Chromatographic Properties of Minimal Aspect Ratio Monolithic Silica

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Chromatographic Properties of Minimal Aspect Ratio Monolithic Silica Columns Takeshi Hara, Shunta Futagami, Wim De Malsche, Sebastiaan Eeltink, Herman Terryn, Gino V. Baron, and Gert Desmet Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02764 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 27, 2017

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Analytical Chemistry

Chromatographic Properties of Minimal Aspect Ratio Monolithic Silica Columns

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Takeshi Hara , Shunta Futagami , Wim De Malsche , Sebastiaan Eeltink , Herman Terryn , Gino V. Baron , Gert 1, Desmet *

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Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium

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Division of Metabolomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan 3

Vrije Universiteit Brussel, Department of Materials and Chemistry, Pleinlaan 2, B-1050 Brussels, Belgium *Corresponding author Tel.: +32 (0) 2 629 3251, Fax.: +32 (0) 2 629 3248, E-mail: [email protected]

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Abstract

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We report on a study wherein we synthesized TMOS-based silica monolithic skeletons in capillaries with

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an i.d. of 5 and 10 µm to produce skeleton structures with very low capillary-to-domain size aspect-

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ratios. These structures include the absolute minimal aspect-ratio case of a monolithic structure whose

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cross-section only contains a single node point. With domain-sized based reduced plate heights running

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as low as hmin = 1.3−1.5 for retained coumarin dyes providing a retention factor of k = 0.6−1.0, the study

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confirms the classic observation that ultra-low aspect ratio columns generate a markedly lower

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dispersion than columns with a larger aspect ratio made in the past by Knox, Jorgenson and Kennedy for

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the packed bed of spheres, but now for silica monoliths. The course of the reduced van Deemter curves,

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and more specifically the ratio of A-term versus C-term band broadening, could be interpreted in terms

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of the width and persistence length of the velocity bias zones in the columns.

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Considering the over-all kinetic performance, it is found that the two best performing structures are also

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the structures with the lowest number of domains or node points, i.e., with the lowest capillary-to-

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domain size aspect-ratio and hence resembling closest to the open-tubular format, which remains

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confirmed as the column format with the best kinetic performance. This is quantified by the fact that the

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minimal impedance values (order of Emin = 100) of the best performing ultra-low aspect ratio monolithic

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columns are still significantly larger than the Emin-values for the reference open-tubular columns (order of

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Emin = 15−20).

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1. Introduction

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In a series of iconic papers, first Knox,1 and later Jorgenson and Kennedy,2 and Jorgenson,3 have

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investigated the effect of the column-to-particle diameter ratio for particle packed bed columns. By

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gradually shrinking the width of the column to such an extent that only a few particles fit onto its

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diameter (some 6−8 in the Knox study, down to some 2 in the Jorgenson study), they found that the

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minimal reduced plate height drops from the typical fully-porous value around 2 till a value of around

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hmin = 1. Making a detailed analysis of the shape of the plots using van Deemter’s model, they essentially

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observed a strong decrease of the eddy-dispersion or A-term band broadening, while the C-term

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contribution remained constant or even slightly increased. The hypothesis they formulated for this

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observation is that, the narrower the capillary, the smaller the radial distance across which large scale

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radial packing heterogeneities can exist. These heterogeneities are the most important of the so-called

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eddy-dispersion. And by reducing the capillary diameter, the distance across which the effect of the

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heterogeneities has to be relaxed by diffusion is reduced as well, such that automatically the degree of

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eddy-dispersion is lowered. Other recent work on the effect of the aspect ratio of packed bed capillary

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columns is that of Bruns et al.,4 who used 1.7 µm particles with 10−75 µm i.d.-capillaries and


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systematically examined the correlation between the aspect ratio and column efficiency as well as that

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between change in packing heterogeneity and the efficiency, using confocal laser microscopy.

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The equivalent of these studies does not exist yet for silica monolith columns, although this type of

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chromatographic support format also clearly suffers from severe heterogeneity effects.5−14 Estimating

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the levels of the A-term contribution, it can even be argued the degree of heterogeneity is larger in

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monolithic columns than in well-packed spherical particle beds.

