- Email: [email protected]
Comprehensive two-dimensional liquid chromatography
CHAPTER
Comprehensive two-dimensional liquid chromatography
16
Francesco Cacciola*, Marina Russo†, Luigi Mondello*,†,‡, Paola Dugo*,†,‡ University of Messina, Messina, Italy* University Campus Bio-Medico of Rome, Rome, Italy† Chromaleont s.r.l., c/o University of Messina, Messina, Italy‡
C HAPTER OUTLINE 16.1 Introduction..................................................................................................... 403 16.2 Fundamentals.................................................................................................. 404 16.3 Instrumental Set-Up and Data Analysis.............................................................. 405 16.4 Novel Stationary Phases................................................................................... 408 16.5 Conclusions and Future Perspectives................................................................ 409 References............................................................................................................... 409
16.1 INTRODUCTION One-dimensional liquid chromatography (1D-LC) is widely applied to the analysis of real-world samples in several fields. However, for the analysis of many real-world samples, for example, biological, food, and environmental, “conventional” 1D-LC methods do not provide rewarding results for enabling compound identification and quantification. Comprehensive two-dimensional high performance liquid chromatography (LC × LC) experienced mostly in the last two decades could be a valuable option. The most general set-up of an LC × LC system consists of two pumps, two columns, injector, interface, and detector. The interface is, in general, a high-pressure two-position switching valve and this device is often referred to as modulator or sampling device. LC × LC methods improve the likelihood for separation of a higher number of sample components since the peak capacity of an LC × LC separation is multiplicative of the product of the peak capacity values of the two single dimensions (n2D = nD1 × nD2) [1–3]. The peak capacities of the two dimensions are shown as the number of adjacent Gaussian profiles that can be accommodated into the space along with the respective separation coordinates. The resolution is represented by rectangular boxes, which divide the separation plane. The total peak capacity, n2D, is therefore approximately equal to the number of such boxes [3]. Liquid Chromatography. http://dx.doi.org/10.1016/B978-0-12-805393-5.00016-6 © 2017 Elsevier Inc. All rights reserved.
403
404
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
In this regard, by careful selection of mobile and stationary phases in the two dimensions to tune orthogonality, the peak capacity of the overall systems can dramatically increase, making group separations of various samples often possible, especially for the interpretation of the LC × LC data. Due to the tremendously higher separation power of LC × LC when compared to “conventional” 1D-LC, the technique has been employed in several fields, for example, polymers, pharmaceuticals and biological mixtures, natural products, environmental and petrochemical samples, and has been the subject of various reviews [4–27].
16.2 FUNDAMENTALS In the last two decades, many works have dealt with all key factors affecting resolution and performance in LC × LC methods. An important role on the dependence of the two dimensions on each other is related to the analysis speed in the second dimension (2D) affecting the first dimension (1D) sampling rate on the peak capacity of the overall LC × LC separations. In 1998, Murphy et al. [28] investigated the effect of the sampling rate on the effective 1D peak width and modeled a Gaussian peak as a histogram profile of the average concentration within every sampling period. The outcome of the research was that approximately three to four modulations per 1D peak (8σ) are needed to avoid serious loss of performance of the LC × LC system as a result of “undersampling” of 1D peaks. Some years later Seeley et al. [29] published a similar work on this topic investigating the sampling frequency for modulators with various duty cycles (2002) introducing the (average) peak broadening factor, σ*, related to selected parameters such as sampling period, τZ, and sampling phase, Φ. τZ is determined by the modulation period or sampling time ts and 1σ of the 1D peak, while Φ represents the way in which the primary peak is cut into aliquots. The sampling phase Φ is the time difference between the center of the sampling cycle nearest to the peak maximum and the peak maximum itself, divided by the sampling period. Peak broadening was less significant when the peak maximum was centered in one of the sampling periods and for low duty cycles. For systems with a duty cycle of 1, as is the case for most LC × LC separations, the peak broadening was practically independent of Φ. Basically, Seeley drew the same conclusions as Murphy, extending his investigations to duty cycles less than 1. Afterwards, Horie et al. [30] demonstrated that modulation periods equal to 2.2–4 are sufficient to minimize “peak broadening”. In addition, they introduced a new parameter, eM (Nobuo factor), which is defined as the ratio of the apparent peak capacity of the first column derived from σ* to its actual peak capacity in the 1D. In 2006 Schoenmakers and co-workers [31] introduced a useful “protocol” for optimizing LC × LC separations based on proper sampling, taking into account suitable chromatographic parameters, such as column dimensions, injection volumes, and flow rates. Carr and co-workers [32–36] poured a great effort towards understanding in LC × LC extending their investigations to the
16.3 Instrumental set-up and data analysis
overall chromatogram. For the determination of the average 1D broadening factor, β, as a function of the sampling time (ts) and the standard deviation of the 1D peaks prior 1σ, 2D statistical overlap theory along with the number of observed peaks in simulated LC × LC separations was used [37]. The effects of the 1D peak capacity and gradient time, and the 2D cycle times on the overall peak capacity of the 2D-LC system was lately reported in 2010 by Potts et al. [35]. It was demonstrated that in fast LC × LC separations (<1 h), the 2D was more important than the 1D in determining overall peak capacity. The benefits on the sampling rate of the 1D effluent attained by the use of multiple 2 D columns have been shown by Fairchild et al. [38] highlighting that peak capacity of the LC × LC system increases, compared to an LC × LC system employing only a single 2D column, while reducing the analysis time.
16.3 INSTRUMENTAL SET-UP AND DATA ANALYSIS In the most common set-up for LC × LC systems, small volume fractions of the effluent from the 1D are transferred via an interface into the 2D. The interface techniques of LC × LC include different types of interface namely dual loop, stop-flow, and vacuum evaporation. Due to its simple structure the dual loop interface is mostly used in LC × LC separations. Last implementations have been recently carried out in Schoenmakers and Stoll's research groups. Schoenmakers and co-workers investigated an actively modulated LC × LC (LC/a × m/LC) aiming to overcome one of the limitations of contemporary LC × LC arising from the combination of diverse 1D and 2D column diameters: the capability of such an approach was evaluated for both SCX × RP and HILIC × RP-LC separations [39–41]. A schematic overview of the modulated LC × LC setup used for yeast analysis is shown in Fig. 16.1. Stoll and coworkers developed a “selective” LC × LC system (sLC × LC) with the aim to break the long-standing link between the timescales of the 1D and 2D separations through novel implementation of existing valve technology [42–44]. A schematic diagram of the instrument configuration for sLC × LC, which allows advantages similar to those derived from off-line LC × LC approach but without most of the major drawbacks of off-line work, is illustrated in Fig. 16.2. Stop-flow mode is applied generally when the analysis speed of the 2D cannot keep up with the sampling frequency of the 1D. A longer 2D column with respect to the commonly employed ones is usually employed to improve resolution as well as peak capacity [45]. Two main disadvantages of such an approach consists of a longer analysis time with respect to continuous LC × LC and potential band broadening phenomena, which may arise for both long parking periods and nonadequate peak focusing on the top of the 2D column [46,47]. Vacuum evaporation was used by Guan and co-workers for alleviating the incompatibility of mobile phases used in NP-LC × RP-LC separations [48]: it allowed to condense the 1D eluents and the 2D solvent redissolved the residents at the inside wall of a loop for further separation
405
406
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
2D
Waste
RP column Wash solvent
FTICR-MS
Waste
1D
2D
SCX column
nano flow in
Make-up flow
FIG. 16.1 Schematic overview of the modulated LC × LC setup used for yeast analysis. Reproduced with permission from Vonk RJ, Gargano AFG, Davydova E, Dekker HL, Eeltink S, de Koning LJ, et al. Comprehensive two-dimensional liquid chromatography with stationary-phase-assisted modulation coupled to high-resolution mass spectrometry applied to proteome analysis of saccharomyces cerevisiae. Anal Chem 2015;87:5387–94 [American Chemical Society].
