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Comprehensive Two-Dimensional Liquid Chromatography - Practical Impacts of Theoretical Considerations

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Comprehensive Two-Dimensional Liquid Chromatography - Practical Impacts of Theoretical Considerations Cent. Eur. J. Chem. • 10(3) • 2012 • 844-875 DOI: 10.2478/s11532-012-0036-z Central European Journal of Chemistry Comprehensive Two-Dimensional Liquid Chromatography - practical impacts of theoretical considerations. A review. Review Article Pavel Jandera Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice 532 10, Czech Republic Received 24 October 2011; Accepted 31 January 2012 Abstract: A theory of comprehensive two-dimensional separations by liquid chromatographic techniques is overviewed. It includes heart-cutting and comprehensive two-dimensional separation modes, with attention to basic concepts of two-dimensional separations: resolution, peak capacity, efficiency, orthogonality and selectivity. Particular attention is paid to the effects of sample structure on the retention and advantages of a multi-dimensional HPLC for separation of complex samples according to structural correlations. Optimization of 2D separation systems, including correct selection of columns, flow-rate, fraction volumes and mobile phase, is discussed. Benefits of simultaneous programmed elution in both dimensions of LCxLC comprehensive separations are shown. Experimental setup, modulation of the fraction collection and transfer from the first to the second dimension, compatibility of mobile phases in comprehensive LCxLC, 2D asymmetry and shifts in retention under changing second-dimension elution conditions, are addressed. Illustrative practical examples of comprehensive LCxLC separations are shown. Keywords: Two-dimensional HPLC • Comprehensive LC • Orthogonality • Selectivity • Sample transfer modulation © Versita Sp. z o.o. 1. Introduction full peak capacity of two-dimensional separation systems offers theoretical possibilities for separations of samples Many fields of modern analytical chemistry, including containing as many as 100 000 analytes. Three- environmental analysis, food analysis, analysis of dimensional separation techniques that offer resolution biological material including biopolymers and low- on the scale of several hundred thousand of compounds molecular compounds, create an ever - increasing still remain a subject of a theoretical consideration for demand for methods enabling analysis of complex future development. This is due to the fact that the samples, especially at low concentration levels. necessary separation speed in the third dimension (3D) Suitability of a separation system for resolving, and the complexity of data handling present problems far identification, and determination of large numbers of above the possibilities of practical solutions among the compounds is characterized by peak capacity. Usually, separation techniques [1]. samples containing up to a few tens of compounds can Even though the main advantage of multi-dimensional be separated on a single column in a uni-dimensional techniques is a dramatic increase in the peak capacity HPLC system. with respect to one-dimensional separations, there Combinations of two or more different separation is another, equally important (even though often not systems can significantly improve the resolution of some fully appreciated), benefit of this technique: by careful compounds with respect to uni-dimensional systems and selection of the separation systems, group separation it can also increase considerably the number of analyzed of various samples, based on a molecular structure of compounds in a complex sample. Two-dimensional (2D) the group members, is usually much easier than the column separations of several hundred of compounds separation in uni-dimensional systems. In that way, have become quite common in the last decade, however, the various structurally related classes of compounds * E-mail: [email protected] 844 P. Jandera Comprehensive Two-Dimensional Liquid Chromatography - practical impacts of theoretical considerations. A review. can be distinguished and located in different areas in greater demand on the instrumentation and method the two-dimensional retention space. For example, development. “Pseudo-multi-dimensional” separations chromatography of macromolecules under critical can be achieved on a single column by using sequential conditions employs size-exclusion chromatography for applications of different selective mobile phases, or on a the separation, according to the molar mass distribution combination of two columns connected in a series using in the first dimension, and normal- or reversed-phase a single mobile phase. HPLC for the separation of species with different end- On-line real-time, two-dimensional separations groups in the second one [2]. employ two separation columns coupled via a modulator Two-dimensional LC separations can be carried interface, most often a switching valve. In the traditional out either in the space or in the time domain [1]. Planar “heart-cutting” setup, one or a few selected fractions are bed techniques such as thin-layer chromatography or collected from the first column effluent and directly re- two-dimensional gel electrophoresis have been used injected into the second dimension separation system for a long time in two-dimensional arrangements. Two- for analysis, while the remaining effluent is by-passed dimensional column separations operating in the time to the waste. The separation power of the off-line domain have been employed mainly in an off-line mode and heart-cutting approaches is usually not sufficient arrangement, in which the fractions eluted from the first for separation of samples containing more than 100 dimension are stored and usually pre-concentrated prior - 200 relevant compounds. To solve this problem, to subsequent introduction onto the second column. The comprehensive 2D chromatographic techniques were resulting data is usually presented as individual, one- introduced, which employ a combination of two different dimensional chromatograms of the second-dimension columns and different separation modes in the first and fractions. in the second dimension. This approach mirrors what is The first on-line, direct real-time coupling of two LC used in the heart-cutting techniques, however, all sample columns was reported more than 30 years ago [3] and compounds - and not only pre-selected fractions - are the development of necessary equipment has greatly subjected to the separation by two different separation advanced since then [4]. The on-line, two-dimensional methods on the two columns in the real analysis time HPLC found practical applications not earlier than [6]. in the last decade, much later than comprehensive In a comprehensive comprehensive LC (LCxLC), GC (GCxGC) [5]. the whole effluent (or its equal proportions) from the The separations in the individual dimensions of first-dimension column is collected in subsequent small- two-dimensional systems are controlled by the same volume fractions, which are then transferred on-line rules, similarly to those used in the separations in into the second-dimension column in multiple repeated uni-dimensional systems. The exception exists in the alternating cycles, before they eventually reach the selection of suitable “orthogonal” combinations of the detector connected at the outlet of the second-dimension first- and second-dimension systems and in the fraction column. The detector records a series of chromatograms transfer conditions between the two dimensions, in for individual fractions sampled from the first column order to preserve the separation and to achieve the best effluent. On one hand, the second-dimension separation separation of the highest number of sample compounds. system with a single-channel detector can be understood These are issues with strong practical implications, as a sample-selective multi-channel detector, much like however, theoretical considerations can establish a a photodiode array UV detector or a mass spectrometer rational basis for the selection of the best 2D separation connected to a single column producing a series of UV conditions. The main problems concerning the two- or mass spectra. On the other hand, a comprehensive dimensional peak capacity and orthogonality, especially 2D separation system, with a spectral multi-channel the advances in fast second-dimension separations, are detector, represents a three- dimensional “hyphenated” addressed in this review. analytical system. The two dimensions are connected on-line via an interface, usually a ten-port or a twelve port switching 2. Two-dimensional experimental valve, or via several six-port switching valves, which setup perform sampling and accumulation of small and narrow fractions from the first column effluent for fast re-injection The off-line techniques do not require any special onto the second column. The valve is equipped with equipment, however, they are labour- and time- two identical-volume sampling loops, one of which is consuming. On-line 2D techniques offer better collecting the effluent from the first dimension, while the possibilities for automation, however, they also create content of the second loop is being transferred onto the 845 846 P. Jandera Comprehensive Two-Dimensional Liquid Chromatography - practical impacts of theoretical considerations. A review. second column and it is subjected to separation in the the bandwidth of a first-dimension peak (Fig. 2A). time the first loop is being filled by a new effluent fraction The individual compounds in the consecutive [1,6]. In the alternating valve operating cycles, the two chromatograms must be identified and distinguished loops
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