Comprehensive multidimensional liquid chromatography: theory and applications

Comprehensive two-dimensional (2D) liquid chromatographic (LC x LC) techniques can be considered innovative methods only recently developed and adopted in many configurations. The revolutionary aspect of comprehensive two-dimensional techniques, with respect to classical multidimensional (MD) chromatography, is that the entire sample is subjected to the 2D advantage. The major benefit is that the separation capacities of each dimension are multiplied, offering a high peak capacity to resolve samples of great complexity. The first part of the present review briefly describes the theoretical and practical aspects related to the development of a multidimensional comprehensive liquid chromatographic method. Applicational experiences in comprehensive liquid chromatography are then described, divided into four groups, according to the HPLC modes used in the two dimensions and to the nature of the samples analyzed.     

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.     

The impact of sampling time on peak capacity and analysis speed in on-line comprehensive two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (2DLC) offers a number of practical advantages over optimized one-dimensional LC in peak capacity and thus in resolving power. The traditional “product rule” for overall peak capacity for a 2DLC system significantly overestimates peak capacity because it neglects under-sampling of the first dimension separation. Here we expand on previous work by more closely examining the effects of the first dimension peak capacity and gradient time, and the second dimension cycle times on the overall peak capacity of the 2DLC system. We also examine the effects of re-equilibration time on under-sampling as measured by the under-sampling factor and the influence of molecular type (peptide vs. small molecule) on peak capacity. We show that in fast 2D separations (less than 1 h), the second dimension is more important than the first dimension in determining overall peak capacity and conclude that extreme measures to enhance the first dimension peak capacity are usually unwarranted. We also examine the influence of sample types (small molecules vs. peptides) on second dimension peak capacity and peak capacity production rates, and how the sample type influences optimum second dimension gradient and re-equilibration times.     

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.     

Effect of first dimension phase selectivity in online comprehensive two dimensional liquid chromatography (LC×LC)

In this study, we examined the effect of first dimension column selectivity in reversed phase (RP) online comprehensive two dimensional liquid chromatography (LC×LC). The second dimension was always a carbon clad metal oxide reversed phase material. The hydrophobic subtraction model (HSM) and the related phase selective triangles were used to guide the selection of six different RP first dimension columns. Various kinds of samples were investigated and thus two different elution conditions were needed to cause full elution from the first dimension columns. We compared LC×LC chromatograms, contours plots, and fcoverage plots by measuring peak capacities, peak numbers, relative spatial coverage, correlation values, etc. The major finding of this study is that the carbon phase due to its rather different selectivity from other reversed phases is reasonably orthogonal to a variety of common types of bonded reversed phases. Thus quite surprisingly the six different first dimension stationary phases all showed generally similar separation patterns when paired to the second dimension carbon phase. This result greatly simplifies the task of choosing the correct pair of phases for RP×RP.     

Comprehensive two-dimensional liquid chromatography

Having nearly exhausted the possibilities for generating peak capacity through improvements in column technology, chromatographers are increasingly looking to alternative ways of maximising chromatographic separation. In recent years there has been increasing activity in the field of comprehensive multidimensional separations to meet analysis demands. Comprehensive two-dimensional liquid chromatography (LC×LC) approaches offer high peak capacity which leads to significantly improved analytical performance over single-column liquid chromatography. There are several closely related avenues available for achieving an LC×LC separation and this review pays special attention to the different valve-based interfaces that have been used to comprehensively couple the first and second dimension columns in LC×LC systems. A brief discussion of column choices for selected applications and the conditions employed is also presented.     

A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography x liquid chromatography

A novel approach for the selection of the operational parameters (linear velocity, column length) for a comprehensive 2D-LC system is discussed. Starting point for the calculations is a given second dimension ((2)D) separation and a desired peak capacity for the 2D system. Using the theory developed here the optimum settings for the first dimension ((1)D) column can be derived. Theory clearly indicates that the choice of the (1)D conditions is basically limited to just one set of column lengths and linear velocities. The new method is tested on a comprehensive two-dimensional liquid chromatography system which uses size-exclusion chromatography (SEC) followed by reversed phase liquid chromatography (RPLC). A novel LC/LC interface, using a six-port valve rather than storage loops, joins the two chromatographic dimensions. From a theoretical comparison of continuous low flow and stop-flow operation the latter method was found to be an attractive mode of interfacing. The common idea that stop-flow operation results in additional band broadening is shown to be incorrect. The new interface design operated in the stop-flow mode permits the use of conventional analytical diameter HPLC columns, 7.8mm for SEC and 4.6mm for RPLC. The reversed phase chromatography utilizes a monolithic C-18 modified silica column, which produces fast and efficient analyses. As test samples complex mixtures of peptides were analyzed.     