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In the present contribution, we report on a study wherein we synthesized TMOS-based (TMOS:

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tetrametoxysilane) silica monolithic skeletons in capillaries with an i.d. of 5 and 10 µm, aiming at

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structures with a capillary-to-domain size aspect-ratio in the order of 5 to 2. The lower bound of this

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range corresponds to a monolith exhibiting only one single node point, hence representing the ultimate

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degree of capillary-to-domain size aspect-ratio reduction that can be achieved. To pursue this, we

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carefully changed the number of domains by very precisely varying the amount of polyethylene glycol

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(PEG) in the sol-gel synthesis mixture. Pioneering work on the generation of silica monolith structures in

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small-aspect ratio spaces has been conducted by Kanamori et al., but these structures were never tested

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chromatographically.15−17

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In the present study, all structures were prepared with the same TMOS concentration. We can hence

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assume the external porosity in all structures is always the same, leaving the domain size as the only

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variable. The chromatographic performance of these structures is compared to that of porous layer

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open-tubular (PLOT) capillaries cladded with a thick mesoporous layer, a geometry which can be

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considered as the special case where the capillary-to-domain size aspect-ratio is equal to one. The PLOT

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data were taken from an earlier recent study.18 Other studies in literature where PLOT columns are

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synthesized in narrow capillaries and subsequently tested for their chromatographic performance are

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those of Forster et al..19−21 Silica monolith columns for LC/MS were produced by Luo et al.,22,23 using

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capillary diameters eventually going as low as 10 µm.

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A key problem to interpret plate height measurements obtained with differently structured monolithic

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beds, or between a particulate column and a monolithic column, is the lack of a theoretically sound

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reduction parameter to determine a physically consistent dimensionless plate height h. Whereas the

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efficiency of spherical particle columns can be conveniently interpreted using the diameter of the

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particles, there is no such well-defined equivalent for silica monolithic beds, for which the so-called

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domain size (= a size measure for the combined skeleton and through-pore) has up till now been the

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most frequently used size measure for silica monolithic beds.24−26 It owes its existence to the fact that, if

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the bed would be perfectly ordered, it would be the size of the unit cell from which the entire structure

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could be reconstructed. Despite the fact the domain size is an ill-defined concept (because of the many

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different ways the size of 3-dimensional objects can be defined if they are not spherical or cubic), and

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has little or no theoretical grounds, it is the best measure currently available. A much cleaner measure 2 ACS Paragon Plus Environment


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for the chromatographic performance is the separation impedance (E), as this omits the need to specify

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a characteristic dimension. It is directly related to the ability of a structure to generate a given efficiency

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in the shortest possible time.27,28 Since the separation impedance is not a pure efficiency measure, but

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also depends on the permeability of the column, both approaches (domain-size based plate height

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analysis and separation impedance plots) can be considered as complementary tools.

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2. Experimental

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2.1. Chemicals and Materials

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Toluene (HPLC grade, > 99.8%), TMOS, 1 M aqueous acetic acid solution for volumetric measurement,

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and polyethylene glycol (PEG) of molecular weight (MW) = 10 000 g/mol were purchased from Sigma-

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Aldrich Co. (Diegem, Belgium). Methanol (HPLC super-gradient grade) was obtained from Biosolve B.V.

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(Valkenswaard, NL). Deionized water was produced in-house with a Milli-Q water purification system

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Merck Millipore (Billerica, MA, USA). Octadecyldimethyl-N,N-dimethylaminosilane (ODS-DMA) was

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obtained from ChemPur Feinchemikalien und Forschungsbedarf GmbH (Karlsrule, DE). Coumarin 440

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(C440: 7-amino-4-methyl-2H-1-benzopyran-2-one), coumarin 460 (C460: 7-(diethylamino)-4-methyl-2H-

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1-benzopyran-2-one), and coumarin 480 (C480: 2,3,6,7-tetrahydro-9-methyl-1H,5H,11H-[1]benzo-

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pyrano-[6,7,8-ij]quinolizin-11-one) were acquired from Vadeno Optical Solutions (Apeldoorn, NL). PTFE

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filters (0.20 µm × 25 mm) were purchased from Macherey-Nagel (Düren, DE). Fused-silica capillaries

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with an inner diameter (i.d.) of 5 µm and 10 µm, possessing an outer diameter of 375 µm were

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purchased from Polymicro Technologies (Phoenix, AZ, USA), which were covered with polyimide coating.

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2.2. Preparation of monolithic silica capillary columns

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Monolithic silica capillary columns with an i.d. of 5 µm and 10 µm were produced using a similar

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preparation procedure as reported previously for the production of TMOS-based monolithic silica columns

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with 100 µm i.d..29 Only the feed compositions are different, as designated with recipe A, B, and C (see

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Table S-1 of the Supporting Information (SI)). Here, 5.6 mL TMOS, 0.900 g urea, 1.180−1.240 g PEG, and 10

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mL 0.01 M aqueous acetic acid solution were applied to produce the mixture sol-solutions using an electric

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balance (accuracy: ±0.1 mg). This read-out precision allows us to control the difference in PEG amount

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between the different recipes to 0.5%. Subsequently, the solutions were charged into fused-silica

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capillaries with 5 or 10 um i.d. (ca. 42 cm in length) by applying a pressure of 50 bar with a HPLC pump

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within 20 min (Note that the gelation takes place in 2 hours for this solution), as described in our earlier

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study on PLOT capillary columns (for detailed information, cf. the literature).18 After filling, the gelation in

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the capillary took place at 25 °C for 20 h with a water bath. The subsequent processes, such as the

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hydrothermal treatment at 95 °C to produce the mesopores and washing the capillary columns with 3 ACS Paragon Plus Environment

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methanol, were carried out according to the protocol described previously for monolithic silica columns.30

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After the washing process, the fabricated capillary columns were dried at 120 °C in an oven for 24 h.