in the 2D. The main pitfall is the potential sample loss risk for volatile components due to evaporation in the interface. More recently, a newly developed vacuum evaporation assisted adsorption interface that allowed fast removal of NP-LC solvent in the vacuum condition and successful solving of the solvent incompatibility problem between NP-LC and RP-LC was constructed for preparative purposes [49]. A proof-of-principle experiment with a novel thermal modulation device with potential use in LC × LC systems has been recently reported by Verstraeten et al. [50]. Based on the thermal desorption concept used in comprehensive two-dimensional gas chromatography (GC × GC) systems, preconcentration of neutral analytes eluting from the 1D was performed in a capillary “trap” column packed with highly retentive porous graphitic carbon particles, placed in an aluminum low-thermal-mass LC heating sleeve. Remobilization of the trapped analytes was achieved by rapidly heating the trap column, by applying temperature ramps up to +1200°C/min. The thermal modulation device is schematically represented in Fig. 16.3. As far as detection is concerned all conventional LC detectors, such as photodiode array, mass spectrometry (MS), and evaporative light scattering detectors, are commonly used in LC × LC; usually, a single detector is installed after the 2 D column, although an additional detector can be used to collect the 1D data, with the 1D separation monitored only during the optimization step. The high speed of the 2D analysis requires a very fast detector acquisition rate to ensure the adequate sampling, which is critical for quantification purposes, otherwise
16.3 Instrumental set-up and data analysis
Load Loop 1 Column I Detector I (Opt) Dilution
Autoinjector
Load Loop 2 Pump I
Column I
Pump II
Detector I (Opt)
A
Dilution
L1
(A)
Pump II A
Dilution
C
L3
Pump II A
C
L3
L2
L6 L5
C
L4
D Waste
Column II Detector II
(B)
Pump I
L1 L2
L5
Autoinjector
B
L6
L4
Detector II
Column I Detector I (Opt)
L1 L2
L6
Column II
Pump I
B
B
L5
Autoinjector
Inject Loop 1
D
L4
Column II Waste
L3
Detector II
D Waste
(C)
FIG. 16.2 Schematic representation of instrument configuration for sLC × LC. Pump I delivers eluent to Column I while Pump II pushes captured fractions of 1D effluent out of sample loops L1 through L6 and delivers eluent to the 2D column. Detector I is optional in the sense that it can be removed to reduce extra-column broadening after 1D elution times of target compounds have been determined. Dilution of 1D effluent was achieved by an external, low-pressure pump T-ed into the 1D flow path to achieve effective on-column focusing in the second dimension. A series of four valves labeled A–D were used to capture and store six fractions of 1D effluent. Example valve configurations required for fraction capture are shown in panels (A and B). Loop L1 is loaded by the 1D flow marked in red (dark grey in print) in panel (A), then valves (B and C) rotate to load L2 shown in panel (B) while the flow from Pump II shown in blue (light grey in print) remains unchanged. Panel (C) shows an example of a flow path required to re-inject captured fractions into the 2D column. Subsequent injection of all six fractions is achieved by simultaneous rotation of valves B and C. Reproduced with permission from Groskreutz SR, Swenson MM, Secor LB, Stoll DR. Selective comprehensive multi-dimensional separation for resolution enhancement in high performance liquid chromatography. Part I: principles and instrumentation. J Chromatogr A 2012;1228:31–40 [Elsevier].
loss in resolution, due to a low number of data points, may occur. Usually, data are visualized in 2D plots or contour plots, where the retention times in the 1D and the 2D dimensions are plotted along the x- and y-axes, respectively, while the color of the spots is a measure for intensity. In LC × LC, quantification can be performed by combining the contribution of each individual 2D “slice” of a 1D
407
408
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
From injector/pump
SiO2
PGC
SiO2
To detector, fraction collector or 2nd column
Low thermal mass heating sleeve
(A) 1st dim. column
Compressed air
SiO2
PGC
SiO2
To detector, fraction collector or 2nd column
Low thermal mass heating sleeve
(B)
Compressed air
FIG. 16.3 Schematic representation of the thermal modulation system consisting of a three-section packed capillary column (silica-PGC-silica) and a low-thermal-mass heating sleeve connected to (A) the injector or pump and (B) the first dimension column. Reproduced with permission from Verstraeten M, Pursch M, Eckerle P, Luong J, Desmet G. Thermal modulation for multidimensional liquid chromatography separations using low-thermal-mass liquid chromatography (LC). Anal Chem 2011;83:7053–60 [American Chemical Society].
chromatographic peak, or by using more advanced algorithms [51–58]. Advanced data analysis techniques, for example, multivariate data analysis, can also be applied to LC × LC data, despite it being more complicated with respect to 1D-LC separations [22–59]. So far, two commercially available software packages, namely Chromsquare and GC Image LC × LC Edition Software specially designed for both visualization and quantification of 2D data, are available on the market.
16.4 NOVEL STATIONARY PHASES The most significant development in the field of novel stationary phases LC × LC has been the introduction of 2D small particles stationary phases with the aim to replace the monolithic types that have been earlier used for the same goal [52,60–64]. The use of monoliths was very advantageous since these columns could be operated at high flow rates without loss of resolution with very brief re-equilibration times and significantly lower backpressures [65]. In 2007, a new particle technology (Fused-Core) was developed to deliver hyperfast chromatographic separations, while avoiding the reliability issues often associated with fast HPLC [66,67]. These stationary phases consisted of a 1.7 μm solid
References
core with a 0.5 μm porous silica shell surrounding it (d.p. = 2.7 μm). The major benefit of these particles was therefore the smaller diffusion path, which reduced axial dispersion of solutes minimizing peak broadening. The higher efficiencies of such stationary phases, capable of high pressure operation, were chosen as 2D of LC × LC systems and several research groups (Mondello, Dugo, Jandera, and Cifuentes) used them in natural products applications [54–56,62,68–81]. Another remarkable development was the introduction of sub-2-μm technology allowing ultrafast analysis and/or high-resolution separations [52,64,74,76,79,82–92]. Besides the selection of stationary phases, mobile phases play a pivotal role in the development of LC × LC methods. In particular, the use of gradient elution conditions in both dimensions was commonly employed in several research works. “Full-in-fraction” gradients, involving an equally generic and steep mobile phase range in each repeated 2D run, is the approach most commonly adopted for LC × LC separations. To enhance the orthogonality degree, three different gradient elution strategies were afterwards investigated [93]. The first one, “segmented in fraction”, used different gradient ranges in different segments of the 2D separation; the second strategy, known as “parallel gradient” implied only a single gradient spanning during the whole 2D separation time adopted simultaneously to the 1D gradient; in the third one “continuously shifting” the initial and final gradient in the 2D is continuously changed over the whole LC × LC analysis. Such optimization approaches applied so far to polyphenol analysis will likely be very fruitful areas of research and will be extended to the analysis of more sample types.
16.5 CONCLUSIONS AND FUTURE PERSPECTIVES Comprehensive liquid chromatography has been extensively exploited in the past two decades with remarkable updates in the last few years with reference to instrumental set-up and availability of novel stationary phases. In particular, the introduction into the market of partially porous particles and sub-2-μm stationary phases, capable of high-pressure and high-temperature fast second-dimension operation, turned out to be a valuable tool for more performant LC × LC separations. Also, the design of different gradients approaches for LC × LC analyses investigated so far was an important implementation and to this regard the use of more robust and flexible software for both qualitative and quantitative purposes is expected to grow in the near future. Recent efforts in the development of transfer devices as well as availability of commercial instrument will expand the use of the technique introduce “routine” use of LC × LC.
REFERENCES [1] Karger BL, Snyder LR, Horvath C. An introduction to separation science. New York, NY: Wiley & Sons; 1973. [2] Giddings JC. Two-dimensional separations: concept and promise. Anal Chem 1984;37:1258A–64A.
409
410
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
[3] Giddings JC. Concepts and comparisons in multidimensional separation. J High Resolut Chrom 1987;10:319–23. [4] Liu Z, Lee ML. Comprehensive two-dimensional separations using microcolumns. J Microcolumn Sep 2000;12:241–54. [5] Evans CR, Jorgenson JW. Multidimensional LC-LC and LC-CE for high-resolution separations of biological molecules. Anal Bioanal Chem 2004;378:1952–61. [6] Guttman A, Varoglu M, Khandurina J. Multidimensional separations in the pharmaceutical arena. Drug Discov Today 2004;9:136–44. [7] Stroink T, Ortiz MC, Bult A, Lingeman H, de Jong GJ, Underberg WJM. On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins. J Chromatogr B 2005;817:49–66. [8] Dixon SP, Pitfield ID, Perrett D. Comprehensive multi-dimensional liquid chromatographic separation in biomedical and pharmaceutical analysis: a review. Biomed Chromatogr 2006;20:508–29. [9] Jandera P. Column selectivity for two-dimensional liquid chromatography. J Sep Sci 2006;29:1763–83. [10] Shalliker RA, Gray MJ. Concepts and practice of multidimensional high-performance liquid chromatography. Adv Chromatogr 2006;44:177–236. [11] Shellie RA, Haddad PR. Comprehensive two-dimensional liquid chromatography. Anal Bioanal Chem 2006;386:405–15. [12] Stoll DR, Li X, Wang X, Carr PW, Porter SEG, Rutan S. Fast comprehensive twodimensional liquid chromatography. J Chromatogr A 2007;1168:3–43. [13] Dugo P, Cacciola F, Kumm T, Dugo G, Mondello L. Comprehensive multidimensional liquid chromatography: theory and applications. J Chromatogr A 2008;1184:353–68. [14] Dugo P, Kumm T, Cacciola F, Dugo G, Mondello L. Multidimensional liquid chromatographic separations applied to the analysis of food samples. J Liq Chromatogr Relat Technol 2008;31:1758–807. [15] Guiochon G, Marchetti N, Mriziq K, Shalliker RA. Implementations of two- dimensional liquid chromatography. J Chromatogr A 2008;1189:109–68. [16] Pol J, Hyotylainen T. Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry. Anal Bioanal Chem 2008;391:21–31. [17] François I, Sandra K, Sandra P. Comprehensive liquid chromatography: fundamental aspects and practical considerations-a review. Anal Chim Acta 2009;641:14–31. [18] Herrero M, Ibanez E, Cifuentes A, Bernal J. Multidimensional chromatography in food analysis. J Chromatogr A 2009;1216:7110–29. [19] Sandra K, Moshir M, D'hondt F, Tuytten R, Verleysen K, Kas K, et al. Highly efficient peptide separations in proteomics. Part 2: bi- and multidimensional liquid-based separation techniques. J Chromatogr B 2009;877:1019–39. [20] Berek D. Two-dimensional liquid chromatography of synthetic polymers. Anal Bioanal Chem 2010;396:421–41. [21] Pierce KM, Kehimkar B, Marney LC, Hoggard JC, Synovec RE. Review of chemometric analysis techniques for comprehensive two dimensional separations data. J Chromatogr A 2012;1255:3–11. [22] Pierce KM, Mohler RE. A review of chemometrics applied to comprehensive twodimensional separations from 2008-2010. Sep Purif Rev 2012;41:143–68. [23] Jandera P. Comprehensive Two-Dimensional Liquid Chromatography-Practical impacts of theoretical considerations. A review. Cent Eur J Chem 2012;10:844–75.
