Elsevier

Analytica Chimica Acta

Volume 991, 23 October 2017, Pages 30-45
Analytica Chimica Acta

Review
3-Dimensional X-ray microtomography methodology for characterization of monolithic stationary phases and columns for capillary liquid chromatography - A tutorial

https://doi.org/10.1016/j.aca.2017.08.040Get rights and content

Highlights

  • Capillary monolithic organic and inorganic columns were prepared.

  • The chromatographic materials and columns were characterized by 3-D X-ray Microtomography (3D-XMT)

  • The results were compared with 2-D imagining techniques: SEM and FESEM and with chromatographic profile.

Abstract

In this tutorial we describe a fast, nondestructive, three-dimensional (3-D) view approach to be used in morphology characterization of capillary monoliths and columns by reconstruction from X-ray microtomography (XMT) obtained by acquiring projection images of the sample from a number of different directions. The method comprises imaging acquisition, imaging reconstruction using specific algorithms and imaging analysis by generation of a 3-D image of the sample from radiographic images. The 3-D images show the morphological data for bulk macropore space and skeleton connectivity of the monoliths and were compared with other images from imaging techniques such as scanning electron microscopy (SEM) and field emission scanning electron microscopy (FESEM) and with chromatographic performance. The 3-D XMT methodology is applicable for organic and inorganic capillary chromatographic monolithic materials and it allows the acquisition of many hundreds (in our case 1001 projections) of longitudinal and cross-sectional images in a single session, resolving morphological details with a 3D-view of the monolithic structure, inclusive inside the column in a sectional structure with volume (three dimensions) when compared to the sectional structure area (with only two dimensions) when using SEM and FESEM techniques.

Introduction

High performance liquid chromatography (HPLC) is the most used analytical separation technique due to its versatility for both quantitative and qualitative analyses of a wide range of compounds, especially in the pharmaceutical industry. For this reason, one of most promising fields in HPLC research and development is related to the synthesis and design of new stationary phases (SP), generating an increasing number of column types, both available commercially and labmade [1], [2].

Miniaturization in chromatography has emerged with capillary liquid chromatography (cLC) and nano-liquid chromatography (nano-LC), as alternatives to HPLC. Columns in both cLC and nano-LC have reduced dimensions, with internal diameters reduced to the order of micrometers (10–500 μm) [3], [4], containing the selected stationary phase, or, recently, confined in noncylindrical conduits used in microfluidic chips [5], in contrast to their larger counterparts for conventional HPLC. Besides miniaturization, cLC or nano-LC presents some advantages as short analysis times, high mass detectivity, low sample volume and reduced use of SP and mobile phases (MP) as well as easy coupling with mass spectrometry [6], [7], [8] and they have been applied for determinations in different areas such as pharmaceuticals, biomedicine, environmental analysis and recently in food control and agricultural research. Recently, porous polymer monoliths, with a hierarchical structure comprising a porous structure (microscale) and a polymer gel structure (nanoscale), created by specific linking chemistry relying on (free) radical cross-linking (co)polymerization, have enabled fabrication of complex chemical analysis systems for “lab-on-a-chip” separations. These micro-devices, presents additional advantages in comparison with cLC and nano-LC using system that are not miniaturized, especially related to coupling with mass spectrometry via porous-polymer assisted nanospray. However there still remain some difficulties related to the preparation of materials under spatial confinement and in accessing their nanostructural heterogeneity by imaging characterization techniques [5], [9].

SP for capillary columns can be derived by organic [10] or inorganic materials [11]. Inorganic materials are mainly represented by silica, the most popular support used in HPLC SP; although materials based on zirconia [12], [13], [14] and titania [15], [16], [17], [18] have already been explored in cLC separations. The organic materials are mainly represented by porous polymers, which can be based on organic polar (methacrylate/acrylate based monoliths, styrene divinylbenzene based monoliths and hypercrosslinked monoliths) or organic non-polar (methacrylate-derived monoliths modified with charged groups, e.g., neutral, anionic, cationic, and zwitterionic monoliths) [19], [20]. According to the type of SP, columns for cLC can be classified as using particles, as in conventional HPLC columns, or monolithic materials [7]. Although a variety of approaches described in the literature for packing particles inside chromatographic columns by HPLC, features of this technology have proven difficult to implement on the capillary scale, particularly because of the technical challenges associated with packing and retaining beads in narrow-bore capillary columns [21], [22]. In this way, capillary columns for cLC use, in the majority of applications, monoliths as SP, are prepared by in-situ reactions. These take into account the fact that downward scalability of monolithic columns is greatly affected by the confinement effect of the capillary wall resulting from the decreased volume-to-surface ratio as the capillary diameter is decreased [23]. Thus, the evaluation of morphological features is a key to understanding interactions and transport processes in porous media and, imaging techniques, especially 3D image reconstructions, could be excellent tools to look inside columns in a more detailed manner.

