Nexera-e - Applications

Comprehensive Two-Dimensional Liquid Chromatograph

Introduction

The need for more powerful and discriminating analytical techniques has grown exponentially over the last decade, in order to handle the high complexity of many natural and synthetic samples.
To this regard, the implementation of multidimensional (MD-LC) and comprehensive two-dimensional liquid chromatography (LCxLC) systems has provided enhanced resolving power for highly complex samples containing hundreds of closely eluting components. The increase in separation power arises from the combination of multiple stationary phases, with different degree of orthogonality (selectivity). Whatever the specific separation technique, the use of mass spectrometry (MS) adds an additional dimension to the analytical LCxLC platform, enabling both reliable identification and the quantitative determination of compounds that may, or may not be, chromatographically fully resolved (Stoll et al., 2007; Dugo et al., 2008; Francois et al., 2009; Guiochon et al., 2009; Donato et al., 2012).

Sampling, Orthogonality, Peak capacity
The great complexity of real-world samples, in fact places a great demand in terms of resolution power, challenging separation scientists on one side, as well as column and hardware manufacturers, on the other. So far, major advances in column phase and technology included perfusive packings, partially porous particles, inorganic-organic hybrids, monoliths, high-temperature columns, sub-2 μm particles, and nanocolumns. In addition, high-purity silica and novel bonding chemistries have made LC columns more efficient and reliable, while commercial instrumentation capable of enhanced sensitivity, reliability, minimal band dispersion or ultra-high pressure operation has been developed, meeting the performance levels required for optimal operation.
Despite such substantial progress, and the considerable efforts made by analysts to develop chromatographic techniques taking full advantage of these developments, the separation efficiency required in many cases overwhelms the capability of any single-column method. As a consequence, much effort has been directed to the development of MD-LC systems, especially in the LCxLC mode, aiming to attain increased peak capacity, nc, viz. the total number of solutes that can be potentially separated, with regularly spaced adjacent peaks filling the available separation space.
The criteria defining LCxLC were postulated roughly two decades ago (Giddings J.C., 1990). The first criterion states that every part of the sample must be subjected to two different separations, that is, the two separation mechanisms must be ‘‘orthogonal’’. According to the second criterion, equal percentages of all the sample components should travel through both the chromatographic columns to reach the detector. The third fundamental requirement is that the separation obtained in the first dimension must not be impaired.
The need for orthogonality can be satisfied by careful choice of two dimensions with a different selectivity, in order to obtain non-correlated retention times in both dimensions. Column selectivity is therefore a primary concern in designing an MD-LC system. A large number of chromatographic separation modes, offering a wide range of selectivities (e.g. related to size, hydrophobicity, charge, etc.) can be exploited. For examples, ion exchange chromatography (IEC), which separate components on the basis of charge, is fairly orthogonal to reversed-phase (RP) LC, which separates on the basis of hydrophobicity; the use of a strong cation-exchange (SCX) column, coupled to either an RP- or ion-pair (IP)-LC turned out to be by far the most used combination of chromatographic separation modes for peptide analysis, delivering high orthogonality according to both charge and hydrophobicity. It must be noted that, usually, the second dimension consists of RPLC, due to its high compatibility in linkage with MS detection.
Beside the choice of stationary phases, selectivity of a separation can be further tuned by adjusting experimental parameters, such as: mobile phase composition and pH value, ion-pairing agents, elution (either isocratic or gradient), flow rate, temperature.
Theoretically, if two or more independent separation mechanisms are combined in a consecutive manner, the peak capacity, nc viz. the total number of solutes that can be potentially separated with regularly spaced adjacent peaks filling the available separation space, dramatically increases as a result of the product of the individual peak capacities: nc1 x nc2 x nc3 x… ncn; however, such an ideal situation is difficult to attain, due to inefficient transfer of separated zones from one dimension to the other, resulting in the remixing of resolving compounds (Giddings, 1984; Giddings, 1987). Theoretical and experimental work carried later showed that, in order to prevent loss of resolution attained in the first dimension, the sampling number per first-dimension peak should be at least three (Murphy et al., 1998). Such a requisite becomes particularly important if the first-dimension contribution towards the entire multidimensional separation is high. At the same time, this requisite poses demand for very fast second dimension separations, or the use of multiple second dimension columns. Additionally, interface dead-volumes and extra-column peak broadening contribute significantly to resolution losses.
More realistically, calculations for the peak capacity should take into account the effects of first- dimension undersampling, the selectivity correlation (orthogonality), and the retention window in both dimensions, which often does not cover the entire gradient duration (Gu et al, 2011; Liu et al., 1995; Camenzuli & Schoenmakers, 2014).