References
[24] Donato P, Cacciola F, Tranchida PQ, Dugo P, Mondello L. Mass spectrometry detection in comprehensive liquid chromatography: basic concepts, instrumental aspects, applications and trends. Mass Spectrom Rev 2012;1:523–59. [25] Bedani F, Kok W, Janssen H-G. A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography × liquid chromatography. J Chromatogr A 2006;1133:126–34. [26] Shi X, Wang S, Yang Q, Lu X, Xu G. Comprehensive two-dimensional chromatography for analyzing complex samples: recent new advances. Anal Methods 2014;6:7112–23. [27] Sarrut M, Crétier G, Heinisch S. Theoretical and practical interest in UHPLC technology for 2D-LC. TrAC-Trend Anal Chem 2014;63:104–12. [28] Murphy RE, Schure MR, Foley JP. Effect of sampling rate on resolution in comprehensive two-dimensional liquid chromatography. Anal Chem 1998;70:1585–94. [29] Seeley JV. Theoretical study of incomplete sampling of the first dimension in comprehensive two-dimensional chromatography. J Chromatogr A 2002;962:21–7. [30] Horie K, Kimura H, Ikegami T, Iwatsuka A, Saad N, Fiehn O, et al. Calculating optimal modulation periods to maximize the peak capacity in two dimensional HPLC. Anal Chem 2007;79:3764–70. [31] Schoenmakers PJ, Vivo-Truyols G, Decrop WMC. A protocol for designing comprehensive two-dimensional liquid chromatography separation systems. J Chromatogr A 2006;1120:282–90. [32] Davis JM, Stoll DR, Carr PW. Effect of first-dimension undersampling on effective peak capacity in comprehensive two-dimensional separations. Anal Chem 2008;80:461–73. [33] Davis JM, Stoll DR, Carr PW. Dependence of effective peak capacity in comprehensive two-dimensional separations on the distribution of peak capacity between the two dimensions. Anal Chem 2008;80:8122–34. [34] Li X, Stoll DR, Carr PW. Equation for peak capacity estimation in two-dimensional liquid chromatography. Anal Chem 2009;81:845–50. [35] Potts LW, Stoll DW, Li X, Carr PW. The impact of sampling time on peak capacity and analysis speed in on-line comprehensive two-dimensional liquid chromatography. J Chromatogr A 2010;1217:5700–9. [36] Gu H, Huang Y, Carr PW. Peak capacity optimization in comprehensive two dimensional liquid chromatography: a practical approach. J Chromatogr A 2011;1218:64–73. [37] Davis JM. Statistical-overlap theory for elliptical zones of high aspect ratio in comprehensive two-dimensional separations. J Sep Sci 2005;28:347–59. [38] Fairchild JN, Horvath K, Guiochon G. Theoretical advantages and drawbacks of on-line, multidimensional liquid chromatography using multiple columns operated in parallel. J Chromatogr A 2009;1216:6210–7. [39] Vonk RJ, Wouters S, Barcaru A, Vivó-Truyols G, Eeltink S, de Koning LJ, et al. Postpolymerization photografting on methacrylate-based monoliths for separation of intact proteins and protein digests with comprehensive two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry. Anal Bioanal Chem 2015;407:3817–29. [40] Vonk RJ, Gargano AFG, Davydova E, Dekker HL, Eeltink S, de Koning LJ, et al. Comprehensive two-dimensional liquid chromatography with stationary-phase-assisted modulation coupled to high-resolution mass spectrometry applied to proteome analysis of saccharomyces cerevisiae. Anal Chem 2015;87:5387–94.
411
412
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
[41] Gargano AFG, Duffin M, Navarro P, Schoenmakers PJ. Reducing dilution and analysis time in online comprehensive two-dimensional liquid chromatography by active modulation. Anal Chem 2016;88:1785–93. [42] Groskreutz SR, Swenson MM, Secor LB, Stoll DR. Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part I: principles and instrumentation. J Chromatogr A 2012;1228:31–40. [43] Davis JM, Stoll DR. Likelihood of total resolution in liquid chromatography: evaluation of one-dimensional, comprehensive two-dimensional, and selective comprehensive twodimensional liquid chromatography. J Chromatogr A 2014;1360:128–42. [44] Larson ED, Groskreutz SR, Harmes DC, Gibbs-Hall IC, Trudo SP, Allen RC, et al. Development of selective comprehensive two-dimensional liquid chromatography with parallel first-dimension sampling and second-dimension separation-application to the quantitative analysis of furanocoumarins in apiaceous vegetables. Anal Bioanal Chem 2013;405:4639–53. [45] Blahovà E, Jandera P, Cacciola F, Mondello L. Two‐dimensional and serial column reversed‐phase separation of phenolic antioxidants on octadecyl‐, polyethyleneglycol‐, and pentafluorophenylpropyl‐silica columns. J Sep Sci 2006;29:555–66. [46] Bedani F, Schoenmakers PJ, Janssen H-G. Theories to support method development in comprehensive two-dimensional liquid chromatography—a review. J Sep Sci 2012;35:1697–711. [47] Sommella E, Cacciola F, Donato P, Campiglia P, Dugo P, Mondello L. Development of an online capillary comprehensive 2D-LC system for the analysis of proteome samples. J Sep Sci 2012;35:530–3. [48] Tian H, Xu J, Guan Y. Comprehensive two-dimensional liquid chromatography (NPLC × RPLC) with vacuum-evaporation interface. J Sep Sci 2008;31:1677–85. [49] Li J-F, Fang H, Yan X, Chang F-R, Wu Z, Wu Y-L, et al. On-line comprehensive twodimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for preparative isolation of toad venom. J Chromatogr A 2016;1456:169–75. [50] Verstraeten M, Pursch M, Eckerle P, Luong J, Desmet G. Thermal modulation for multidimensional liquid chromatography separations using low-thermal-mass liquid chromatography (LC). Anal Chem 2011;83:7053–60. [51] Kivilompolo M, Hyötyläinen T. Comprehensive two-dimensional liquid chromatography in analysis of Lamiaceae herbs: characterisation and quantification of antioxidant phenolic acids. J Chromatogr A 2007;1145:155–64. [52] Kivilompolo M, Hyotylainen T. Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages. J Sep Sci 2008;31:3466–72. [53] Mondello L, Herrero M, Kumm T, Dugo P, Cortes H, Dugo G. Quantification in comprehensive two-dimensional liquid chromatography. Anal Chem 2008;80:5418–24. [54] Dugo P, Cacciola F, Donato P, Airado-Rodriguez D, Herrero M, Mondello L. Comprehensive two-dimensional liquid chromatography to quantify polyphenols in red wines. J Chromatogr A 2009;1216:7483–7. [55] Russo M, Cacciola F, Bonaccorsi I, Dugo P, Mondello L. Determination of flavanones in Citrus juices by means of one- and two-dimensional liquid chromatography. J Sep Sci 2011;34:681–7. [56] Cook DW, Rutan SC, Stoll DR, Carr PW. Two dimensional assisted liquid chromatography—a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation, dual detectors, and multivariate curve resolution. Anal Chim Acta 2015;859:87–95.
