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A New Dimension in Separation Science: Comprehensive Two-Dimensional Gas Chromatography

A New Dimension in Separation Science: Comprehensive Two-Dimensional Gas Chromatography
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  Gas Chromatography  DOI: 10.1002/anie.201200842 A New Dimension in Separation Science:Comprehensive Two-Dimensional Gas Chromatography Cornelia Meinert and Uwe J. Meierhenrich* Dedicated to Professor Volker Schurig analytical methods · complex samples ·gas chromatography · mass spectrometry 1. Introduction Gas chromatography (GC) is a technique that has beenused in separation science since 1951. [1] Several years later,the invention of capillary columns [2] (i.e., wall-coated open-tubular columns) allowed analytes to be highly resolved assharp and often base-line-separated signals. Owing to thispioneering development—a quantum leap in separationscience—approximately 400000 GC instruments are current-ly in operation worldwide. The second quantum leap inchromatographic sciences occurred very recently, which wasthe introduction of multidimensional gas chromatography. Bycoupling two stationary phases with different selectivity inseries, it becomes possible to resolve analytes that cannot beseparated by conventional one-dimensional (1D) GC. Multi-dimensional GC separations are classified either as heart-cutting two-dimensional (2D) GC or as comprehensive two-dimensional gas chromatography (GCGC). [3] Heart-cutting2D GC selectively transfers a subset of analytes froma primary column ( 1 D, first dimension) to a secondary column( 2 D, second dimension) using a valve or Deans switch device.An individual segment of the primary column effluentintroduced to the secondary column is referred to asa heart-cut. Heart-cutting 2D GC is best suited for isolatingand analyzing target compounds in complex mixtures and hascontributed significantly to enhancing resolution in GC. Incontrast, GCGC—the most recent and most powerful two-dimensional gas chromatographic technique—passes theentire sample through both stationary phases. The key toGCGC resides in the “modulation process”: the manner inwhich sequential segments of the first dimension are contin-ually transferred to the second dimension by a modulator.Today, comprehensive two-dimensional gas chromatog-raphy is beginning to be successfully applied in advancedlaboratories to detect and/or quantify trace-level constituentsand contaminants in various types of samples. However, thereal strength of GCGC is in separating complex samples.The most prominent applications include the analyses of crude oils in petrochemistry, [4] soil, water, and air samples inenvironmental chemistry, [5] nutrient samples in food chemis-try, [6] essential oils, [7] absolutes in the aroma and perfumeindustry, [8,9] and a wide variety of metabolites in biochem-istry. [10] Coupling a GCGC with a mass spectrometer orflame-ionization detector makes this analytical approachextremely powerful for the qualitative and quantitativedetermination of targeted and non-targeted substances.This Minireview is not focused on the most recentapplications of GCGC techniques but rather highlightsthe state-of-the-art in comprehensive two-dimensional gaschromatography. Instrumental designs developed to realizecontinuous multidimensional gas chromatography are re-viewed as well asrecent advances and limitations ofGCGC. T  he introduction and development of comprehensive two-dimen- sional gas chromatography offers greatly enhanced resolution andidentification of organic analytes in complex mixtures compared toany one-dimensional separation technique. Initially promoted by theneed to resolve highly complex petroleum samples, the techniquesenormous separation power and enhanced ability to gather informa-tion has rapidly attracted the attention of analysts from all scientific fields. In this Minireview, we highlight the fundamental theory, recent advances, and future trends in the instrumentation and application of comprehensive two-dimensional column separation. [*] Dr. C. Meinert, Prof. Dr. U. J. MeierhenrichInstitut de Chimie de Nice ICN, UMR CNRS 7272, Universit deNice-Sophia Antipolis, Facult des SciencesParc Valrose, 06108 Nice (France)E-mail: uwe.meierhenrich@unice.frHomepage:    AngewandteMinireviews  U. J. Meierhenrich and C. Meinert 2   2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  Angew. Chem. Int. Ed.  2012 ,  51 , 2–13         These are not the final page numbers!  