Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography

A new approach to high speed, comprehensive online dual gradient elution 2DLC (LCxLC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the "analytic dynamic range" problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.     

Peak capacity optimization in comprehensive two dimensional liquid chromatography: a practical approach

In this work we develop a practical approach to optimization in comprehensive two dimensional liquid chromatography (LC x LC) which incorporates the important under-sampling correction and is based on the previously developed gradient implementation of the Poppe approach to optimizing peak capacity. The Poppe method allows the determination of the column length, flow rate as well as initial and final eluent compositions that maximize the peak capacity at a given gradient time. It was assumed that gradient elution is applied in both dimensions and that various practical constraints are imposed on both the initial and final mobile phase composition in the first dimension separation. It was convenient to consider four different classes of solute sets differing in their retention properties. The major finding of this study is that the under-sampling effect is very important and causes some unexpected results including the important counter-intuitive observation that under certain conditions the optimum effective LC x LC peak capacity is obtained when the first dimension is deliberately run under sub-optimal conditions. In addition, we found that the optimum sampling rate in this study is rather slower than reported in previous studies and that it increases with longer first dimension gradient times.     

Trilinearity deviation ratio: A new metric for chemometric analysis of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry data

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC–TOFMS) is a well-established instrumental platform for complex samples. However, chemometric data analysis is often required to fully extract useful information from the data. We demonstrate that retention time shifting from one modulation to the next, Δ²t_R, is not sufficient alone to quantitatively describe the trilinearity of a single GC × GC–TOFMS run for the purpose of predicting the performance of the chemometric method parallel factor analysis (PARAFAC). We hypothesize that analyte peak width on second dimension separations, ²W_b, also impacts trilinearity, along with Δ²t_R. The term trilinearity deviation ratio, TDR, which is Δ²t_R normalized by ²W_b, is introduced as a quantitative metric to assess accuracy for PARAFAC of a GC × GC–TOFMS data cube. We explore how modulation ratio, M_R, modulation period, P_M, temperature programming rate, T_ramp, sampling phase (in-phase and out-of-phase), and signal-to-noise ratio, S/N, all play a role in PARAFAC performance in the context of TDR. Use of a P_M in the 1–2 s range provides an optimized peak capacity for the first dimension separation (500–600) for a 30 min run, with an adequate peak capacity for the second dimension separation (12–15), concurrent with an optimized two-dimensional peak capacity (6000–7500), combined with sufficiently low TDR values (0–0.05) to facilitate low quantitative errors with PARAFAC (0–0.5%). In contrast, use of a P_M in the 5 s or greater range provides a higher peak capacity on the second dimension (30–35), concurrent with a lower peak capacity on the first dimension (100–150) for a 30 min run, and a slightly reduced two-dimensional peak capacity (3000–4500), and furthermore, the data are not sufficiently trilinear for the more retained second dimension peaks in order to directly use PARAFAC with confidence.     

Comprehensive supercritical fluid chromatography × reversed phase liquid chromatography for the analysis of the fatty acids in fish oil

The separation of the phenacyl esters of the fatty acids originating from a fish oil extract by means of a comprehensive analysis using silver-ion (SI) supercritical fluid chromatography (SFC) and reversed phase liquid chromatography (RP-LC) in the first and second dimensions, respectively, is described. The combination ensured a high orthogonality and peak capacity, particularly when compared to the comprehensive RP-LC × 2RP-LC separation achieved by using a configuration with two columns in parallel in the second dimension. The construction of the SI-SFC × RP-LC interface consists of two two-position/ten-port switching valves, of which one is equipped with two loops packed with octadecyl silica (ODS) particles. Compared to the SFC × RP-LC configuration described in an earlier publication, the peak capacity in the second dimension was increased. Water was not only added as make-up fluid to the SFC effluent to ensure analyte focusing, but also as rinsing medium of the loops prior to the transfer of the fractions to the second dimension. In the SFC dimension, high efficiency and loadability were obtained by coupling two wide-bore columns (4.6 mm ID) in series. Evaporative light scattering (ELSD) and ultraviolet (UV) detection with standard and high-pressure flow cells were evaluated in terms of data acquisition speed and suppression of signal interferences originating from the supercritical carbon dioxide (CO₂) expansion.     

