Liquid Chromatography

Recent Advances in Two-dimensional Liquid Chromatography

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Two-Dimensional Liquid Chromatography (2D-LC) has become a relatively mature research, development and application area due to significant advances made in hardware and software in recent years. 2D-LC is typically applied for the separation of very complex samples or when target components are very hard to resolve with a one-dimensional liquid chromatography (1D) separation. Details on the 2D-LC technology have recently been reviewed by Stoll and Carr in a tutorial article [1]. This present review article, covering publications from early 2016 until mid-2017, highlights recent applications using different 2D separation modes, such as comprehensive analysis and various types of heart-cutting. It also includes the latest developments in terms of modulation, i.e. the interfaces between the 1st and 2nd dimension, and software related to 2D-LC method development. 

Comprehensive 2D-LC

In comprehensive 2D-LC, often referred to as LC x LC, the entire effluent from the first dimension (1D) is transferred to the second dimension (2D) and the sample is subjected to separation in both dimensions. Depending on how the 1D effluent is transferred to 2D, LC x LC can be further categorised into ‘offline’ and ‘online’ mode. Online LC x LC is the focus in this review, because in contrast to offline LC x LC, online LC x LC is faster, easier to automate, and is less vulnerable to sample loss or degradation. Compared to offline LC x LC, online LC x LC is more difficult to employ and has much higher requirements on hardware and software. Recent advances in 2D-LC instrumentation and software, especially recent commercial offerings have significantly reduced the time and expertise required for the development of industrial applications and was critical for the proliferation of the technique.  

The biggest advantage of LC x LC to one-dimensional LC (1D-LC) is the greatly increased peak capacity (nc). In order to maximise the peak capacity, selecting stationary and mobile phases with different separation mechanisms for the two dimensions to achieve orthogonal separations is the key. Orthogonality in chromatography is a measure of how different the selectivity is between two separation dimensions. LC techniques encompass a wide variety of separation mechanisms, such as reversed phase (RPLC), normal phase (NPLC), hydrophilic interaction liquid chromatography (HILIC), ion exchange (IEC), size exclusion (SEC), etc. To achieve orthogonal separations, different combinations can be used with careful consideration of mobile phase compatibility, column dimension and flow rate match, and column regeneration if gradient elution is used. In this review, some recently reported LC x LC applications are highlighted about the combination of different separation mechanisms.

Among the different separation modes, RPLC x RPLC techniques were mostly practiced since fully compatible mobile phases are employed. Separation between both dimensions is achieved by using combinations of RP columns with different phase chemistry and selectivity. Recent publications include using cyano propyl stationary phase with C18 columns for phenolic samples [2], diol and C18 columns for phenolic compounds in canes [3], pentafluoro phenyl (PFP) with C18 columns for metabolite profiling [4], amino with C18 columns for bioactive compounds in fruits [5], and C18 with phenyl-hexyl columns  for Chinese medicines [6], etc. Fast chiral separations have been employed in second dimension for separation of pharmaceuticals and synthetic intermediates for chiral and achiral drugs and metabolites as well as constitutional and stereo isomers [7]. Although RP x RP is not considered the most orthogonal 2D separation, advanced instrument features such as shift gradient for second dimension separation have been applied to maximise the utilisation of the two-dimensional separation space. An example 2D shift gradient program is given in Figure 1 and a comparison of LC x LC separation of red wine using full gradient and shift gradient is shown in Figure 2 [2]. The shift gradient accounts for changes in polarity of constituents eluting from 1D and enables better separation in 2D. Besides different combinations of stationary phase chemistry, using different mobile phase pH for the two dimensions in RPLC x RPLC mode was also demonstrated to be an effective way to gain orthogonality for peptide samples due to the zwitterionic nature of the peptide [8].  

Figure 2. RPLC x RPLC analysis of red wine with a cyano column in 1D and a C18 column in 2D under optimised full gradient program (A) and shift gradient program (B). Adapted from reference [2].

