Modulation optimization when using a splitter pump after the first dimension in comprehensive two- dimensional liquid chromatography.

The rapid growth in the use of two dimensional liquid chromatography (2D-LC) applied to the analysis of moderately to highly complex mixtures, has been fueled by continuous improvements in performance and robustness of the instrument components, as well as the ease-of-use of software necessary for controlling the 2D-LC instrument hardware, and analysis of the large data files that result from this type of work. This work has focused on the evaluation of the performance of an online full comprehensive mode (LC×LC), when an active modulation is implemented using a flow splitter pump placed after the 1D effluent. Two different types of splitting pumps were evaluated: a binary ultra-high pressure liquid chromatography (UHPLC) pump and a high precision syringe pump. We analyzed the performance (reproducibility in peak area and retention times and the 2D peak dispersion) as a function of the location of the active pump Before or After the modulation valve, and the influence of connecting tubes (based on internal diameter and length) necessary between the interface, waste, and the splitting pump. The effect on the flow direction on the filling and flushing of the injection loops at the modulation valve was also analyzed for each pump. In this study, we demonstrate that flow-splitting LCxLC assembly can be performed using either a UHPLC binary pump or a simple syringe pump. Flow splitting after the first dimension is a straightforward strategy to: (i) independently select the 1D column and flow rates with respect to the second dimension; (ii) consciously dilute the eluate according to the solvent characteristics of the second dimension, thereby avoiding 2D peak distortions; and (iii) adapt the injected amount to the second column according to the relative concentration of the components in a complex sample. However, careful consideration of the system setup is necessary. It is demonstrated how experimental results can be significantly affected in terms of peak broadening and reproducibility if optimization of the interface is not taken into account. In addition, under the optimized conditions, the reproducibility in peak area and dispersion in the 2D dimension were evaluated as a function of the amount of sample transferred in terms of percentage of filled loop.

Simultaneous analysis of cannabinoids and terpenes in Cannabis sativa inflorescence using full comprehensive two-dimensional liquid chromatography coupled to smart active modulation.

Nowadays, the higher peak capacity achievable by comprehensive two-dimensional liquid chromatography (LC×LC) for the analysis of vegetal samples is well-recognized. In addition, numerous compounds may be present in very different amounts. Cannabinoids and terpenes represent the main components of Cannabis sativa inflorescence samples, whose quantities are relevant for many application purposes. The analyses of both families are performed by different methods, at least two different separation methodologies, mainly according to their chemical characteristics and concentration levels. In this work, concentration differences and sample complexity issues were addressed using an LC×LC method that incorporates an optimized modulation strategy, namely smart active modulation, for the simultaneous analysis of cannabinoids and terpenes. The system was built by interposing an active flow splitter pump between both dimensions. This set up aimed to exploit the known advantages of LC×LC. In addition, here we proposed to use the splitter pump for online control over the splitting ratio to facilitate the selective dilution of different eluted fractions containing compounds with highly different concentrations. This work represents the first application and demonstration of smart active modulation (SAM) in LC×LC to simultaneously determine analytes with significant differences in concentration levels present in complex samples. The proposed method was tested with eight different strains, from which fingerprints were taken, and numerous cannabinoids and terpenes were identified in these samples. With this strategy, between 49 and 54 peaks were obtained in the LC×LC chromatograms corresponding to different strains. THCA-A was the main component in six strains, while CBDA was the main component in the other two strains. The main terpenes found were myrcene (in five strains), limonene (in two strains), and humulene (in one strain). Additionally, numerous other cannabinoids and terpenes were identified in these samples, providing valuable compositional information for growers, as well as medical and recreational users. The SAM strategy here proposed is simple and it can be extended to other complex matrices.

Determination of polycyclic aromatic hydrocarbons in surface waters by high performance liquid chromatography previous to preconcentration through solid-phase extraction by using polymeric monoliths.

A simple, sensitive and reproducible solid-phase extraction method using plastic cartridges containing a monolithic sorbent (m-SPE), coupled to reverse phase liquid chromatography analysis, aiming to determine fifteen polycyclic aromatic hydrocarbons in surface water samples, was developed. The sorbent was easily prepared through a thermal polymerization reaction by using a mixture of n-butyl methacrylate as non-polar monomer and ethylene glycol dimethacrylate as crosslinker contained in a typical Polypropylene syringe cartridge. The effect of different parameters (type of hydrophobic monomer, elution solvent, sample volume, sorbent amount and sorbent load capacity) on the extraction efficiency was optimized. The optimal conditions were achieved by using n-butyl methacrylate as monomer, tetrahydrofurane (THF) as solvent for sorbent cleaning, THF:acetone (1:1) as elution solvent, 25.00 mL of sample volume, 600 µL of the polymerization mixture and 60 µg L-1 as sample loading capacity. Finally, the sorbent charge capacity, the reusability of the cartridges and the extraction efficiency of the m-SPE monolith, as compared with a typical C8 cartridge, were evaluated. Under the optimized experimental conditions, enrichment factors were between 76 and 103, relative recovery factors from 78 to 103%, accuracy values in the range of 58 to 98%, and inter-batch reproducibility values from between 2 and 10%, were obtained. The limits of detection and quantification were obtained by two different procedures: the signal to noise (S/N) ratios (3 and 10, respectively) and the IUPAC convention. The lowest LOD and LOQ values, obtained with the S/N ratios, were between 0.02 and 1.00 µg L-1, respectively whereas with the IUPAC convention the values were between 0.07 and 5 µg L-1. Using this procedure, several PAHs could be detected in the surface water sample taken from a river stream located in La Plata city (Buenos Aires Province, Argentina).

Comprehensive two-dimensional liquid chromatographic method (Chiral × Achiral) for the simultaneous resolution of pesticides.