Instrumental Set-up and Method Development
Several strategies have been implemented by the researchers in designing LCxLC systems. The transfer of fractions, from the first to the second dimension, can be performed either in the off- or in the on-line mode; both techniques are widely employed and both present distinct pros and cons.
In off-line methods (LC/LC), fractions from the first chromatographic step are collected via a fraction collector, separated from the solvent by evaporation, re-dissolved and then re-injected onto the second column. The most striking advantage of such an approach derives from the great flexibility in coupling any separation modes, in terms of mobile phases and buffer compatibility, time and duration. The separation power can be maximized by independent optimization of each dimension, while sensitivity can be tuned by regulating the sample concentration injected in both dimensions.
Main drawbacks consist in the potential loss of sample or degradation during solvent evaporation, possible sample contamination or artefacts formation, difficulties in automation. Moreover, such methods are obviously more time-consuming and suffer from low analytical reproducibility. Discontinuous off-line methods via fraction collections have been advantageously employed in LC/LC separations of tryptic digests, when non-volatile buffers salts such as NaCl or KCl, or high amounts of organic solvent were needed to elute peptides in the first, SCX dimension, prior to the second RP one.
On-line approaches are more technically challenging and difficult to operate, with respect to off-line methods; on the other hand, they bring in the great advantage of fully-automated solute transfer between the two separation dimensions, with no flow interruption. As a consequence, they allow for greater analytical repeatability. Additionally, the reduced sample handling makes operation with unstable compounds more feasible and minimizes losses, an issue of particular relevance when dealing with limited sample amounts. A number of factors have to be taken into consideration, when developing an on-line 2D method, since the more stringent requirements render the coupling of different separation modes far more complicated with specific interfaces and software needed. Column type and dimensions, particle size, mobile phase flow rate and composition used in the two dimensions, need to be optimized both in terms of compatibility, and nc generation. The optimization of solvents and/or temperature gradients can help to increase the peak capacity, in both dimensions, while the mobile phase composition (eluting strength), the fraction volume and the transfer frequency will ultimately affect peak focusing at the head of the second-dimension column.
Mobile phase composition and viscosity can also generate excessive backpressure and contribute to band broadening phenomena.
The majority of on-line LCxLC configurations developed are based on the use of two-position, 10-, 8-, or 6-port switching valves, equipped with two sample loops. The two loops must be of identical volumes, and are alternately filled with the effluent from the first column; loop size is dependent on the mobile-phase volume per sampling period, and should at least be equal to the volume of a single fraction. The sampling period must, in turn, be equal to the time available for analysis in the second dimension, since the loop content is continuously re-injected onto the secondary column. For such reasons, the separation in 2D needs to be fast, a requirement that can be accomplished through the use of short columns, packed with small size particles, and with short re-equilibration times. Such columns are usually operated under gradient conditions, to minimize wrap-around phenomena and peak broadening; however, such operated conditions require re-conditioning steps at the end of each modulation cycle, sometimes resulting in higher background detection noise, since the mobile phase composition is modified rapidly. The use of monolithic columns and partially porous particles, or high temperature separations have proved to be very helpful in addressing such an issue. Both chromatographic dimensions can be operated under isocratic or gradient conditions; however, to avoid incompatibility issues between the two dimensions of the on-line set-up, the use of narrow- or micro-bore primary columns is mandatory to reduce mobile phase miscibility and the occurrence of possible solvent strength mismatch, which both affect system compatibility. The low flow-rates, typically employed with such columns, also reduces band broadening and help effective peak focusing at the head of the 2D column, thanks to the minimized mobile-phase strength, connected to the transfer from 1D to 2D. In contrast, wider bore columns (4.6 mm I.D.) have higher loading capacities, but require flow splitting prior to the interface, with consequent loss of sample. Alternatively, they can be operated at sub-optimum flow rates, the drawback in this case being a reduction of separation efficiency.
In the so-called “packed loop interface”, storage loops are replaced with trap columns packed with stationary phase; while the first trap column is loaded with 1D effluent, the sample components trapped in the previous cycle are released from the second trap loop, for subsequent 2D separation. Such a configuration allows re-concentration of the analytes, which are focused on the trap column prior to further chromatography; as a consequence, narrow sample bands can be introduced onto the second column. Since trapping efficiency and rapid desorption are inversely related, satisfactory method optimization of the operating parameters requires a weak solvent in 1D, to allow efficient focusing, while a strong solvent is necessary in 2D, to allow fast desorption. Parallel analytical columns can be used in alternative to trap columns, equipped with individual sample loops. Such a choice poses the stringent requirement of identical retention behaviour and efficiency, since analyses are performed on consecutive fractions in parallel, and the two resulting chromatograms are to be matched to generate a contour plot.
In the stop-flow technique, a valve is used to interface the two columns, without any storage loops or trap columns. At every switch of the valve, the flow in 1D is stopped, and the fraction collected is transferred for separation in 2D. After 2D separation, the primary flow and separation are restarted, to provide a new fraction. This type of approach is obviously more time demanding than on-line methods based on continuous flow, and its use is therefore limited to situations where only a limited number of 1D fractions need to be collected.