Optimisation of LC x LC method can be laborious as many parameters can affect the overall performance of the LC x LC methods, such as column dimensions, particles sizes, flow rates, modulation time, 2D injection volume, gradient-elution program, temperature, etc. Modulation describes the event of sample collection from 1D and subsequent injection onto 2D. For LC x LC modulation time typically takes 30s – 1.5 min, which means the 2D separation needs to be very fast. To make the LC x LC method development more practical and less time-consuming, simulation and computer programs have been developed for the prediction of 2D-LC method performance [9, 10]. These models have been successfully applied to RP x RP type systems.

Another very promising combination of separation mechanisms for LC x LC involves the use of HILIC for 1D and RP conditions for 2D. Although the hyphenation of HILIC and RP in LC x LC is more complicated due to the mismatch of solvent strength used in each dimension and the relatively long column equilibration time for HILIC separation, HILIC x RP is considered more orthogonal compared to RP x RP and have been reported several times for metabolite profiling, bio-active compounds and phenolics and flavonoid compounds [11-14]. Strategies such as using trapping column to replace the loop or using micro-flow rate to reduce 2D injection volume have been exploited to minimise the strong solvent going from 1D to 2D column [12, 13, 15]. Alternating 1D separation mode between HILIC and RP using zwitterionic monolithic micro columns combined with a shift gradient for 2D has been reported as a novel system to obtain three-dimensional data in a relatively short time for the separation of flavones and related polyphenolic compounds [12].

Normal phase and reversed phase combinations (NP x RP) are also considered highly orthogonal, but the coupling of these two separation modes is more challenging due to the incompatibility of 1D and 2D mobile phases. Most of the reported NP x RP applications were not carried out in full comprehensive mode [16, 17]. Online NPLC x RPLC using vacuum evaporation assisted adsorption interface was recently reported for the isolation of toad venom. On-line solvent exchange within the two dimensions was achieved to eliminate the problem with incompatible mobile phases [18]. Separation of triblock copolymers composed of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks with regard to block composition has been reported using NP x RP with LC at critical conditions (LCCC) of PEO block and interactions of PPO block for 1D, and LCCC of PPO block and interactions of PEO block for 2D [19]. Two short columns of identical stationary phase are used for trapping the 1D analyte a few times before injecting onto the 2D column in order to conduct the separation in a comprehensive manner with a relatively slow second dimension separation. Using this technique, full resolution of all oligomers was accomplished (Figure 3).

Another combination of orthogonal separation mechanisms that have proven to be highly effective, especially for compounds containing both ionic and hydrophobic moieties, is ion exchange chromatography (IEC) with RPLC.  IEC x RPLC has been reported for applications ranging from synthetic dyes to proteins and monoclonal antibodies [20-22]. Due to the long equilibration time of IEC stationary phase and the time constraint of 2D separation, IEC is only deemed possible as first dimension in LC x LC. [21] 

A unique combination of separation mechanisms worth mentioning is the recently reported LC x LC analysis of nanoparticles by using hydrodynamic chromatography (HDC) in 1D and SEC in 2D to gain information on particle size and hydrodynamic radius simultaneously (Figure 4) [23]. Online dissolution of the particles eluting from the HDC column was exploited using the Agilent Jet Weaver V35 or V100 mixer and tetrahydrofuran (THF) as solvent before injecting onto the 2D SEC column.

As the needs for discovering new compounds (e.g. biomarkers, bioactives) continue to increase, using advanced separation techniques such as 2D-LC with high-end mass spectrometers for the identification, fractionation and isolation of complex samples such as crude natural products and extracts, biological membrane and fluids has become a promising and attractive approach [24-26]. Separation of microbial metabolites by online analytical and preparative LC x LC and isolation of milligram quantities of metabolites has been reported recently [27] and a review article on preparative 2D separations was also recently published [25].