Monoliths as SP for chromatography are medias in formats that can be compared to a single large “particle” that does not contain interparticular voids [21]. As a result, all the MP must flow through the SP and convective flow greatly accelerates the rate of mass transfer, in contrast to diffusion, which is the typical driving force for mass transfer within the pores of particulate stationary phases during chromatographic separation process. This enables a substantial increase in the speed of the separation [9], [21], [22], [24]. Silica monoliths have a hierarchically structured permanent pore space, with a typical bimodal pore size distribution. On the other hand, polymer monoliths exhibit a monomodal pore-size distribution and a difficult-to-discern mesoporous and potentially microporous structure [25].

The use of monoliths as SP for cLC is a good alternative to the traditional particulate SP due to the simplicity of the in-situ preparation, with no need for separate packing process. Even more important, the excellent permeability of monolithic columns to flow gives low back-pressure to the cLC system compared to columns with particles [26], [27], [28], [29], [30], [31]. Also, monolithic capillary columns do not need frits for isolation of the SP, which makes the packing process easier and cheaper, with possibility to produce polymeric monoliths (polar or non-polar) [10], [19], [20] or inorganic monoliths (silica, zirconia, titania) [11], [12], [13], [14], [15], [16], [17], [18].

For cLC, monoliths are synthesized inside the capillary column by free radical crosslinking (co)polymerization processes, by the sol-gel process (SGP) with inorganic precursors or by click-chemistry reactions [32], [33], [34], [35] to produce hybrid inorganic-organic porous polymer monoliths. The latter are more uniform in terms of their nanoscale composition and physicochemical properties, including separation profile, than a network derived by a free-radical (co)polymerization process [35] and are one of the new trends in nanoseparations and in the microfluidics and nanofluidics fields, with emphasis on the separation of biomolecules and pharmaceutical compounds. In addition, there have recently been several publications reporting on the use of special procedures and/or reagents during synthesis to generate mesopores in the organic polymeric skeleton [31] on the use of surfactants as template molecules [36], on early termination of the polymerization reaction [37], [38] and on hyper-crosslinking of the monolith using Friedel-Crafts reaction as the second step in monolith development [39].

Taking into account chemical properties, inorganic silica-based capillary monoliths [40] demonstrate good solvent resistance, excellent mechanical stabilities and higher separation efficiencies for separation of small molecules when compared to polymer-based monoliths [25]. On the other hand, the stability of silica-based SP depends on the chromatographic conditions. Silica does not withstand high (>9) or low (<2) pH in the MP, especially at high temperatures [41], [42]. Thus, alternative inorganic materials [43] for chromatography are a research area of great interest. Metal oxides, such as zirconia (ZrO2) [44], [45] or titania (TiO2) [46], [47], [48], have been produced and evaluated as SP.

ZrO2 is used in liquid phase chromatography as a stationary phase support due to its great mechanical stability, its resistance to high temperatures and its resistance over a wide range of pH [44], [45], making zirconia monoliths interesting materials to be explored in capillary and nano-LC [12], [13], [14].

TiO2 presents proprieties and retention mechanisms similar to silica, however, it has considerably higher chemical stability [46], [47], [48]. Due to its amphoteric and polar properties, caused by the presence of titanols in structure [43], it could be more explored as SP, especially for application in hydrophilic interaction chromatography (HILIC) [49], [50] as an alternative to silica based SP. The use of this metal oxide as a monolithic SP could present benefits related to its chemical properties as well as from the chemical resistance of the packing material, making titania monoliths as well as zirconia monoliths strong candidates to be explored in capillary and nano-LC. The bed structure of a SP is an important parameter, since interferes directly in chromatographic performance. Specifically, for monolithic columns, the morphologies of the monoliths vary among themselves. The skeleton of a monolith may be a globular or fused mass with no distinct microglobules, depending upon the monomer and porogen compositions. The morphology also differs between inorganic silica monoliths and organic polymeric monoliths. Inorganic silica monoliths have a significant fraction of small mesopores in the skeleton when compared to organic polymeric monoliths [31], [51]. On the other hand, polymer monoliths, feature “gel porosity” in contact with the MP used in LC. This gel porosity is accessible to small solutes and absent in the dry state of the monoliths [25].

