Due to increasing demands for product quality from regulatory environment and increased sensitivity of HPLC detectors, analytics in general is getting more and more complex. The visibility of components in a sample is constantly increasing. Therefore, the requirements for separation science to resolve such complex mixtures are raised to a new level and to its limits. New, automated, industy 4.0 compatible approaches must be implemented to meet the new separation scientific challenges. Multidimensional separation techniques are necessary for quick and easy solutions. In this lecture the possibilities and limits of separation science concerning plate count (peak capacity, respectively), selectivity and speed are demonstrated and possibilities to overcome separation challenges for HPLC are discussed. A workflow to achieve orthogonal selectivity is demonstrated in some complex application areas. Finally a vision for the HPLC lab of the future is given.

Due to increasing demands for product quality from regulatory environment and increased sensitivity of HPLC detectors, analytics in general is getting more and more complex. The visibility of components in a sample is constantly increasing. Therefore, the requirements for separation science to resolve such complex mixtures are raised to a new level and to its limits. New, automated, industy 4.0 compatible approaches must be implemented to meet the new separation scientific challenges. Multidimensional separation techniques are necessary for quick and easy solutions. In this lecture the possibilities and limits of separation science concerning plate count (peak capacity, respectively), selectivity and speed are demonstrated and possibilities to overcome separation challenges for HPLC are discussed. A workflow to achieve orthogonal selectivity is demonstrated in some complex application areas. Finally a vision for the HPLC lab of the future is given.

Due to increasing demands for product quality from regulatory environment and increased sensitivity of HPLC detectors, analytics in general is getting more and more complex. The visibility of components in a sample is constantly increasing. Therefore, the requirements for separation science to resolve such complex mixtures are raised to a new level and to its limits. New, automated, industy 4.0 compatible approaches must be implemented to meet the new separation scientific challenges. Multidimensional separation techniques are necessary for quick and easy solutions. In this lecture the possibilities and limits of separation science concerning plate count (peak capacity, respectively), selectivity and speed are demonstrated and possibilities to overcome separation challenges for HPLC are discussed. A workflow to achieve orthogonal selectivity is demonstrated in some complex application areas. Finally a vision for the HPLC lab of the future is given.

Hydrophilic interaction liquid chromatography (HILIC) appears today as an attractive technique for analysis of polar and/or ionizable compounds. Although HILIC chromatography is known to provide valuable retention and selectivity of polar compounds and provide highly compatible conditions for coupling with mass spectrometry, it often is avoided due to issues surrounding robustness and repeatability. On the contrary, the separation of hydrophobic compounds is achieved by highly reproducible reversed phase liquid chromatography (RPLC). Amphiphilic compounds bearing both hydrophobic and hydrophilic properties, however, are in between. Furthermore, the analysis of complex samples necessitates a broad coverage of polarities even when applying selective high-resolution mass spectrometry detection. Therefore, chromatographic methods will be presented to address the mentioned challenges using the example of lipids. Given the chemical diversity of lipids and their biological relevance, suitable methods for lipid profiling and quantification are demanded to reduce sample complexity and analysis times. An online coupling of HILIC dedicated to class-specific separation of polar lipid classes to RPLC for nonpolar lipid analysis was developed. In this setup, the orthogonal HILIC and RP separations were performed serially, offering the full separation space in one analytical run. Furthermore, heart-cut online two-dimensional LC can be used for enrichment and tailored analysis of minor lipid classes. For example, a fast HILIC method was developed for the separation of cardiolipins and their oxidation products from other phospholipid classes, but more important from nonpolar lipid classes, such as triacylglycerols and cholesterol. Those classes can negatively affect the electrospray ionization and the chromatography. For the heart-cut approach, the cardiolipin fraction was selectively transferred to a loop using a six-port valve followed by the transfer to a RP column in second dimension. On the RP column, the transferred cardiolipin fraction including the oxidation products were separated according to the hydrophobicity of fatty acid residues. Matrix effects were reduced significantly compared to the onedimensional approach. The sole application of either RPLC or HILIC offers complementary modes of separation to address todays need for complex sample analysis. By two-dimensional approaches, the information content and the peak capacity can be magnified.