Heart-cutting 2D-LC

Heart-cutting (HC) techniques are used for target analysis of one or multiple components, mostly in complex sample matrices [1]. With the respective hardware, 2D-LC can be executed in single or multiple heart-cutting modes. In many cases a single HC experiment can be sufficient to resolve co-elution of two critical components. Recent examples include metabolite profiling of vitamin D [28] and quantitative determination N-nitrosoamines in tobacco smoke [29]. Both applications have used tandem MS as the detection mode. A combined approach using HC and column switching has been reported for simultaneous metabolomics and lipidomics analysis [30]. 

Heart-cutting 2D-LC is already being considered as a tool for impurity analysis in pharmaceutical QC laboratories (see Figure 5) [31]. An in-depth method validation has shown good accuracy, sensitivity and robustness. Additional applications include a validated HC 2D-LC method for determination of vancomycin in human plasma [32] and clone selection for monoclonal antibodies (mAb) [33].

Multiple heart-cutting (MHC) 2D-LC is very powerful when several components are co-eluting in the 1D separation. Hereby fractions from 1D are stored in loops and are analysed subsequently in 2D. An advantage of multiple heart-cutting versus LC x LC operation is the decoupling of 2D and 1D. 2D analysis times are no longer constrained to match with the modulation frequency, allowing for somewhat slower and more optimised 2D runs. Method development effort is therefore reduced for MHC compared with that of LC x LC. 

Recent examples include analysis of impurities in peptides and proteins by 2D-LC/MS [34]. The 1D separation has a high salt content which presents a huge challenge for MS detection. The use of MHC eliminates the salt from 1D and led to superior MS performance, as suppression effects are significantly reduced in 2D. Additional applications of MHC are presented for the separation of target constituents in natural products-derived pesticides [35] and the determination of pyrrolizidine alkaloid isomers and their N-oximes with Q-TOF/MS detection [36]. Also, quantification of drugs in human plasma [37], determination of secondary metabolites in flowers [38] and advanced structural profiling of Chinese medicine formulas are described [6].

A related heart-cutting mode is called high-resolution sampling (or selective comprehensive 2D-LC [1]) – it is typically applied when only very narrow cuts can be made because of reduced solvent compatibility or when peak purity is studied. In such cases three or more consecutive cuts are made across one single peak [35]. On the other hand, MHC was applied in a low resolution mode (continuous MHC with modulation time of four min) for a 2D-LC ion mobility TOF MS investigation of a Ginkgo plant extract [39]. The low resolution in 1D creates single peaks for MS, which facilitates structure analysis.


The modulator is the interface between the two chromatographic dimensions. It transfers fractions of 1D effluent onto 2D.  In most cases a 10-port valve or an 8-port duo valve is used. Some research activities are on-going in search for alternative or improved modulation strategies.  

Compatibility of 1st and 2nd dimension mobile phases is sometimes an issue, as strong 1D eluents (methanol, acetonitrile, THF) can cause significant peak broadening and distortion or even sample breakthrough in 2D. Active modulation (AM) uses dilution of 1D eluent with weaker mobile phase [40] and subsequent trapping on solid-phase extraction (SPE) cartridges [41]. Higher loop filling factors can be used which leads to shorter run times. For a separation of aromatic ethoxylates analysis time could be reduced by a factor of five from 200 min to 40 min when AM was applied [41].

Stoll and colleagues presented an improved valve-based modulation strategy recently, called active solvent modulation (ASM) [42]. The 8-port duo-valve contains an additional loop which is used for temporary on-line dilution of 1D effluent with 2D eluent.  Solvent mismatch problems are reduced and much higher sensitivity can be obtained in 2D.  This is achieved by peak focusing with the low eluent strength mobile phase for the time when ASM is activated (see Figure 6).

An alternative concept for modulation was presented by Fornells and coworkers.  Modulation can be executed via evaporation of 1D mobile phase in microfluidic channels using a vacuum pump [43]. At this point, semi-volatile components were partially evaporated which led to reduced recovery.

