The use of phospholipid modified column for the determination of lipophilic properties in high performance liquid chromatography.

A new chromatographic stationary phase obtained by coating a reversed phase amide column with phosphatidylcholine based liposomes solution to yield a phospholipid modified column (PLM). The modification is achieved by the dynamic coating method which recycles the coating solution through the column in a closed loop for a period of 24 h. The chromatographic properties of the new column have changed significantly as compared to the original amide column due to the phospholipid coating. A good correlation was observed between n-octanol/water logP values and the logarithm of the retention factor obtained on the PLM column for a large number of solutes. In addition the PLM column was characterized using the linear solvation energy relationship (LSER). The values of the LSER system constants for the PLM column were calculated and were found to be very close to those of the n-octanol/water extraction system thus suggesting that the PLM column can be used for the estimation of n-octanol/water partition coefficient and serve as a possible alternative to the shake-flask method for lipophilicity determination. In addition, the results suggest that the PLM column can provide an alternative to other phospholipid-based column such as the IAM and the DPC columns.

Characterization of Ascentis RP-Amide column: Lipophilicity measurement and linear solvation energy relationships.

This study investigates lipophilicity determination by chromatographic measurements using the polar embedded Ascentis RP-Amide stationary phase. As a new generation of amide-functionalized silica stationary phase, the Ascentis RP-Amide column is evaluated as a possible substitution to the n-octanol/water partitioning system for lipophilicity measurements. For this evaluation, extrapolated retention factors, log k'w, of a set of diverse compounds were determined using different methanol contents in the mobile phase. The use of n-octanol enriched mobile phase enhances the relationship between the slope (S) of the extrapolation lines and the extrapolated log k'w (the intercept of the extrapolation),as well as the correlation between log P values and the extrapolated log k'w (1:1 correlation, r2 = 0.966).In addition, the use of isocratic retention factors, at 40% methanol in the mobile phase, provides a rapid tool for lipophilicity determination. The intermolecular interactions that contribute to the retention process in the Ascentis RP-Amide phase are characterized using the solvation parameter model of Abraham.The LSER system constants for the column are very similar to the LSER constants of the n-octanol/water extraction system. Tanaka radar plots are used for quick visual comparison of the system constants of the Ascentis RP-Amide column and the n-octanol/water extraction system. The results all indicate that the Ascentis RP-Amide stationary phase can provide reliable lipophilic data.

Atomic-force-controlled capillary electrophoretic nanoprinting of proteins.

The general nanoprinting and nanoinjection of proteins on non-conducting or conducting substrates with a high degree of control both in terms of positional and timing accuracy is an important goal that could impact diverse fields from biotechnology (protein chips) to molecular electronics and from fundamental studies in cell biology to nanophotonics. In this paper, we combine capillary electrophoresis (CE), a separation method with considerable control of protein movement, with the unparalleled positional accuracy of an atomic force microscope (AFM). This combination provides the ability to electrophoretically or electroosmotically correlate the timing of protein migration with AFM control of the protein deposition at a high concentration in defined locations and highly confined volumes estimated to be 2 al. Electrical control of bovine serum albumin printing on standard protein-spotting glass substrates is demonstrated. For this advance, fountain pen nanolithography (FPN) that uses cantilevered glass-tapered capillaries is amended with the placement of electrodes on the nanopipette itself. This results in imposed voltages that are three orders of magnitude less than what is normally used in capillary electrophoresis. The development of atomic-force-controlled capillary electrophoretic printing (ACCEP) has the potential for electrophoretic separation, with high resolution, both in time and in space. The large voltage drop at the tip of the tapered nanopipettes allows for significant increases in concentration of protein in the small printed volumes. All of these attributes combine to suggest that this methodology should have a significant impact in science and technology.

Effect of n-octanol in the mobile phase on lipophilicity determination by reversed-phase high-performance liquid chromatography on a modified silica column.