Hydrophilic interaction liquid chromatography (HILIC) appears today as an attractive technique for analysis of polar and/or ionizable compounds. Although HILIC chromatography is known to provide valuable retention and selectivity of polar compounds and provide highly compatible conditions for coupling with mass spectrometry, it often is avoided due to issues surrounding robustness and repeatability. On the contrary, the separation of hydrophobic compounds is achieved by highly reproducible reversed phase liquid chromatography (RPLC). Amphiphilic compounds bearing both hydrophobic and hydrophilic properties, however, are in between. Furthermore, the analysis of complex samples necessitates a broad coverage of polarities even when applying selective high-resolution mass spectrometry detection. Therefore, chromatographic methods will be presented to address the mentioned challenges using the example of lipids. Given the chemical diversity of lipids and their biological relevance, suitable methods for lipid profiling and quantification are demanded to reduce sample complexity and analysis times. An online coupling of HILIC dedicated to class-specific separation of polar lipid classes to RPLC for nonpolar lipid analysis was developed. In this setup, the orthogonal HILIC and RP separations were performed serially, offering the full separation space in one analytical run. Furthermore, heart-cut online two-dimensional LC can be used for enrichment and tailored analysis of minor lipid classes. For example, a fast HILIC method was developed for the separation of cardiolipins and their oxidation products from other phospholipid classes, but more important from nonpolar lipid classes, such as triacylglycerols and cholesterol. Those classes can negatively affect the electrospray ionization and the chromatography. For the heart-cut approach, the cardiolipin fraction was selectively transferred to a loop using a six-port valve followed by the transfer to a RP column in second dimension. On the RP column, the transferred cardiolipin fraction including the oxidation products were separated according to the hydrophobicity of fatty acid residues. Matrix effects were reduced significantly compared to the onedimensional approach. The sole application of either RPLC or HILIC offers complementary modes of separation to address todays need for complex sample analysis. By two-dimensional approaches, the information content and the peak capacity can be magnified.

Hydrophilic interaction liquid chromatography (HILIC) appears today as an attractive technique for analysis of polar and/or ionizable compounds. Although HILIC chromatography is known to provide valuable retention and selectivity of polar compounds and provide highly compatible conditions for coupling with mass spectrometry, it often is avoided due to issues surrounding robustness and repeatability. On the contrary, the separation of hydrophobic compounds is achieved by highly reproducible reversed phase liquid chromatography (RPLC). Amphiphilic compounds bearing both hydrophobic and hydrophilic properties, however, are in between. Furthermore, the analysis of complex samples necessitates a broad coverage of polarities even when applying selective high-resolution mass spectrometry detection. Therefore, chromatographic methods will be presented to address the mentioned challenges using the example of lipids. Given the chemical diversity of lipids and their biological relevance, suitable methods for lipid profiling and quantification are demanded to reduce sample complexity and analysis times. An online coupling of HILIC dedicated to class-specific separation of polar lipid classes to RPLC for nonpolar lipid analysis was developed. In this setup, the orthogonal HILIC and RP separations were performed serially, offering the full separation space in one analytical run. Furthermore, heart-cut online two-dimensional LC can be used for enrichment and tailored analysis of minor lipid classes. For example, a fast HILIC method was developed for the separation of cardiolipins and their oxidation products from other phospholipid classes, but more important from nonpolar lipid classes, such as triacylglycerols and cholesterol. Those classes can negatively affect the electrospray ionization and the chromatography. For the heart-cut approach, the cardiolipin fraction was selectively transferred to a loop using a six-port valve followed by the transfer to a RP column in second dimension. On the RP column, the transferred cardiolipin fraction including the oxidation products were separated according to the hydrophobicity of fatty acid residues. Matrix effects were reduced significantly compared to the onedimensional approach. The sole application of either RPLC or HILIC offers complementary modes of separation to address todays need for complex sample analysis. By two-dimensional approaches, the information content and the peak capacity can be magnified.