Longitudinal thermal modulation for LC x LC was recently demonstrated [44]. A dual stage set-up for trapping and remobilisation on micro columns similar to that of GC x GC has been used. The modulator was applied to separate constituents in red wine and a peak width reduction of 30-60% was achieved due to peak focusing.

Fractionised sampling and stacking (FSS) was introduced as an interface for heart-cutting 2D-LC [45]. Here a combination of valves was used to dilute strong effluent from a 1D HILIC separation with weak eluent used for RP in 2D.

Pulsed elution 2D-LC was introduced by the group of Nielsen, Denmark [46]. A sequence of pulses of increasing elution strength is generated in 1D. Between the pulses 1D is kept at non-elution conditions using low elution strength mobile phase. The effluent from the first dimension is actively modulated using trap columns and subsequently analysed in the second dimension. With this approach enhanced compatibility of 1D and 2D is obtained. Long run times were required for this proof-of-concept study – 500-900 min when no-elution times of 5-10 min are applied.

In summary, many new applications and technical developments were seen during the last 18 months. In our opinion 2D-LC will continue to be a very active research area. We expect further advances to be made in hardware and software to facilitate 2D-LC method development. These developments will result in improved qualitative and quantitative measurements for a large variety of chemical and biochemical applications.


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6. X. Qiao, Q. Wang, W. Song, Y. Qian, Y. Xiao, R. An, D.A. Guo, M. Ye, A chemical profiling solution for Chinese medicine formulas using comprehensive and loop-based multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, J. Chromatogr. A, 1438 (2016) 198-204.

7. C.L. Barhate, E.L. Regalado, N.D. Contrella, J. Lee, J. Jo, A.A. Makarov, D.W. Armstrong, C.J. Welch, Ultrafast Chiral Chromatography as the Second Dimension in Two Dimensional Liquid Chromatography Experiments, Anal.Chem., 89 (2017) 3545-3553.

8. K. Sandra, G. Vanhoenacker, I. Vandenheede, M. Steenbeke, M. Joseph, P. Sandra, Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine, J. Chromatogr. B, 1032 (2016) 119-130.

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10. D.R. Stoll, R.W. Sajulga, B.N. Voigt, E.J. Larson, L.N. Jeong, S.C. Rutan, Simulation of elution profiles in liquid chromatography - II: Investigation of injection volume overload under gradient elution conditions applied to second dimension separations in two-dimensional liquid chromatography, J. Chromatogr. A, 1523 (2017) 162-172.

11. L. Montero, A.P. Sanchez-Camargo, V. Garcia-Canas, A. Tanniou, V. Stiger-Pouvreau, M. Russo, L. Rastrelli, A. Cifuentes, M. Herrero, E. Ibanez, 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, 1428 (2016) 115-125.

12. T. Hajek, P. Jandera, M. Stankova, P. Cesla, 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, 1446 (2016) 91-102.

13. E. Sommella, O.H. Ismail, F. Pagano, G. Pepe, C. Ostacolo, G. Mazzoccanti, M. Russo, E. Novellino, F. Gasparrini, P. Campiglia, Development of an improved online comprehensive hydrophilic interaction chromatography x reversed-phase ultra-high-pressure liquid chromatography platform for complex multiclass polyphenolic sample analysis, J. Sep. Sci., 40 (2017) 2188-2197.

14. L. Montero, E. Ibanez, M. Russo, R. di Sanzo, L. Rastrelli, A.L. Piccinelli, R. Celano, A. Cifuentes, M. Herrero, 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, 913 (2016) 145-159.

15. F. Cacciola, S. Farnetti, P. Dugo, P.J. Marriott, L. Mondello, Comprehensive two-dimensional liquid chromatography for polyphenol analysis in foodstuffs, J. Sep Sci., 40 (2017) 7-24.

16. L. Yang, P. Lv, W.P. Ai, L.N. Li, S.S. Shen, H.G. Nie, Y.B. Shan, Y. Bai, Y.N. Huang, H.W. Liu, Lipidomic analysis of plasma in patients with lacunar infarction using normal-phase/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry, Anal. Bioanal. Chem., 409 (2017) 3211-3222.

