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Discusses a model for the propagation of a finite concentration zone in a chromatographic column for the case of a single component sample, and explores operating conditions of ion-exchange chromatography for the production of albumin from bovine serum and plasma. Focus is on theoretical, instrument

The Thermodynamic and Kinetic Basis of Liquid Chromatography X. Li, A.M. Hupp, and V.L. McGuffin Applications of Ionic Liquids in Extraction, Chromatography, and Electrophoresis C.F. Poole Applications of Capillary Electrophoresis to Molecular Recognition and Analysis of In-Capillary Enzyme-Mediated Transformations J. Zavaleta, D.B. Chinchilla, A. Brown, A. Ramirez, V. Calderon, T. Sogomonyan, R. Montes, and F.A. Gomez Aptamers as Molecular Recognition Elements in Chromatographic Separations D. Hutanu and V.T. Remcho Advances in High-Throughput High-Performance Liquid Chromatography and "Purification-Friendly" Combinatorial Library Design Strategies S. Schefzick Biological, Clinical, and Forensic Analysis Using Comprehensive Two-Dimensional Gas Chromatography N.H. Snow Determination of Endocrine Disrupting Chemicals Found in Environmental Samples by Gas Chromatography/ Mass Spectrometry J.D. Stuart Index

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 log P 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.

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, log k ′ w ( o ) , obtained on the above column with the n-octanol-modified mobile phase were very well correlated (1:1 correlation) with literature values of log P (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 log k ′ w ( o ) values and calculated values of the logarithm of the distribution coefficient at pH 7.0 (log D7.0) for ionized acidic and basic drugs (r2 = 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.

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.

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.

We describe here the use of gold nanoparticles in conjunction with chip-based capillary electrophoresis to improve the selectivities between solutes and to increase the efficiency of the separation. We coated the microchannel wall of a microfluidic device with a layer of poly(diallyldimethylammonium chloride) (PDADMAC) and then collected on it citrate-stabilized gold nanoparticles. The resolutions and the plate numbers of the solutes were doubled in the presence of the gold nanoparticles. Such selectivity improvements reflect changes in the observed mobility accrued from interactions of solutes with the particle surface. The electrochemical detection and the quantitation of the solutes were not effected by the PDADMAC and the gold nanoparticles.

System peaks, which occur in chromatophy with multicomponent mobile phases, car be used for the continuous on-line monitoring of chemical reactions. The mobile phase reservoir is the chemical reactor, and the mobile phase, which contains the reaction mixture, is recycled continuously through the column. At predetermined times, a neat solvent, normally the solvent of the reaction, is injected. The system peaks that result reflect the status of the reaction. We describe here the use of system peaks to follow the progress of two reactions: titration of aniline and hydrolysis of aspirin. In the case of aniline titration, we obtained a titration curve by plotting the capacity ratio of the aniline system peak versus the acid volume added during the titration. The experimental endpoint agreed very well with the theoretical equivalence point. A plot of the capacity factor as a function of mobile phase pH behaves similarly to such plots in conventional chromatography. In the hydrolysis of aspirin, we followed the progress of the reaction by monitoring the formation of salicylic acid via its system peak. The data allowed us to calculate the apparent hydrolysis rate constant. The value obtained is in excellent agreement with literature values

A study was made to establish whether the size of system peaks, which occur in chromatography with multi-component mobile phases, is a linear function of the concentration of the components. In the two chromatographic systems studied, it was found that the size of the system peaks is proportional to concentration. Using the fact that the injection of concentrations lower than that in the mobile phase results in negative system peaks whereas the injection of higher concentrations yields positive peaks, a simple method to determine the concentration of the components in the mobile phase is described. The approach seems to be robust although the retention times of some mobile phase components might be affected by the concentration of other components. The results presented indicate that system peaks can be used to monitor continuously various streams such as rivers, waste waters and physiological fluids.