Silica based monoliths exhibit a distinct bimodal pore size distribution with macropores (flow-through pores) of approximately 2 μm diameter and mesopores (diffusional pores) of approximately 10 nm diameter and for this type of monoliths, pores are classified as closed pores and opened pores [52], [53], [54]. Since closed pores do not participate in adsorption/desorption phenomena, which are the main process in chromatography, their definition is basically theoretical. On the other hand, open pores participate in adsorption/desorption phenomena and are classified into two main groups: blind pores and connected pores. The shape of pores can be cylindrical, slit-shaped, ink-bottle-shaped/cone-shaped [54] (Fig. 1).

The morphology and the pore structure of a monolithic chromatographic bed are very important features in the design and characterization of SP since these aspects directly influence the hydrodynamic properties (e.g., flow properties), thermodynamic properties (e.g., loadability) and the mass transfer kinetics (e.g., efficiency), since all are correlated with the chromatographic performance of stationary phase [55], [56], [57], [58], [59], [60]. For inorganic monoliths, such as silica, the most favorable pore configuration for mass transfer kinetics in the porous material are highly connected cylindrical pores, since cylindrical pore format has been used almost universally in pore size characterization of different materials for chromatography [61]. The most unfavorable pore configurations are blind-ink-bottle-shaped pores that usually contribute only negligibly to the mass transport [54].

In terms of imaging techniques like scanning electron microscopy (SEM) and field emission scanning electron microscopy (FESEM) rapidly provide direct information about monolith morphology from bulk or cross sectional capillary images and continues to be widely used for qualitative characterization of the polymeric monolithic structure [62]. However, SEM or FESEM images offer no reliable quantitative analysis of macropore interfaces for silica monoliths, since the geometry from the thick focal depth is projected onto a single two dimensional (2-D) image, and thus lacks the required morphological details [63], [64]. Atomic force microscopy (AFM) using topography imaging, in conjunction with noninvasive Raman imaging, generate a three-dimensional (3-D) pore structure reconstruction of polymeric monolithic bead-based media [59]. Confocal laser scanning microscopy (CLSM) [65], [66] applied to SP characterization, presents a fast, non-destructive, and quantitative method composed of column pretreatment, image acquisition, image processing, and statistical analysis (by the use of algorithms) of the image data used for characterize the hybrid silica macropore space morphology of monoliths (rods and particle-packed beds) structure, radially resolved, including silica monoliths with macropore sizes already entering submicron dimensions (ca. 0.8 μm) [66]. CLSM supplies a thin (ca.150 nm) focal plane and 3-D images can be reconstructed via computer algorithms by stacking the series of 2-D CLSM images [63] although smaller, in the mesopore space of monoliths (2–50 nm) are more difficult to access [67]. In addition, the macropore space of the silica monolith samples have been also reconstructed using focused ion beam scanning electron microscopy (FIB-SEM) followed by a quantitative assessment of geometrical and topological properties based on chord length distributions (CLDs) and branch-node analysis of the pore network, respectively, presenting average macropore sizes from the micrometer down to the submicrometer range [68]. Serial block face scanning electron microscopy (SBF-SEM) [69] was used for study the morphology of porous polymers and determines their transport characteristics and thus their efficiency in numerous applications, using a large-volume reconstruction and analysis of skeleton and void space of the monolith statistically evaluated to extract key structural parameters relevant to mass transport, and to quantify finite-size effects or evaluating the structural features of a methacrylatebased polymer monolith from the pore scale to the column scale [70].

X-ray microtomography (XMT) is a radiographic imaging technique that can produce 3D images of a material's internal scanned sample structure at a spatial resolution of μm of a from a series of X-ray shadow images which in tomographic terminology are called projections [71], [72], [73], [74].

