Editorial
For reprint orders, please contact [email protected]
How can we improve ion-exchange separations in LC? “…by decreasing the particle size and increasing the pressure rating an increase of a factor of 2 in efficiency can be achieved, without sacrificing analysis time.” Keywords: column technology • high-pressure ion-exchange chromatography • method development • performance limits • separation efficiency
Ion-exchange chromatography (IEX) emerged as an analytical technique for the separation of ions and ionizable analytes in 1975, when Small et al. described a novel approach to suppress counter ions in the mobile phase prior to conductivity detection [1] . Nowadays, dedicated instrumentation is available and compatible with normal bore (4.6 mm i.d.) and capillary (400 μm i.d.) column formats. Whereas the technology was initially applied only for the analysis of small inorganic anions and cations, its scope has expanded and IC is now commonly applied to analyze a wide range of species, including organic acids and aliphatic amines, and a wide range of biomolecules including antibodies, intact proteins, peptides and carbohydrates, among others. To analyze these analytes in contemporary sample mixtures with a large dynamic range as encountered in emerging fields, such as biotechnology research and clinical diagnostics, the demands on separation efficiency have increased significantly [2,3] . The objective of this editorial is to provide a short overview of the IEX performance limits that can be achieved with current instrumentation and column technology, and to raise discussion on how to realize the next leap in performance optimization in IC. The resolution (R s ) of two Gaussian peaks separated in a chromatographic experiment depends on the plate number (N ), the retention factor of the most retained compound (k), and the selectivity factor (α), according to:
10.4155/BIO.14.170 © 2014 Future Science Ltd
To improve IEX separations, considerable effort has been spent on tuning α via optimization of the stationary-phase chemistry and architecture [4] . By tuning the stationary-phase surface of the resins and eluent conditions different IC mechanisms, that is, Coulomb interactions, ion exclusion and ion pairing, can be utilized to realize the desired critical-pair separation. Both silica- and polymer-based resins have been developed, the latter being stable at high pH mobile phases. An additional advantage of polymerbased resins is the better biocompatibility (e.g., no undesired interactions with residual silanol groups), and hence reduced carry over [5] . Outstanding overviews describing the latest stationary-phase developments to tune the selectivity can be found in the literature [4,6–8] . Whereas major improvements in N and analysis time have been realized in HPLC by the introduction of ultra-high-pressure systems in combination with columns packed with sub-2-μm particles, the developments in the field of IEX separations have lagged behind. Conventional columns are packed with particle diameters between 6 and 10 μm, and the column length varies typically between 150 and 250 mm, yielding approximately 13,000 plates in isocratic mode. Only recently columns packed with 4-μm particle technology have become commercially available. The separation efficiency in terms of
Bioanalysis (2014) 6(15), 2021–2023
Sebastiaan Eeltink Author for correspondence: Department of Chemical Engineering, Vrije Universiteit Brussel; Pleinlaan 2, B-1050, Brussels, Belgium Tel.: + 32 2629 3324 seeltink@ vub.ac.be
Erwin R Kaal Analysis R&D, DSM Food Specialities B.V., Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands
part of
ISSN 1757-6180
2021
Editorial Eeltink & Kaal plate height (H), which is directly related to N through the column length (N = L/H), is often visualized as a function of mobile-phase velocity (u0 ). Columns packed with smaller particles yield lower H values (better separation efficiency), and the optimum flow velocity increases. Hence, separations yielding higher efficiencies can be achieved in shorter analysis times, at the expense of higher operating pressures. However, a plate-height curve does not incorporate information of column permeability, one of the key drivers for the development of new stationary-phase supports. Towards ultra-high-pressure IEX In a landmark paper published in 1996 by Professor Hans Poppe “Some reflections on speed and efficiency of modern chromatographic methods” the performance limits of different (pressure