Use of the kinetic plot method to analyze commercial high-temperature liquid chromatography systems. I: Intrinsic performance comparison.

The present study aimed at mapping the separation speed potential of a critical pair on commercial high-temperature HPLC (HT-HPLC) supports at elevated temperatures. For this purpose, band broadening and pressure drop measurements were conducted on three different commercial HT-HPLC columns operated at various elevated temperatures but by keeping the same retention factor. The plate height data were subsequently transformed into a plot showing the minimal required analysis time needed to yield a given required effective plate number. For the considered RPLC alkylbenzene separations, it was found that the maximal gain in separation speed of the critical pair that can be obtained by varying the operating temperature from T=30 to 120 degrees C can be expected to be of the order of a factor of 3-4, if using an individually optimized column length for each considered temperature and if no secondary adsorption effects occur at the lower temperature. This gain factor, remaining more or less constant over the most relevant range of plate numbers, largely paralleled the reduction of the mobile phase viscosity accompanying the temperature increase.

Use of the kinetic plot method to analyze commercial high-temperature liquid chromatography systems. II. Practically constrained performance comparison.

Using a set of experimentally determined plate height data obtained on three commercial high-temperature HPLC supports, and evaluating their isocratic separation speed potential under the application of a set of instrumental constraints, a qualitative map of the practically achievable critical pair separation speed potential of high-temperature HPLC has been established. The obtained data show that the gain in separation speed is more strongly affected by the instrumental limitations in the high-temperature range than it is for the low temperatures. For the presently considered case of alkylbenzene separations, the potential gain in analysis time that can be obtained by going from T=30 to 120 degrees C in the presence of a typical set of instrumental limitations nevertheless remains of the order of a factor of 2-4. The study also shows that improvements on the instrumentation side (increased detector frequency, pumping flow rate, smaller extra-column volumes, ...) are indispensable to fully benefit from the high temperature advantages for all separations requiring less than 10,000 effective theoretical plates.

Optimization of the coupling of high-temperature liquid chromatography and flame ionization detection application to the separations of alcohols.

The feasibility of coupling high-temperature liquid chromatography (HTLC) to flame ionization detection (FID) has been studied. FID parameter values (hydrogen flow-rate, air flow-rate and FID temperature), typically set in gas chromatography are rarely suitable for liquid chromatography. Best values depend obviously on the water flow rate which is defined depending on both column temperature and column internal diameter. The FID parameters were optimized according to the water flow-rate by means of an experimental design. The potential of the method is shown with some alcohol separations and the value of increasing column temperature while reducing the column diameter is highlighted.

Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase liquid chromatography: application to method development.

Optimizing separation of ionizable compounds in order to find robust conditions has become an important part of method development in liquid chromatography. This work is an attempt to explain the observed variations of retention of acid and basic compounds with the organic modifier content in the mobile phase, according to various factors: the type of modifier, the type of buffer, the temperature and of course the type of solute. This is done by considering the variation of the so-called chromatographic pKa which refers to the pH measured in the aqueous medium and is determined from retention data. A procedure is described that accurately relates, from nine experiments, retention to solvent composition and pH. The limits of such a procedure are evaluated and two examples of optimized separations of basic compounds are given.

Use of optimization software to determine rugged analysis conditions in high-performance liquid chromatography.

The ruggedness evaluation of an analytical method is now generally required for further validation. By considering ruggedness at an early stage of method development, major disappointments and amount of work could be avoided. This work shows that the optimization software OSIRIS can be helpful for the chromatographer during a method development, as it takes into account the method ruggedness. The ruggedness of the analysis conditions is then evaluated all along the selectivity optimization procedure. This optimization software belongs to the interpretive methods that consist of predicting the optimum conditions by modeling first the solute retention over the parameter space using a minimum number of preliminary runs. The choice of a response function is studied. This response function must be able to take into account several individual criteria: analysis time, minimal resolution and ruggedness of each parameter. Some optimum separations, determined using a ruggedness criteria or not, are given and compared in terms of long term repeatability.

Effect of temperature in reversed phase liquid chromatography.

The high temperature liquid chromatography (HTLC) reveals interesting chromatographic properties but even now, it misses some theoretical aspects concerning the influence of high temperature on thermodynamic and kinetic aspects of chromatography: such a knowledge is very essential for method development. In this work, the effect of temperature on solute behavior has been studied using various stationary phases which are representative of the available thermally stable materials present on the market. The thermodynamic properties were evaluated by using different mobile phases: acetonitrile-water, methanol-water and pure water. The obtained results were discussed on the basis of both type of mobile phases and type of stationary phases. Type of mobile phase was found to play an important role on the retention of solutes. The kinetic aspect was studied at various temperatures ranging from ambient temperature to high temperature (typically from about 30 to 200 degrees C) by fitting the experimental data with the Knox equation and it was shown that the efficiency is improved significantly when the temperature is increased. In this paper, we also discussed the problem of temperature control for thermostating columns which may represent a significant source of peak broadening: by taking into account the three main parameters such as heat transfer, pressure drop and band broadening resulting from the preheating tube, suitable rules are set up for a judicious choice of the column internal diameter.

Fractalkine/CX3CL1 enhances GABA synaptic activity at serotonin neurons in the rat dorsal raphe nucleus.

Serotonin (5-hydroxytryptamine; 5-HT) has an important role in mood regulation, and its dysfunction in the central nervous system (CNS) is associated with depression. Reports of mood and immune disorder co-morbidities indicate that immune-5-HT interactions may mediate depression present in immune compromised disease states including HIV/AIDS, multiple sclerosis, and Parkinson's disease. Chemokines, immune proteins that induce chemotaxis and cellular adhesion, and their G-protein coupled receptors distribute throughout the CNS, regulate neuronal patterning, and mediate neuropathology. The purpose of this study is to investigate the neuroanatomical and neurophysiological relationship between the chemokine fractalkine/CX3CL1 and its receptor CX3CR1 with 5-HT neurons in the rat midbrain raphe nuclei (RN). Immunohistochemistry was used to examine the colocalization of CX3CL1 or CX3CR1 with 5-HT in the RN, and whole-cell patch-clamp recordings in rat brain slices were used to determine the functional impact of CX3CL1 on 5-HT dorsal raphe nucleus (DRN) neurons. Greater than 70% of 5-HT neurons colocalize with CX3CL1 and CX3CR1 in the RN. CX3CL1 localizes as discrete puncta throughout the cytoplasm, whereas CX3CR1 concentrates to the perinuclear region of 5-HT neurons and exhibits microglial expression. CX3CL1 and CX3CR1 also colocalize with one another on individual RN cells. Electrophysiology studies indicate a CX3CL1-mediated enhancement of spontaneous inhibitory postsynaptic current (sIPSC) amplitude and dose-dependent increase of evoked IPSC (eIPSC) amplitude without affecting eIPSC paired-pulse ratio, a finding observed selectively in 5-HT neurons. CX3CL1's effect on eIPSC amplitude is blocked by pretreatment with an anti-CX3CL1 neutralizing antibody. Thus, CX3CL1 enhances postsynaptic GABA receptor number or sensitivity on 5-HT DRN neurons under conditions of both spontaneous and synaptically-evoked GABA release. CX3CL1 may indirectly inhibit 5-HT neurotransmission by increasing the sensitivity of 5-HT DRN neurons to GABA inputs. Therapies targeting CX3CL1 may treat serotonin related mood disorders, including depression experienced by patients with compromised immune systems.