Reversed-phase Chromatographic Retention Research Articles

Trimethylation Enhancement Using Diazomethane (TrEnDi) Enables Enhanced Detection of Glufosinate and 3-(Methylphosphinico)propionic Acid from Complex Canola Samples.

Over the past century, agriculture practices have transitioned from manual cultivation to the use of an array of chemical herbicides for weed control including phosphinothricin, or glufosinate (GLUF). Consequently, the potential for long-term residual GLUF exposure in the food chain has increased, highlighting the need for improved analytical strategies for its detection, as well as the detection of its main breakdown product 3-(methylphosphinico)propionic acid (MPPA). Chemical derivatization strategies have been developed to improve the detection of GLUF and MPPA via liquid chromatography tandem mass spectrometry analyses. Herein, we employ trimethylation enhancement using diazomethane (TrEnDi) for the first time as a means to confer analytical advantages via quantitatively derivatizing these analytes into permethylated GLUF ([GLUFTr]+) and MPPA ([MPPATr+H]+). Comparing [GLUFTr]+ and [MPPATr+H]+ to underivatized counterparts, TrEnDi yields 2.8-fold and 1.7-fold improvements in reversed-phase chromatographic retention, respectively, while MS-based sensitivity is enhanced 4.1-fold and 11.0-fold, respectively. Successful analyte derivatization (with >99% yields) was further demonstrated on a commercial herbicide solution imparting consistent analytical enhancements. To investigate the benefits of TrEnDi in a bona fide agricultural scenario, simple aqueous extractions from distinct parts of field-grown canola plants were performed to quantify GLUF and MPPA before and after TrEnDi derivatization. In their underivatized forms, GLUF and MPPA were undetectable in all field samples, whereas [GLUFTr]+ and [MPPATr+H]+ were readily quantifiable using the same analysis conditions. Our results demonstrate that TrEnDi continues to be a useful tool to enhance the analytical characteristics of organic molecules that are traditionally difficult to detect.

Mechanistic Modeling of Reversed-Phase Chromatography of Insulins within the Temperature Range 10-40 °C.

In the many published theories on the retention in reversed-phase chromatography (RPC), the focus is generally on the effect of the concentration of the mobile phase modulator(s), although temperature is known to have a significant influence both on the retention and on the selectivity between the adsorbates. The aim of this study was to investigate and model the combined effects of the temperature and the modulator concentrations on RPC of three insulin variants. KCl and ethanol were used as mobile phase modulators, and the experiments were performed on two different adsorbents, with C18 and C4 ligands. The temperature dependence was investigated for the interval 10–40 °C and at two different concentrations of each modulator. The model is derived from the expression for the adsorption equilibrium, which assumes that ethanol is adsorbed to the ligands and displaced by the insulin molecules, similar to the displacement of counterions in the steric mass-action model for ion-exchange chromatography. A good model fit to the new linear-range retention data was achieved by only adding and calibrating three parameters for the temperature dependence of the equilibrium. We found that a lower temperature results in a longer retention time for all adsorbates, adsorbents, and modulator concentrations used in this study, indicating that the adsorption process is enthalpy-driven. A comparison of the different contributions to the temperature dependence revealed that the large contribution from the equilibrium constant is dampened by the significant contributions of the opposite sign from the changes in activity coefficients of insulins and ethanol. Neglect of these effects when comparing different adsorbents and modulators might yield incorrect conclusions because the equilibrium constant varies with both, whereas the activity coefficients should be independent of the adsorbent. As expected, the conditions that promote higher retention also give a higher selectivity between the adsorbates. Nonetheless, in relation to its effect on the retention, the influence of the KCl concentration on the selectivity was significantly stronger than that of the temperature or that of the ethanol concentration.

