Improving peak shapes with counter gradients in two-dimensional high performance liquid chromatography

To achieve the greatest peak capacity in two-dimensional high performance liquid chromatography (2D-HPLC) a gradient should be operated in both separation dimensions. However, it is known that when an injection solvent that is stronger than the initial mobile phase composition is deleterious to peak performance, thus causing problems when cutting a portion from one gradient into another. This was overcome when coupling hydrophilic interaction with reversed phase chromatography by introducing a counter gradient that changed the solvent strength of the second dimension injection. It was found that an injection solvent composition of 20% acetonitrile in water gave acceptable results in one-dimensional simulations with an initial composition of 5% acetonitrile. When this was transferred to a 2D-HPLC separation of standards it was found that a marked improvement in peak shape was gained for the moderately retained analytes (phenol and dimethyl phthalate), some improvement for the weakly retained caffeine and very little change for the strongly retained n-propylbenzene and anthracene which already displayed good chromatographic profiles. This effect was transferred when applied to a 2D-HPLC separation of a coffee extract where the indecipherable retention profile was transformed to a successful application multidimensional chromatography with peaks occupying 71% of the separation space according to the geometric approach to factor analysis.

Contributions to high performance liquid chromatography

High performance liquid chromatography (HPLC) is an enabling science with application in all scientific disciplines requiring analysis or purification. The research described here details performance aspects of the chromatography column, which lead to a new design concept in the chromatography column. Studies were also undertaken to characterise selectivity leading to new stationary phases. Research on fluid dynamics in packed beds showed how a mismatch in solvent viscosities between the injection plug and mobile pahse influences the performance of on-line multidimentional HPLC. Selectivity id detection was also examined in an effort to better understand sample analysis. 

Investigating retention characteristics of a mixed-mode stationary phase and the enhancement of monolith selectivity for high-performance liquid chromatography

The synthesis and chromatographic behavior of an analytical size mixed-mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3-cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance π-type bonding within a single separation environment. Selectivity studies were undertaken using n-alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the π-type selectivities afforded by the two different ligands on the mixed-mode column.

Unilateral bicep curl hemodynamics: low-pressure continuous vs high-pressure intermittent blood flow restriction

 Light-load exercise training with blood flow restriction (BFR) increases muscle strength and size. However, the hemodynamics of BFR exercise appear elevated compared with non-BFR exercise. This questions the suitability of BFR in special/clinical populations. Nevertheless, hemodynamics of standard prescription protocols for BFR and traditional heavy-load exercise have not been compared. We investigated the hemodynamics of two common BFR exercise methods and two traditional resistance exercises. Twelve young males completed four unilateral elbow flexion exercise trials in a balanced, randomized crossover design: (a) heavy load [HL; 80% one-repetition maximum (1-RM)]; (b) light load (LL; 20% 1-RM); and two other light-load trials with BFR applied (c) continuously at 80% resting systolic blood pressure (BFR-C) or (d) intermittently at 130% resting systolic blood pressure (BFR-I). Hemodynamics were measured at baseline, during exercise, and for 60-min post-exercise. Exercising heart rate, blood pressure, cardiac output, and rate–pressure product were significantly greater for HL and BFR-I compared with LL. The magnitude of hemodynamic stress for BFR-C was between that of HL and LL. These data show reduced hemodynamics for continuous low-pressure BFR exercise compared with intermittent high-pressure BFR in young healthy populations. BFR remains a potentially viable method to improve muscle mass and strength in special/clinical populations.