2. The Fundamentals of Multidimensional GasChromatography The invention of comprehensive multidimensional sepa-ration techniques is a milestone development in chromatog-raphy history. Since the first proposal of this technique byGiddings [11] in 1984, great advances have been made both intheory and practice. The first comprehensive two-dimensionalgas chromatogram was recorded for an oil sample in 1991, [12] and immediately attracted attention. Complex real-life sam-ples, which often contain several thousand components andisomers spread over a wide concentration and volatility range,are a routine challenge. The complete resolution and iden-tification of each individual component has often been anunrealistic task, particularly using classical 1D GC. Therevolutionary aspect of the new GCGC methodology isthat the entire sample is resolved on two distinct capillarycolumns of complementary selectivity, which results inenhanced peak resolution. The increased resolution incombination with a third selective detection dimension (e.g.,mass spectrometry) currently makes the GCGC method-ology the most powerful analytical device for the analysis of volatile and semi-volatile organic compounds. Details regard-ing GCGC and the applicability of GCGC for variouscomplex matrices have been described in depth in articlesproviding a background to the fundamentals of this tech-nique. [13,14] In ideal two-dimensional gas chromatography, the peakcapacity,  n c , becomes the product of the peak capacity in eachdimension as shown in Equation (1). [15] n c,GC  GC  ¼ 1 n c2 n c  ð 1 Þ The  n c  is the maximum number of peaks a chromato-graphic system can resolve in an arbitrary time interval witha predetermined lowest acceptable resolution,  R S . An  R S  1.5is considered to be adequate for most applications, whichcorresponds to a standard deviation ( s  ) as a measure of peakwidth of 6 s  . 2.1. Comprehensive Separation The modulation process that connects two capillarycolumns containing stationary phases of different polarity isof crucial importance in GCGC. Although this modulationprocess can be achieved through a variety of differentapproaches, the underlying principle remains basically thesame: the modulator, placed between the two columns,ensures that (ideally) all of the effluent from the first columnis periodically trapped and re-injected as very sharp bandsonto the secondary column. Peaks eluting from the firstdimension column are sliced into several segments, each of which is eluted through the second dimension column. Insome systems, the second column is housed in a separate ovento allow more flexible and independent control of thetemperature. Finally, enhanced resolution is achieved becausecompounds undergo two independent separations, whichensures that peak overlap arising from equivalent elutiontimes on both columns is less likely.The time required for one modulation cycle is defined asthe modulation period ( P  M ) and is generally 2–8 s. Modu-lation has to be faster than the peak elution width at baselineon the  1 D column, so that multiple second dimension sub-peaks (slices) are obtained and the peak resolution in  1 D isnot degraded. Effective comprehensive GCGC perfor-mance is achieved with at least three or four modulationsper 1D peak. [13] Based on the modulation period, the time/response data stream is converted into a two-dimensionalretention plane (contour plot) spanned by the two retentiontime axes (1D time2D time). [12] The effect of this continu-ous process is shown in Figure 1b.Since GCGC relies upon fast analysis of accumulatedsub-peaks, it is usual to apply a short (1–2 m)  2 D column of narrow inner diameter (I.D.; normally 0.1 mm) with a rela-tively thin film thickness to achieve complete elution of pulsed sub-peaks in the second dimension, while the  1 Dcolumn is a conventional capillary column (length approx-imately 25 m) to give a normal GC elution. Ideally, analyteselute from the second column within the time frame of themodulation period, thereby preventing any potential overlapwith peaks from a subsequent modulation event, which isa phenomenon called wrap-around. Wrap-around signals areusually avoided; however, can be accepted if compoundsshowing wrap-around do not co-elute or interfere with solutesfrom the next modulation.As visualized in Figure 1, the effects from the modulatorcompress the analytes before they are released into thesecondary column, that is, they have a much smaller width atbase. The focusing effect combined with minimal band Uwe J. Meierhenrich studied chemistry atthe Philipps University of Marburg. After completing his Ph.D. at the University of  Bremen with Prof. Wolfram H.