LCxLC-MS: Capabilities and Requirements
When designing an on-line LCxLC-MS technique, the detector response and the limited dynamic range of the instrument must be carefully considered. Tubing and valves used may, in fact, cause significant dilution and negatively affect the MS response, with the overall dilution factor being multiplicative of the dilution factors in each dimension. The use of trapping columns for fraction collection may alleviate such an issue, since analyte re-concentration occurs.
More stringent requirements are posed on the second dimension, which represent the back-end separation prior to the MS system, with regards to the column type, mobile phase composition and flow rate. RP chromatography is the most common choice, since typical mobile phases at the end of an RP separation contain high percentages of organic solvents and volatile additives, which are beneficial for MS ionization. With regards to column dimension, miniaturization (use of a microbore, 1.0 mm i.d. or capillary, 0.1-0.5 mm i.d. column) is also beneficial as far as dilution and sensitivity are concerned; in addition, reduced mobile-phase flow rates (μL to the nL range) avoids flow splitting prior to the MS source.
Another simple way to perform LCxLC-MS separations consists in the direct coupling of two or more columns, with orthogonal selectivities (typically based on SCX and RP materials) which are tandem-packed into a single capillary. Fractions are eluted from the first column with a series of pulsed steps of increasing elution strength, then followed by a continuous gradient, for analyte elution from the secondary column; the latter can be directly interfaced with an ESI (electrospray)/MS system. Minimizing peak dispersion is beneficial for direct MS detection, though such bi- or triphasic stationary phase segments can be applied in on-line set-up, because of the problems arising from limited compatibility, reproducibility, and flexibility.
The high selectivity of MS detection provides an enhanced identification capability, in discriminating non-isobaric compounds which are not (completely) chromatographically resolved. Mass spectrometry allows for quantitative determination to be carried out very accurately, precisely, and with high sensitivity (at the pg level), using isotopically-labeled compounds as internal standards. Unlike the UV detector, MS systems can also be employed with non-absorbing analytes, and can be operated in the full scan mode (TIC) or, more specifically, in tandem MS (MS-MS) experiments or in the selected ion monitoring (SIM) mode. SIM operation is preferred for the development of selective and sensitive quantitative assays, while tandem MS data, generated by using soft ionization techniques, provide structural information which can help in the identification of unknown analytes (Gross, 2004). However, the quantification, or even the detection of a target trace component, in SIM mode, can be difficult in the presence of high background ions with the same m/z values; in such cases, constant neutral loss or precursor ion scanning techniques help in distinguishing the ions of interest from unspecific matrix components simply by monitoring only those m/z values which originate from a characteristic fragmentation pattern. The so-called “selected reaction monitoring” (SRM) or “multiple reaction monitoring” (MRM) mode enhances selectivity and lowers detection limits, therefore reducing sample consumption; additionally, the SRM approach can also decrease analysis times by reducing the need for clean-up procedures.
The interest toward the implementation of LCxLC-MS systems is thus obvious, however a number of requirements and incompatibilities need to be addressed, make direct linkage of the two techniques a complicated issue.
The first obvious requirement is that of an interface, its primary purpose being the removal of the mobile phase before the analyte enters the high vacuum of the mass spectrometer. Second, the majority of analytes amenable to LC separation are low-volatility, thermally-labile compounds and, therefore, are not suited to either CI or EI ionization. The LC pump must be pulse-free, to deliver the mobile phase at a constant flow rate, which in turn must be compatible with the type of interface adopted, apart from the LC column inner diameter; such a requirement is necessary to provide a stable TIC trace of background ions. Mobile phase parameters such as surface tension, boiling point, and conductivity are likely to affect the operation of the interface, as well as the presence of non-volatile buffers (potassium or sodium phosphate) additives which are deposited onto the probe during evaporation of the mobile phase. These limitations in turn pose some restraints on the choice of the second-dimension stationary phase, for which a popular and straighforward option, consists in reversed-phase chromatography. To avoid a negative effect on the performance of the two separate instruments a number of requirements should be met by the LC-MS interface including: high ionization efficiency (to ensure high sample transfer to the MS); low chemical background (to minimize interference with the analysis); repeatable quantitative information (wide dynamic range at low limits of detection); no degradation of LC performance (especially in the case of multi-component mixtures); no reaction with the analytes (during transfer and introduction into the MS); ability to generate ions over a wide range of molecular weights. Operation of the interface should be compatible with the widest range of possible chromatographic conditions: mobile phase composition (0 to 100% aqueous), buffers and additives (no precipitation should occur), mobile-phase flow rates (nL to mL min-1 range), and gradient elution. The last requirement is mandatory in LCxLC-MS platforms, which are typically implemented to attain the separation of complex mixtures, the components of which may exhibit a broad range of polarities; hence, gradient elution is likely to be used in one or both of the chromatographic dimensions. On the mass spectrometer side, operation of interface should obviously not compromise the full capabilities of the MS detector, in terms of vacuum requirements, ionization modes, scanning rates, and resolution. The interface should ideally enable the generation of a mass spectrum for any component eluting from the LC system. In contrast to GC-MS, no single LC-MS interface possesses similar capabilities. As a consequence, attempts on coupling LC to MS have led to a variety of interfaces, as well as new ionization techniques.
Atmospheric pressure ionization (API) was the first technique to directly interface LC with MS, followed by thermospray (TSP) ionization and the particle beam interface (PBI). Nowadays, CF-FAB, API, TSP, PBI, and the moving belt have been almost entirely replaced by ESI, MALDI, atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI). LC-nanoESI operation has become feasible in recent years with the development of interfaces that are suitable for linkage with capillary-type LC columns, operated in the L-to-nL flow range. At the time of its introduction, nanoESI was operated in the direct infusion mode, with the sample being delivered, without prior separation, to the ion source via an injection valve; current configurations use gold-coated capillaries or automated chips, and allow analyte detection down to the femtomoles level, thus boosting the sensitivity of LC-MS techniques.
In LCxLC-MS platforms, a fast MS acquisition rate is often necessary, since the second-dimension separation can be very rapid, and peak widths characterized by a few seconds or less are not unusual. To adequately sample the 2D effluent, the mass analyzer should be capable of acquiring at least 6-10 data points per peak. In parallel, with the progress in interface technology, the older sector machines have now been replaced by ion trapping instruments, quadrupoles, and time of flight systems. A variety of hybrid instruments, of later development, are commercially- available, and are characterized by high resolution, enhanced sensitivity, as well as increased mass accuracy over a wide dynamic range; among such systems, the ion mobility TOF, quadrupole TOF (Q-TOF), ion trap-TOF (IT-TOF), and linear ion trap-Fourier transform ion cyclotron resonance (FT-ICR), can be mentioned. The unprecedented accuracy, speed and resolution of the most advanced devices currently marketed have definitely concurred to bring mass spectrometry to a central role in both basic and applied present-day research.
Quadrupole-type instruments have been the most commonly used, in conjunction with LC or LCxLC, because of simplicity of use, relatively low cost and ruggedness; however, traditional quadrupoles offer low resolution (typically 1 Da) and present a limited mass range limited. On the other hand, ultimate generation single-quadrupole instrumentations allow for high speed scanning and ultrafast polarity switching, for high-speed analyses. It must be emphasized that the small size and the possibility to perform tandem MS, make quadrupole analyzers ideal for benchtop LC-MS and LC-MSn applications.
TOF instruments present the advantage of high scan speed, high resolution (using a reflectron), and virtually no limit on mass range (simultaneous detection of all ions in the mass range occurs, with no need for scanning). Maximum sensitivity is obtained, irrespective if a single ion or a full mass spectrum is analyzed; furthermore, the high mass resolution allows the prediction of precise chemical formulae, in the identification of unknown compounds. TOF systems are also ideal as a second stage in tandem MS experiments, in combination with either an ion-trap (IT-TOF) or a quadrupole (Q-TOF).
From the quantitative standpoint, the linear dynamic range depends on the type of source employed; ESI is characterized by a dynamic range over 2-3 orders of magnitude, and currently represents the most common choice for routine LC-MS users in quantitative analysis. However, APCI and APPI techniques offer greater sensitivity and wider dynamic range (4-5 orders of magnitude), though their use for large biomolecules is precluded.