17. R. Weng, S.S. Shen, C. Burton, L. Yang, H.G. Nie, Y.L. Tian, Y. Bai, H.W. Liu, Lipidomic profiling of tryptophan hydroxylase 2 knockout mice reveals novel lipid biomarkers associated with serotonin deficiency, Anal. Bioanal. Chem., 408 (2016) 2963-2973.

18. J.F. Li, H. Fang, X. Yan, F.R. Chang, Z. Wu, Y.L. Wu, Y.K. Qiu, On-line comprehensive two-dimensional normal-phase liquid chromatography x reversed-phase liquid chromatography for preparative isolation of toad venom, J. Chromatogr. A, 1456 (2016) 169-175.

19. M.I. Malik, S. Lee, T. Chang, Comprehensive two-dimensional liquid chromatographic analysis of poloxamers, J. Chromatogr. A, 1442 (2016) 33-41.

20. Z. Huang, G.Q. Yan, M.X. Gao, X.M. Zhang, Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma, Anal. Chem., 88 (2016) 2440-2445.

21. B.W.J. Pirok, J. Knip, M.R. van Bommel, P.J. Schoenmakers, Characterization of synthetic dyes by comprehensive two-dimensional liquid chromatography combining ion-exchange chromatography and fast ion-pair reversed-phase chromatography, J. Chromatogr.  A, 1436 (2016) 141-146.

22. M. Sorensen, D.C. Harmes, D.R. Stoll, G.O. Staples, S. Fekete, D. Guillarme, A. Beck, Comparison of originator and biosimilar therapeutic monoclonal antibodies using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry, MAbs, 8 (2016) 1224-1234.

23. B.W.J. Pirok, N. Abdulhussain, T. Aalbers, B. Wouters, R.A.H. Peters, P.J. Schoenmakers, Nanoparticle Analysis by Online Comprehensive Two-Dimensional Liquid Chromatography combining Hydrodynamic Chromatography and Size-Exclusion Chromatography with Intermediate Sample Transformation, Anal. Chem. 89, (2017) 9167-9174.

24. A. Corgier, M. Sarrut, G. Cretier, S. Heinisch, Potential of Online Comprehensive Two-Dimensional Liquid Chromatography For Micro-Preparative Separations of Simple Samples, Chromatographia, 79 (2016) 255-260.

25. L. Marlot, K. Faure, Preparative two dimensional separations involving liquid-liquid chromatography, J. Chromatogr. A, 1494 (2017) 1-17.

26. X.Y. Ouyang, P.E.G. Leonards, Z. Tousova, J. Slobodnik, J. de Boer, M.H. Lamoreet, Rapid Screening of Acetylcholinesterase Inhibitors by Effect-Directed Analysis Using LC x LC Fractionation, a High Throughput in Vitro Assay, and Parallel Identification by Time of Flight Mass Spectrometry, Anal. Chem., 88 (2016) 2353-2360.

27. X. Yan, L.J. Wang, Z. Wu, Y.L. Wu, X.X. Liu, F.R. Chang, M.J. Fang, Y.K. Qiu, New on-line separation workflow of microbial metabolites via hyphenation of analytical and preparative comprehensive two-dimensional liquid chromatography, J. Chromatogr. B, 1033 (2016) 1-8.

28. A. Mena-Bravo, F. Priego-Capote, M.D. Luque de Castro, Two-dimensional liquid chromatography coupled to tandem mass spectrometry for vitamin D metabolite profiling including the C3-epimer-25-monohydroxyvitamin D3, J. Chromatogr. A, 1451 (2016) 50-57.

29. M. Chen, L. Wang, H. Dong, X. Shao, D. Wu, B. Liu, X. Zhang, C. Chen, Quantitative method for analysis of tobacco-specific N-nitrosamines in mainstream cigarette smoke by using heart-cutting two-dimensional liquid chromatography with tandem mass spectrometry, J. Sep. Sci., 40 (2017) 1920-1927.