-P. Thie-mann, he identified amino acids in artificial comets at the Max Planck Institute for Solar System Research in Katlenburg-Lindauand at C.B.M. in Orlans in preparation for the Rosetta comet mission. In 2005, hebecame a full Professor at the University of  Nice-Sophia Antipolis. He was awarded theHorst Pracejus Prize from the GDCh in2011 for his work on chirality and enantio-selective chromatography.Cornelia Meinert studied chemistry at theUniversities of Rostock and Leipzig. Shereceived her Ph.D. on characterizing com-plex environmental mixtures using effect-directed analysis and preparative capillaryGC at the Helmholtz Centre for Environ-mental Research in Leipzig. In 2009, shebecame a postdoctoral research fellow inthe Meierhenrich group at the University of  Nice. Her current research focuses on thesrcin of biomolecular asymmetry, especiallyenantiomer separation using GCxGC techniques. Gas Chromatography  Angewandte Chemie 3  Angew. Chem. Int. Ed.  2012 ,  51 , 2–13  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers!    broadening on the short secondary column results in narrowpeaks with enhanced peak height and thus in improveddetection limits. [16,17] Typical enhancements of the signal-to-noise ratio are on the order of 5–10-times depending on thedetector acquisition rate, modulator system, secondary col-umn characteristics (length, internal diameter, and stationaryphase), gas velocity, and temperature program. Other majorbenefits in trace analysis of 2D GC over 1D GC are struc-tured chromatograms, which reduce the possibility of inter-ference through peak overlap and/or co-extracted matriximpurities. Additionally, the noise is minimal so a true surfacearea for peak integration is available. 2.2. Structured Separation Space in GCGC Chromatograms Apart from superior resolution of an analyte fromchromatographic noise and co-eluting compounds, a compre-hensive GCGC analysis reveals the structural properties of analytes through the clustering of structurally related homo-logues, congeners, and isomers in the 2D plot. [18,19] Figure 2highlights the important features of the 2D chromatogramand the following chemical trends can be determined toa further extent: well-resolved regions of saturated, cyclic-saturated, olefinic, heteroatomic, and aromatic compounds.Straight-chain and branched hydrocarbons are separated byretention time along the lower part of the second dimension,whereas the two- and three-ring aromatics (e.g., phenan-threne, biphenyl, dibenzothiophene, dibenzofuran, and thealkylated isomeric forms of each) can be observed at the topof the chromatogram as a result of their stronger interactionand higher relative retention on the secondary polar column.Within the group of geometrical isomers, patterns of elutionof the different alkylated species can be determined. Gen-erally, group-type separation can be helpful in the identifica-tion of unknowns and, in some cases, preferred over real 2Dseparation, in which the overall separation of adjacent peaksis the major goal.In GCGC, several parameters have a profound influ-ence on the overall separation efficiency and have to beoptimized before analytical performance. Dallge et al. andOng et al. [20] published optimization procedures and Beenset al. [21,22] developed programs that can predict the outcomeof GCGC separation based on the thermodynamics of theseparation. Further studies have used computer modeling topredict and optimize the separation space in GCGC, basedon the enthalpy ( D H  i ) and entropy ( D S i ) from experimentallydetermined retention times of target compounds. [23] However,the combined increase in experimental parameters—therelationship and interplay—causes the determination of optimal analytical settings for GCGC to be difficult. [24] Inaddition to optimal modulation settings, the primary opera-tional parameters to be considered are the chemistry of thestationary phases (type, film thickness), column dimensions(length, diameter), gas flow rate, outlet pressure conditions,temperature regime for both columns, and the detectorsettings. For example, suboptimal column selection can resultin a loss of selectivity and overall efficiency of the separationprocess. Classically, the first dimension column has a non-polar stationary phase to separate a wide range of compoundsin various matrices based on the partition coefficientsbetween the mobile and stationary phases of analytes. Thesecond column is usually polar and short to give