A float mechanism of retention in reversed-phase chromatography

A float mechanism of retention in reversed-phase HPLC is proposed as an alternative to the known mechanisms of the distribution and hydrophobic expulsion of sorbate to the surface of a sorbent. Experimental data that the sorption of a flavylium structure is poorly sensitive to the position of OH groups, and that the retention of anthocyanins depends on the length of bonded alkyl radicals of reversed phase, form the basis of the proposed hypothesis. It is noted that the retention of anthocyanins depends on the orientation of hydroxyl groups in carbohydrate radicals, due to which the chromatographic behavior of anthocyanins is different for glucosides and galactosides, for arabinosides and xylosides, and so on. In other words, retention is a reliable indicator of the composition of a carbohydrate fragment. It is concluded that carbohydrate radicals serve as unique floats, while flat flavilic ions penetrate into the bonded phase. The existence of floats is the main reason for the lower efficiency (of the number of theoretical plates) of the peaks of anthocyanins. It is shown that if two carbohydrate radicals are present at different sites of aglycone (a two-float sorbate), the peaks of the substance are characterized by substantial additional broadening.

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa.

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1% (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 μM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients.

Artificial neural network modelling of pharmaceutical residue retention times in wastewater extracts using gradient liquid chromatography-high resolution mass spectrometry data

The modelling and prediction of reversed-phase chromatographic retention time (tR) under gradient elution conditions for 166 pharmaceuticals in wastewater extracts is presented using artificial neural networks for the first time. Radial basis function, multilayer perceptron and generalised regression neural networks were investigated and a comparison of their predictive ability for model solutions discussed. For real world application, the effect of matrix complexity on tR measurements is presented. Measured tR for some compounds in influent wastewater varied by >1min in comparison to tR in model solutions. Similarly, matrix impact on artificial neural network predictive ability was addressed towards developing a more robust approach for routine screening applications. Overall, the best neural network had a predictive accuracy of <1.3min at the 75th percentile of all measured tR data in wastewater samples (<10% of the total runtime). Coefficients of determination for 30 blind test compounds in wastewater matrices lay at or above R2=0.92. Finally, the model was evaluated for application to the semi-targeted identification of pharmaceutical residues during a weeklong wastewater sampling campaign. The model successfully identified native compounds at a rate of 83±4% and 73±5% in influent and effluent extracts, respectively. The use of an HRMS database and the optimised ANN model was also applied to shortlisting of 37 additional compounds in wastewater. Ultimately, this research will potentially enable faster identification of emerging contaminants in the environment through more efficient post-acquisition data mining.

Enhancement of capillary electrochromatographic separation performance by conductive polymer in a layer-by-layer fabricated graphene stationary phase

In this work, we fabricated a novel graphene-based capillary column for open-tubular capillary electrochromatography (OT-CEC) by a layer-by-layer strategy. To immobilize graphene onto the inner surface of silica capillary, a bio-inspired method was first used to functionalize the capillary surface with a layer of polydopamine (PDA). Graphene oxide (GO) was then introduced and can covalently react with polydopamine, realizing immobilization of graphene as a result. To enhance the modification efficiency of polydopamine, a conductive polymer, polyaniline (PANI) was introduced to be a sub-layer; polydopamine was then introduced following with GO, to generate a multilayer GO-PDA-PANI@capillary. Interestingly, separation efficiency of the graphene-based capillary was enhanced significantly by using conductive PANI as a sub-layer. The morphology of different layers modified on the capillary column was characterized by scanning electron microscopy (SEM). The electroosmotic flow (EOF) characteristics of capillaries modified with different layers were also investigated by varying the pH value of mobile phase. GO-PDA-PANI@capillary showed good separation efficiency towards alkylbenzenes by OT-CEC mode, with theoretic plate numbers up to 133,918 for benzene. The separation was found to follow a reversed-phase chromatographic retention mechanism. Repeatability of the GO-PDA-PANI@capillary was studied, with relative standard deviations for intra-day and inter-day runs less than 2.89%, and column-to-column runs less than 6.17%. The separation performance of GO-PDA-PANI@capillary was also compared with that of the reported graphene modified capillary.