The results of the present work show that the physical shape of the injection end of the capillary in CE has a major effect on the observed efficiencies. Straight-edge capillaries yield much more efficient electropherograms than do slanted edge capillaries. Also, injections into straight-edge capillaries give more symmetrical peaks. Indications are that the effect of the inlet shape is independent of molecular size. Moments analysis shows that the increase in size of the injected plug. due to the slanted shape of the capillary inlet, is not sufficient to explain the large decrease in efficiency.

Electroosmotic flow in capillary zone electrophoresis must be controlled for precise and reproducible performance. We found that the addition of any one of several classes of compounds such as amines, amino acids or organic acids to the running buffer stabilizes electroosmotic flow. With the additives in the buffer, the precision of migration times improved to better than 1%. The reproducibilities improved to better than 5%. The system is very stable at various conditions for small molecules and for proteins at very acidic or very basic conditions.

Calixarenes are macrocyclic oligomers having the shape of a conical vase. Its inner cavity can accomodate various guest molecules, in similarity to other macrocyclic molecules such as crown ethers and cyclodextrins. Thus, calixarenes can be used to manipulate selectivities in chromatography and in capillary electrophoresis. We report here initial results that demonstrate the value of calixarenes as selectivity modifiers in capillary electrophoresis. Retention behaviors of chlorinated phenols, benzenediols, and toluidines in the presence of p-sulfonate calix[6]arene was investigated

A new approach to simulate the movement of bands through a chromatographic column is presented. Similar to the Craig distribution model, the mass balance equation is divided into two equations describing two successive processes. The first equation includes two effects: solute diffusion in the mobile phase and migration of the solute band with the mobile phase. The second equation deals with the distribution of the solute between phases, i.e., the adsorption isotherm. The partial differential equations are integrated numerically over time and space using two methods. The first approach is a finite difference method. In the second approach, the propagation operator is expanded in a Chebyshev series, where large time steps can be used. The rate of adsorption and desorption is determined by the size of the time increment. By varying the size of the time step, it is possible to study kinetic effects. The influences of sample size, injection width, rate of mass transfer, and mobile phase velocity on the elution profile were studied. Simulations using the modified Craig approach with either of the two numerical procedures showed that the solutes behaved in the chromatographically expected manner. Moreover, with linear adsorption isotherms, direct relationships between HETP, as well as retention times, and experimental parameters could be established.

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.

An expression is derived for the plate height in capillary electrophoresis for cases where hydrostatic flow (HF) is present. HF will occur whenever the buffer levels at both ends of the capillary are not at the same height. Whereas the plate height equation for an HF-free system has only a molecular diffusion term, the plate equation in the presence of HF has an additional term which is a resistance to mass transfer term. The second term is a function of capillary radius, the hydrostatic velocity, the solute diffusion coefficient and the electrophoretic velocity. Unlike in chromatography, the mass transfer term usually does not increase with increasing solute velocity. Nonetheless, the contribution of this additional term to the total plate height can be substantial for wide capillaries and large solute molecules. We calculated maximum allowable buffer height differences, Δ h max , for a given loss in plate height. It was found that for large molecules and wide capillaries, Δ h max , can be less than 1 mm, making severe demands on the instrumental design. With small solutes and narrow capillaries, the requirement for extract buffer levelling at both ends of the capillary is less acute.

The separation and determination of various monosaccharides and oligosaccharides by different HPLC techniques, published in the period from the second part of 1983 up to December 1987, are reviewed. However, several earlier works of particular significance are also covered. This review includes a systematic description of the important elements in an advanced HPLC system for analysis of saccharides: stationary phases for columns, specific mobile phases, the use of precolumn or postcolumn derivatization and the choise of detector for each specific analysis. the most significant developments of the last decade are the increasing use of bonded-phase

Selectivity and retention surfaces were developed for the rapid high-performance liquid chromatographic separation of dideoxyribonucleosides which are promising antiretroviral agents. In this study, the dideoxyribonucleosides 2′,3′-dideoxyadenosine, 2′,3′-dideoxyinosine, 2′,3′-dideoxycytidine and 3′-deoxythymidine were used as model compounds and 2′-azido-3′-deoxythymidine (AZT) and AZT-5′-phosphate (AZT-5′-P) as experimental text compounds. The interdependence of the experimental variables, column temperature, mobile phase pH and percentage of methanol, and the effects of these variables on the reversed-phase liquid chromatographic (RPLC) behavior of the dideoxy compounds were investigated. The condition for optimal resolution of these compounds, as determined from the surfaces, were mobile phases of pH 5.0 and either 14% methanol at 40°C or 15% methanol at 30°C. These surfaces served as guidelines in determining conditions for preparative and analytical scale RPLC separations of AZT from AZT-5′-P.

