Peak modeling approach to accurate assignment of first-dimension retention times in comprehensive two-dimensional chromatography

Modeling of first-dimension retention of peaks based on modulation phase and period allows reliable prediction of the modulated peak distributions generated in the comprehensive two-dimensional chromatography experiment. By application of the inverse process, it is also possible to use the profile of the modulated peaks (their heights or areas) to predict the shape and parameters of the original input chromatographic band (retention time, standard deviation, area) for the primary column dimension. This allows an accurate derivation of the firstdimension retention time (RSD 0.02%) which is equal to that for the non-modulated experiment, rather than relying upon the retention time of the major modulated peak generated by the modulation process (RSD 0.16%). The latter metric can produce a retention time that differs by at least the modulation period employed in the experiment, which displays a discontinuity in the retention time vs modulation phase plot at the point of the 180° out-ofphase modulation. In contrast, the new procedure proposed here gives a result that is essentially independent of modulation phase and period. This permits an accurate value to be assigned to the first-dimension retention. The proposed metric accounts for the time on the seconddimension, the phase of the distribution, and the holdup time that the sampled solute is retained in the modulating interface. The approach may also be based on the largest three modulated peaks, rather than all modulated peaks. This simplifies the task of assigning the retention time with little loss of precision in band standard deviation or retention time, provided that these peaks are not all overloaded in the first or second dimension.

Exploring two-dimensional chromatography and chemiluminescence selectivity for complex sample analysis

 Human, animal and plant samples contain a considerable number of chemical substances critical for supporting their existence. This thesis presents improved techniques to find and analyse these components to help us understand more about their roles in the complex jigsaw of life.

The analysis of café espresso using two-dimensional reversed phase–reversed phase high performance liquid chromatography with UV-absorbance and chemiluminescence detection

In this study, an activity based screening technique combining two-dimensional liquid chromatography (2DHPLC) with UV-absorbance and chemiluminescence detection was applied to study “Ristretto”, "Decaffeinatto” and “Volluto” espresso coffees. This technique, which coupled the separation power of 2DHPLC with the sensitivity and selectivity of the chemiluminescence detection, offers great potential for screening complex samples for antioxidant compounds. Detailed information regarding the complexity of the sample, and the variation between these three coffees could be obtained using this multidimensional-hyphenated method of analysis.

Effects of variation in modulator temperature during cryogenic modulation in comprehensive two-dimensional gas chromatography

Many modulation systems in comprehensive 2D GC (GC×GC) are based on cryogenic methods. High trapping temperatures in these systems can result in ineffective trapping of the more volatile compounds, whilst temperatures that are too low can prevent efficient remobilisation of some compounds. To better understand the trapping and release of compounds over a wide range of volatilities, we have investigated a number of different constant temperature modulator settings, and have also examined a constant temperature differential between the cryo-trap and the chromatographic oven. These investigations have led us to modify the temperature regulation capabilities of the longitudinally modulated cryogenic system (LMCS). In contrast to the current system, where the user sets a constant temperature for the cooling chamber, the user now sets the temperature difference between the cryo-trap and the chromatographic oven. In this configuration, the cooling chamber temperature increases during the chromatographic run, tracking the oven temperature ramp. This produces more efficient, volatility-dependent modulation, and increases the range of volatile compounds that can be analysed under optimal trap-and-release conditions within a single analytical run. This system also reduces cryogenic fluid consumption.

Peak picking and the assessment of separation performance in two-dimensional high performance liquid chromatography

An algorithm was developed for 2DHPLC that automated the process of peak recognition, measuring their retention times, and then subsequently plotting the information in a two-dimensional retention plane. Following the recognition of peaks, the software then performed a series of statistical assessments of the separation performance, measuring for example, correlation between dimensions, peak capacity and the percentage of usage of the separation space. Peak recognition was achieved by interpreting the first and second derivatives of each respective one-dimensional chromatogram to determine the 1D retention times of each solute and then compiling these retention times for each respective fraction ‘cut’. Due to the nature of comprehensive 2DHPLC adjacent cut fractions may contain peaks common to more than one cut fraction. The algorithm determined which components were common in adjacent cuts and subsequently calculated the peak maximum profile by interpolating the space between adjacent peaks. This algorithm was applied to the analysis of a two-dimensional separation of an apple flesh extract separated in a first dimension comprising a cyano stationary phase and an aqueous/THF mobile phase as the first dimension and a second dimension comprising C18-Hydro with an aqueous/MeOH mobile phase. A total of 187 peaks were detected.

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.