Preparation and evaluation of pepsin affinity organic polymer capillary monolithic column

The protein modified monolithic column in affinity capillary electrochromatography (CEC) has attracted considerable attention over the past decades because of its great enantioseparation ability. A porous polymethacrylate ester-based capillary monolithic column poly (glycidyl methacrylate-co-ethyleneglycol dimethacrylate) (poly (GMA-co-EDMA)) was prepared by in situ co-polymerization. The process was initiated thermally by azobisisobutyronitrile (AIBN). The polymerization mixture contained GMA as the function monomer and EDMA as the crosslinking agent with 1,4-butanediol and 1-propanol as the binary porogen solvent. Under the optimized reaction conditions, including the proportion of monomer and porogens, reaction temperature etc, the column exhibited a uniform structure, sufficient permeability and excel- lent pressure resistance. The separation of alkyl benzenes on the column was mainly based on typical reversed-phase chromatographic retention mechanism. The reproducibility and stability were good with RSDs less than 9. 0%. A pepsin functionalized organic polymer monolith was prepared by covalently bonded pepsin to poly(GMA-co-EDMA) monolith with glutaraldehyde as a spacer based on the activity of epoxide group. The enantioseparation performance of the pepsin affinity monolith for basic enantiomers has been investigated by CEC. Nefopam, amlodipine, citalopram and chlorpheniramine were resolved, and baseline separations of nefopam, amlodipine, citalopram were achieved. The influences of pH, operating voltage, temperature and sample quantity used on the chiral separation were studied. The chiral recognition mechanism of enantiomers on the monolithic column in CEC is discussed. This work developed a new method for the prepataion and application of protein affinity monolith in CEC.

Determination of partition coefficients n-octanol/water for treosulfan and its epoxy-transformers: An example of a negative correlation between lipophilicity of unionized compounds and their retention in reversed-phase chromatography

For the last decade an alkylating agent treosulfan (TREO) has been successfully applied in clinical trials in conditioning prior to hematopoietic stem cell transplantation. Pharmacological activity of the pro-drug depends on its epoxy-transformers, monoepoxide (S,S-EBDM) and diepoxide (S,S-DEB), which are formed in a non-enzymatic consecutive reaction accompanied by a release of methanesulfonic acid. In the present study partition coefficient n-octanol/water (POW) of TREO as well as its biologically active epoxy-transformers was determined empirically (applying a classical shake-flask method) and in silico for the first time. In vitro the partition was investigated at 37°C in the system composed of the pre-saturated n-octanol and 0.05 M acetate buffer pH 4.4 adjusted with sodium and potassium chloride to ionic strength of 0.16 M. Concentration of the analytes was quantified by reversed-phase high performance liquid chromatography (RP-HPLC) method in which retention time increased from S,S-DEB to TREO. It was shown that neither association nor dissociation of the tested compounds in the applied phases occurred. Calculated logPOW (TREO: -1.58±0.04, S,S-EBDM: -1.18±0.02, S,S-DEB: -0.40±0.03) indicate the hydrophilic character of the all three entities, corresponding to its pharmacokinetic parameters described in the literature. Experimentally determined logPOW of the compounds were best comparable to the values predicted by algorithm ALOGPs. Interestingly, the POW values determined in vitro as well as in silico were inversely correlated with the retention times observed in the endcapped RP-HPLC column. It might be explained by the fact that a cleavage of methansulfonic acid from a small molecule of TREO generates significant changes in the molecular structure. Consequently, despite the common chemical origin, TREO, S,S-EBDM and S,S-DEB do not constitute a 'congeneric' series of compounds. We concluded that this might occur in other low-weight species, therefore measurement of their POW by RP-HPLC had to be applied with a special care.