The analysis of inorganic anions found in residues of low explosives present a difficult forensic problem. Current methods are neither quantitative nor sensitive. While the liquid chromatographic separation of these anions is relatively easy, their detection is difficult since they cannot be detected by conventional high-performance liquid chromatography detectors. We report a method that a can detect inorganic anions, which are commonly found in combustion residues of low explosives, by reversed-phase ion-pair liquid chromatography, using indirect UV detection. The ion-pair reagent is benzyltributylammonium chloride. In addition, the phosphate buffer mobile phase contains hexane sulfonate. There is little sample preparation since no interferences were encountered while testing real samples from explosive residues. Detection limits are low and selectivity is high compared with existing techniques. Analysis time is short and the separation can be repeated immediately.

The second derivative of a chromatographic signal can aid in the recognition of composite peaks. In the case of a two-component composite, the second derivative has three maxima and two minima. The time position of the second of the three maxima is indicative of the point where the peak envelope of each component cross each other. This second maximum can be used to trigger the end of integration of the first component and the beginning of the integration of the second component. In theory, the second derivative approach works well. The present paper investigates the limits of the method. It was found that, depending on the separation between the two components in the composite, there is a peak height ratio below which the method fails to recognize the existence of a composite system. The greater the separation, the smaller the height ratio. At 4{sigma} separation, the minimum height ratio that the method can still recognize as a composite is around 10{sup {minus}5}. However, the useful range of the approach may extend to a lower range of height ratios due to the small changes in the area with relatively large changes in the peak height ratio. Noise also affects the usefulmore » range of the method. In general, as the noise increases, the method fails at higher peak height ratios. A surface describing the useful range of the method is given as a function of the signal-to-noise ratio, the separation and the height ratio.« less

The complex formation between β-cyclodextrin and nitrophenol isomers was studied with the aid of reversed phase liquid chromatography. The effects of temperature, pH, and methanol content on the formation constants of the nitrophenol/β-cyclodextrin complexes were investigated. The complex formation constants were found to decrease with increasing temperatures. This decrease in formation constant was independent of the pH of the mobile phase. From the temperature dependence of the formation constants we were able to extract enthalpies and entropies of the complex formation. The values calculated in this work agree favorably with published values. Three pHs were examined: 3.57, 4.65, and 5.6. The formation constants of the ortho and meta isomers were highest at the lowest pH and leveled off at the two higher pHs. The para isomer showed a minimum in the formation constant at pH 4.65. This behavior was independent of the pH of the mobile phase.

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.

We describe here the use of gold nanoparticles to manipulate the selectivity between solutes in capillary electrophoresis. Two different gold-based nanoparticles were added to the run buffer. In one case, the nanoparticles were stabilized with citrate ions, but in another study, the gold nanoparticles were capped with mercaptopropionate ions (thiol-stablized). Citrate-stabilized gold nanoparticles were used in conjunction with capillaries treated with poly(diallyldimethylammonium chloride) (PDADMAC). The positively charged PDADMAC layer on the capillary walls adsorbs the negatively charged gold nanoparticles. The model solutes that were used to study the effect of the presence of the citrate-stabilized gold nanoparticles are structural isomers of aromatic acids and bases. The presence of the PDADMAC layer and the PDADMAC plus the gold nanoparticles changes both the electroosmotic mobility and the observed mobility of the solutes. These changes in the mobilities influence the observed selectivities and the separations of the system. Thiol-stabilized gold nanoparticles were used without PDADMAC in the capillary. The model solutes studied in this part are various aromatic amines. In this case as well, the presence of the gold nanoparticles modifies the electroosmotic mobility and the observed mobility of the solutes. These changes in the mobilities are manifested in selectivity alterations. The largest change in the selectivities occurs at low concentrations of the gold nanoparticles in the run buffer. The presence of nanoparticles improves the precision of the analysis and increases the separation efficiency.