References
[57] Beccaria M, Costa R, Sullini G, Grasso E, Cacciola F, Dugo P, et al. Determination of the triacylglycerol fraction in fish oil by comprehensive liquid chromatography techniques with the support of gas chromatography and mass spectrometry data. Anal Bioanal Chem 2015;407:5211–25. [58] Donato P, Rigano F, Cacciola F, Schure M, Farnetti S, Russo M, et al. Comprehensive two-dimensional liquid chromatography-tandem mass spectrometry for the simultaneous determination of wine polyphenols and target contaminants. J Chromatogr A 2016;1458:54–62. [59] Bailey HP, Rutan SC. Comparison of chemometric methods for the screening of comprehensive two-dimensional liquid chromatographic analysis of wine. Anal Chim Acta 2013;770:18–28. [60] Cacciola F, Jandera P, Blahovà E, Mondello L. Development of different comprehensive two-dimensional systems for the separation of phenolic antioxidants. J Sep Sci 2006;29:2500–13. [61] Cacciola F, Jandera P, Hajdù Z, Česla P, Mondello L. Comprehensive two-dimensional liquid chromatography with parallel gradients for separation of phenolic and flavone antioxidants. J Chromatogr A 2007;1149:73–87. [62] Dugo P, Cacciola F, Herrero M, Donato P, Mondello L. Use of partially porous column as second dimension in comprehensive two-dimensional system for analysis of polyphenolic antioxidants. J Sep Sci 2008;31:3297–308. [63] Krauze-Baranowska M, Glod D, Kula M, Majdan M, Halasa R, Matkowski A, et al. Chemical composition and biological activity of Rubus idaeus shoots—a traditional herbal remedy of Eastern Europe. BMC Complement Altern Med 2014;14:480–92. [64] Hájek T, Jandera P, Staňková M, Česla P. Automated dual two-dimensional liquid chromatography approach for fast acquisition of three-dimensional data using combinations of zwitterionic polymethacrylate and silica-based monolithic columns. J Chromatogr A 2016;1446:91–102. [65] Lubda D, Cabrera K, Kraas W, Shafer C, Cunningham D. New developments in the application of monolithic HPLC columns. LC GC Eur 2001;14:730–4. [66] Way W. The reporter USA 2007. 25. 20076–7. [67] Way WK. The reporter Europe 2007. 28. 20073–4. [68] Hájek T, Škeřiková V, Česla P, Vyňuchalová K, Jandera P. Multidimensional LC × LC analysis of phenolic and flavone natural antioxidants with UV-electrochemical coulometric and MS detection. J Sep Sci 2008;31:3309–28. [69] Dugo P, Cacciola F, Donato P, Assis RA, Caramão EB, Mondello L. High efficiency liquid chromatography techniques coupled to mass spectrometry for the characterization of mate extracts. J Chromatogr A 2009;1216:7213–21. [70] Mondello L, Beccaria M, Donato P, Cacciola F, Dugo G, Dugo P. Comprehensive twodimensional liquid chromatography with evaporative light-scattering detection for the analysis of triacylglycerols in Borago officinalis. J Sep Sci 2011;34:688–92. [71] Cacciola F, Donato P, Giuffrida D, Torre G, Dugo P, Mondello L. Ultra high pressure in the second dimension of a comprehensive two-dimensional liquid chromatographic system for carotenoid separation in chili red peppers. J Chromatogr A 2012;1255:244–51. [72] Mazzi Leme G, Cacciola F, Donato P, Cavalheiro AJ, Dugo P, Mondello L. Continuos vs. Segmented second-dimension system gradients for comprehensive two-dimensional liquid chromatography of sugarcane (Saccharum spp.). Anal Bioanal Chem 2014;406: 4315–24. [73] Jandera P, Hájek T, Česla P. Comparison of various second-dimension gradient types in comprehensive two-dimensional liquid chromatography. J Sep Sci 2010;33:1382–97.
413
414
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
[74] Jandera P, Hájek T, Staňková M, Vynuchalová K, Česla P. Optimization of comprehensive two-dimensional gradient chromatography coupling in-line hydrophilic interaction and reversed phase liquid chromatography. J Chromatogr A 2012;1268:91–101. [75] Česla P, Hájek T, Jandera P. Optimization of two-dimensional gradient liquid chromatography separations. J Chromatogr A 2009;1216:3443–57. [76] Hájek T, Jandera P. Columns and optimum gradient conditions for fast seconddimension separations in comprehensive two-dimensional liquid chromatography. J Sep Sci 2012;35:1712–22. [77] Montero L, Herrero M, Prodanov M, Ibanez E, Cifuentes A. Characterization of grape seed procyanidins by comprehensive two-dimensional hydrophilic interaction x reversed phase liquid chromatography coupled to diode array detection and tandem mass spectrometry. Anal Bioanal Chem 2013;405:4627–38. [78] Montero L, Herrero M, Ibanez E, Cifuentes A. Profiling of phenolic compounds from different apple varieties using comprehensive two-dimensional liquid chromatography. J Chromatogr A 2013;1313:275–83. [79] Montero L, Herrero M, Ibanez E, Cifuentes A. Separation and characterization of phlorotannins from brown algae Cystoseira abies-marina by comprehensive two-dimensional liquid chromatography. Electrophoresis 2014;35:1644–51. [80] Montero L, Sánchez-Camargo AP, García-Canas V, Tanniou A, Stiger-Pouvreau V, Russo M, et al. Anti-proliferative activity and chemical characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry of phlorotannins from the brown macroalga Sargassum muticum collected on North-Atlantic coasts. J Chromatogr A 2016;1428:115–25. [81] Montero L, Ibanez E, Russo M, di Sanzo R, Rastrelli L, Piccinelli AL, et al. Metabolite profiling of licorice (Glycyrrhiza glabra) from different locations using comprehensive two-dimensional liquid chromatography coupled to diode array and tandem mass spectrometry detection. Anal Chim Acta 2016;913:145–59. [82] Kivilompolo M, Oburka V, Hyotylainen T. Comprehensive two-dimensional liquid chromatography in the analysis of antioxidant phenolic compounds in wines and juices. Anal Bioanal Chem 2008;391:373–80. [83] Cacciola F, Delmonte P, Jaworska K, Dugo P, Mondello L, Rader J. Employing ultra high pressure liquid chromatography as the second dimension in a comprehensive two-dimensional system for analysis of Stevia rebaudiana extracts. J Chromatogr A 2011;1218:2012–8. [84] Krol-Kogus B, Glod D, Krauze-Baranowska M, Matlawska I. Application of one- and two-dimensional high-performance liquid chromatography methodologies for the analysis of C-glycosylflavones from fenugreek seeds. J Chromatogr A 2014;1367:48–56. [85] Kalili KM, de Villiers A. Off-line comprehensive 2-dimensional hydrophilic interaction × reversed phase liquid chromatography analysis of procyanidins. J Chromatogr A 2009;1216:6274–84. [86] Kalili KM, de Villiers A. Off-line comprehensive two-dimensional hydrophilic interaction x reversed phase liquid chromatographic analysis of green tea phenolics. J Sep Sci 2010;33:853–63. [87] Beelders T, Kalili KM, Joubert E, Beer D, de Villiers A. Comprehensive twodimensional liquid chromatographic analysis of rooibos (Aspalathus linearis) phenolics. J Sep Sci 2015;35:1808–20. [88] Kalili KM, de Villiers A. Systematic optimisation and evaluation of on-line, off-line and stop-flow comprehensive hydrophilic interaction chromatography × reversed phase liq-
References
[89]
[90] [91] [92]
[93]
uid chromatographic analysis of procyanidins. Part II: application to cocoa procyanidins. J Chromatogr A 2013;1289:69–79. Kalili KM, Vestner J, Stander MA, de Villiers A. Toward unraveling grape tannin composition: application of online hydrophilic interaction chromatography × reversed-phase liquid chromatography-time-of-flight mass spectrometry for grape seed analysis. Anal Chem 2013;85:9107–15. Jandera P, Staňkova M, Hájek T. New zwitterionic polymethacrylate monolithic columns for one- and two-dimensional microliquid chromatography. J Sep Sci 2013;36:2430–40. Willemse CM, Stander MA, Tredoux AGJ, de Villiers A. Comprehensive two-dimensional liquid chromatographic analysis of anthocyanins. J Chromatogr A 2014;1359:189–201. Willemse CM, Stander MA, Vestner J, Tredoux AGJ, de Villiers A. Comprehensive twodimensional hydrophilic interaction chromatography (HILIC) × reversed-phase liquid chromatography coupled to high-resolution mass spectrometry (RP-LC-UV-MS) analysis of anthocyanins and derived pigments in red wine. Anal Chem 2015;87:12006–15. Li D, Jakob C, Schimitz O. Practical considerations in comprehensive two-dimensional liquid chromatography systems (LCxLC) with reversed-phases in both dimensions. Anal Bioanal Chem 2015;407:153–67.