In the more than forty years that encompass its birth and development, ion chromatography (IC) has undergone enormous changes. While in its earliest embodiments IC was focused primarily on the analysis of inorganic anions, today IC plays an important role in the analysis of organic and inorganic anions and cations. Although separations of ions by ion-exchange chromatography prevail, other liquid chromatography techniques such as ion-exclusion chromatography, reversed-phase liquid chromatography in the ion-suppression mode, and even hydrophilic interaction and mixed-mode liquid chromatography are also used today. Thus, the definition of the term ion chromatography became much broader over the years to be an umbrella term today for all liquid chromatographic techniques that are suitable for separating and detecting ionic and ionisable species. Although the method of ion chromatography is well matured and widely accepted today, the past decade has seen a number of exciting developments in ion chromatography that further established this analytical technique. Of particular importance are the new 4 µm ion-exchange packing materials. The pathway of method speedup in ion chromatography follows the one used in conventional HPLC with the transition to UHPLC techniques. In ion chromatography, however, the pathway of using smaller particle sizes and smaller column formats can only be followed to a certain extent due to the limited back pressure tolerance of metal-free components in the fluidic system of IC instruments. Progress in the design of cation exchangers has been made toward new stationary phases for improved separations of amines. Over the past decade, electrolytic eluent generation (RFIC) has been well established as an alternative to manually prepared eluents. RFIC not only facilitates the use of gradient elution techniques in ion chromatography, but also provides the user with more consistent data. Electrolytic eluent generation was also a prerequisite for the development of Capillary Ion Chromatography. As an analytical tool, capillary IC offers several important advantages, including higher mass sensitivity, compatibility with smaller sample volumes, and significant reduction of eluent consumption and associated waste disposal. In addition, the very small flow rates used in capillary IC enable the permanent operation of the system over a long period of time, thus eliminating time-consuming and error prone steps such as system startup and equilibration as well as manual eluent preparation. The enhanced stability of capillary IC systems increases laboratory productivity, as fewer calibration sequences are required and the system can quickly be verified for system performance by just running check standards, which is of utmost importance for the pharmaceutical industry. For the majority of applications, conductivity detection with suppression devices represents the most versatile detection system augmented today with a variety of other detection modes such as UV/Vis and amperometry. A growing number of applications are based on hyphenation, thus coupling ion chromatography with ICP-OES, ICP-MS, and ESI-MS. The advantage of coupling ICP with ion chromatography includes the ability to separate and detect metals with different oxidation states. The analytical interest in chemical speciation is based on the fact that the oxidation state of an element determines toxicity, environmental behavior, and biological effects. Hyphenation with ESI-MS provides the analyst with mass-selective information. Challenging applications such as the determination of haloacetic acids and polar pesticides at trace levels by IC-ESI-MS/MS or the identification and the quantification of metabolites by coupling capillary IC to an Orbitrap MS clearly demonstrate the need for MS hyphenation to achieve the required sensitivity and selectivity. Amino acids and carbohydrates are also analyzed by anion-exchange chromatography. In combination with integrated pulsed amperometry as a direct detection method for amino acids and carbohydrates, anion-exchange chromatography revolutionized these two application areas. Thus, ion chromatography has become almost indispensable for the analysis of low- and high-molecular weight inorganic and organic anions and cations.