Since XMT is a non-destructive, noninvasive and powerful technique which provides 3D information of materials it is consequently a technique very attractive in Materials Science since the relation between macroscopic properties and the micro-structure of a material is very frequently required as several disciplines including physics, medicine, biology, mineral processing, geology and powder technology [75]. On the other hand, until the moment, there is no studies with applications of 3-D XMT in SP characterization, which could allow studying SP structure inside the capillary column without cutting it, ideal for applications with focus to observe the heterogeneity of materials, especially monoliths, prepared by in-situ reactions inside the column.

A significant milestone in XMT imaging was the use of synchrotron radiation as an X-ray source, which brought significant enhancements to the imaging that can be produced, since the emitted light which results from the bending of a high-energy electron beam due to a magnetic field is many orders of magnitude greater in brightness than that emitted by conventional X-ray source. As a result the high flux allows resolving very subtle variations in absorptivity and therefore internal structure [71] and which could be particularly interesting in the characterization of SP for chromatography. The analog for this phenomenon could be the difference between taking a photograph in low light, where the resulting image can be grainy, and taking a photograph with an accompanying flash, where the resulting image has much higher contrast, making a parallel between XMT images based on the use of synchrotron radiation and SEM and FESEM images. Additional advantages of synchrotron radiation include X-ray beam collimation, which simplifies the tomographic reconstruction algorithm, and the tunability of the X-ray energy to a narrow energy band [71].

The process for acquiring 3-D XMT images involves the rotation of a sample in an X-ray beamline while the detector collects projections (radio-graphs) for each angle. A scintillator positioned between the sample and the detector transforms X-rays into visible light. Thereafter, reconstruction algorithms generate a 3-D image of the sample from the radiographic images [76], [77], [78].

The aim of this tutorial is describing the fundamental principles and practical operational details of the 3-D XMT methodology for characterization of monoliths and columns for cLC, by obtaining 3-D images using X-rays generated from synchrotron radiation of both inorganic and organic chromatographic materials (bulk and columns).

Section snippets

Materials and methods

For 3-D XMT characterization were used polar SiO2 and ZrO2 monolithic chromatographic materials (bulk and columns) which were prepared as following procedure described in a previously publication [14]. A new polar inorganic monolith to be used as chromatographic material for cLC based in TiO2 was prepared and reaction optimized as described in the next sections.

Results and discussion

The results obtained with the LNLS beamline were full 3-D XMT images of the monoliths and capillary columns. The structure of the bulk and the capillaries were compared with the results obtained with other imaging techniques: scanning electron microscopy (SEM), field emission scanning electron microscopy (FESEM) and with the chromatographic profile described in the chromatographic characterization for TiO2 capillary monolithic columns and with other results for inorganic capillary monolithic

Conclusions

Although the lack of quantitative information results, which in our view is a clear need for future work in this area, 3-D XMT methodology described here represents a interesting and non-destructive source for pore structure information that can help to elucidate chromatographic performance observations covering both retention and resolution aspects. In addition, the results presented herein demonstrate that X-rays from synchrotron radiation can be applied for imaging techniques and are an

Acknowledgements

The authors acknowledge financial support and fellowships from the Brazilian Agencies FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Processes 2012/23518-2 and 2013/25933-0), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Process 307101/2011-8, Brazilian Synchrotron Light Laboratory (LNLS) Proposal IMX-17042. The LNLS staffs, especially MSc Francis O'Dowd and PhD Nathaly L. Archilha are also acknowledged. This is a contribution of INCTBio (Instituto Nacional

Carla Grazieli Azevedo da Silva, born in the Brazil, obtained her B.S. from Federal University of Rio Grande do Sul in 2004, her M.Sc. in Chemistry from Federal University of Rio Grande do Sul in 2007 and her Ph.D. in Sciences from University of Campinas in 2013, with an internship at Université D' Orléans in 2012. She did a post-doctorate at the University of Campinas (2013–2016) and in the University of São Paulo (2016) in Analytical Chemistry. She is currently an Associate Professor of the

References (99)

  • E. Lesellier et al.

    Advantages of the use of monolithic stationary phases for modeling the retention in sub/supercritical chromatography application to cis/trans-β-carotene separation

    J. Chromatogr. A

    (2003)
  • T.J. Causon et al.