415
Comprehensive two-dimensional liquid chromatography
16
Francesco Cacciola*, Marina Russo†, Luigi Mondello*,†,‡, Paola Dugo*,†,‡ University of Messina, Messina, Italy* University Campus Bio-Medico of Rome, Rome, Italy† Chromaleont s.r.l., c/o University of Messina, Messina, Italy‡
C HAPTER OUTLINE 16.1 Introduction..................................................................................................... 403 16.2 Fundamentals.................................................................................................. 404 16.3 Instrumental Set-Up and Data Analysis.............................................................. 405 16.4 Novel Stationary Phases................................................................................... 408 16.5 Conclusions and Future Perspectives................................................................ 409 References............................................................................................................... 409
16.1 INTRODUCTION One-dimensional liquid chromatography (1D-LC) is widely applied to the analysis of real-world samples in several fields. However, for the analysis of many real-world samples, for example, biological, food, and environmental, “conventional” 1D-LC methods do not provide rewarding results for enabling compound identification and quantification. Comprehensive two-dimensional high performance liquid chromatography (LC × LC) experienced mostly in the last two decades could be a valuable option. The most general set-up of an LC × LC system consists of two pumps, two columns, injector, interface, and detector. The interface is, in general, a high-pressure two-position switching valve and this device is often referred to as modulator or sampling device. LC × LC methods improve the likelihood for separation of a higher number of sample components since the peak capacity of an LC × LC separation is multiplicative of the product of the peak capacity values of the two single dimensions (n2D = nD1 × nD2) [1–3]. The peak capacities of the two dimensions are shown as the number of adjacent Gaussian profiles that can be accommodated into the space along with the respective separation coordinates. The resolution is represented by rectangular boxes, which divide the separation plane. The total peak capacity, n2D, is therefore approximately equal to the number of such boxes [3]. Liquid Chromatography. http://dx.doi.org/10.1016/B978-0-12-805393-5.00016-6 © 2017 Elsevier Inc. All rights reserved.
403
404
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
In this regard, by careful selection of mobile and stationary phases in the two dimensions to tune orthogonality, the peak capacity of the overall systems can dramatically increase, making group separations of various samples often possible, especially for the interpretation of the LC × LC data. Due to the tremendously higher separation power of LC × LC when compared to “conventional” 1D-LC, the technique has been employed in several fields, for example, polymers, pharmaceuticals and biological mixtures, natural products, environmental and petrochemical samples, and has been the subject of various reviews [4–27].
16.2 FUNDAMENTALS In the last two decades, many works have dealt with all key factors affecting resolution and performance in LC × LC methods. An important role on the dependence of the two dimensions on each other is related to the analysis speed in the second dimension (2D) affecting the first dimension (1D) sampling rate on the peak capacity of the overall LC × LC separations. In 1998, Murphy et al. [28] investigated the effect of the sampling rate on the effective 1D peak width and modeled a Gaussian peak as a histogram profile of the average concentration within every sampling period. The outcome of the research was that approximately three to four modulations per 1D peak (8σ) are needed to avoid serious loss of performance of the LC × LC system as a result of “undersampling” of 1D peaks. Some years later Seeley et al. [29] published a similar work on this topic investigating the sampling frequency for modulators with various duty cycles (2002) introducing the (average) peak broadening factor, σ*, related to selected parameters such as sampling period, τZ, and sampling phase, Φ. τZ is determined by the modulation period or sampling time ts and 1σ of the 1D peak, while Φ represents the way in which the primary peak is cut into aliquots. The sampling phase Φ is the time difference between the center of the sampling cycle nearest to the peak maximum and the peak maximum itself, divided by the sampling period. Peak broadening was less significant when the peak maximum was centered in one of the sampling periods and for low duty cycles. For systems with a duty cycle of 1, as is the case for most LC × LC separations, the peak broadening was practically independent of Φ. Basically, Seeley drew the same conclusions as Murphy, extending his investigations to duty cycles less than 1. Afterwards, Horie et al. [30] demonstrated that modulation periods equal to 2.2–4 are sufficient to minimize “peak broadening”. In addition, they introduced a new parameter, eM (Nobuo factor), which is defined as the ratio of the apparent peak capacity of the first column derived from σ* to its actual peak capacity in the 1D. In 2006 Schoenmakers and co-workers [31] introduced a useful “protocol” for optimizing LC × LC separations based on proper sampling, taking into account suitable chromatographic parameters, such as column dimensions, injection volumes, and flow rates. Carr and co-workers [32–36] poured a great effort towards understanding in LC × LC extending their investigations to the
16.3 Instrumental set-up and data analysis
overall chromatogram. For the determination of the average 1D broadening factor, β, as a function of the sampling time (ts) and the standard deviation of the 1D peaks prior 1σ, 2D statistical overlap theory along with the number of observed peaks in simulated LC × LC separations was used [37]. The effects of the 1D peak capacity and gradient time, and the 2D cycle times on the overall peak capacity of the 2D-LC system was lately reported in 2010 by Potts et al. [35]. It was demonstrated that in fast LC × LC separations (<1 h), the 2D was more important than the 1D in determining overall peak capacity. The benefits on the sampling rate of the 1D effluent attained by the use of multiple 2 D columns have been shown by Fairchild et al. [38] highlighting that peak capacity of the LC × LC system increases, compared to an LC × LC system employing only a single 2D column, while reducing the analysis time.
16.3 INSTRUMENTAL SET-UP AND DATA ANALYSIS In the most common set-up for LC × LC systems, small volume fractions of the effluent from the 1D are transferred via an interface into the 2D. The interface techniques of LC × LC include different types of interface namely dual loop, stop-flow, and vacuum evaporation. Due to its simple structure the dual loop interface is mostly used in LC × LC separations. Last implementations have been recently carried out in Schoenmakers and Stoll's research groups. Schoenmakers and co-workers investigated an actively modulated LC × LC (LC/a × m/LC) aiming to overcome one of the limitations of contemporary LC × LC arising from the combination of diverse 1D and 2D column diameters: the capability of such an approach was evaluated for both SCX × RP and HILIC × RP-LC separations [39–41]. A schematic overview of the modulated LC × LC setup used for yeast analysis is shown in Fig. 16.1. Stoll and coworkers developed a “selective” LC × LC system (sLC × LC) with the aim to break the long-standing link between the timescales of the 1D and 2D separations through novel implementation of existing valve technology [42–44]. A schematic diagram of the instrument configuration for sLC × LC, which allows advantages similar to those derived from off-line LC × LC approach but without most of the major drawbacks of off-line work, is illustrated in Fig. 16.2. Stop-flow mode is applied generally when the analysis speed of the 2D cannot keep up with the sampling frequency of the 1D. A longer 2D column with respect to the commonly employed ones is usually employed to improve resolution as well as peak capacity [45]. Two main disadvantages of such an approach consists of a longer analysis time with respect to continuous LC × LC and potential band broadening phenomena, which may arise for both long parking periods and nonadequate peak focusing on the top of the 2D column [46,47]. Vacuum evaporation was used by Guan and co-workers for alleviating the incompatibility of mobile phases used in NP-LC × RP-LC separations [48]: it allowed to condense the 1D eluents and the 2D solvent redissolved the residents at the inside wall of a loop for further separation
405
406
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
2D
Waste
RP column Wash solvent
FTICR-MS
Waste
1D
2D
SCX column
nano flow in
Make-up flow
FIG. 16.1 Schematic overview of the modulated LC × LC setup used for yeast analysis. Reproduced with permission from Vonk RJ, Gargano AFG, Davydova E, Dekker HL, Eeltink S, de Koning LJ, et al. Comprehensive two-dimensional liquid chromatography with stationary-phase-assisted modulation coupled to high-resolution mass spectrometry applied to proteome analysis of saccharomyces cerevisiae. Anal Chem 2015;87:5387–94 [American Chemical Society].