In the more than forty years that encompass its birth and development, ion chromatography (IC) has undergone enormous changes. While in its earliest embodiments IC was focused primarily on the analysis of inorganic anions, today IC plays an important role in the analysis of organic and inorganic anions and cations. Although separations of ions by ion-exchange chromatography prevail, other liquid chromatography techniques such as ion-exclusion chromatography, reversed-phase liquid chromatography in the ion-suppression mode, and even hydrophilic interaction and mixed-mode liquid chromatography are also used today. Thus, the definition of the term ion chromatography became much broader over the years to be an umbrella term today for all liquid chromatographic techniques that are suitable for separating and detecting ionic and ionisable species. Although the method of ion chromatography is well matured and widely accepted today, the past decade has seen a number of exciting developments in ion chromatography that further established this analytical technique. Of particular importance are the new 4 µm ion-exchange packing materials. The pathway of method speedup in ion chromatography follows the one used in conventional HPLC with the transition to UHPLC techniques. In ion chromatography, however, the pathway of using smaller particle sizes and smaller column formats can only be followed to a certain extent due to the limited back pressure tolerance of metal-free components in the fluidic system of IC instruments. Progress in the design of cation exchangers has been made toward new stationary phases for improved separations of amines. Over the past decade, electrolytic eluent generation (RFIC) has been well established as an alternative to manually prepared eluents. RFIC not only facilitates the use of gradient elution techniques in ion chromatography, but also provides the user with more consistent data. Electrolytic eluent generation was also a prerequisite for the development of Capillary Ion Chromatography. As an analytical tool, capillary IC offers several important advantages, including higher mass sensitivity, compatibility with smaller sample volumes, and significant reduction of eluent consumption and associated waste disposal. In addition, the very small flow rates used in capillary IC enable the permanent operation of the system over a long period of time, thus eliminating time-consuming and error prone steps such as system startup and equilibration as well as manual eluent preparation. The enhanced stability of capillary IC systems increases laboratory productivity, as fewer calibration sequences are required and the system can quickly be verified for system performance by just running check standards, which is of utmost importance for the pharmaceutical industry. For the majority of applications, conductivity detection with suppression devices represents the most versatile detection system augmented today with a variety of other detection modes such as UV/Vis and amperometry. A growing number of applications are based on hyphenation, thus coupling ion chromatography with ICP-OES, ICP-MS, and ESI-MS. The advantage of coupling ICP with ion chromatography includes the ability to separate and detect metals with different oxidation states. The analytical interest in chemical speciation is based on the fact that the oxidation state of an element determines toxicity, environmental behavior, and biological effects. Hyphenation with ESI-MS provides the analyst with mass-selective information. Challenging applications such as the determination of haloacetic acids and polar pesticides at trace levels by IC-ESI-MS/MS or the identification and the quantification of metabolites by coupling capillary IC to an Orbitrap MS clearly demonstrate the need for MS hyphenation to achieve the required sensitivity and selectivity. Amino acids and carbohydrates are also analyzed by anion-exchange chromatography. In combination with integrated pulsed amperometry as a direct detection method for amino acids and carbohydrates, anion-exchange chromatography revolutionized these two application areas. Thus, ion chromatography has become almost indispensable for the analysis of low- and high-molecular weight inorganic and organic anions and cations.