Fast, comprehensive two-dimensional liquid chromatography

The absolute need to improve the separating power of liquid chromatography, especially for multi-constituent biological samples, is becoming increasingly evident. In response, over the past few years, there has been a great deal of interest in the development of two-dimensional liquid chromatography (2DLC). Just as 1DLC is preferred to 1DGC based on its compatibility with biological materials we believe that ultimately 2DLC will be preferred to the much more highly developed 2DGC for such samples. The huge advantage of 2D chromatographic techniques over 1D methods is inherent in the tremendous potential increase in peak capacity (resolving power). This is especially true of comprehensive 2D chromatography wherein it is possible, under ideal conditions, to obtain a total peak capacity equal to the product of the peak capacities of the first and second dimension separations. However, the very long timescale (typically several hours to tens of hours) of comprehensive 2DLC is clearly its chief drawback. Recent advances in the use of higher temperatures to speed up isocratic and gradient elution liquid chromatography have been used to decrease the time needed to do the second dimension LC separation of 2DLC to about 20s for a full gradient elution run. Thus, fast, high temperature LC is becoming a very promising technique. Peak capacities of over 2000 and rates of peak capacity production of nearly 1 peak/s have been achieved. In consequence, many real samples showing more than 200 peaks with signal to noise ratios of better than 10:1 have been run in total times of under 30 min. This report is not intended to be a comprehensive review of 2DLC, but is deliberately focused on the issues involved in doing fast 2DLC by means of elevating the column temperature; however, many issues of broader applicability will be discussed.     

Handling within run retention time shifts in two-dimensional chromatography data using shift correction and modeling

The use of PARAFAC for modeling GC × GC-TOFMS peaks is well documented. This success is due to the trilinear structure of these data under ideal, or sufficiently close to ideal, chromatographic conditions. However, using temperature programming to cope with the general elution problem, deviations from trilinearity within a run are more likely to be seen for the following three cases: (1) compounds (i.e., analytes) severely broadened on the first column hence defined by many modulation periods, (2) analytes with a very high retention factor on the second column and likely wrapped around in that dimension, or (3) with fast temperature program rates. This deviation from trilinearity is seen as retention time-shifted peak profiles in subsequent modulation periods (first column fractions). In this report, a relaxed yet powerful version of PARAFAC, known as PARAFAC2 has been applied to handle this shift within the model step by allowing generation of individual peak profiles in subsequent first column fractions. An alternative approach was also studied, utilizing a standard retention time shift correction to restore the data trilinearity structure followed by PARAFAC. These two approaches are compared when identifying and quantifying a known analyte over a large concentration series where a certain shift is simulated in the successive first column fractions. Finally, the methods are applied to real chromatographic data showing severely shifted peak profiles. The pros and cons of the presented approaches are discussed in relation to the model parameters, the signal-to-noise ratio and the degree of shift.     

Novel oxidation reaction of prochlorperazine with bromate in the presence of synergistic activators and its application to trace determination by flow injection/spectrophotometric method

Valve-based comprehensive two-dimensional gas chromatography (GC × GC) is one of the most compact, robust, and inexpensive GC × GC instrument designs. The major drawback of a valve-based modulation configuration lies in diminished detection sensitivity. This loss in sensitivity is because under typical operating conditions the fraction of the first column (i.e., column 1) effluent transferred to the second column (i.e., column 2) is likely to be ∼5-10%. To address this loss in sensitivity, we report the development of a unique total-transfer (i.e., 100%) valve-based GC × GC, without adding complexity to the instrumentation. The new instrument design relies upon simply blocking one of the appropriate ports of the high-speed six-port diaphragm valve that is used as the modulator between columns 1 and 2. The modulation period and difference in head pressure between columns 1 and 2 are found to be the two primary variables that are controlled to provide good detection sensitivity and 100% mass transfer from column 1 to column 2. The detection sensitivity is better with a longer the modulation period. A limit of detection of 0.03 ng/μl was obtained for octane. This sensitive GC × GC configuration is also shown to provide acceptable separation peak capacity, with good separations achieved for real complex samples: gasoline and Eucalyptus oil, where compounds were spread out over much of the two-dimensional separation space. In principle, this total-transfer, valve-based GC × GC is more portable and less expensive than currently available GC × GC instrumentation.     

