challenge. An artificial neural network method was performed to link directly between ternary solvent strength parameters and predicted resolution, which were determined by accurate prediction results of retention time and a peak width of half height, and to visualize appropriate ternary mobile phase compositions as a range of resolution over 1.5 on the contour profile. By using mixtures of similar pharmaceutical compounds in case studies, we verified a possibility of prediction to find the optimal range of condition. Observed chromatographic results on the optimal condition mostly matched with the prediction and the average of difference between observed and predicted resolution were approximately 0.3. This means that enough accuracy for prediction could be achieved by the proposed procedure. Consequently, the procedure to search the optimal range of ternary solvent strength achieving an appropriate separation is provided by using the resolution Design Space based on accurate prediction.

High-Performance Liquid Chromatographic Enantioseparation of Atropisomeric Biphenyls on Seven Chiral Stationary Phases

The HPLC enantioseparation of eight racemic atropisomeric biphenyls on commercially available polymeric Chiralcel OD-H, Lux Cellulose-1, Lux Cellulose-2, Chiralcel OJ, Lux Amylose-2 and Chiralpak OT(+) and on the brush-type Whelk-O1 columns, both in normal-phase mode and in polar organic solvent mode, has been investigated. The attempts to enantioseparate the selected biphenyls on Whelk-O1 were unsuccessful. All compounds were well resolved on almost one of the polymeric columns. Lux Cellulose-2 showed to be suitable for enantioseparation of all biphenyls. The effect of mobile phase, temperature, type of chiral selector and analyte structure on enantioseparation are examined and discussed. 2-Propanol and ethanol were employed as mobile phase modifiers and their influence on the retention and enantioseparation was investigated. Also a ternary mobile phase (n-hexane/2-propanol/methanol 91:6:3) was employed to test the separation of the eight biphenyls. In same cases, the elution with pure ethanol provided good enantioseparation in shorter elution times. The experimental data evidenced the complemental chiral recognition capabilities of polysaccharide-based CSPs. Noteworthy, Lux Cellulose-1 and Chiralcel OD-H contain the same chiral selector, but the first one exhibited higher retention factors. The evaluation of chromatographic data provided information about the chiral recognition mechanisms. In this regard, we report on the effect of ortho and meta biphenyl substituents on the retention and enantioseparation. In addition, computational evaluation of electrostatic potentials of analytes furnished a very interesting piece of information about the enantioseparability as well as the chiral recognition mechanisms on the evaluated chiral selectors. Keywords: Atropisomeric biphenyls, Enantiomeric separation, High-performance liquid chromatography, Molecular recognition, Polymetacrylate stationary phases, Polysaccharide stationary phases, catalysts, asymmetric synthesis, enantiomers, Melanoma Cell, analogues, chiral biphenyls, nuclear magnetic resonance, Chromatographic Screening, buffer

Separation of Some Aromatic Amino Acid Enantiomers with Pressurized Planar Electrochromatography and TLC

Influence of buffer pH, buffer concentration and ratio of methanol to acetonitrile in ternary mobile phase on migration distance of enantiomers of phenylalanine, tyrosine and DOPA in pressurized planar electrochromatography (PPEC) and thin-layer chromatography (TLC) systems is presented and compared. The applied operating variables in PPEC system more strongly influence on separation selectivity of investigated solutes than those in TLC one. Higher separation efficiency of PPEC system relative to TLC one is also demonstrated.

Evaluation of Ternary Mobile Phases for the Analysis of Carbonyl Compound Derivatives Using High-Performance Liquid Chromatography

In this study, the feasibility of ternary mobile phases was examined in a high-performance liquid chromatography (HPLC)-based analysis of carbonyl compounds (CCs). To test the performance of different ternary phases, the liquid phase standards containing a 15 aldehyde/ketone-DNPH(o) mix were analyzed through a series of five-point calibration experiments. For this comparison, three types of ternary mobile phases were prepared initially by mixing water (W) with two of the following three organic solvents: isopropanol (I), methanol (M), and tetrahydrofuran (T). The resulting three types of ternary phases (named as WIM, WTM, and WIT) were tested and evaluated in relation to the water content or in terms of methanol-to-water ratio (M/W). The results derived by the three ternary phases revealed that the optimal resolution was attained near maximum water content, while those of WIT consistently suffered from poor resolution problems. The relative performances of WIM and WTM phases, if assessed by three key operating parameters (sensitivity, retention time, and resolution), were found to be reliable for most selected CCs with the decreasing M/W ratio.