fastseparation in the second dimension based on differentpolarity interactions (i.e., dipole–dipole, hydrogen-bonding,and polarizability effects).A separation is considered truly orthogonal if crossinformation (or synentropy) across both dimensions is zero [25] (i.e., the retention of molecules on each column must beindependent). Minimizing synentropy is important in multi-dimensional separations because increased synentropy leadsto an increased part of the separation space that is inacces-sible. Sample constituents tend to cluster along a diagonal inthe 2D retention time plane. By minimizing any correlationbetween the selectivities of the two stationary phases, theefficiency of GCGC separations can be enhanced. There-fore, an increased percentage of separation-space usage isgained. The maximization of orthogonality of different sta-tionary-phase combinations has been described using con-stants of a solvation parameter model, such as the Abrahammodel. [26] A separation-space evaluation method based onDenaulays triangulation algorithms was proposed by Semardet al., [27] and then further used to optimize the selection of column sets, geometric parameters of the secondary column, Figure 1.  Illustration of a GCGC chromatogram. a) Chromatographicpeaks ( a ,  b , and  g ) eluted from a typical apolar  1 D column sequentiallysliced into distinct fractions during a defined modulation period ( P  M ).Non-resolved analytes are often better resolved on a (generally polar)short micro-bore  2 D column. b) The data stream from the detector isthen plotted based on the modulation in a 2D contour color plotformat or c) directly showing signal intensities in 3D presentation asconical peaks.    Angewandte Minireviews  U. J. Meierhenrich and C. Meinert 4   2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  Angew. Chem. Int. Ed.  2012 ,  51 , 2–13         These are not the final page numbers!  and other experimental conditions (e.g., gas flow and temper-ature).Even though non-polarpolar column combinations areoften stated as the preferred approach for GCGC, non-classical conditions, that is, polarmoderately non-polarcolumn combinations [28] as well as liquid crystalline, [29] ionicliquid, [30] and enantioselective stationary phases, [31–34] aregaining attention and can lead to high-resolution and group-type separations. Moreover, Omais and co-workers [35] recent-ly suggested that the non-polarpolar column combination isnot a necessary condition to achieve large peak distribution inthe 2D plane, and non-classical conditions can occasionallyprovide a large occupation of 2D space. Thus, there are nofixed rules for the combination of column-phase types. Thefinal decision regarding column selection will depend on thesamples composition and be based on fundamental consid-erations, such as “has an acceptable separation been ach-ieved?”  3. Milestones in the GCGC Techniques  3.1. Cutting the First Dimension Chromatogram into Fine Slices:Pneumatic versus Thermal Modulation Various types of modulators have been designed and havedemonstrated their suitability for GCGC measurements. Inprinciple, there are two approaches to achieve modulation:pneumatic [36] and thermal. [12,37–40] Generally thermal modu-lation provides a greater degree of sensitivity enhancement.Mondello et al. [41] recently listed all the GCGC modulatorsdeveloped and included the main characteristics, so ourMinireview focuses only on the most significant devices anddevelopments.Pioneering work by Liu and Phillips [12] used an on-columntwo-stage thermal-desorption modulator, which was heatedby a resistive film painted onto the capillary surface andcooled by ambient air. However, this modulator was difficultto operate and only had a short lifetime. The first reliableheated modulator, which was also the first to be commercial-ized, was developed in 1999 by Phillips et al. [37] The rotating Figure 2.  