References
Camenzuli, M., Schoenmakers, P.J., Anal. Chim. Acta 838 (2014) 93.
Donato, P., Cacciola, F., Tranchida, P.Q., Dugo, P., Mondello, L. 2012 Mass Spectrom. Rev. 31 (2012) 523.
Dugo P., Cacciola F., Kumm T., Dugo G., Mondello L., J. Chromatogr. A 1184 (2008) 353.
Francois I., Sandra K., Sandra P., Anal Chim Acta 641 (2009) 14.
Guiochon G., Marchetti M., Mriziq K., Shalliker R.A., J. Chromatogr. A 1189 (2009) 109.
Giddings J.C. Anal. Chem. 56 (1984) 1258A.
Giddings J.C., J. High Res. Chromatogr. 10 (1987) 319.
Gross J.H. (2004). Mass spectrometry. A textbook. Berlin Heidelberg:Springer-Verlag
Gu, H., Huang, Y., Carr, P.W., J. Chromatogr. A 1218 (2011) 64.
Liu Z., Patterson D.G., Lee M., Anal. Chem. 67 (1995) 3840.
Murphy R.E., Schure M.R., Foley J.P., Anal. Chem. 70 (1998) 4353-4360.
Stoll D.R., Li X., Wang X., Carr P.W., Porter S.E.G., Rutan S.C., J. Chromatogr. A 1168 (2007) 3.