vs electro-driven) separation techniques were visualized in a single plot [9] . This approach directly provides information concerning the time required to generate a specific N for all possible column-length and particle-size combinations, while operating at the maximum system pressure [10,11] . Recently, we have applied this approach to visualize the kinetic performance limits for 7- and 4-μm particle-packed capillary IC columns operated in isocratic mode, while applying high-pressure and high-temperature conditions [12] . A 40% increase in N could be achieved by increasing the maximum system pressure from 20 to 35 MPa and applying columns packed with 4- instead of 7-μm particles. Via coupling of capillary columns and applying the maximum pressure 20,000 plates were recorded for a baseline separation of seven ions within only 7.5 min [12] . The next leap in performance in ion chromatography will probably be realized when ultra-high-pressure IC instrumentation and columns packed with, for example, sub-3-μm particles become available. When assuming that columns can be packed equally well with smaller particles, the kinetic plot extrapolation allows prediction of the effect of particle size and operating pressure on performance limits. It can be estimated that the analysis time can be reduced with a factor of 2.5 while maintaining the same plate number when using columns packed with 2.5-μm particles at a column pressure of 70 MPa and scaling the column length, instead of using columns packed with 4-μm particles operated at conventional pressures (P = 35 MPa). Similarly, by decreasing the particle size and increasing the pressure rating an increase of a factor of 2 in efficiency can be achieved, without sacrificing analysis time. It should be noted, however, that to maintain these high separation efficiencies, it is important that extra-column band broadening is reduced. This may imply a redesign of the current instrumentation to overcome the need for
2022
Bioanalysis (2014) 6(15)
connection tubing and further miniaturization of suppressor technology applied prior to detection. Furthermore, the use of high-end engineering plastics needs to be explored that can resist higher operating pressure and that are inert to aggressive eluents, such as high pH mobile phases. Novel column structures Theoretical predictions have shown that the separation efficiency can be increased up to one order of magnitude when the structure inhomogeneity is alleviated [13] . A step-change in column fabrication may be achieved in the long run with the application of novel techniques, such as three-dimensional printing [14] . The two most promising morphologies currently applied in the UHPLC field are the fused-core particlepacked columns and monolithic stationary phases. Fused-core-particle technology, with a diameter of a few microns and a shell thickness of a few hundreds nanometers, was recently reintroduced in the HPLC providing unparalleled separations with reduced plate heights in the range of 1.2–1.5. Compared with fullyporous particles the particle-size distribution is much narrower, and a 40% reduction of the eddy-diffusion contribution (A-term) to band broadening has been reported [15] . Furthermore, depending on the core-toparticle-diameter ratio longitudinal diffusion (B-term) is lowered by approximately 25%, and the stationaryphase mass transfer (Cs-term) is reduced by a factor of 2 [15] .
“Kinetic plots provide insights into the best
column length/particle size combination to achieve the highest separation efficiency in the shortest possible analysis time.
”
Rigid polymer-based monolithic columns, composed of macroporous interconnected polymer network, were introduced in the early 1990s for protein separations [16] , and functionalized monolithic materials have also been employed for large bio-molecule IEX separations [17] . Whereas the porosity of packed columns is fixed, monolithic stationary phases have the potential to perform intrinsically better than packed columns, since the both macropores and polymer microglobule size can be optimized independently from each other [18] . Hence, where the porosity of a column with a sphere packing is fixed at approximately 40%, this parameter can be tuned for monoliths. Still, in order to precision engineer homogeneous monolithic materials with the desired pore structure on a nanoscale level novel synthesis approaches need to be pursued.
future science group
How can we improve ion-exchange separations in LC?