In this study, we show that the addition of n-octanol to the mobile phase improves the chromatographic determination of lipophilicity parameters of xenobiotics (neutral solutes, acidic, neutral and basic drugs) on a Phenomenex Gemini C18 column. The Gemini C18 column is a new generation hybrid silica-based column with an extended pH range capability. The wide pH range (2-12) afforded the examination of basic drugs and acidic drugs in their neutral form. Extrapolated retention factor values, [Formula: see text] , obtained on the above column with the n-octanol-modified mobile phase were very well correlated (1:1 correlation) with literature values of logP (logarithm of the partition coefficient in n-octanol/water) of neutral compounds and neutral drugs (69). In addition, we found good linear correlations between measured [Formula: see text] values and calculated values of the logarithm of the distribution coefficient at pH 7.0 (logD(7.0)) for ionized acidic and basic drugs (r(2)=0.95). The Gemini C18 phase was characterized using the linear solvation energy relationship (LSER) model of Abraham. The LSER system constants for the column were compared to the LSER constants of n-octanol/water extraction system using the Tanaka radar plots. The comparison shows that the two methods are nearly equivalent.

Organically modified sols as pseudostationary phases for microchip electrophoresis.

We demonstrate that the selectivity of microchip electrophoresis separations is greatly improved by the presence of organically modified silica (Ormosil) sols in the run buffer. A negatively-charged N-(trimethoxysilylpropyl)ethylenediamine triacetic-acid (TETT)-based sol is used for improving the selectivity between nitroaromatic explosives and a methyltrimethoxysilane (MTMOS)-based sol is employed for enhancing the microchip separation of environmental pollutants, aminophenols. These sols are added to the run buffer and act as pseudostationary phases. Their presence in the run buffer changes the apparent mobility of studied solutes, and leads to a higher resolution. The observed mobilities changes reflect the interactions between the Ormosil sols and the solutes. Relevant experimental variables have been characterized and optimized. The diverse chemistry of Ormosil sols should be extremely useful for tailoring the selectivity of a wide range of electrophoresis microchip separations.

Organically modified silica sol-mediated capillary electrophoresis.

We describe in this paper the use of ormosil (organically modified silica) sols as additives to the run buffer for selectivity manipulations between solutes in capillary electrophoresis. CE systems that contain sol additives in the run buffer can be thought of as pseudocapillary electrochromatography. Three sols based on different types of silanes were studied. Methyltrimethoxysilane (MTMOS)-based sol was found to improve selectivities between various aromatic acids. Aminopropyltrimethoxysilane (APS) sol interacts differently with structural isomers of aromatic acids than does MTMOS. At low pH with APS sol in the run buffer, neutral solutes can be separated, as well. The separation of the neutral solutes seems to be facilitated by the formation of hydrogen bonds between the solutes and the APS sol. APS and N-[3-(trimethoxysilyl)propyl]-ethylenediamine (EDAS) affect the separation of the same compounds differently, thus indicating that even small changes of the functional groups of the sol have pronounced effect on the interactions between the sols and the solutes.

Temperature-dependent refractive index issues using a UV-visible detector in high-performance liquid chromatography.

While studying the properties of some potential, UV-detection compatible, mobile phase modifiers for reversed-phase liquid chromatography (RPLC) we encountered serious detector baseline problems. The most severe problem was a periodic saw-tooth-shaped drift. This drift was most noticeable when using water-THF and water-dioxolane mobile phases. We demonstrate that the baseline problems are related to temperature fluctuation in a water bath used to maintain a constant column temperature. The temperature fluctuations change the refractive index (RI) of the mobile phase, which in turn affects the amount of light reaching the detector. Our results show that the magnitude of the baseline drift is related to the RI value of the mobile phases. The baseline problems can be alleviated using a different temperature controller whose feedback mechanism assures smoother operation. Since there is no single and complete source of information in the literature concerning the RI values of typical mobile phases in RPLC, we decided to measure them as a function of the % modifier (between 0 and 100%) and the temperature (between 25 degrees C and 50 degrees C). Knowing the RI values allows us to calculate the excess volume of a binary liquid system.