Development of a switchable multidimensional/comprehensive two-dimensional gas chromatographic analytical system

In this study, a new system for analysis using a dual comprehensive two-dimensional gas chromatography/targeted multidimensional gas chromatography (switchable GC × GC/targeted MDGC) analysis was developed. The configuration of this system not only permits the independent operation of GC, GC × GC and targeted MDGC analyses in separate analyses, but also allows the mode to be switched from GC × GC to targeted MDGC any number of times through a single analysis. By incorporating a Deans switch microfluidics transfer module prior to a cryotrapping device, the flow stream from the first dimension column can be directed to either one of two second dimension columns in a classical heart-cutting operation. Both second columns pass through the cryotrap to allow solute bands to be focused and then rapidly remobilized to the respective second columns. A short second column enables GC × GC operation, whilst a longer column is used for targeted MDGC. Validation of the system was performed using a standard mixture of compounds relevant to essential oil analysis, and then using compounds present at different abundances in lavender essential oil. Reproducibility of retention times and peak area responses demonstrated that there was negligible variation in the system over the course of multiple heart-cuts, and proved the reliable operation of the system. An application of the system to lavender oil, as a more complex sample, was carried out to affirm system feasibility, and demonstrate the ability of the system to target multiple components in the oil. The system was proposed to be useful for study of aroma-impact compounds where GC × GC can be incorporated with MDGC to permit precise identification of aroma-active compounds, where heart-cut multidimensional GC-olfactometry detection (MDGC-O) is a more appropriate technology for odour assessment.     

An experimental study of sampling time effects on the resolving power of on-line two-dimensional high performance liquid chromatography

The experimental effects of sampling time on the resolving power of on-line LC×LC were investigated. The first dimension gradient time ((1)t(g)) and sampling time (t(s)) were systematically varied ((1)t(g)=5, 12, 24 and 49 min; t(s)=6, 12, 21 and 40s). The resolving power of on-line LC×LC was evaluated in terms of two metrics namely the numbers of observed peaks and the effective 2D peak capacities obtained in separations of extracts of maize seeds. The maximum effective peak capacity and number of observed peaks of LC×LC were achieved at sampling times between 12 and 21s, at all first dimension gradient times. In addition, both metrics showed that the "crossover" time at which fully optimized 1DLC and LC×LC have equal resolving power varied somewhat with sampling time but is only about 5 min for sampling times of 12 and 21s. The longest crossover time was obtained when the sampling time was 6s. Furthermore, increasing the first dimension gradient time gave large improvements in the resolving power of LC×LC relative to 1DLC. Finally, comparisons of the corrected and effective 2D peak capacities as well as the number of peaks observed showed that the impact of the coverage factor is quite significant.     

Effects of first dimension eluent composition in two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC×LC) has received a great deal of attention during the past few years because of its extraordinary resolving power. The biggest advantage of this technique is that very high peak capacities can be generated in a relatively short time. Numerous approaches to maximize the peak capacity in LC×LC have been employed. In this work we investigate the impact of the first dimension mobile phase on selectivity. LC×LC has several potential advantages over one-dimensional LC (1DLC) in that unconventional solvents, at least in reversed-phase LC, can be used. For example, solvents which strongly adsorb in the UV in the first dimension are not problematic in LC×LC. This so because the UV detector is placed after the second dimensional column, as pulses of the first dimension eluent arrive at the second dimensional column, they elute well before the solutes of interest and therefore do not interfere at all with detection of solute peaks. So far, the most widely used solvents in reversed-phase 1DLC are methanol and acetonitrile. However, the "UV advantage" of 2DLC allows us to employ UV active solvents, such as acetone. We compare their differential selectivities to that of acetonitrile for the separation of 23 indole acetic acids of interest in plant biology. We also apply them to the separation of a maize seed extract, a very complex sample. In both sample sets, mobile phase composition can be an important parameter to increase the orthogonality of the two dimensions and thus, to increase the effective peak capacity of LC×LC.     