in the 2D. The main pitfall is the potential sample loss risk for volatile components due to evaporation in the interface. More recently, a newly developed vacuum evaporation assisted adsorption interface that allowed fast removal of NP-LC solvent in the vacuum condition and successful solving of the solvent incompatibility problem between NP-LC and RP-LC was constructed for preparative purposes [49]. A proof-of-principle experiment with a novel thermal modulation device with potential use in LC × LC systems has been recently reported by Verstraeten et al. [50]. Based on the thermal desorption concept used in comprehensive two-dimensional gas chromatography (GC × GC) systems, preconcentration of neutral analytes eluting from the 1D was performed in a capillary “trap” column packed with highly retentive porous graphitic carbon particles, placed in an aluminum low-thermal-mass LC heating sleeve. Remobilization of the trapped analytes was achieved by rapidly heating the trap column, by applying temperature ramps up to +1200°C/min. The thermal modulation device is schematically represented in Fig. 16.3. As far as detection is concerned all conventional LC detectors, such as photodiode array, mass spectrometry (MS), and evaporative light scattering detectors, are commonly used in LC × LC; usually, a single detector is installed after the 2 D column, although an additional detector can be used to collect the 1D data, with the 1D separation monitored only during the optimization step. The high speed of the 2D analysis requires a very fast detector acquisition rate to ensure the adequate sampling, which is critical for quantification purposes, otherwise
16.3 Instrumental set-up and data analysis
Load Loop 1 Column I Detector I (Opt) Dilution
Autoinjector
Load Loop 2 Pump I
Column I
Pump II
Detector I (Opt)
A
Dilution
L1
(A)
Pump II A
Dilution
C
L3
Pump II A
C
L3
L2
L6 L5
C
L4
D Waste
Column II Detector II
(B)
Pump I
L1 L2
L5
Autoinjector
B
L6
L4
Detector II
Column I Detector I (Opt)
L1 L2
L6
Column II
Pump I
B
B
L5
Autoinjector
Inject Loop 1
D
L4
Column II Waste
L3
Detector II
D Waste
(C)
FIG. 16.2 Schematic representation of instrument configuration for sLC × LC. Pump I delivers eluent to Column I while Pump II pushes captured fractions of 1D effluent out of sample loops L1 through L6 and delivers eluent to the 2D column. Detector I is optional in the sense that it can be removed to reduce extra-column broadening after 1D elution times of target compounds have been determined. Dilution of 1D effluent was achieved by an external, low-pressure pump T-ed into the 1D flow path to achieve effective on-column focusing in the second dimension. A series of four valves labeled A–D were used to capture and store six fractions of 1D effluent. Example valve configurations required for fraction capture are shown in panels (A and B). Loop L1 is loaded by the 1D flow marked in red (dark grey in print) in panel (A), then valves (B and C) rotate to load L2 shown in panel (B) while the flow from Pump II shown in blue (light grey in print) remains unchanged. Panel (C) shows an example of a flow path required to re-inject captured fractions into the 2D column. Subsequent injection of all six fractions is achieved by simultaneous rotation of valves B and C. Reproduced with permission from Groskreutz SR, Swenson MM, Secor LB, Stoll DR. Selective comprehensive multi-dimensional separation for resolution enhancement in high performance liquid chromatography. Part I: principles and instrumentation. J Chromatogr A 2012;1228:31–40 [Elsevier].
loss in resolution, due to a low number of data points, may occur. Usually, data are visualized in 2D plots or contour plots, where the retention times in the 1D and the 2D dimensions are plotted along the x- and y-axes, respectively, while the color of the spots is a measure for intensity. In LC × LC, quantification can be performed by combining the contribution of each individual 2D “slice” of a 1D
407
408
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
From injector/pump
SiO2
PGC
SiO2
To detector, fraction collector or 2nd column
Low thermal mass heating sleeve
(A) 1st dim. column
Compressed air
SiO2
PGC
SiO2
To detector, fraction collector or 2nd column
Low thermal mass heating sleeve
(B)
Compressed air
FIG. 16.3 Schematic representation of the thermal modulation system consisting of a three-section packed capillary column (silica-PGC-silica) and a low-thermal-mass heating sleeve connected to (A) the injector or pump and (B) the first dimension column. Reproduced with permission from Verstraeten M, Pursch M, Eckerle P, Luong J, Desmet G. Thermal modulation for multidimensional liquid chromatography separations using low-thermal-mass liquid chromatography (LC). Anal Chem 2011;83:7053–60 [American Chemical Society].
chromatographic peak, or by using more advanced algorithms [51–58]. Advanced data analysis techniques, for example, multivariate data analysis, can also be applied to LC × LC data, despite it being more complicated with respect to 1D-LC separations [22–59]. So far, two commercially available software packages, namely Chromsquare and GC Image LC × LC Edition Software specially designed for both visualization and quantification of 2D data, are available on the market.
16.4 NOVEL STATIONARY PHASES The most significant development in the field of novel stationary phases LC × LC has been the introduction of 2D small particles stationary phases with the aim to replace the monolithic types that have been earlier used for the same goal [52,60–64]. The use of monoliths was very advantageous since these columns could be operated at high flow rates without loss of resolution with very brief re-equilibration times and significantly lower backpressures [65]. In 2007, a new particle technology (Fused-Core) was developed to deliver hyperfast chromatographic separations, while avoiding the reliability issues often associated with fast HPLC [66,67]. These stationary phases consisted of a 1.7 μm solid
References
core with a 0.5 μm porous silica shell surrounding it (d.p. = 2.7 μm). The major benefit of these particles was therefore the smaller diffusion path, which reduced axial dispersion of solutes minimizing peak broadening. The higher efficiencies of such stationary phases, capable of high pressure operation, were chosen as 2D of LC × LC systems and several research groups (Mondello, Dugo, Jandera, and Cifuentes) used them in natural products applications [54–56,62,68–81]. Another remarkable development was the introduction of sub-2-μm technology allowing ultrafast analysis and/or high-resolution separations [52,64,74,76,79,82–92]. Besides the selection of stationary phases, mobile phases play a pivotal role in the development of LC × LC methods. In particular, the use of gradient elution conditions in both dimensions was commonly employed in several research works. “Full-in-fraction” gradients, involving an equally generic and steep mobile phase range in each repeated 2D run, is the approach most commonly adopted for LC × LC separations. To enhance the orthogonality degree, three different gradient elution strategies were afterwards investigated [93]. The first one, “segmented in fraction”, used different gradient ranges in different segments of the 2D separation; the second strategy, known as “parallel gradient” implied only a single gradient spanning during the whole 2D separation time adopted simultaneously to the 1D gradient; in the third one “continuously shifting” the initial and final gradient in the 2D is continuously changed over the whole LC × LC analysis. Such optimization approaches applied so far to polyphenol analysis will likely be very fruitful areas of research and will be extended to the analysis of more sample types.
16.5 CONCLUSIONS AND FUTURE PERSPECTIVES Comprehensive liquid chromatography has been extensively exploited in the past two decades with remarkable updates in the last few years with reference to instrumental set-up and availability of novel stationary phases. In particular, the introduction into the market of partially porous particles and sub-2-μm stationary phases, capable of high-pressure and high-temperature fast second-dimension operation, turned out to be a valuable tool for more performant LC × LC separations. Also, the design of different gradients approaches for LC × LC analyses investigated so far was an important implementation and to this regard the use of more robust and flexible software for both qualitative and quantitative purposes is expected to grow in the near future. Recent efforts in the development of transfer devices as well as availability of commercial instrument will expand the use of the technique introduce “routine” use of LC × LC.
REFERENCES [1] Karger BL, Snyder LR, Horvath C. An introduction to separation science. New York, NY: Wiley & Sons; 1973. [2] Giddings JC. Two-dimensional separations: concept and promise. Anal Chem 1984;37:1258A–64A.
409
410
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
[3] Giddings JC. Concepts and comparisons in multidimensional separation. J High Resolut Chrom 1987;10:319–23. [4] Liu Z, Lee ML. Comprehensive two-dimensional separations using microcolumns. J Microcolumn Sep 2000;12:241–54. [5] Evans CR, Jorgenson JW. Multidimensional LC-LC and LC-CE for high-resolution separations of biological molecules. Anal Bioanal Chem 2004;378:1952–61. [6] Guttman A, Varoglu M, Khandurina J. Multidimensional separations in the pharmaceutical arena. Drug Discov Today 2004;9:136–44. [7] Stroink T, Ortiz MC, Bult A, Lingeman H, de Jong GJ, Underberg WJM. On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins. J Chromatogr B 2005;817:49–66. [8] Dixon SP, Pitfield ID, Perrett D. Comprehensive multi-dimensional liquid chromatographic separation in biomedical and pharmaceutical analysis: a review. Biomed Chromatogr 2006;20:508–29. [9] Jandera P. Column selectivity for two-dimensional liquid chromatography. J Sep Sci 2006;29:1763–83. [10] Shalliker RA, Gray MJ. Concepts and practice of multidimensional high-performance liquid chromatography. Adv Chromatogr 2006;44:177–236. [11] Shellie RA, Haddad PR. Comprehensive two-dimensional liquid chromatography. Anal Bioanal Chem 2006;386:405–15. [12] Stoll DR, Li X, Wang X, Carr PW, Porter SEG, Rutan S. Fast comprehensive twodimensional liquid chromatography. J Chromatogr A 2007;1168:3–43. [13] Dugo P, Cacciola F, Kumm T, Dugo G, Mondello L. Comprehensive multidimensional liquid chromatography: theory and applications. J Chromatogr A 2008;1184:353–68. [14] Dugo P, Kumm T, Cacciola F, Dugo G, Mondello L. Multidimensional liquid chromatographic separations applied to the analysis of food samples. J Liq Chromatogr Relat Technol 2008;31:1758–807. [15] Guiochon G, Marchetti N, Mriziq K, Shalliker RA. Implementations of two- dimensional liquid chromatography. J Chromatogr A 2008;1189:109–68. [16] Pol J, Hyotylainen T. Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry. Anal Bioanal Chem 2008;391:21–31. [17] François I, Sandra K, Sandra P. Comprehensive liquid chromatography: fundamental aspects and practical considerations-a review. Anal Chim Acta 2009;641:14–31. [18] Herrero M, Ibanez E, Cifuentes A, Bernal J. Multidimensional chromatography in food analysis. J Chromatogr A 2009;1216:7110–29. [19] Sandra K, Moshir M, D'hondt F, Tuytten R, Verleysen K, Kas K, et al. Highly efficient peptide separations in proteomics. Part 2: bi- and multidimensional liquid-based separation techniques. J Chromatogr B 2009;877:1019–39. [20] Berek D. Two-dimensional liquid chromatography of synthetic polymers. Anal Bioanal Chem 2010;396:421–41. [21] Pierce KM, Kehimkar B, Marney LC, Hoggard JC, Synovec RE. Review of chemometric analysis techniques for comprehensive two dimensional separations data. J Chromatogr A 2012;1255:3–11. [22] Pierce KM, Mohler RE. A review of chemometrics applied to comprehensive twodimensional separations from 2008-2010. Sep Purif Rev 2012;41:143–68. [23] Jandera P. Comprehensive Two-Dimensional Liquid Chromatography-Practical impacts of theoretical considerations. A review. Cent Eur J Chem 2012;10:844–75.