Applicable type of compounds

LCxLC and LCxLC-MS techniques can be applied to several research fields. The majority of the applications developed so far deals with proteins (intact or digested), food products, pharmaceutical, biological, organic and environmental samples. Moreover, a few applications report on synthetic and natural polymers and oligomers.

LCxLC of Intact and Digested Proteins
The term ‘‘proteomics’’ deals with the characterization, identification and quantification of proteins in cells, tissues, or biological fluids and, also involves the assay of protein interactions, to perform a given cellular task, or as key players in a number of diseases. To further complicate the situation, different environmental, biological (even individual), pharmacological, and disease factors ultimately affect protein expression, and determine statistically significant variations.
Traditionally, 2D-GE followed by MS identification has been at the forefront in this field, given its excellent resolving power for intact proteins. However, such a technique is affected by several disadvantages that led comprehensive liquid chromatography coupled to mass spectrometry to have a central role in the field of proteomic research as a convenient alternative to the more laborious, less comprehensive gel-based methods.
Different separation modes have been exploited to deliver a wide range of selectivities in the 1D, such SCX or strong-anion exchange (SAX), size exclusion chromatography (SEC), or hydrophilic interaction liquid chromatography (HILIC). In contrast, RP LC is usually employed in the 2D, due to its high MS-compatibility. The most common RP stationary phases used are octadecylsilica (ODS), commercially available in a variety of column lengths, internal diameters, particle and pore size, as well as pH stabilities, and hydrophobicity. Hydrophobic interactions are responsible for peptide separation under gradient conditions, typically employing acetonitrile (ACN) as the organic modifier, and trifluoroacetic acid (TFA) or formic acid (FA) as ion-pair reagents. The use of low percentages of organic solvent allows effective on-line desalting and concentration of peptides at the same time, providing excellent MS compatibility.
On one hand, intact proteins are difficult to handle, making their LC separation and subsequent MS/MS detection quite challenging. On the other hand, their digestion with a proteolytic enzyme (usually, trypsin) dramatically increases sample complexity. As a consequence, LCxLC improves fractionation of the peptides, according to their charge and hydrophobicity, increases ionization efficiency, and reduces the complexity of the sample prior to MS detection. Furthermore, the separation of low-abundant peptides, from more abundant species, ultimately result in an overall increase in dynamic range, addressing typical MS detection issues, such as ion suppression and undersampling.