    Critical differences in chromatographic properties of silica- and polymer-based monoliths

    J. Chromatogr. A

    (2014)
  • G. Guiochon

    Monolithic columns in high-performance liquid chromatography

    J. Chromatogr. A

    (2007)
  • J. Zhang et al.

    Ultratrace liquid chromatography/mass spectrometry analysis of large peptides with post-translational modifications using narrow-bore poly(styrene-divinylbenzene) monolithic columns and extended range proteomic analysis

    J. Chromatogr. A

    (2007)
  • P. Aggarwal et al.

    Monolithic bed structure for capillary liquid chromatography

    J. Chromatogr. A

    (2012)
  • M. Wolter et al.

    In-situ functionalized monolithic polysiloxane-polymethacrylate composite materials from polythiol-ene double click reaction in capillary column format for enantioselective nano-high-performance liquid chromatography

    J. Chromatogr. A

    (2017)
  • P. Zhang et al.

    Facile one-pot preparation of a novel imidazolium-based monolith by thiol-ene click chemistry for capillary liquid chromatography

    Electrophoresis

    (2016)
  • F. Alves et al.

    Radical-mediated step-growth: preparation of hybrid polymer monolithic columns with fine control of nanostructural and chromatographic characteristics

    J. Chromatogr. A

    (2015)
  • I. Nischang et al.

    On the separation of small molecules by means of nano-liquid chromatography with methacrylate-based macroporous polymer monoliths

    J. Chromatogr. A

    (2010)
  • I. Nischang et al.

    Towards porous polymer monoliths for the efficient, retention-independent performance in the isocratic separation of small molecules by means of nano-liquid chromatography

    J. Chromatogr. A

    (2010)
  • J. Urban et al.

    Hypercrosslinking: new approach to porous polymer monolithic capillary columns with large surface area for the highly efficient separation of small molecules

    J. Chromatogr. A

    (2010)
  • J. Nawrocki

    The silanol group and its role in liquid chromatography

    J. Chromatogr. A

    (1997)
  • H.A. Claessens et al.

    Review on the chemical and thermal stability of stationary phases for reversed-phase liquid chromatography

    J. Chromatogr. A

    (2004)
  • J. Nawrocki et al.

    Chemistry of zirconia and its use in chromatography

    J. Chromatogr. A

    (1993)
  • J. Nawrocki et al.

    Part II. Chromatography using ultra-stable metal oxide-based stationary phases for HPLC

    J. Chromatogr. A

    (2004)
  • V. Zizkovsky et al.

    Titania-based stationary phase in separation of ondansetron and its related compounds

    J. Chromatogr. A

    (2008)
  • P. Jandera

    Stationary and mobile phases in hydrophilic interaction chromatography: a review

    Anal. Chim. Acta

    (2011)
  • B.A. Grimes et al.

    Pore structural characterization of monolithic silica columns by inverse size-exclusion chromatography

    J. Chromatogr. A

    (2007)
  • A. Sáfrány et al.

    Control of pore formation in macroporous polymers synthesized by single-step g-radiation-initiated polymerization and cross-linking

    Polymer

    (2005)
  • K. Hormann et al.

    Morphology and separation efficiency of a new generation of analytical silica monoliths

    J. Chromatogr. A

    (2012)
  • D. Hlushkou et al.

    Comparison of first and second generation analytical silica monoliths by pore-scale simulations of eddy dispersion in the bulk region

    J. Chromatogr. A

    (2013)
  • K. Hormann et al.

    Mass transport properties of second-generation silica monoliths with mean mesopore size from 5 to 25 nm

    J. Chromatogr. A

    (2014)
  • D. Lubda et al.

    Comprehensive pore structure characterization of silica monoliths with controlled mesopore size and macropore size by nitrogen sorption, mercury porosimetry, transmission electron microscopy and inverse size exclusion chromatography

    J. Chromatogr. A

    (2005)
  • R. Skudas et al.

    Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

    J. Chromatogr. A

    (2007)
  • Y. Yao et al.

    Determination of pore size distributions of porous chromatographic adsorbents by inverse size-exclusion chromatography

    J. Chromatogr. A

    (2004)
  • I.J. Nischang

    Porous polymer monoliths: morphology, porous properties, polymer nanoscale gel structure and their impact on chromatographic performance

    J. Chromatogr. A

    (2013)
  • S. Bruns et al.