30. S. Wang, L. Zhou, Z. Wang, X. Shi, G. Xu, Simultaneous metabolomics and lipidomics analysis based on novel heart-cutting two-dimensional liquid chromatography-mass spectrometry, Anal. Chim. Acta, 966 (2017) 34-40.

31. S.H. Yang, J. Wang, K. Zhang, Validation of a two-dimensional liquid chromatography method for quality control testing of pharmaceutical materials, J. Chromatogr. A, 1492 (2017) 89-97.

32. Y. Sheng, B. Zhou, High-throughput determination of vancomycin in human plasma by a cost-effective system of two-dimensional liquid chromatography, J. Chromatogr. A, 1499 (2017) 48-56.

33. K. Sandra, M. Steenbeke, I. Vandenheede, G. Vanhoenacker, P. Sandra, The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection, J. Chromatogr. A, 1523  (2017) 283-292.

34. P. Petersson, K. Haselmann, S. Buckenmaier, Multiple heart-cutting two dimensional liquid chromatography mass spectrometry: Towards real time determination of related impurities of bio-pharmaceuticals in salt based separation methods, J. Chromatogr. A, 1468 (2016) 95-101.

35. M. Pursch, P. Lewer, S. Buckenmaier, Resolving Co-Elution Problems of Components in Complex Mixtures by Multiple Heart-Cutting 2D-LC, Chromatographia, 80 (2017) 31-38.

36. M.G.M. van de Schans, M.H. Blokland, P.W. Zoontjes, P.P.J. Mulder, M.W.F. Nielen, Multiple heart-cutting two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry of pyrrolizidine alkaloids, J. Chromatogr. A, 1503 (2017) 38-48.

37. V.M.F. Goncalves, P. Rodrigues, C. Ribeiro, M.E. Tiritan, Quantification of alprenolol and propranolol in human plasma using a two-dimensional liquid chromatography (2D-LC), J. Pharm. Biomed. Anal., 141 (2017) 1-8.

38. M. Kula, D. Glod, M. Krauze-Baranowska, Application of on-line and off-line heart-cutting LC in determination of secondary metabolites from the flowers of Lonicera caerulea cultivar varieties, J. Pharm. Biomed. Anal., 131 (2016) 316-326.

39. S. Stephan, C. Jakob, J. Hippler, O.J. Schmitz, A novel four-dimensional analytical approach for analysis of complex samples, Anal. Bioanal. Chem., 408 (2016) 3751-3759.

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41. A.F.G. Gargano, M. Duffin, P. Navarro, P.J. Schoenmakers, Reducing Dilution and Analysis Time in Online Comprehensive Two-Dimensional Liquid Chromatography by Active Modulation, Anal. Chem., 88 (2016) 1785-1793.

42. D.R. Stoll, K. Shoykhet, P. Petersson, S. Buckenmaier, Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography, Anal. Chem., 89 (2017) 9260-9267.

43. E. Fornells, B. Barnett, M. Bailey, R.A. Shellie, E.F. Hilder, M.C. Breadmore, Membrane assisted and temperature controlled on-line evaporative concentration for microfluidics, J. Chromatogr. A, 1486 (2017) 110-116.

44. M.E. Creese, M.J. Creese, J.P. Foley, H.J. Cortes, E.F. Hilder, R.A. Shellie, M.C. Breadmore, Longitudinal On-Column Thermal Modulation for Comprehensive Two-Dimensional Liquid Chromatography, Anal. Chem., 89 (2017) 1123-1130.

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46. S.S. Jakobsen, J.H. Christensen, S. Verdier, C.R. Mallet, N.J. Nielsen, Increasing Flexibility in Two-Dimensional Liquid Chromatography by Pulsed Elution of the First Dimension: A Proof of Concept, Anal. Chem., 89 (2017) 8723-8730.


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