Conclusion IEX provides unique separation selectivity and is well suited to separate a wide range of biomolecules, including antibodies, proteins and polypeptides. With the recent introduction of capillary column technology and instrumentation new applications have become within reach. For example, the biological activity of biomolecules present in minute amounts of samples (as encountered in the field of clinical diagnostics) can now be assessed by applying native separation and fractionation conditions. An additional advantage is the flow rate compatibility with electrospray mass-spectrometric detection (after eluent suppression), enhancing the ionization efficiency and hence improving detection limits. The field of IEX will benefit from the developments in the field of UHPLC. Kinetic plots provide insights into the best column length/particle size combination
to achieve the highest separation efficiency in the shortest possible analysis time. We anticipate that a significant gain in efficiency and separation speed can be achieved with further increasing the pressure tolerance of instrumentation and the development of columns packed with sub-3-μn (core shell) particles and improved monolithic structures. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
References 1
Small H, Stevens T, Baumann WC. Novel ion exchange chromatographic method using conductimetric detection. Anal. Chem. 47, 1801–1809 (1975).
the kinetic performance of LC supports with a different size of morphology. Anal. Chem. 77, 4058–4070 (2005). 11
Vaast A, Broeckhoven K, Dolman S, Desmet G, Eeltink S. Comparison of the gradient kinetic performance of silica monolithic capillary columns with columns packed with 3 μm porous and 2.7 μm fused-core silica particles. J. Chromatogr. A 1228, 270–275 (2012).
12
Wouters B, Bruggink C, Desmet G, Agroskin Y, Pohl C, Eeltink S. Capillary ion chromatography at high pressure and temperature. Anal. Chem. 84, 7212–7217 (2012).
2
Haddad PR, Nesterenko PN, Buchberger W. Recent developments and emerging directions in ion chromatography. J. Chromatogr. A 1184, 456–473 (2008).
3
Lucy CA, Wahab MF. Advances in high-speed and highresolution ion chromatography. LC-GC 31, 38–42 (2013).
4
Pohl C. Recent developments in ion-exchange columns for ion chromatography. LC-GC 31, 16–22 (2013).
13
5
Dolman S, Eeltink S, Vaast A, Pelzing M. Investigation of carryover of peptides in nano-liquid chromatography/ mass spectrometry using packed and monolithic capillary columns. J. Chromatogr. B 912, 56–63 (2013).
Billen J, Desmet G. Understanding and design of existing and future chromatographic support formats. J. Chromatogr. A 1168, 73–99 (2007).
14
Fee C, Nawada S, Dimartino S. 3D printed porous media columns with fine control of column packing morphology. J. Chromatogr. A 1333, 18–24 (2014).
15
Guiochon G, Gritti F. Shell particles, trials, tribulations and triumphs. J. Chromatogr. A 1218, 1915–1938 (2011).
16
Wang QC, Svec F, Frechet JMJ. Reversed-phase chromatography of small molecules and peptides on a continuous rod of macroporous poly(styrene-codivinylbenzene). J. Chromatogr. A 669, 230–235 (1994).
17
Nordborg A, Hilder EF, Haddad PR. Monolithic phases for ion chromatography. Ann. Rev. Anal. Chem. 4, 197– 226 (2011).
18
Eeltink S, Herrero-Martinez JM, Rozing GP, Schoenmakers PJ, Kok WT. Tailoring the morphology of methacrylate ester-based monoliths for optimum efficiency in liquid chromatography. Anal. Chem. 77, 7342–7347 (2005).
6
Chambers SD, Glenn KM, Lucy CA. Developments in ion chromatography using monolithic columns. J. Sep. Sci. 30, 1628–1645 (2007).
7
Paull B, Nesterenko PN. Novel ion chromatographic stationary phases for the analysis of complex matrices. Analyst 130, 134–146 (2005).
8
Mansour FR, Danielson ND. Multimodal liquid chromatography of small molecules. Anal. Meth. 5, 4955–4972 (2013).
9
Poppe H. Some reflections on speed and efficiency of modern chromatographic methods. J. Chromatogr. A 778, 3–21 (1996).