Analysis of polyethyleneoxide macromonomers by liquid chromatography along the critical adsorption line

Polyoxyethylene macromonomers are analyzed by one-dimensional liquid chromatography under different conditions, depending on the required information. These samples may contain polyethylene glycol (PEG) and the corresponding di(meth)acrylate besides the desired mono(meth)acrylate. The molar mass distribution (MMD) of the PEG and the monoester can be obtained by liquid adsorption chromatography (LAC) on a reversed-phase column in acetone-water with a gradient from 10% to 20% acetone. The MMD of the diesters can be obtained with isocratic elution by liquid chromatography at critical conditions (LCCC) on a reversed-phase column in 31% acetone, or using size-exclusion conditions for PEG and LAC conditions for the end groups, which is the case in 40-55% acetone. The absolute amount of the series with different functionality can be obtained by LCCC in ternary mobile phases consisting of acetone, methanol, and water along the critical adsorption line. Under such conditions, all series elute as narrow peaks (regardless their MMD), which can easily be integrated and quantified.

Hypercrosslinking: New approach to porous polymer monolithic capillary columns with large surface area for the highly efficient separation of small molecules

Monolithic polymers with an unprecedented surface area of over 600 m 2/g have been prepared from a poly(styrene- co-vinylbenzyl chloride- co-divinylbenzene) precursor monolith that was swollen in 1,2-dichloroethane and hypercrosslinked via Friedel-Crafts reaction catalyzed by ferric chloride. Both the composition of the reaction mixture used for the preparation of the precursor monolith and the conditions of the hypercrosslinking reaction have been varied using mathematical design of experiments and the optimized system validated. Hypercrosslinked monolithic capillary columns contain an array of small pores that make the column ideally suited for the high efficiency isocratic separations of small molecules such as uracil and alkylbenzenes with column efficiencies reproducibly exceeding 80,000 plates/m for retained compounds. The separation process could be accelerated while also improving peak shape through the use of higher temperatures and a ternary mobile phase consisting of acetonitrile, tetrahydrofuran, and water. As a result, seven compounds were well separated in less than 2 min. These columns also facilitate separations of peptide mixtures such as a tryptic digest of cytochrome c using a gradient elution mode which affords a sequence coverage of 93%. A 65 cm long hypercrosslinked capillary column used in size exclusion mode with tetrahydrofuran as the mobile phase afforded almost baseline separation of toluene and five polystyrene standards.

HPTLC METHOD FOR QUANTIFICATION OF VALERENIC ACID IN AYURVEDIC DRUG JATAMANSI AND ITS SUBSTITUTES

Objective of the present study was quantification of valerenic acid in rhizome of three plant species which is generally traded under the name of Jatamansi. A simple, rapid, cost-effective and accurate high performance thin layer chromatographic method has been developed for quantification of valerenic acid in Valeriana jatamansi, Nardostachys jatamansi, and Selinum vaginatum, which is one of the stable compounds and designated as a key marker compound. Separation and quantification of valerenic acid was achieved by HPTLC using ternary mobile phase of toluene: ethyl acetate: formic acid (80:20:5 v/v/v) on precoated silica gel 60F254 aluminum plate and densitometric determination was carried out in λ280 absorption-reflectance UV mode by deuterium lamp.

Evaluation of ternary mobile phases for reversed-phase liquid chromatography: Effect of composition on retention mechanism

The effect of varying mobile phase composition across a ternary space between two binary compositions is examined, on four different reversed-phase stationary phases. Examined stationary phases included endcapped C8 and C18, as well as a phenyl phase and a C18 phase with an embedded polar group (EPG). Mobile phases consisting of 50% water and various fractions of methanol and acetonitrile were evaluated. Retention thermodynamics are assessed via use of the van’t Hoff relationship, and retention mechanism is characterized via LSER analysis, as mobile phase composition was varied from 50/50/0 water/methanol/acetonitrile to 50/0/50 water/methanol acetonitrile. As expected, as the fraction of acetonitrile increases in the mobile phase, retention decreases. In most cases, the driving force for this decrease in retention is a reduction of the enthalpic contribution to retention. The entropic contribution to retention actually increases with acetonitrile content, but not enough to overcome the reduction in the enthalpic contribution. In a similar fashion, as methanol is replaced with acetonitrile, the v, e, and a LSER system constants change to favor elution, while the s and c constants change to favor retention. The b system constant did not show a monotonic change with mobile phase composition. Overall changes in retention across the mobile phase composition range varied, based on the identity of the stationary phase and the composition of the mobile phase.