Unambiguous determination of isobaric histone modifications by reversed-phase retention time and high-mass accuracy

Methylation and acetylation of lysines are crucial posttranslational modifications that regulate gene transcription and have been shown to be misregulated in many forms of cancers. Western blot, immunoprecipitation, and immunofluorescence are commonly used to characterize histone acetylation and methylation. However, these approaches are limited by the availability, site specificity, and cross-reactivity of antibodies. Mass spectrometry is emerging as an additional powerful tool for histone characterization. The isobaric nature of trimethylation and acetylation (42.0470 and 42.0106 Da, respectively) confounds histone characterization by means other than high-resolution/high-mass accuracy mass spectrometry. In this study, we adapted methodology that exploits difference in the relative retention time of acetylated and methylated peptides to unequivocally distinguish between these two modifications even with low-mass accuracy mass spectrometers. The approach was tested on tryptic digest of Saccharomyces cerevisiae histones. We found that acetylation resulted in increased retention in reversed-phase chromatography, whereas methylation, including trimethylation, showed little change in retention. For example, the acetylated forms of peptide 27KSAPSTGGVKKPHR 40 eluted at 15.63 min, whereas the methylated forms eluted at 13.89 min. In addition, the effect of acetylation was cumulative as observed in the case of peptide 9KSTGGKAPR 17, whose unmodified, monoacetylated, and diacetylated isoforms eluted at 7.43, 10.47, and 16.49 min, respectively. The modification patterns of the peptides in question were subsequently verified by high-mass accuracy tandem mass spectrometry.

Modeling the combined effect of temperature and organic modifier content on reversed-phase chromatographic retention: Effectiveness of derived models in isocratic and isothermal mode retention prediction

Models considering simultaneously mobile phase organic content and column temperature were developed in this study by an extension of different equations describing the influence of temperature on solute retention. This extension was achieved by two methods: a semi-thermodynamic and a direct combination of equations expressed separately the dependence of the retention upon each of these factors. The above approaches gave a great number of expressions for the logarithm of the solute retention factor in terms of both temperature and organic content in the mobile phase, ln k( T, φ), determined from the dependence of the standard enthalpy of the retention process on T. From the final expressions of ln k( T, φ) we tested only those with the minimum number of adjustable parameters, i.e. those that correspond to a constant standard enthalpy of the retention process. For this test we examined the retention behaviour of a sample of alkylbenzenes in aqueous acetonitrile eluents. These compounds exhibit ln k versus 1/ T plots with a very small curvature. We found that a new equation for ln k( T, φ) based on the adsorption model for retention performs better than all the others. The average percentage prediction error ranges from 0.7 to 1.4%.

Evaluation of the influence of residual silanol groups on the total retention in reversed-phase chromatography

It was demonstrated that the characteristics of stationary phases for reversed-phase chromatography can be compared by analyzing the retention of several compounds belonging to a homologous series during elution with one and the same composition of mobile phase or the retention of a nonpolar compound during elution with several mobile phases of different compositions. The slope of the corresponding linear correlations can be used to characterize the hydrophobicity of the columns (stationary phases) under study. To analyze the polar interactions between the sorbate and the stationary phase it is advantageous to study the retention of a polar substance in several (at least in two) mobile phases of different compositions. The degree of displacement of the linear correlation relative to that for nonpolar substances can be considered a measure of the influence of residual silanol groups on the total retention characteristics.

Fundamentals of Reversed Phase Chromatography: Thermodynamic and Exothermodynamic Treatment

Reversed phase chromatography (RPC) is the most popular branch of HPLC for the analysis and purification of a wide variety of substances. Despite significant advances in both our knowledge and understanding of the fundamental principles governing the retention behavior in RPC, there is considerable debate in the literature regarding the mechanism of retention. This review addresses the theoretical foundation of the chromatographic technique, with an emphasis on thermodynamic and exothermodynamic treatment of retention equilibrium, as well as their implication on the mechanistic aspects of RPC retention. A unified and rigorous treatment based on the solvophobic theory is reviewed in terms of its ability to shed light on the physicochemical underpinnings of the retention in RPC, and to quantitatively predict the retention behavior of nonpolar compounds, acids and bases, and peptides and proteins. Also highlighted are areas of future challenges in the theory and practice of RPC, potentially leading to a better quantitative understanding and use of the popular technique.