In the more than forty years that encompass its birth and development, ion chromatography (IC) has undergone enormous changes. While in its earliest embodiments IC was focused primarily on the analysis of inorganic anions, today IC plays an important role in the analysis of organic and inorganic anions and cations. Although separations of ions by ion-exchange chromatography prevail, other liquid chromatography techniques such as ion-exclusion chromatography, reversed-phase liquid chromatography in the ion-suppression mode, and even hydrophilic interaction and mixed-mode liquid chromatography are also used today. Thus, the definition of the term ion chromatography became much broader over the years to be an umbrella term today for all liquid chromatographic techniques that are suitable for separating and detecting ionic and ionisable species. Although the method of ion chromatography is well matured and widely accepted today, the past decade has seen a number of exciting developments in ion chromatography that further established this analytical technique. Of particular importance are the new 4 µm ion-exchange packing materials. The pathway of method speedup in ion chromatography follows the one used in conventional HPLC with the transition to UHPLC techniques. In ion chromatography, however, the pathway of using smaller particle sizes and smaller column formats can only be followed to a certain extent due to the limited back pressure tolerance of metal-free components in the fluidic system of IC instruments. Progress in the design of cation exchangers has been made toward new stationary phases for improved separations of amines. Over the past decade, electrolytic eluent generation (RFIC) has been well established as an alternative to manually prepared eluents. RFIC not only facilitates the use of gradient elution techniques in ion chromatography, but also provides the user with more consistent data. Electrolytic eluent generation was also a prerequisite for the development of Capillary Ion Chromatography. As an analytical tool, capillary IC offers several important advantages, including higher mass sensitivity, compatibility with smaller sample volumes, and significant reduction of eluent consumption and associated waste disposal. In addition, the very small flow rates used in capillary IC enable the permanent operation of the system over a long period of time, thus eliminating time-consuming and error prone steps such as system startup and equilibration as well as manual eluent preparation. The enhanced stability of capillary IC systems increases laboratory productivity, as fewer calibration sequences are required and the system can quickly be verified for system performance by just running check standards, which is of utmost importance for the pharmaceutical industry. For the majority of applications, conductivity detection with suppression devices represents the most versatile detection system augmented today with a variety of other detection modes such as UV/Vis and amperometry. A growing number of applications are based on hyphenation, thus coupling ion chromatography with ICP-OES, ICP-MS, and ESI-MS. The advantage of coupling ICP with ion chromatography includes the ability to separate and detect metals with different oxidation states. The analytical interest in chemical speciation is based on the fact that the oxidation state of an element determines toxicity, environmental behavior, and biological effects. Hyphenation with ESI-MS provides the analyst with mass-selective information. Challenging applications such as the determination of haloacetic acids and polar pesticides at trace levels by IC-ESI-MS/MS or the identification and the quantification of metabolites by coupling capillary IC to an Orbitrap MS clearly demonstrate the need for MS hyphenation to achieve the required sensitivity and selectivity. Amino acids and carbohydrates are also analyzed by anion-exchange chromatography. In combination with integrated pulsed amperometry as a direct detection method for amino acids and carbohydrates, anion-exchange chromatography revolutionized these two application areas. Thus, ion chromatography has become almost indispensable for the analysis of low- and high-molecular weight inorganic and organic anions and cations.

Over decades chiral separation technologies have developed into seemingly mature analytical techniques. A large number of chiral stationary phases are nowadays commercially available and allow separation of all kind of enantiomers or stereoisomers. Chiral stationary phases (CSPs) based on polysaccharide selectors are dominating the field, followed by glycopeptide antibiotics CSPs. A variety of alternatives exist if these columns fail. Industry has established extended automated screening programs, both by HPLC but also SFC, to figure out the most suitable chiral column for specific application problems. High throughput is a serious demand in those column screens because in initial drug discovery a large number of potential drug entities must be screened. To cope with this demand, most of the successful selector chemistries have meanwhile been transferred from standard 5 µm fully porous particle (FPP) technologies to superficially porous particle (SPP) or sub-2µm FPP particle technologies. While enantioselectivity was mostly maintained, the new modern particle technologies allow much faster separations. Especially SPP technologies seem to be advantageous and chiral separations at sub-second speed have been illustrated. Subminute separations are of course a great benefit not only in high-throughput screening but also for enantioselective 2D-LC. Fast chiral separations with such modern columns can be coupled to non-enantioselective RPLC separations in the first dimension in order to separate complex mixtures of enantiomers by comprehensive RP×chiral or specific difficult to separate mixtures of stereoisomers by multiple heart cut RP-chiral separation systems. Such a setup has shown applicability for enantiomer separations of the entire set of proteinogenic amino acids and some isobaric/isomeric congeners in the context of non-ribosomal peptides and therapeutic peptides, respectively. We have established such a multiple heart cut enantioselective RP-chiral separation system with SPP type chiral column based on quinine carbamate selector. Twenty five (25) amino acids have been enantioresolved in a run time of about 100 min. Meanwhile the run time could be reduced to 60 min. Also comprehensive chiral×chiral 2DLC setup has been established. It gives particular elution patterns of amino acid enantiomers which may allow straightforward data processing. If such analysis is performed in presence of real matrix, hyphenation of such separation systems to mass spectrometry is mandatory. Enantioselective matrix effects, however, may complicate the MS analysis and lead to inaccurate data. Solutions are discussed in this presentation.