Determination of neurotransmitters and their metabolites using one- and two-dimensional liquid chromatography with acidic potassium permanganate chemiluminescence detection

High-performance liquid chromatography with chemiluminescence detection based on the reaction with acidic potassium permanganate and formaldehyde was explored for the determination of neurotransmitters and their metabolites. The neurotransmitters norepinephrine and dopamine were quantified in the left and right hemispheres of rat hippocampus, nucleus accumbens and prefrontal cortex, and the metabolites vanillylmandelic acid, 3,4-dihydrophenylacetic acid, 5-hydroxyindole-3-acetic acid and homovanillic acid were identified in human urine. Under optimised chemiluminescence reagent conditions, the limits of detection for these analytes ranged from 2.5 × 10−8 to 2.5 × 10−7 M. For the determination of neurotransmitter metabolites in urine, a two-dimensional high-performance liquid chromatography (2D-HPLC) separation operated in heart-cutting mode was developed to overcome the peak capacity limitations of the one-dimensional separation. This approach provided the greater separation power of 2D-HPLC with analysis times comparable to conventional one-dimensional separations.

Off-line two-dimensional liquid chromatography for metabolomics: an example using Agaricus bisporus mushrooms exposed to UV irradiation

It has previously been shown that irradiation with UV light increases the vitamin D content of certain mushroom species, but the effect on other nutrients is unknown, and is difficult to assess due to the complexity of the sample matrix. Here, an offline reversed phase × reversed phase two-dimensional liquid chromatography methodology was developed and applied to Agaricus bisporus mushrooms in order to demonstrate the potential of the technique and assess the effect of UV irradiation on the mushroom’s metabolic profile. The method allowed the detection of 158 peaks in a single analytical run. A total of 51 compounds including sugars, amino acids, organic and fatty acids and phenolic compounds were identified using certified reference standards. After irradiation of the mushrooms with UV for 30 s the number of peaks detected decreased from 158 to 150; 47 compounds increased in concentration while 72 substances decreased. This is the first time that two-dimensional liquid chromatography has been carried out for the metabolomic analysis of mushrooms. The data provide an overview of the gain/loss of nutritional value of the mushrooms following UV irradiation and demonstrate that the increased peak capacity and separation space of two-dimensional liquid chromatography has great potential in metabolomics.

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.

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%.

Screening of cannabinoids in industrial-grade hemp using two-dimensional liquid chromatography coupled with acidic potassium permanganate chemiluminescence detection

Widely known for its recreational use, the cannabis plant also has the potential to act as an antibacterial agent in the medicinal field. The analysis of cannabis plants/products in both pharmacological and forensic studies often requires the separation of compounds of interest and/or accurate identification of the whole cannabinoid profile. In order to provide a complete separation and detection of cannabinoids, a new two-dimensional liquid chromatography method has been developed using acidic potassium permanganate chemiluminescence detection, which has been shown to be selective for cannabinoids. This was carried out using a Luna 100 Å CN column and a Poroshell 120 EC-C18 column in the first and second dimensions, respectively. The method has utilized a large amount of the available separation space with a spreading angle of 48.4° and a correlation of 0.66 allowing the determination of more than 120 constituents and mass spectral identification of ten cannabinoids in a single analytical run. The method has the potential to improve research involved in the characterization of sensitive, complex matrices.

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.

Blind column selection protocol for two-dimensional high performance liquid chromatography

The selection of two orthogonal columns for two-dimensional high performance liquid chromatography (LC×LC) separation of natural product extracts can be a labour intensive and time consuming process and in many cases is an entirely trial-and-error approach. This paper introduces a blind optimisation method for column selection of a black box of constituent components. A data processing pipeline, created in the open source application OpenMS®, was developed to map the components within the mixture of equal mass across a library of HPLC columns; LC×LC separation space utilisation was compared by measuring the fractional surface coverage, fcoverage. It was found that for a test mixture from an opium poppy (Papaver somniferum) extract, the combination of diphenyl and C18 stationary phases provided a predicted fcoverage of 0.48 and was matched with an actual usage of 0.43. OpenMS®, in conjunction with algorithms designed in house, have allowed for a significantly quicker selection of two orthogonal columns, which have been optimised for a LC×LC separation of crude extractions of plant material.

In-silico optimisation of two-dimensional high performance liquid chromatography for the determination of Australian methamphetamine seizure samples

In-silico optimisation of a two-dimensional high performance liquid chromatography (2D-HPLC) separation protocol has been developed for the interogation of methamphetamine samples including model, real world seizure, and laboratory synthesised samples. The protocol used Drylab® software to rapidly identify the optimum separation conditions from a library of chromatography columns. The optimum separation space was provided by the Phenomonex Kinetex PFP column (first dimension) and an Agilent Poroshell 120 EC-C18 column (second dimension). To facilitate a rapid 2D-HPLC analysis the particle packed C18 column was replaced with a Phenomenex Onyx Monolithic C18 withought sacrificing separation performance. The Drylab® optimised and experimental separations matched very closely, highlighting the robust nature of HPLC simulations. The chemical information gained from an intermediate methamphetamine sample was significant and complimented that generated from a pure seizure sample. The influence of the two-dimensional separation on the analytical figures of merit was also investigated. The limits of detection for key analytes in the second dimension determined for methamphetamine (4.59 × 10-⁴ M), pseudoephedrine (4.03 × 10-4 M), caffeine (5.16 × 10-⁴ M), aspirin (9.32 × 10-4 M), paracetamol (5.93 × 10-4 M) and procaine (2.02 × 10-3 M).