Reversed-phase chromatographic retention behavior of some fibrate-type antihyperlipidemic drugs

Relationships between R M values and mobile phase composition have been examined for six fibrate-type drugs — bezafibrate, ciprofibrate, clofibrate, clofibric acid, fenofibrate, and gemfibrozil. They were separated in horizontal chambers on RP18 plates by use of mobile phases containing phosphate buffer and different amounts of six modifiers — acetone, acetonitrile, dioxane, isopropanol, methanol, and tetrahydrofuran. Plates were visualized under UV irradiation at λ = 254 nm, and scanned with a densitometer in multi-wavelength scan mode. Optimum modifier concentrations were also investigated on RP8 and CN plates for comparison. The linearity of relationships between R M and modifier volume fraction, molar fraction, and the logarithm of the molar fraction was calculated. Most results fitted the Snyder-Soczewinski equation with r > 0.95; for almost half r > 0.99. Separation of all the drugs was achieved on RP18 plates with mobile phase containing 70% dioxane in pH 7.60 phosphate buffer.

Estimation of Size for 1−2 nm Nanoparticles Using an HPLC Electrochemical Detector of Double Layer Charging

Reversed-phase HPLC chromatographic separations of Au monolayer protected clusters (MPCs) in the size range 1−2 nm (diameter) are detected electrochemically (ECLC) on the basis of double layer charging of the metal-like nanoparticles by the detector electrode. Discrete double-layer charging peaks are detected for the nanoparticles with resolution similar to that of the photodiode array detector. The ECLC experiment further seeks not solely to detect the nanoparticle peaks, but to estimate the nanoparticle sizes from the electrochemical responses. Hydrodynamic current−potential curves of nanoparticles eluted in different peaks were obtained by repeating chromatographic separations at different working electrode potentials; the E1/2 separations between the voltammetric waves give estimates of cluster capacitance and, thus, size. Other approaches to core size determination are discussed, including determination based upon relative values of optical absorbance and current for each eluted peak. The data show that, at least for alkanethiolate MPCs, the elution order on the HPLC column employed is small clusters first and that there are significant differences in reversed-phase chromatographic retention times for clusters with apparently very similar core sizes over the range of 1−2-nm diameter.

Normal- and reversed-phase chromatography ofpara-substituted propanoic acid amides

The chromatographic behavior of newly synthesized para -substituted propanoic acid amides has been studied in normal- and reversed-phase thin-layer chromatography. The retention mechanism on different TLC supports was investigated. The retention constants determined for the amides are discussed in terms of the physicochemical properties of the solute and the stationary and mobile phases. R M,w , derived by extrapolation from retention in reversed-phase chromatography, was correlated with hydrophobic parameters π and log P , calculated by the Rekker fragmental method and by use of the commercial program ACD/log P .

Solvophobic Theory and Normalized Free Energies of Nonpolar Substances in Reversed Phase Chromatography

A simplified version of the solvophobic theory is employed to reexamine a large set of retention data with nonpolar and weakly polar eluites in reversed phase chromatography (RPC) to test certain predictions by the theory and to clarify further the roles of the mobile and the stationary phase in the retention process. The free energy of retention in RPC is expressed in terms of the nonpolar surface area of the eluite, the pertinent interfacial tensions, and the energetics of eluite−stationary phase interactions in the gas phase. Within this framework changes in retention free energy per unit water accessible nonpolar surface area are evaluated in the entire range of the organic modifier concentration by using thermodynamic data for the retention of nonpolar eluites on alkyl silica bonded phase in gas chromatography and for the transfer of nonpolar solutes from the hydroorganic solvent to the gas phase. The normalized retention free energy parameters thus obtained according to the theory are in excellent a...

Effect of the eluent pH on the thermospray molecular ion intensity of nucleosides

The protonated molecular ion intensities of 15 nucleosides obtained by thermospray ionisation have been measured using 0.1 M ammonium acetate mobile phase at neutral and acidic pH. To explain the dependence of the molecular ion intensity on the mobile phase pH, the hydrophobicity, the pK a values and reversed-phase high-performance liquid chromatographic retention data (log k′) at neutral and acidic pH values were studied. Significant correlations (above 95% probability level) were found between the change in the protonated molecular ion intensity and the hydrophobicity as well as the p K a values of the compounds. The reversed-phase chromatographic retention parameter (log k′) obtained at pH 3.5, showed significant correlation together with the p K a values to the molecular ion intensity change caused by decreasing the mobile phase pH. None of the investigated nucleosides showed an increased molecular io intensity change at low pH when more than 5% methanol was present in the mobile phase.