10
Desmet G, Clicq D, Gzil P. Geometry-independent plate height representation methods for the direct comparison of
future science group
Editorial
www.future-science.com
2023
For reprint orders, please contact [email protected]
How can we improve ion-exchange separations in LC? “…by decreasing the particle size and increasing the pressure rating an increase of a factor of 2 in efficiency can be achieved, without sacrificing analysis time.” Keywords: column technology • high-pressure ion-exchange chromatography • method development • performance limits • separation efficiency
Ion-exchange chromatography (IEX) emerged as an analytical technique for the separation of ions and ionizable analytes in 1975, when Small et al. described a novel approach to suppress counter ions in the mobile phase prior to conductivity detection [1] . Nowadays, dedicated instrumentation is available and compatible with normal bore (4.6 mm i.d.) and capillary (400 μm i.d.) column formats. Whereas the technology was initially applied only for the analysis of small inorganic anions and cations, its scope has expanded and IC is now commonly applied to analyze a wide range of species, including organic acids and aliphatic amines, and a wide range of biomolecules including antibodies, intact proteins, peptides and carbohydrates, among others. To analyze these analytes in contemporary sample mixtures with a large dynamic range as encountered in emerging fields, such as biotechnology research and clinical diagnostics, the demands on separation efficiency have increased significantly [2,3] . The objective of this editorial is to provide a short overview of the IEX performance limits that can be achieved with current instrumentation and column technology, and to raise discussion on how to realize the next leap in performance optimization in IC. The resolution (R s ) of two Gaussian peaks separated in a chromatographic experiment depends on the plate number (N ), the retention factor of the most retained compound (k), and the selectivity factor (α), according to:
10.4155/BIO.14.170 © 2014 Future Science Ltd
To improve IEX separations, considerable effort has been spent on tuning α via optimization of the stationary-phase chemistry and architecture [4] . By tuning the stationary-phase surface of the resins and eluent conditions different IC mechanisms, that is, Coulomb interactions, ion exclusion and ion pairing, can be utilized to realize the desired critical-pair separation. Both silica- and polymer-based resins have been developed, the latter being stable at high pH mobile phases. An additional advantage of polymerbased resins is the better biocompatibility (e.g., no undesired interactions with residual silanol groups), and hence reduced carry over [5] . Outstanding overviews describing the latest stationary-phase developments to tune the selectivity can be found in the literature [4,6–8] . Whereas major improvements in N and analysis time have been realized in HPLC by the introduction of ultra-high-pressure systems in combination with columns packed with sub-2-μm particles, the developments in the field of IEX separations have lagged behind. Conventional columns are packed with particle diameters between 6 and 10 μm, and the column length varies typically between 150 and 250 mm, yielding approximately 13,000 plates in isocratic mode. Only recently columns packed with 4-μm particle technology have become commercially available. The separation efficiency in terms of
Bioanalysis (2014) 6(15), 2021–2023
Sebastiaan Eeltink Author for correspondence: Department of Chemical Engineering, Vrije Universiteit Brussel; Pleinlaan 2, B-1050, Brussels, Belgium Tel.: + 32 2629 3324 seeltink@ vub.ac.be
Erwin R Kaal Analysis R&D, DSM Food Specialities B.V., Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands
part of
ISSN 1757-6180
2021
Editorial Eeltink & Kaal plate height (H), which is directly related to N through the column length (N = L/H), is often visualized as a function of mobile-phase velocity (u0 ). Columns packed with smaller particles yield lower H values (better separation efficiency), and the optimum flow velocity increases. Hence, separations yielding higher efficiencies can be achieved in shorter analysis times, at the expense of higher operating pressures. However, a plate-height curve does not incorporate information of column permeability, one of the key drivers for the development of new stationary-phase supports. Towards ultra-high-pressure IEX In a landmark paper published in 1996 by Professor Hans Poppe “Some reflections on speed and efficiency