LCxLC of Food Products
Food products are complex mixtures containing both organic and inorganic constituents; their analysis is generally directed to the assessment of food safety and authenticity, the control of a technological process, the determination of nutritional values and the detection of molecules with a possible beneficial or a toxic effect on human health. As a consequence, one of the most stringent demands of food chemistry is directed toward the continuous improvement and development of powerful analytical techniques, aiming to allow analysis of the main components of food samples, as well as of the minor components.
To unambiguously identify such molecules, LCxLC hyphenated to MS represents a useful tool, especially when commercial standards are not available.
LCxLC systems, developed and applied to the analysis of food compounds, employed the combination of NPxRP, RPxRP and HILICxRP separation modes. In NP mode, two different separation methods were explored: adsorption with polar inorganic packing material, generally silica, and partition, in which a polar organic phase is chemically bonded to the silica substrate, such as cyanopropyl (CN) or diol groups. Among NP-LCxRP-LC approaches, a partition mechanism can be considered also for the combination of silver ion chromatography in combination with non aqueous reversed phase chromatography (SICxNARP-LC) employed for triacylglycerol analysis. On the other hand, HILICxRP-LC and RP-LCxRP-LC set-up by using different stationary phase chemistries have been employed for analysis of phospholipids in milk samples and polyphenols in various food-related products.

LCxLC of Pharmaceutical, Biological, Organic and Environmental Compounds
Over the last few years, pharmaceuticals have been gaining an increasing interest, with the main attention devoted to the assessment of pharmaceutically active compounds, as well as their isolation from all potential impurities and/or degradation products. Pharmaceutical impurities can be classified as organic, inorganic, and residual solvents used for regulatory purposes and can originate from alterations of raw material, or as synthetic intermediates, under either reaction (temperature, pH) or storage conditions (hydrolysis, oxidation, ring opening, etc.), leading to very complex mixtures. For such a reason, conventional LC–MS systems often fail in the detection of such impurities, thus requiring more powerful alternatives such as LCxLC mainly with RP stationary phases in both dimensions, in combination with MS, for unambiguous determination.

LCxLC of Synthetic and Natural Polymers and Oligomers
Liquid chromatography represents a powerful tool for the molecular characterization of polymers, the latter having a multivariate distribution in molecular characteristics, such as molecular weight, chain architecture, chemical composition and functionality. The LC characterization of polymers can be divided into three different modes: size exclusion, critical conditions, and interaction modes. Size-exclusion chromatography is the most widely employed technique in the molecular characterization of polymers, due to its high speed, simplicity of use and wide applicability. For the analysis of synthetic and natural polymers and oligomers, LCxLC approaches in combination with MALDI-TOF-MS were investigated. With regards to the first dimension, different separation modes have been used namely, NP-GPEC (normal phase gradient polymer elution chromatography), LC-CC (liquid chromatography at critical conditions) and RPLC, whereas for the second dimension, a SEC column has been employed in all cases. While polymer separation on an SEC column is exclusively based on molecular weight, the LC-CC technique separates polymers in relation to their molecular characteristics, such as specific composition or functionality.


 

Why and where LCxLC and LCxLC/MS is required

Comprehensive LC is a viable tool for analysis of complex food samples thanks to the practical selection of separation systems with low selectivity correlations in the two dimensions.
The increasingly high interest in the last decade can be traced back to the commercial availability of ready-to-use LCxLC systems equipped with dedicated software for both method development and data handling, allowing qualitative and quantitative purposes. Further noteworthy improvements will be the exploiting of novel separation materials with very fine particle size, e.g. sub-2 μm, and the development of analytical methods capable to reduce 2D analysis time for increased sample throughput and to minimize the sensitivity loss with respect to conventional (monodimensional) systems. To this purpose, the use of narrow-bore or micro-bore columns in 2D may help to avoid flow-rate splitting from the LC system prior to the MS probe, and thus reduce the dilution.
The implementation of LCxLC and, to a greater extent, LCxLC-MS analytical platforms can be of great help in addressing a number of important issues:
 


  • Attain more robust quantification (reliable data to be obtained from baseline-resolved peaks)

  • Perform (semi-)preparative analysis (for isolation of compounds of interest for further use)

  • Avoid sample extraction/purification (MS provides an added separation/selectivity dimension)

  • Reduce false positives (isobaric compounds are time-resolved prior to MS detection)

  • Allow for targeted and untargeted/post-targeted analysis (reduce sample complexity and improve the quality of MS spectra)
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