Effect of mobile phase additives on peptide retention in reversed-phase chromatography with pellicular and totally porous sorbents

The effect of two mobile phase additives, trifluoroacetic acid and phosphoric acid, on the energetics of peptide retention in reversed-phase chromatography was investigated using Hy-Tach C 18 micropellicular and Vydac C 4 and C 18 totally porous stationary phases. The effect of the relatively low phase ratio of columns packed with micropellicular sorbents was also examined. The logarithmic retention factors, of two model peptides, Ac-RGGGGLGLGK-amide and Ac-RGAGGLGLGK-amide, were evaluated with different columns and additives in a practical range of eluent strength. The dependence of the logarithmic retention factor on the concentration of acetonitrile in the mobile phase was linear in all cases. The higher sensitivity of the retention to the organic modifier concentration in the case of the Hy-Tach C 18 column is attributed to the relatively low phase ratio of this column. Pairwise plots of the logarithmic retention factors were linear. The plots of data obtained with the two additives has unit slopes and thus reveal homoenergetic retention behavior. On the other hand data obtained on two different columns manifest homooenergetic retention, the slopes of plots are different from unity. The analysis has yielded consistent results and validated the assumption that the retention free energy can be divided into two components arising from mobile phase and stationary phase contributions. The approach also allowed an estimation of the relative phase ratios of the columns and the Vydac C 18 column was found to have an 3 and 8 times higher phase ratio than the Vydac C 4 and the Hy-Tach C 18 column, respectively.

Relationship between the retention characteristics and physicochemical parameters of some steroid drugs in reversed-phase high-performance liquid chromatography

The retention times of thirteen steroidal drugs were determined on an ODS column using methanol-0.025 M K 2HPO 4 mixtures as eluent. A linear correlation was calculated between the capacity factor (log k′) and the concentration of methanol in the eluent. Principal component analysis was applied to find the physico-chemical parameters of solutes influencing their retention behaviour. The visualization of results was carried out by non-linear mapping, cluster analysis and varimax rotation. Each steroidal drug showed regular retention behaviour on ODS, the retention time decreasing monotonously with increasing concentration of methanol in the eluent. Principal component analysis demonstrated that five background components contain most (90.67%) of the information present in the eleven original variables. The chromatographic parameters form a loose cluster with the Taft's steric constant that these parameters may also have a considerable influence on the reversed-phase chromatographic retention of these drugs.

A Hydrophobicity Scale Based on the Migration Index from Microemulsion Electrokinetic Chromatography of Anionic Solutes

A migration index (MI) concept, previously introduced as a novel scale for measuring the hydrophobicity of neutral solutes, was applied to anionic solutes. The required parameters for determining the MI were measured by both microemulsion electrokinetic chromatography with a sodium dodecyl sulfate/1-butanol/heptane/buffer solution and capillary zone electrophoresis with 1-butanol added to the buffer. The obtained MI values of anionic solutes correlated very well with the logarithm of the partition coefficients between 1-octanol and water (log P), whereas the reversed-phase chromatographic retention parameter (log k‘w), which is also used as a hydrophobicity scale, correlated very little with log P for the tested anionic solutes. Moreover, the linear relationship between the MI and log P of the anionic solutes was consistent with that of neutral solutes. Although the relationship between the MI and log P of solutes having carboxylic groups deviated from those of others, the MI provided better regression to bioactivities such as human skin permeability than log P in quantitative structure−activity relationship studies. Therefore, this system would be an appropriate biochemical model. In addition, because measurement of the MI is easy, rapid, and reproducible, and the required sample amount is nanogram size, the MI is expected to be a useful and universal hydrophobicity scale.