of modern chromatographic methods” the performance limits of different (pressure vs electro-driven) separation techniques were visualized in a single plot [9] . This approach directly provides information concerning the time required to generate a specific N for all possible column-length and particle-size combinations, while operating at the maximum system pressure [10,11] . Recently, we have applied this approach to visualize the kinetic performance limits for 7- and 4-μm particle-packed capillary IC columns operated in isocratic mode, while applying high-pressure and high-temperature conditions [12] . A 40% increase in N could be achieved by increasing the maximum system pressure from 20 to 35 MPa and applying columns packed with 4- instead of 7-μm particles. Via coupling of capillary columns and applying the maximum pressure 20,000 plates were recorded for a baseline separation of seven ions within only 7.5 min [12] . The next leap in performance in ion chromatography will probably be realized when ultra-high-pressure IC instrumentation and columns packed with, for example, sub-3-μm particles become available. When assuming that columns can be packed equally well with smaller particles, the kinetic plot extrapolation allows prediction of the effect of particle size and operating pressure on performance limits. It can be estimated that the analysis time can be reduced with a factor of 2.5 while maintaining the same plate number when using columns packed with 2.5-μm particles at a column pressure of 70 MPa and scaling the column length, instead of using columns packed with 4-μm particles operated at conventional pressures (P = 35 MPa). Similarly, by decreasing the particle size and increasing the pressure rating an increase of a factor of 2 in efficiency can be achieved, without sacrificing analysis time. It should be noted, however, that to maintain these high separation efficiencies, it is important that extra-column band broadening is reduced. This may imply a redesign of the current instrumentation to overcome the need for
2022
Bioanalysis (2014) 6(15)
connection tubing and further miniaturization of suppressor technology applied prior to detection. Furthermore, the use of high-end engineering plastics needs to be explored that can resist higher operating pressure and that are inert to aggressive eluents, such as high pH mobile phases. Novel column structures Theoretical predictions have shown that the separation efficiency can be increased up to one order of magnitude when the structure inhomogeneity is alleviated [13] . A step-change in column fabrication may be achieved in the long run with the application of novel techniques, such as three-dimensional printing [14] . The two most promising morphologies currently applied in the UHPLC field are the fused-core particlepacked columns and monolithic stationary phases. Fused-core-particle technology, with a diameter of a few microns and a shell thickness of a few hundreds nanometers, was recently reintroduced in the HPLC providing unparalleled separations with reduced plate heights in the range of 1.2–1.5. Compared with fullyporous particles the particle-size distribution is much narrower, and a 40% reduction of the eddy-diffusion contribution (A-term) to band broadening has been reported [15] . Furthermore, depending on the core-toparticle-diameter ratio longitudinal diffusion (B-term) is lowered by approximately 25%, and the stationaryphase mass transfer (Cs-term) is reduced by a factor of 2 [15] .
“Kinetic plots provide insights into the best
column length/particle size combination to achieve the highest separation efficiency in the shortest possible analysis time.
”
Rigid polymer-based monolithic columns, composed of macroporous interconnected polymer network, were introduced in the early 1990s for protein separations [16] , and functionalized monolithic materials have also been employed for large bio-molecule IEX separations [17] . Whereas the porosity of packed columns is fixed, monolithic stationary phases have the potential to perform intrinsically better than packed columns, since the both macropores and polymer microglobule size can be optimized independently from each other [18] . Hence, where the porosity of a column with a sphere packing is fixed at approximately 40%, this parameter can be tuned for monoliths. Still, in order to precision engineer homogeneous monolithic materials with the desired pore structure on a nanoscale level novel synthesis approaches need to be pursued.
future science group
How can we improve ion-exchange separations in LC?