DryLab® optimised two-dimensional high performance liquid chromatography for differentiation of ephedrine and pseudoephedrine based methamphetamine samples

In-silico optimised two-dimensional high performance liquid chromatographic (2D-HPLC) separations of a model methamphetamine seizure sample are described, where an excellent match between simulated and real separations was observed. Targeted separation of model compounds was completed with significantly reduced method development time. This separation was completed in the heart-cutting mode of 2D-HPLC where C18 columns were used in both dimensions taking advantage of the selectivity difference of methanol and acetonitrile as the mobile phases. This method development protocol is most significant when optimising the separation of chemically similar chemical compounds as it eliminates potentially hours of trial and error injections to identify the optimised experimental conditions. After only four screening injections the gradient profile for both 2D-HPLC dimensions could be optimised via simulations, ensuring the baseline resolution of diastereomers (ephedrine and pseudoephedrine) in 9.7 min. Depending on which diastereomer is present the potential synthetic pathway can be categorized.

Study on nanocellulose by high pressure homogenization in homogeneous isolation

Nanocellulose from cotton cellulose was prepared by high pressure homogenization (HPH) in ionic liquids (1-butyl-3-methylimidazolium chloride ([Bmim]Cl). The nanocellulose possessed narrow particle size distribution, with diameter range of 10–20 nm. Weight average molecular weight (Mw) of nanocellulose treated by HPH was lower (173.8 kDa) than the one ILs treated cellulose (344.6 kDa). X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), and Solid-state CP/MAS 13C NMR measurements were employed to study the mechanism of structural changes, which suggested that network structure between cellulose chains were destructed by the shearing forces of HPH in combination with ionic liquids. The intermolecular and intra-molecular hydrogen bonds of cellulose were further destroyed, leading to the long cellulose molecular chains being collapsed into short chains. Therefore, the nanocellulose could provide desired properties, such as lower thermal stability and strong water holding capacity. Results indicated that it had great potential in the applications for packaging, medicines, cosmetics and tissue engineering.

Protocols for finding the most orthogonal dimensions for two-dimensional high performance liquid chromatography

The selection of two high performance liquid chromatography (HPLC) columns with vastly different retention mechanisms is vital for performing effective two-dimensional (2D-) HPLC. This paper reports on a systematic method to select a pair of HPLC columns that provide the most different separations for a given sample. This was completed with the aid of a HPLC simulator that predicted retention profiles on the basis of real experimental data, which is difficult when the contents of sample matrices are largely-or completely-unknown. Peaks from the same compounds must first be matched between chromatograms to compare the retention profiles and optimised 2D-HPLC column selection. In this work, two methods of matching peaks between chromatograms were explored and an optimal pair of chromatography columns was selected for 2D-HPLC. First, a series of 17 antioxidants were selected as an analogue for a coffee extract. The predicted orthogonality of the standards was 39%, according to the fractional surface coverage 'bins' method, which was close to the actual space utilisation of the standard mixture, 44%. Moreover, the orthogonality for the 2D-HPLC of coffee matched the predicted value of 38%. The second method employed a complex sample matrix of urine to optimise the column selections. Seven peaks were confidently matched between chromatograms by comparing relative peak areas of two detection strategies: UV absorbance and potassium permanganate chemiluminescence. It was found that the optimal combinations had an orthogonality of 35% while the actual value was closer to 30%.

Assessing the detectability of antioxidants in two-dimensional high-performance liquid chromatography

This paper explores the analytical figures of merit of two-dimensional high-performance liquid chromatography for the separation of antioxidant standards. The cumulative two-dimensional high-performance liquid chromatography peak area was calculated for 11 antioxidants by two different methods--the areas reported by the control software and by fitting the data with a Gaussian model; these methods were evaluated for precision and sensitivity. Both methods demonstrated excellent precision in regards to retention time in the second dimension (%RSD below 1.16%) and cumulative second dimension peak area (%RSD below 3.73% from the instrument software and 5.87% for the Gaussian method). Combining areas reported by the high-performance liquid chromatographic control software displayed superior limits of detection, in the order of 1 × 10(-6) M, almost an order of magnitude lower than the Gaussian method for some analytes. The introduction of the countergradient eliminated the strong solvent mismatch between dimensions, leading to a much improved peak shape and better detection limits for quantification.