Section of a GCGC-ToF-MS chromatogram of a diesel fuel using a non-polarpolar column set. Isomers line up as bands in the 2Dchromatogram and congener groups or homologues appear as separate bands. Analytes at the lower separation space of the GCGCchromatogram express less-polar interactions with the  2 D column. Image used with permission from LECO Corporation. Similar chromatogramscan be obtained by the use of chromatographic GCGC systems supplied by Agilent, Shimadzu or Thermo Scientific. Gas Chromatography  Angewandte Chemie 5  Angew. Chem. Int. Ed.  2012 ,  51 , 2–13  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers!    thermal modulator (thermal sweeper), shown in Figure 3a,was able to trap and re-inject a sample using a slotted heaterconnected perpendicular to a rotating shaft that periodicallypassed along a segment of a thick-film chromatographiccolumn, which acted as a trap. The major drawback of thethermal sweeper is that the operationtemperature ofthe ovenmust be approximately 100   C lower than the maximumallowed temperature of the stationary phase in the modu-lation capillary. Today, the thermal sweeper and relatedmodulation techniques are no longer used.More efficient modulators using cryogenically cooledliquids, such as carbon dioxide, nitrogen, or air wereintroduced in the late 1990s. The first cryogenic modulatorwas described by Kinghorn and Marriott. [38] The longitudi-nally modulated cryogenic system (LMCS) uses expandingliquid carbon dioxide to cryogenically trap and focus theanalytes in the first centimeters of the second column(Figure 3b). Re-injection is achieved by moving the modu-lator longitudinally away and heating the trapped fraction bymeans of the ambient oven air. Several types of contemporarycryogenic jet-based modulators [39] (Figure 3c) rely on carbondioxide or liquid nitrogen for cooling conventional bench-scale GCGC systems without any moving parts.The principle parameter of the second class of modulators(valve-based modulators) relies on flow switching. A segmentof the migrating peak is sampled, and upon switching thevalve, pushed with high gas flow into  2 D in a differential flowarrangement. Valve-based modulators are very simple indesign and were first introduced by Synovec and co-workersin 1998. [36a] However, early valve-based modulators send onlya part of the effluent from the first column to the secondcolumn through. Therefore, use of the valve-based modula-tion technique was limited to relatively concentrated samples.The latest developments in valve/flow modulators, such asdifferential-flow modulation using a microfluidic Deansswitch, [42] valve-switching modulation, [43] pulsed-flow modu-lation, [44] and capillary-flow technology, have allowed valve/flow modulators to become competitive with thermal mod-ulators. [45] Thermal modulation mostly provides better reso-lution and is less restricted in column/flow combinations;however, a major drawback is the availability and relativelylarge consumption of liquid nitrogen or carbon dioxide. Noveldifferential flow modulation techniques are effective over theentire volatility range without requiring adjustments andcryogens. In addition, they allow portable and on-line processinstrumentation to be possible.  3.2. Data Handling by Adequate Software In the early history of GCGC, advanced data processingmethods were absent. The first automated data-handling-plus-interpretation results of one 2D-chromatogram requiredapproximately 7 h. [46] The huge amount of informationgenerated in comprehensive GCGC applications causesthe corresponding difficulties in data-handling to becomeimmediately evident. The development of software algo-rithms that integrate all 2D peaks and subsequently identifyand summarize the peak areas of each compound aftermodulation have contributed greatly to the increased use andavailability of GCGC systems. In a recent Review, Zenget al. [47] detailed how state-of-the-art chemometric techniquesaid analysts in data interpretation and analysis of GCGC Figure 3.  Illustration of the operational principles of various types of modulators. a) The thermal sweeper system (column A), placed be-tween the first and second dimension, accumulates solutes from the 1 D until the slotted heater (H), rotated by the modulator (M), focusesand finally expels the solute onto the secondary column. b) Longitudi-nally modulated cryogenic system (LMCS). At T, LMCS is in thetrapping position, and R is the release position when the LMCS ismoved toward the injection direction to expose the trapped soluteband to oven temperature. c) Cryogenic jet-based modulator. A cold-jet(depicted on the right-hand side) traps analytes eluting from  1 D.Subsequently a hot-jet switches on, the trapped solute band heats uprapidly and the analytes are released into  2 D while the left-hand sidecold-jet is switched on to prevent sample breakthrough of   1 D; after thetrap-release cycle the next modulation is started.    Angewandte Minireviews  U. J. Meierhenrich and C. Meinert 6   2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  Angew. Chem. Int. Ed.  2012 ,  51 , 2–13         These are not the final page numbers!
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