Conclusion IEX provides unique separation selectivity and is well suited to separate a wide range of biomolecules, including antibodies, proteins and polypeptides. With the recent introduction of capillary column technology and instrumentation new applications have become within reach. For example, the biological activity of biomolecules present in minute amounts of samples (as encountered in the field of clinical diagnostics) can now be assessed by applying native separation and fractionation conditions. An additional advantage is the flow rate compatibility with electrospray mass-spectrometric detection (after eluent suppression), enhancing the ionization efficiency and hence improving detection limits. The field of IEX will benefit from the developments in the field of UHPLC. Kinetic plots provide insights into the best column length/particle size combination
to achieve the highest separation efficiency in the shortest possible analysis time. We anticipate that a significant gain in efficiency and separation speed can be achieved with further increasing the pressure tolerance of instrumentation and the development of columns packed with sub-3-μn (core shell) particles and improved monolithic structures. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
References 1
Small H, Stevens T, Baumann WC. Novel ion exchange chromatographic method using conductimetric detection. Anal. Chem. 47, 1801–1809 (1975).
the kinetic performance of LC supports with a different size of morphology. Anal. Chem. 77, 4058–4070 (2005). 11
Vaast A, Broeckhoven K, Dolman S, Desmet G, Eeltink S. Comparison of the gradient kinetic performance of silica monolithic capillary columns with columns packed with 3 μm porous and 2.7 μm fused-core silica particles. J. Chromatogr. A 1228, 270–275 (2012).
12
Wouters B, Bruggink C, Desmet G, Agroskin Y, Pohl C, Eeltink S. Capillary ion chromatography at high pressure and temperature. Anal. Chem. 84, 7212–7217 (2012).
2
Haddad PR, Nesterenko PN, Buchberger W. Recent developments and emerging directions in ion chromatography. J. Chromatogr. A 1184, 456–473 (2008).
3
Lucy CA, Wahab MF. Advances in high-speed and highresolution ion chromatography. LC-GC 31, 38–42 (2013).
4
Pohl C. Recent developments in ion-exchange columns for ion chromatography. LC-GC 31, 16–22 (2013).
13
5
Dolman S, Eeltink S, Vaast A, Pelzing M. Investigation of carryover of peptides in nano-liquid chromatography/ mass spectrometry using packed and monolithic capillary columns. J. Chromatogr. B 912, 56–63 (2013).
Billen J, Desmet G. Understanding and design of existing and future chromatographic support formats. J. Chromatogr. A 1168, 73–99 (2007).
14
Fee C, Nawada S, Dimartino S. 3D printed porous media columns with fine control of column packing morphology. J. Chromatogr. A 1333, 18–24 (2014).
15
Guiochon G, Gritti F. Shell particles, trials, tribulations and triumphs. J. Chromatogr. A 1218, 1915–1938 (2011).
16
Wang QC, Svec F, Frechet JMJ. Reversed-phase chromatography of small molecules and peptides on a continuous rod of macroporous poly(styrene-codivinylbenzene). J. Chromatogr. A 669, 230–235 (1994).
17
Nordborg A, Hilder EF, Haddad PR. Monolithic phases for ion chromatography. Ann. Rev. Anal. Chem. 4, 197– 226 (2011).
18
Eeltink S, Herrero-Martinez JM, Rozing GP, Schoenmakers PJ, Kok WT. Tailoring the morphology of methacrylate ester-based monoliths for optimum efficiency in liquid chromatography. Anal. Chem. 77, 7342–7347 (2005).
6
Chambers SD, Glenn KM, Lucy CA. Developments in ion chromatography using monolithic columns. J. Sep. Sci. 30, 1628–1645 (2007).
7
Paull B, Nesterenko PN. Novel ion chromatographic stationary phases for the analysis of complex matrices. Analyst 130, 134–146 (2005).
8
Mansour FR, Danielson ND. Multimodal liquid chromatography of small molecules. Anal. Meth. 5, 4955–4972 (2013).
9
Poppe H. Some reflections on speed and efficiency of modern chromatographic methods. J. Chromatogr. A 778, 3–21 (1996).
10
Desmet G, Clicq D, Gzil P. Geometry-independent plate height representation methods for the direct comparison of
future science group
Editorial
www.future-science.com
2023