A graphical method for understanding the kinetics of peak capacity production in gradient elution liquid chromatography

A novel graphical method for assessing the compromise between conditional peak capacity and separation speed for packed bed columns under gradient conditions has been developed and applied to the separation of peptides. This approach is analogous to and complements the conventional "Poppe plot" used to study plate count in isocratic separations. The use of the new plot can assist the design of appropriate column formats (e.g. particle size and column length) for both dimensions in gradient elution two-dimensional liquid chromatography (2DLC). Particularly for the second dimension of 2DLC, we find that smaller particles provide faster separations even though fast separations based on particles smaller than 2 microm are practically limited by the required miniscule column length. We also find that high temperatures strongly enhance the kinetics of peak capacity production whereas higher pressures help achieve larger absolute peak capacities albeit at the cost of longer analysis time.     

Measurement of retention in comprehensive two-dimensional gas chromatography using flow modulation with methane dopant

Flow modulation of methane-doped carrier gas is used to visualize the second dimension hold-up time in GC × GC continuously throughout the run. This provides an internal reference of hold-up time and presents a straightforward means of examining retention in each dimension of GC × GC. Retention factors on similar and dissimilar column pairs are examined. Stationary phase bleed is shown to be retained by the second dimension column.     

Effects of the gradient profile, sample volume and solvent on the separation in very fast gradients, with special attention to the second-dimension gradient in comprehensive two-dimensional liquid chromatography

Gradient elution provides significant improvement in peak capacity with respect to isocratic conditions and therefore should be used in comprehensive two-dimensional LC×LC, both in the first and in the second dimension, where, however, gradients are limited to a short time period available for separation, usually 1 min or less. Gradient conditions spanning over a broad mobile phase composition range in each second-dimension fraction analysis are used with generic "full in fraction" (FIF) gradients. "Segment in fraction" (SIF) gradients cover a limited gradient range adjusted independently to suit changing lipophilicity range of compounds transferred to the second dimension during the first-dimension gradient run and to provide regular coverage of the two-dimensional retention space. Optimization of the gradient profiles is important tool for achieving high two-dimensional peak capacity and savings of the separation time in comprehensive LC×LC. Calculations based on the well-established gradient-elution theory can be used to predict the elution times and bandwidths in fast gradients, taking into account increased probability of pre-gradient or post-gradient elution. The fraction volumes transferred into the second dimension may significantly affect the second-dimension bandwidths, especially at high elution strength of the fraction solvent, which may cause even band distortion or splitting in combined normal-phase (HILIC)-RP systems, but also in some two-dimensional RP-RP systems. In the present work, the effects of the fast gradient profile, of the sample volume and solvent on the elution time and bandwidths were investigated on a short column packed with fused-core porous-shell particles, providing narrow bandwidths and fast separations at moderate operating pressure.     

Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques

The overall peak capacity in comprehensive two-dimensional liquid chromatographic (LC x LC) separation can be considerably increased using efficient columns and carefully optimized mobile phases providing large differences in the retention mechanisms and separation selectivity between the first and the second dimension. Gradient-elution operation and fraction-transfer modulation by matching the retention and the elution strength of the mobile phases in the two dimensions are useful means to suppress the band broadening in the second dimension and to increase the number of sample compounds separated in LC x LC. Matching parallel gradients in the first and second dimension eliminate the necessity of second-dimension column re-equilibration after the independent gradient runs for each fraction, increase the use of the available second-dimension separation time and can significantly improve the regularity of the coverage of the available retention space in LC x LC separations, especially with the first- and second-dimension systems showing partial selectivity correlations. Systematic development of an LC x LC method with parallel two-dimensional gradients was applied for separation of phenolic acids and flavone compounds. Several types of bonded C18, amide, phenyl, pentafluorophenyl and poly(ethylene glycol) columns were compared using the linear free energy relationship method to find suitable column combination with low correlation of retention of representative standards. The phase systems were optimized step-by-step to find the mobile phases and gradients providing best separation selectivity for phenolic compounds. The optimization of simultaneous parallel gradients in the first and second dimension resulted in significant improvement in the utilization of the available two-dimensional retention space.     

Enhanced resolution comprehensive two-dimensional gas chromatography applied to the analysis of roasted coffee volatiles

The present research is based on the full exploitation of the separation power of a 0.05 mm internal diameter (ID) capillary, as a comprehensive two-dimensional (2D) GC (GC × GC) secondary column, with the objective of attaining very high-resolution second dimension separations. The aim was achieved by using a split-flow system developed in previous research [P.Q. Tranchida, A. Casilli, P. Dugo, G. Dugo, L. Mondello, Anal. Chem. 79 (2007) 2266], and a dual-oven GC × GC instrument. The column combination employed consisted of a polar 30 m × 0.25 mm ID column connected, by means of a T union, to a detector-linked high-resolution 1.1 m × 0.05 mm ID apolar analytical column and to a 0.33 m × 0.05 mm ID retention gap; the latter was connected to a manually operated split valve. As previously demonstrated, the use of a split valve enables the regulation of gas flows through both analytical columns, generating the most appropriate gas linear velocities. Comprehensive 2D GC experiments were carried out on Arabica roasted coffee volatiles (previously extracted by means of solid-phase microextraction) with the split-valve closed (equal to what can be defined as conventional GC × GC) and with the split-valve opened at various degrees. The reasons why it is absolutely not effective to use a 0.05 mm ID column as second dimension in a conventional GC × GC instrument will be discussed and demonstrated. On the contrary, the use of a 0.05 mm ID column as second dimension, under ideal conditions in a split-flow, twin-oven system, will also be illustrated and discussed.     

Evaluation of 1.0 mm i.d. column performances on ultra high pressure liquid chromatography instrumentation

The present study focuses on the evaluation of 1.0 mm i.d. (internal diameter) columns on a commercial Ultra-High Pressure system. These systems have been developed specifically to operate columns with small volumes, typically 2.1 mm i.d., by reducing extra-column volume dispersion. The use of columns with smaller i.d. results in a reduced solvent consumption and required sample volume. The evaluation of the columns was carried out with samples containing neutral and pharmaceutical compounds. In isocratic mode, the extra-column volume produced additional band broadening leading to poor performances compared to equivalent 2.1 mm i.d. columns. By increasing the length of the column, the influence of the extra-column bandspreading could be reduced and 75,000 plates were obtained when four columns were coupled. In gradient mode, the effect of the extra-column contribution on efficiency was limited and about 80% of the performance of the 2.1 mm i.d. columns was obtained. Optimum conditions in gradient mode were further investigated by changing flow rate, gradient time and column length. A different approach of the calculation of peak capacity was also considered for the comparison of the influence of these different parameters.     