References
[24] Donato P, Cacciola F, Tranchida PQ, Dugo P, Mondello L. Mass spectrometry detection in comprehensive liquid chromatography: basic concepts, instrumental aspects, applications and trends. Mass Spectrom Rev 2012;1:523–59. [25] Bedani F, Kok W, Janssen H-G. A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography × liquid chromatography. J Chromatogr A 2006;1133:126–34. [26] Shi X, Wang S, Yang Q, Lu X, Xu G. Comprehensive two-dimensional chromatography for analyzing complex samples: recent new advances. Anal Methods 2014;6:7112–23. [27] Sarrut M, Crétier G, Heinisch S. Theoretical and practical interest in UHPLC technology for 2D-LC. TrAC-Trend Anal Chem 2014;63:104–12. [28] Murphy RE, Schure MR, Foley JP. Effect of sampling rate on resolution in comprehensive two-dimensional liquid chromatography. Anal Chem 1998;70:1585–94. [29] Seeley JV. Theoretical study of incomplete sampling of the first dimension in comprehensive two-dimensional chromatography. J Chromatogr A 2002;962:21–7. [30] Horie K, Kimura H, Ikegami T, Iwatsuka A, Saad N, Fiehn O, et al. Calculating optimal modulation periods to maximize the peak capacity in two dimensional HPLC. Anal Chem 2007;79:3764–70. [31] Schoenmakers PJ, Vivo-Truyols G, Decrop WMC. A protocol for designing comprehensive two-dimensional liquid chromatography separation systems. J Chromatogr A 2006;1120:282–90. [32] Davis JM, Stoll DR, Carr PW. Effect of first-dimension undersampling on effective peak capacity in comprehensive two-dimensional separations. Anal Chem 2008;80:461–73. [33] Davis JM, Stoll DR, Carr PW. Dependence of effective peak capacity in comprehensive two-dimensional separations on the distribution of peak capacity between the two dimensions. Anal Chem 2008;80:8122–34. [34] Li X, Stoll DR, Carr PW. Equation for peak capacity estimation in two-dimensional liquid chromatography. Anal Chem 2009;81:845–50. [35] Potts LW, Stoll DW, Li X, Carr PW. The impact of sampling time on peak capacity and analysis speed in on-line comprehensive two-dimensional liquid chromatography. J Chromatogr A 2010;1217:5700–9. [36] Gu H, Huang Y, Carr PW. Peak capacity optimization in comprehensive two dimensional liquid chromatography: a practical approach. J Chromatogr A 2011;1218:64–73. [37] Davis JM. Statistical-overlap theory for elliptical zones of high aspect ratio in comprehensive two-dimensional separations. J Sep Sci 2005;28:347–59. [38] Fairchild JN, Horvath K, Guiochon G. Theoretical advantages and drawbacks of on-line, multidimensional liquid chromatography using multiple columns operated in parallel. J Chromatogr A 2009;1216:6210–7. [39] Vonk RJ, Wouters S, Barcaru A, Vivó-Truyols G, Eeltink S, de Koning LJ, et al. Postpolymerization photografting on methacrylate-based monoliths for separation of intact proteins and protein digests with comprehensive two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry. Anal Bioanal Chem 2015;407:3817–29. [40] Vonk RJ, Gargano AFG, Davydova E, Dekker HL, Eeltink S, de Koning LJ, et al. Comprehensive two-dimensional liquid chromatography with stationary-phase-assisted modulation coupled to high-resolution mass spectrometry applied to proteome analysis of saccharomyces cerevisiae. Anal Chem 2015;87:5387–94.
411
412
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
[41] Gargano AFG, Duffin M, Navarro P, Schoenmakers PJ. Reducing dilution and analysis time in online comprehensive two-dimensional liquid chromatography by active modulation. Anal Chem 2016;88:1785–93. [42] Groskreutz SR, Swenson MM, Secor LB, Stoll DR. Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part I: principles and instrumentation. J Chromatogr A 2012;1228:31–40. [43] Davis JM, Stoll DR. Likelihood of total resolution in liquid chromatography: evaluation of one-dimensional, comprehensive two-dimensional, and selective comprehensive twodimensional liquid chromatography. J Chromatogr A 2014;1360:128–42. [44] Larson ED, Groskreutz SR, Harmes DC, Gibbs-Hall IC, Trudo SP, Allen RC, et al. Development of selective comprehensive two-dimensional liquid chromatography with parallel first-dimension sampling and second-dimension separation-application to the quantitative analysis of furanocoumarins in apiaceous vegetables. Anal Bioanal Chem 2013;405:4639–53. [45] Blahovà E, Jandera P, Cacciola F, Mondello L. Two‐dimensional and serial column reversed‐phase separation of phenolic antioxidants on octadecyl‐, polyethyleneglycol‐, and pentafluorophenylpropyl‐silica columns. J Sep Sci 2006;29:555–66. [46] Bedani F, Schoenmakers PJ, Janssen H-G. Theories to support method development in comprehensive two-dimensional liquid chromatography—a review. J Sep Sci 2012;35:1697–711. [47] Sommella E, Cacciola F, Donato P, Campiglia P, Dugo P, Mondello L. Development of an online capillary comprehensive 2D-LC system for the analysis of proteome samples. J Sep Sci 2012;35:530–3. [48] Tian H, Xu J, Guan Y. Comprehensive two-dimensional liquid chromatography (NPLC × RPLC) with vacuum-evaporation interface. J Sep Sci 2008;31:1677–85. [49] Li J-F, Fang H, Yan X, Chang F-R, Wu Z, Wu Y-L, et al. On-line comprehensive twodimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for preparative isolation of toad venom. J Chromatogr A 2016;1456:169–75. [50] Verstraeten M, Pursch M, Eckerle P, Luong J, Desmet G. Thermal modulation for multidimensional liquid chromatography separations using low-thermal-mass liquid chromatography (LC). Anal Chem 2011;83:7053–60. [51] Kivilompolo M, Hyötyläinen T. Comprehensive two-dimensional liquid chromatography in analysis of Lamiaceae herbs: characterisation and quantification of antioxidant phenolic acids. J Chromatogr A 2007;1145:155–64. [52] Kivilompolo M, Hyotylainen T. Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages. J Sep Sci 2008;31:3466–72. [53] Mondello L, Herrero M, Kumm T, Dugo P, Cortes H, Dugo G. Quantification in comprehensive two-dimensional liquid chromatography. Anal Chem 2008;80:5418–24. [54] Dugo P, Cacciola F, Donato P, Airado-Rodriguez D, Herrero M, Mondello L. Comprehensive two-dimensional liquid chromatography to quantify polyphenols in red wines. J Chromatogr A 2009;1216:7483–7. [55] Russo M, Cacciola F, Bonaccorsi I, Dugo P, Mondello L. Determination of flavanones in Citrus juices by means of one- and two-dimensional liquid chromatography. J Sep Sci 2011;34:681–7. [56] Cook DW, Rutan SC, Stoll DR, Carr PW. Two dimensional assisted liquid chromatography—a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation, dual detectors, and multivariate curve resolution. Anal Chim Acta 2015;859:87–95.