Ultrafast liquid chromatography/ultraviolet and liquid chromatography/tandem mass spectrometric analysis

Optimal liquid chromatography/mass spectrometric [LC/MS(/MS)] analysis depends on both the LC selectivity and the electrospray efficiency. Here, we outline a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. To achieve ultrafast LC separation with little sacrifice in peak capacity, one needs to start with a column that provides a good peak capacity at short gradient run times; secondly, it is important to use high flow rates to achieve a good gradient peak capacity. Following this strategy, it was possible to baseline-resolve a mixture (containing acidic, neutral, and basic pharmaceutical analytes) in seconds. By coupling the selectivity provided by fast LC separation with the specificity of MS/MS detection, it is possible to separate and identify a wide range of analytes in 1-min gradient analyses. Also, the impact of mobile phase pH on both the chromatographic selectivity and the MS/MS sensitivity is demonstrated.     

Comprehensive two-dimensional chromatography in food analysis

Comprehensive two-dimensional (2D) chromatographic techniques can be considered innovative methods, only quite recently developed. Since their introduction to the chromatographic community, these techniques have been used in several fields and have gained an excellent reputation as valuable and powerful analytical tools. The revolutionary aspect of comprehensive multidimensional (MD) techniques, in respect to classical MD chromatography, is that the entire sample is subjected to the 2D advantage. The resulting unprecedented separating capacity makes these approaches prime choices when analysts are challenged with highly complex mixtures. Furthermore, in the case of automated systems, instrumental analysis times are roughly the same as in monodimensional applications. The present review reports various comprehensive chromatographic applications on different food matrices. The GC x GC section highlights two fundamental aspects for component separation/identification: the exceptional peak capacity and the formation of group types on the 2D space plane. The LC x LC section reports the employment in food analysis of a recently developed multidimensional normal-phase (NP)-reversed-phase (RP) high performance liquid chromatography (HPLC) system. Also reported are comprehensive LC x GC and packed column supercritical fluid chromatography (pSFC x pSFC) applications in this field.     

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.     

二维液相色谱接口技术

二维液相色谱具有峰容量大、分辨率高、分析速度快等优点,已经成为复杂样品分离分析的重要工具。两种分离模式的转换通常需要经过一个特殊接口来完成,接口是二维液相色谱系统的核心,也是限制二维液相色谱应用的瓶颈;两种流动相不互溶时,接口尤为重要。本文针对二维液相色谱接口技术近期的发展和应用进行总结。引用文献51篇。     

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.     

Use of shift gradient in the second dimension to improve the separation space in comprehensive two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC?×?LC) has received much attention because it offers much higher peak capacities than separation in a single dimension. The advantageous peak capacity makes it attractive for the separation of complex samples. Various gradient methods have been used in LC?×?LC systems. The use of continuous shift gradient is advantageous because it combines the peak compression effect of full gradient mode and the tailed gradient program in parallel gradient mode. Here, a comparison of LC?×?LC analysis of Chinese herbal medicine with full gradient mode and shift gradient mode in the second dimension was performed. A correlation between the first and second dimensions was found in full gradient mode, and this was significantly reduced with shift gradient mode. The orthogonality increased by 43.7 %. The effective peak distribution area increased significantly, which produced better separation.     

Gruppenspezifische Analyse von Citratcyclusintermediaten in der modernen Flüssigkeits-Chromatographie im unteren Nanoval-Bereich

The analysis of TCA intermediates and related compounds with modern liquid chromatography (LC) is described. Short-chain carboxylic acids are detected by adding a compound specific indicator (o-nitro-phenol) to the eluent. Without gradient elution, the noise level of the chromatogram baseline does not exceed 1×10^?3 O.D. and the peak area for 10 nanoequivalent acetic acid is 25 absorbance seconds. With repeated applications of sample to column, deviations of 5% of the medium peak area value are not exceeded (range of confidence 95%). Preparation of the biological samples is achieved by ether extraction with a simple column-chromatography apparatus, which at the same time, preseparates critical acids otherwise not completely separable by elution without gradient. The application of LC to the analysis of biological material is demonstrated by a chromatogram of 10 μl of human serum.     