Over decades chiral separation technologies have developed into seemingly mature analytical techniques. A large number of chiral stationary phases are nowadays commercially available and allow separation of all kind of enantiomers or stereoisomers. Chiral stationary phases (CSPs) based on polysaccharide selectors are dominating the field, followed by glycopeptide antibiotics CSPs. A variety of alternatives exist if these columns fail. Industry has established extended automated screening programs, both by HPLC but also SFC, to figure out the most suitable chiral column for specific application problems. High throughput is a serious demand in those column screens because in initial drug discovery a large number of potential drug entities must be screened. To cope with this demand, most of the successful selector chemistries have meanwhile been transferred from standard 5 µm fully porous particle (FPP) technologies to superficially porous particle (SPP) or sub-2µm FPP particle technologies. While enantioselectivity was mostly maintained, the new modern particle technologies allow much faster separations. Especially SPP technologies seem to be advantageous and chiral separations at sub-second speed have been illustrated. Subminute separations are of course a great benefit not only in high-throughput screening but also for enantioselective 2D-LC. Fast chiral separations with such modern columns can be coupled to non-enantioselective RPLC separations in the first dimension in order to separate complex mixtures of enantiomers by comprehensive RP×chiral or specific difficult to separate mixtures of stereoisomers by multiple heart cut RP-chiral separation systems. Such a setup has shown applicability for enantiomer separations of the entire set of proteinogenic amino acids and some isobaric/isomeric congeners in the context of non-ribosomal peptides and therapeutic peptides, respectively. We have established such a multiple heart cut enantioselective RP-chiral separation system with SPP type chiral column based on quinine carbamate selector. Twenty five (25) amino acids have been enantioresolved in a run time of about 100 min. Meanwhile the run time could be reduced to 60 min. Also comprehensive chiral×chiral 2DLC setup has been established. It gives particular elution patterns of amino acid enantiomers which may allow straightforward data processing. If such analysis is performed in presence of real matrix, hyphenation of such separation systems to mass spectrometry is mandatory. Enantioselective matrix effects, however, may complicate the MS analysis and lead to inaccurate data. Solutions are discussed in this presentation.

Over decades chiral separation technologies have developed into seemingly mature analytical techniques. A large number of chiral stationary phases are nowadays commercially available and allow separation of all kind of enantiomers or stereoisomers. Chiral stationary phases (CSPs) based on polysaccharide selectors are dominating the field, followed by glycopeptide antibiotics CSPs. A variety of alternatives exist if these columns fail. Industry has established extended automated screening programs, both by HPLC but also SFC, to figure out the most suitable chiral column for specific application problems. High throughput is a serious demand in those column screens because in initial drug discovery a large number of potential drug entities must be screened. To cope with this demand, most of the successful selector chemistries have meanwhile been transferred from standard 5 µm fully porous particle (FPP) technologies to superficially porous particle (SPP) or sub-2µm FPP particle technologies. While enantioselectivity was mostly maintained, the new modern particle technologies allow much faster separations. Especially SPP technologies seem to be advantageous and chiral separations at sub-second speed have been illustrated. Subminute separations are of course a great benefit not only in high-throughput screening but also for enantioselective 2D-LC. Fast chiral separations with such modern columns can be coupled to non-enantioselective RPLC separations in the first dimension in order to separate complex mixtures of enantiomers by comprehensive RP×chiral or specific difficult to separate mixtures of stereoisomers by multiple heart cut RP-chiral separation systems. Such a setup has shown applicability for enantiomer separations of the entire set of proteinogenic amino acids and some isobaric/isomeric congeners in the context of non-ribosomal peptides and therapeutic peptides, respectively. We have established such a multiple heart cut enantioselective RP-chiral separation system with SPP type chiral column based on quinine carbamate selector. Twenty five (25) amino acids have been enantioresolved in a run time of about 100 min. Meanwhile the run time could be reduced to 60 min. Also comprehensive chiral×chiral 2DLC setup has been established. It gives particular elution patterns of amino acid enantiomers which may allow straightforward data processing. If such analysis is performed in presence of real matrix, hyphenation of such separation systems to mass spectrometry is mandatory. Enantioselective matrix effects, however, may complicate the MS analysis and lead to inaccurate data. Solutions are discussed in this presentation.