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry for metabonomics: Biomarker discovery for diabetes mellitus

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC-TOFMS) coupled with pattern recognition methods was applied to analyze plasma from diabetic patients and healthy controls. After sample preparation and GC × GC-TOFMS analysis, collected data were transformed, the peak alignment between different chromatograms was performed to generate the metabolites’ peak table, then orthogonal signal correction filtered partial least-squares discriminant analysis (OSC-PLSDA) was carried out to model the data and discover metabolites with a significant concentration change in diabetic patients. With the method above, diabetic patients and healthy controls could be correctly distinguished based on the metabolic abnormity in plasma. Five potential biomarkers including glucose, 2-hydroxyisobutyric acid, linoleic acid, palmitic acid and phosphate were identified. It was found that elevated free fatty acids were essential pathophysiological factors in diabetes mellitus which reflected either the hyperglycemia or the deregulation of fatty acids metabolism. These potential biomarkers in plasma, e.g. palmitic acid, linoleic acid and 2-hydroxybutyric acid might be helpful in the diagnosis or further study of diabetes mellitus. This study shows the practicability and advantage of GC × GC-TOFMS coupled with data analysis and mining for metabonomics in biomarker discovery.     

Headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry for the determination of volatile compounds from marine salt

In this work, a methodology to characterise the volatile and semi-volatile compounds from marine salt by headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC/TOFMS) was developed. Samples from two saltpans of Aveiro, in Portugal, with diverse locations, obtained over three years (2004, 2005, and 2007) were analysed. A 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane SPME fibre was used. The volatiles present in the headspace of the solid salt samples (crystals) were equilibrated overnight at 60 °C and extracted for 60 min prior to injection in the GC × GC/TOFMS. 157 compounds, distributed over the chemical groups of hydrocarbons, aldehydes, esters, furans, haloalkanes, ketones, ethers, alcohols, terpenoids, C₁₃ norisoprenoids, and lactones were detected across the samples. Furans, haloalkanes and ethers were identified for the first time in marine salt. The large number of co-elutions on the first column that were resolved by the GC × GC system revealed the complexity of marine salt volatile composition. The existence of a structured 2D chromatographic behaviour according to volatility, in the first dimension (¹D), and primarily polarity, in the second dimension (²D), was demonstrated, allowing more reliable identifications. The resolution and sensitivity of GC × GC/TOFMS enabled the separation and identification of a higher number of volatile compounds compared to GC–qMS, allowing a deeper characterisation of this natural product.     

A new measure of orthogonality for multi-dimensional chromatography

Multi-dimensional chromatographic techniques, such as (comprehensive) two-dimensional liquid chromatography and (comprehensive) two-dimensional gas chromatography, are increasingly popular for the analysis of complex samples, such as protein digests or mineral oils. The reason behind the popularity of these techniques is the superior performance, in terms of peak-production rate (peak capacity per unit time), that multi-dimensional separations offer compared to their one-dimensional counterparts. However, to fully utilize the potential of multi-dimensional chromatography it is essential that the separation mechanisms used in each dimension be independent of each other. In other words, the two separation mechanisms need to be orthogonal. A number of algorithms have been proposed in the literature for measuring chromatographic orthogonality. However, these methods have their limitations, such as reliance on the division of the separation space into bins, need for specialist software or requirement of advanced programming skills. In addition, some of the existing methods for measuring orthogonality include regions of the separation space that do not feature peaks. In this paper we introduce a number of equations which provides information on the spread of the peaks within the separation space in addition to measuring orthogonality, without the need for complex computations or division of the separation space into bins.     

Critical comparison of performances of superficially porous particles and sub-2 μm particles under optimized ultra-high pressure conditions

The performance of 2.7 μm superficially porous particles at 600 bar and sub-2 μm fully porous particles at 1000 bar were compared by the Poppe plot method. Theoretical Poppe plots were first constructed for each stationary phase to compare their kinetic performance at different analysis times. The theory was then verified by experiments under the optimized conditions identified from the Poppe plot calculation. We found that the 2.7 μm superficially porous particles at 600 bar can provide similar performance compared to the sub-2 μm fully porous particles at ultra-high pressure (1000 bar) when analysis times are very short (e.g. sub-minute). As analysis time increases, the superficially porous particles start to outperform the sub-2 μm particles and can give much higher efficiencies (e.g. > 2 times higher plate count) at very long analysis times (>3 h). The comparison was extended to gradient elution of a mixture of pharmaceutical interest by constructing gradient peak capacity Poppe plots and similar behavior was observed.