    Morphological analysis of physically reconstructed capillary hybrid silica monoliths and correlation with separation efficiency

    J. Chromatogr. A

    (2011)
  • K. Hormann et al.

    Analytical silica monoliths with submicron macropores: current limitations to a direct morphology–column efficiency scaling

    J. Chromatogr. A

    (2013)
  • T. Müllner et al.

    Finite-size effects in the 3D reconstruction and morphological analysis of porous polymers

    Mat. Today

    (2014)
  • E.N. Landis et al.

    X-ray microtomography

    Mater. Charact.

    (2010)
  • R. Moreno-Atanasio et al.

    Combining X-ray microtomography with computer simulation for analysis of granular and porous materials

    Particuology

    (2010)
  • L. Salvo et al.

    X-ray micro-tomography an attractive characterization technique in materials science

    Nucl. Instr. Meth. Phys. Res. B

    (2003)
  • X. Fu et al.

    Investigation of particle packing in model pharmaceutical powders using X-ray microtomography and discrete element method

    Powder Technol.

    (2006)
  • M. Kim et al.

    Enantioseparation of chiral acids and bases on a clindamycin phosphate-modified zirconia monolith by capillary electrochromatography

    J. Chromatogr. A

    (2012)
  • A.P. Kumar et al.

    Enantioseparation on cellulose dimethylphenylcarbamate-modified zirconia monolithic columns by reversed-phase capillary electrochromatography

    J. Chromatogr. A

    (2010)
  • G. Sena et al.

    Ecdysis period of Rhodnius prolixus head investigated using phase contrast synchrotron microtomography

    Phys. Med.

    (2016)
  • M. Krumm et al.

    Reducing non-linear artifacts of multimaterial objects in industrial 3D computed tomography

    NDT&E Int.

    (2008)
  • S. Rocchi et al.

    Enantiomers separation by nano-liquid chromatography: use of a novel sub-2μm vancomycin silica hydride stationary phase

    J. Chromatogr. A

    (2015)
  • D. Gharbharana et al.

    Tuning preparation conditions towards optimized separation performance of thermally polymerized organo-silica monolithic columns in capillary liquid chromatography

    J. Chromatogr. A

    (2015)
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    Carla Grazieli Azevedo da Silva, born in the Brazil, obtained her B.S. from Federal University of Rio Grande do Sul in 2004, her M.Sc. in Chemistry from Federal University of Rio Grande do Sul in 2007 and her Ph.D. in Sciences from University of Campinas in 2013, with an internship at Université D' Orléans in 2012. She did a post-doctorate at the University of Campinas (2013–2016) and in the University of São Paulo (2016) in Analytical Chemistry. She is currently an Associate Professor of the Federal University of Mato Grosso. Her present research activities involve preparation and characterization of new stationary phases. She has published more than 15 papers in indexed journals. In 2014 she was nominated by the American Chemical Society (ACS) as a member of the “Next Generation Ambassadors of Chemistry”, an award received by top 15 Brazilian young scientists.

    Carla Beatriz Grespan Botolli, born in the Brazil, obtained her B.S. in Industrial Chemistry from Federal University of Santa Maria in 1996 and her Ph.D. in Sciences from University of Campinas in 2002. She did a post-doctorate at University of Campinas (2002–2004). She joined the Institute of Chemistry of the State University of Campinas in 2004. Her present research activities principally involve development of monolithic stationary phases for liquid and capillary chromatography. She has published more than 30 papers in indexed journals as well as 2 book chapters and supervised more than 10 theses and dissertations.

    Carol Hollingworth Collins, born in the United States, obtained her B.S. from Bates College in 1952 and her Ph.D. in Physical and Organic Chemistry from Iowa State University in 1958. She did a post-doctorate at the University of Wisconsin (1958–1961) in these same areas. Subsequently she worked at Brookhaven National Laboratory (USA) and at the Université de Louvain and several nuclear centers (State University of New York, Taiwan, Philippines and Indonesia). She joined the Institute of Chemistry of the State University of Campinas in 1974 and became a professor emerita in 2012. Her present research activities involve high performance liquid chromatography. She has published more than 200 papers in indexed journals as well as 15 book chapters, coordinated the publication of two books on chromatography and supervised more than 50 theses and dissertations. She is a Titular member of the Brazilian Academy of Science.

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