We have modified a reversed-phase (RP8) column by passing through it an aqueous solution of phosphatidylcholine-based liposomes. The phospholipids from the liposomes adsorb onto the octyl chain of the stationary phase, thus altering the nature of the stationary phase and of the chromatographic interactions. The properties of the phospholipid-modified column were investigated using solutes belonging to several chemical classes. We found that the retention factors of negatively and positively charged solutes decreased as the amount of phospholipid in the column was increased. For the solutes studied here the extent of the decrease was smaller for the positive solutes. With neutral solutes, the retention factors of some (benzenediols) increased markedly while with others (ketones) the retention factors decreased. The selectivities between the various solutes on the phospholipid-modified column were different than on the original reversed-phase column. The retention behavior of the solutes can be explained in terms of (1) effective shielding of the hydrophobic part of the stationary phase by the polar head groups of the phospholipids and (2) hydrogen bond formation between the solutes and the carbonyl oxygens as well as the non-ester phosphate oxygens in the polar head groups of the phospholipids.

A relatively simple method for extraction of free fatty acids from cerebrospinal fluid with aminopropyl bonded-phase columns, and the estimation of palmitic acid (C16:0) and stearic acid (C18:0) concentrations by high-performance liquid chromatographic analysis is described. The values of C16:0 and C18:0 in patients with non-neurological disorders lie within a narrow range, with a mean (+/- S.D.) of 4.02 +/- 0.33 micrograms/ml for C16:0 and 2.72 +/- 0.39 micrograms/ml for C18:0.

Summary In order to assess the viability of the photodiode array detector, its noise characteristics and linear range have been studied. It was found that at worst the peak noise is ±0.001 a.u. but more typical values are ±6·10 −4 a.u. With increased signal averaging, the noise values can be decreased to ±2·10 −4 a.u. The detector was found to have a linear range between 2·10 3 and 6.5·10 3 , depending on the wavelength. The versatility of the diode array detector was demonstrated by deconvoluting a multisolute chromatographic peak and determining the amounts of each solute in the mixture. The agreement between the amounts injected and those calculated was within several percent.

A new bonded phase, containing a Co(en)33+ moiety, was prepared in order to study the chromatographic behavior of solutes capable of forming outersphere complexes with cobalt compounds. The test solutes were nucleotides and nucleosides. It was found that solute—statoinary phase interactions could be quite strong resulting in long retention times. This was particularly so with triphosphate nucleotide. To overcome this difficulty Mg(II) was added to the mobile phase. Under these conditions selectivities, efficiencies and analysis times were greatly improved. The behavior of the Co(en)33+ phase was compared to that of a C18 column and a diamine system. The importance of adding Mg(II) to the mobile phase is demonstrated on all systems.

Phase-transfer catalysis is applied for the separation of carboxylate salts in aqueous solutions.

It is shown that the retention indices of polar solutes are affected by the amount of stationary phase in the column. The retention indices increase with decreasing stationary phase loadings. The reasons for this behavior are most likely due to the existence of mixed retention mechanisms. At low amounts of stationary phases, adsorption on the support or column walls can take place. Studies were made using four supports, two of which were Chromosorb W and the other two Chromosorb P. The effects of changing the liquid phase loadings are more pronounced when using Chromosorb P.

The theory of resolution optimization by isochronal analysis is further extended to the cases involving simultaneous changes in mobile phase velocity and column temperature, and in mobile phase composition and temperature. In each case, the resolution equation, when written in terms of isochronal conditions, shows that improvements can be obtained without sacrificing analysis time. Several subcases, which relate to the dependence of the selectivity factors on the changed parameters, are described. For each system, the contributions of the various parameters to the resolution are examined. The results of these discussions are plotted as resolution surfaces, on which lines of constant analysis time are indicated. The experimental routes that chromatographers must take in order to apply isochronal analysis are described.