This work demonstrates the potential of two-dimensional liquid chromatography (2D-LC) to increase the resolution capacity of multiple pesticides in a single analysis of samples that contain both chiral and achiral compounds. The setup is based on the combination of a chiral column in the first dimension and an achiral column in the second dimension using the on-line full comprehensive mode (LC × LC). This method was optimized for the separation of 24 pesticides (17 chiral and 7 achiral). The 2D-LC system was built with an active flow splitter pump in order to easily adjust the volume of sample transferred to the second dimension and to select and optimize independently the flow rate in the first dimension. The first-dimension optimization involved the comparison of enantioresolution abilities of three different polysaccharides chiral stationary phases as well as different elution conditions, while in the second-dimension parameters like stationary phase and organic modifier were explored. Other experimental variables that influence the two-dimensional peak capacity (orthogonality, sampling frequency, shift gradients, etc.) are discussed.

Synchronized gradient elution in capillary liquid chromatography.

The synchronization of injection valve operation and gradient elution in capillary liquid chromatography (cHPLC) is studied. Focus is placed on the cHPLC systems which rely on the splitting of a primary flow to provide the much smaller secondary flow required at the injection device and analytical column. Owing to the tiny secondary flow rates, synchronization is necessary to achieve proper optimization of gradient elution methods. Otherwise, there is a risk of having the analytes totally or partially eluted in the initial isocratic conditions, and there is no control on the actual gradient profile reaching the column. Synchronization is first achieved by switching back the valve to bypass after injection. This is important to save time, and to avoid the gradient slope to be reduced by mixing within the internal volume of the injector (a 47% of slope reduction, in the conditions used in this work). Valve switching to bypass should be produced immediately after the arrival of the end of the sample plug to the valve (tV). Fine system synchronization is further achieved by starting the gradient at the match time (tM), which is the time required to match the arrival of both the gradient front and the end of the sample plug to the valve, and therefore also to the column inlet. Synchronization of these two events requires starting the gradient either before or even after the injection, thus to prevent a late or an early arrival of the gradient front to the injection valve, respectively. Owing to their dependence with the backpressure, both tV and tM should be measured in the presence of the column at the initial gradient conditions. Simple experiments designed to measure tV and tM are described. With synchronization according to the techniques described in this work, control on the real gradient elution conditions at the column location is maintained, the analysis time is reduced and efficiency improves. The effects of synchronization are illustrated by injecting a mixture of alkylbenzenes. At 1µL min(-1), valve switching to bypass reduced analysis time from ca. 36 to 12min (butylbenzene), and improved peak symmetry (from 2.00 to 0.94 for methylbenzene) and efficiency (the average apparent plate count increased approximately 60%). Synchronization according to the match time further improved efficiency (approximately, up to 120%).

Determinations of gas-liquid partition coefficients using capillary chromatographic columns. Alkanols in squalane.

This study focused on an investigation into the experimental quantities inherent in the determination of partition coefficients from gas-liquid chromatographic measurements through the use of capillary columns. We prepared several squalane - (2,6,10,15,19,23-hexamethyltetracosane) - containing columns with very precisely known phase ratios and determined solute retention and hold-up times at 30, 40, 50 and 60°C. We calculated infinite dilution partition coefficients from the slopes of the linear regression of retention factors as a function of the reciprocal of the phase ratio by means of fundamental chromatographic equations. In order to minimize gas-solid and liquid-solid interface contributions to retention, the surface of the capillary inner wall was pretreated to guarantee a uniform coat of stationary phase. The validity of the proposed approach was first tested by estimating the partition coefficients of n-alkanes between n-pentane and n-nonane, for which compounds data from the literature were available. Then partition coefficients of sixteen aliphatic alcohols in squalane were determined at those four temperatures. We deliberately chose these highly challenging systems: alcohols in the reference paraffinic stationary phase. These solutes exhibited adsorption in the gas-liquid interface that contributed to retention. The corresponding adsorption constant values were estimated. We fully discuss here the uncertainties associated with each experimental measurement and how these fundamental determinations can be performed precisely by circumventing the main drawbacks. The proposed strategy is reliable and much simpler than the classical chromatographic method employing packed columns.

Effect of temperature and solvent composition on acid dissociation equilibria, I: Sequenced (s)(s)pKa determination of compounds commonly used as buffers in high performance liquid chromatography coupled to mass spectroscopy detection.

A new automated and rapid potentiometric method for determining the effect of organic-solvent composition on pK(a) has been developed. It is based on the measurements of pH values of buffer solutions of variable solvent compositions using a combined glass electrode. Additions of small volumes of one precisely thermostated solution into another, both containing exactly the same analytical concentrations of the buffer components, can produce continuous changes in the solvent composition. Two sequences of potential measurements, one of increasing and the other of decreasing solvent content, are sufficient to obtain the pK(a) values of the acidic compound within the complete solvent-composition range in about 2h. The experimental design, procedures, and calculations needed to convert the measured pH into the thermodynamic pK(a) values are thoroughly discussed. This rapid and automated method allows the systematic study of the effect of solvent compositions and temperatures on the pK(a). It has been applied to study the dissociation constants of two monoprotic acids: formic acid and triethylamine:HCl in acetonitrile/water mixtures within the range from 0 to 90% (v/v) at temperatures between 20°C and 60°C. These volatile compounds are frequently used to control the pH of the mobile phase in HPLC, especially in methods coupled to mass-spectrometry detection. The obtained pK(a) values are in excellent agreement with those previously reported. The results were fitted to empirical functions between pK(a) and temperature and composition. These equations, which can be used to estimate the pK(a) of these substances at any composition and temperature, would be highly useful in practical work during chromatographic method development.