A practical approach to maximizing peak capacity by using long columns packed with pellicular stationary phases for proteomic research

Maximization of peak capacity is a very important step in developing one-dimensional separations of complex samples. In recent work, it was shown that the use of small particles in combination with the new technique of ultrahigh pressure liquid chromatography (UHPLC) was able to generate very high peak capacities. Here we show the ability of conventional HPLC instrumentation to give comparable peak capacities to those obtained in UHPLC for the important case of complex mixtures of peptides but at much lower pressures by using a 60 cm long set of columns packed with 5 microm pellicular (superficially porous) particles. We first show, in complete agreement with the well known results of the theory of isocratic separations that, when time is not limiting, the best peak capacities in gradient elution chromatography are obtained by using large particles and the longest column that can be operated at the pump's pressure limit. Two different types of 5 microm particles (superficially porous and totally porous) were compared for their efficiency in gradient chromatography of peptides. We find that the pellicular material gave about 50% higher peak capacity compared to the analogous porous material. A 60 cm column set packed with pellicular particles was made by connecting short columns in series; a peak capacity of about 460 was obtained in 4 h at room temperature. Increasing the column temperature to 70 degrees C reduced the analysis time to 2 h and further increased the peak capacity to more than 500. The number of peaks observed in the separation of bovine serum albumin tryptic peptides was greatly increased and the separation quality was significantly improved.     

Determination of flavanones in Citrus juices by means of one- and two-dimensional liquid chromatography

1-D and 2-D comprehensive (LC×LC) liquid chromatography methods have been developed and compared for the separation and quantification of flavanones in various Citrus juices. 1-D analyses were carried out on a superficially porous C18 column, whereas the 2-D LC approach was composed of a polyethylene glycol silica narrow-bore column packed with totally porous particles in the first dimension (D1) and a superficially porous C18 column in the second dimension (D2). Low-selectivity correlations were ensured by the complementary separation mechanisms offered by the D1 and D2 columns. Quantification was carried out both manually and by means of a software capable of detecting and quantifying each peak from the 2-D plot. Limit of detection (LOD) values as low as 0.023 μg/mL were obtained for hesperidin used as reference material for 1-D LC analyses, whereas values as high as 0.432 μg/mL were obtained by comprehensive LC. This discrepancy can be traced back to the minor sensitivity experienced in comprehensive LC due to both sample dilution in D1 and the high flow rates employed in D2. On the other hand, the separation capabilities of the LC×LC approach allowed to reduce the interferences coming from the matrix and to achieve the separation of some critical pairs, e.g. hesperidin/naringin difficult to accomplish in 1-D LC.     

Comprehensive two-dimensional liquid chromatography to quantify polyphenols in red wines

A comprehensive two-dimensional liquid chromatography method has been applied for the quantification of polyphenols in red wines and compared to the most commonly employed conventional LC approach. Such methodology comprised the use of a microbore conventional HPLC column packed with totally porous particles in the first dimension and a partially porous column of conventional diameter in the second dimension. Even though a good number of applications in comprehensive LC have been reported, quantification experiments have been rarely described. To this regard, the advantages of comprehensive LC together with the employment of dedicated software capable of detecting and quantifying each peak from the 2D plot, have been taken into account for quantifying the most representative polyphenols in three different commercial Sicilian red wine samples. The optimized method has been validated in terms of linearity, sensitivity, detection and quantification limits. LODs as low as 0.02 ppm were obtained using the one-dimensional HPLC-DAD method, whereas values lower than 0.10 ppm were obtained by comprehensive LC. However, comprehensive LC allowed the quantification of a higher number of compounds with RSD lower than 10% thanks to its improved resolving power. The separation capabilities of comprehensive LC allowed the analysis of complex natural samples without any pre-treatment to effectively reduce the interferences coming from the matrix.     

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.