References
[57] Beccaria M, Costa R, Sullini G, Grasso E, Cacciola F, Dugo P, et al. Determination of the triacylglycerol fraction in fish oil by comprehensive liquid chromatography techniques with the support of gas chromatography and mass spectrometry data. Anal Bioanal Chem 2015;407:5211–25. [58] Donato P, Rigano F, Cacciola F, Schure M, Farnetti S, Russo M, et al. Comprehensive two-dimensional liquid chromatography-tandem mass spectrometry for the simultaneous determination of wine polyphenols and target contaminants. J Chromatogr A 2016;1458:54–62. [59] Bailey HP, Rutan SC. Comparison of chemometric methods for the screening of comprehensive two-dimensional liquid chromatographic analysis of wine. Anal Chim Acta 2013;770:18–28. [60] Cacciola F, Jandera P, Blahovà E, Mondello L. Development of different comprehensive two-dimensional systems for the separation of phenolic antioxidants. J Sep Sci 2006;29:2500–13. [61] Cacciola F, Jandera P, Hajdù Z, Česla P, Mondello L. Comprehensive two-dimensional liquid chromatography with parallel gradients for separation of phenolic and flavone antioxidants. J Chromatogr A 2007;1149:73–87. [62] Dugo P, Cacciola F, Herrero M, Donato P, Mondello L. Use of partially porous column as second dimension in comprehensive two-dimensional system for analysis of polyphenolic antioxidants. J Sep Sci 2008;31:3297–308. [63] Krauze-Baranowska M, Glod D, Kula M, Majdan M, Halasa R, Matkowski A, et al. Chemical composition and biological activity of Rubus idaeus shoots—a traditional herbal remedy of Eastern Europe. BMC Complement Altern Med 2014;14:480–92. [64] Hájek T, Jandera P, Staňková M, Česla P. Automated dual two-dimensional liquid chromatography approach for fast acquisition of three-dimensional data using combinations of zwitterionic polymethacrylate and silica-based monolithic columns. J Chromatogr A 2016;1446:91–102. [65] Lubda D, Cabrera K, Kraas W, Shafer C, Cunningham D. New developments in the application of monolithic HPLC columns. LC GC Eur 2001;14:730–4. [66] Way W. The reporter USA 2007. 25. 20076–7. [67] Way WK. The reporter Europe 2007. 28. 20073–4. [68] Hájek T, Škeřiková V, Česla P, Vyňuchalová K, Jandera P. Multidimensional LC × LC analysis of phenolic and flavone natural antioxidants with UV-electrochemical coulometric and MS detection. J Sep Sci 2008;31:3309–28. [69] Dugo P, Cacciola F, Donato P, Assis RA, Caramão EB, Mondello L. High efficiency liquid chromatography techniques coupled to mass spectrometry for the characterization of mate extracts. J Chromatogr A 2009;1216:7213–21. [70] Mondello L, Beccaria M, Donato P, Cacciola F, Dugo G, Dugo P. Comprehensive twodimensional liquid chromatography with evaporative light-scattering detection for the analysis of triacylglycerols in Borago officinalis. J Sep Sci 2011;34:688–92. [71] Cacciola F, Donato P, Giuffrida D, Torre G, Dugo P, Mondello L. Ultra high pressure in the second dimension of a comprehensive two-dimensional liquid chromatographic system for carotenoid separation in chili red peppers. J Chromatogr A 2012;1255:244–51. [72] Mazzi Leme G, Cacciola F, Donato P, Cavalheiro AJ, Dugo P, Mondello L. Continuos vs. Segmented second-dimension system gradients for comprehensive two-dimensional liquid chromatography of sugarcane (Saccharum spp.). Anal Bioanal Chem 2014;406: 4315–24. [73] Jandera P, Hájek T, Česla P. Comparison of various second-dimension gradient types in comprehensive two-dimensional liquid chromatography. J Sep Sci 2010;33:1382–97.
413
414
CHAPTER 16 Comprehensive two-dimensional liquid chromatography
[74] Jandera P, Hájek T, Staňková M, Vynuchalová K, Česla P. Optimization of comprehensive two-dimensional gradient chromatography coupling in-line hydrophilic interaction and reversed phase liquid chromatography. J Chromatogr A 2012;1268:91–101. [75] Česla P, Hájek T, Jandera P. Optimization of two-dimensional gradient liquid chromatography separations. J Chromatogr A 2009;1216:3443–57. [76] Hájek T, Jandera P. Columns and optimum gradient conditions for fast seconddimension separations in comprehensive two-dimensional liquid chromatography. J Sep Sci 2012;35:1712–22. [77] Montero L, Herrero M, Prodanov M, Ibanez E, Cifuentes A. Characterization of grape seed procyanidins by comprehensive two-dimensional hydrophilic interaction x reversed phase liquid chromatography coupled to diode array detection and tandem mass spectrometry. Anal Bioanal Chem 2013;405:4627–38. [78] Montero L, Herrero M, Ibanez E, Cifuentes A. Profiling of phenolic compounds from different apple varieties using comprehensive two-dimensional liquid chromatography. J Chromatogr A 2013;1313:275–83. [79] Montero L, Herrero M, Ibanez E, Cifuentes A. Separation and characterization of phlorotannins from brown algae Cystoseira abies-marina by comprehensive two-dimensional liquid chromatography. Electrophoresis 2014;35:1644–51. [80] Montero L, Sánchez-Camargo AP, García-Canas V, Tanniou A, Stiger-Pouvreau V, Russo M, et al. Anti-proliferative activity and chemical characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry of phlorotannins from the brown macroalga Sargassum muticum collected on North-Atlantic coasts. J Chromatogr A 2016;1428:115–25. [81] Montero L, Ibanez E, Russo M, di Sanzo R, Rastrelli L, Piccinelli AL, et al. Metabolite profiling of licorice (Glycyrrhiza glabra) from different locations using comprehensive two-dimensional liquid chromatography coupled to diode array and tandem mass spectrometry detection. Anal Chim Acta 2016;913:145–59. [82] Kivilompolo M, Oburka V, Hyotylainen T. Comprehensive two-dimensional liquid chromatography in the analysis of antioxidant phenolic compounds in wines and juices. Anal Bioanal Chem 2008;391:373–80. [83] Cacciola F, Delmonte P, Jaworska K, Dugo P, Mondello L, Rader J. Employing ultra high pressure liquid chromatography as the second dimension in a comprehensive two-dimensional system for analysis of Stevia rebaudiana extracts. J Chromatogr A 2011;1218:2012–8. [84] Krol-Kogus B, Glod D, Krauze-Baranowska M, Matlawska I. Application of one- and two-dimensional high-performance liquid chromatography methodologies for the analysis of C-glycosylflavones from fenugreek seeds. J Chromatogr A 2014;1367:48–56. [85] Kalili KM, de Villiers A. Off-line comprehensive 2-dimensional hydrophilic interaction × reversed phase liquid chromatography analysis of procyanidins. J Chromatogr A 2009;1216:6274–84. [86] Kalili KM, de Villiers A. Off-line comprehensive two-dimensional hydrophilic interaction x reversed phase liquid chromatographic analysis of green tea phenolics. J Sep Sci 2010;33:853–63. [87] Beelders T, Kalili KM, Joubert E, Beer D, de Villiers A. Comprehensive twodimensional liquid chromatographic analysis of rooibos (Aspalathus linearis) phenolics. J Sep Sci 2015;35:1808–20. [88] Kalili KM, de Villiers A. Systematic optimisation and evaluation of on-line, off-line and stop-flow comprehensive hydrophilic interaction chromatography × reversed phase liq-
References
[89]
[90] [91] [92]
[93]
uid chromatographic analysis of procyanidins. Part II: application to cocoa procyanidins. J Chromatogr A 2013;1289:69–79. Kalili KM, Vestner J, Stander MA, de Villiers A. Toward unraveling grape tannin composition: application of online hydrophilic interaction chromatography × reversed-phase liquid chromatography-time-of-flight mass spectrometry for grape seed analysis. Anal Chem 2013;85:9107–15. Jandera P, Staňkova M, Hájek T. New zwitterionic polymethacrylate monolithic columns for one- and two-dimensional microliquid chromatography. J Sep Sci 2013;36:2430–40. Willemse CM, Stander MA, Tredoux AGJ, de Villiers A. Comprehensive two-dimensional liquid chromatographic analysis of anthocyanins. J Chromatogr A 2014;1359:189–201. Willemse CM, Stander MA, Vestner J, Tredoux AGJ, de Villiers A. Comprehensive twodimensional hydrophilic interaction chromatography (HILIC) × reversed-phase liquid chromatography coupled to high-resolution mass spectrometry (RP-LC-UV-MS) analysis of anthocyanins and derived pigments in red wine. Anal Chem 2015;87:12006–15. Li D, Jakob C, Schimitz O. Practical considerations in comprehensive two-dimensional liquid chromatography systems (LCxLC) with reversed-phases in both dimensions. Anal Bioanal Chem 2015;407:153–67.
415