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After the fabrication of bare-silica columns, in-situ functionalization of the silica surface was carried out

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with 20:80% (v/v) ODS-DMA/toluene mixture solution following a procedure similar to that used in the

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proceeding study on monolithic silica columns.30

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2.3. Measurements

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The LC instrument consisted of a LPG-3400M pump (Thermo Fisher Scientific, Germering, DE), a

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Rheodyne 7125 manual injector with a 5 µL sample loop (IDEX Health & Science GmbH) with a home-

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build T-split injection flow system, a fluorescence microscope IX-71 using the U-RFT-T lamp power

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supply (Olympus, Tokyo, JP), and a charge-coupled device camera C4742-95-12ERG (Hamamatsu

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Photonics, Shizuoka, JP). During the measurements, split ratios were maintained around 1/250 000 for

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the 5 µm i.d.-capillary columns, and around 1/65 000 for the 10 µm i.d.-capillaries, respectively, in order

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to achieve picoliter injection volumes and nanoliter flow rates, as explained in the literature.18,31 For the

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on-column fluorescence detection, the excitation wavelength was set at 360−420 nm with a XF1075

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387AF28 filter (Omega Optical Inc., VT, USA) and fluorescence wavelength was at 400−500 nm with

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MF460-80 filter (Thorlabs Elliptec GmbH, Dortmund, DE), respectively. Data acquisition rate was set to

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provide at least more than 20 points to draw each peak by assuming peak width is 4σ. 2 mM coumarin

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compounds, dissolved in the solvent with a mobile phase composition, were utilized to examine

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column performance under the aforementioned LC set-up conditions using 70:30% (v/v)

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methanol/water as mobile phase. The fluorescence microscope images were processed with MatLab

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2012b software (Mathworks, MA, USA), to visualize the chromatograms. Theoretical plate numbers

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were calculated with half width at half maximum (FWHM).

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For the scanning electron microscopy (SEM) measurements, 6 cm-capillary segments were cut out from

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the 40 cm-fabricated capillary columns, in order to divide them into three pieces of 2 cm-capillaries for

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the observation (i.e. residual 34 cm-capillaries were used for column test in HPLC, and 6 cm-capillaries

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for SEM measurement.) Then, a thin gold coating with around 8 nm was applied using a sputter coater

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(208 HR, Cressington Scientific instruments Ltd, Watford, UK). SEM images of macroporous structure

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were obtained using a field-emission scanning electron microscope JSM-7100F from JEOL Ltd. (Tokyo,

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JP).

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3. Results and discussion

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3.1 Geometrical characterization and retention factors



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Fig. 1 shows representative SEM pictures of the obtained structure for each of the different synthesis

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mixtures A, B, C in the 5 and 10 µm capillaries. The SEMs very clearly show that the geometry of the

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formed monolithic structures is very sensitive to the amount of PEG added to the sol-gel mixture. While

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this amount only varies from 1.180 g (recipe A) over 1.200 g (recipe B) to 1.240 g (recipe C), the number

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of branches and node points making up the silica skeleton increases prominently. Concomitantly, also

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the number of parallel flow-through channels into which the cross-sectional area of the capillaries is

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divided increases.

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Important information about the structures’ geometry can also be obtained from the retention factors.

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Interestingly, the retention factors we measured for C460 and C480 for the A, B and C-mixtures and

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capillary diameters are, within the experimental variability, very similar (see Table 1 : 3.3% CV for kC460

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and 3.5% CV for kC480). To interpret this, it should first of all be noted that it is well known from a series

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of other studies of monolithic silica prepared with PEG as the phase-separation inducer, that the

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resulting volumetric fraction of silica material is nearly exclusively determined by the concentration of

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TMOS in the sol-gel mixture.25,29,30,32 Secondly, a previous study where we studied PLOT columns

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produced with varying TMOS content also clearly showed that, independently of the TMOS

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concentration, the produced layers always have the same mesoporous structure (i.e., the same amount

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of stationary phase per volume of silica support) and that our ODS-modification procedure is very

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reproducible and provides a constant C18 coverage.18 This could be concluded from the fact that the

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retention factors obtained in that study scaled directly with the amount of stationary phase per volume

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of silica support, or, in other words, that the retention equilibrium constant obtained with our coating

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procedure is independent of the volume of deposited silica. The creation of a monolithic structure inside

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a capillary could also be seen as a way to increase the loadability of the capillary over that of a PLOT

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column.18 However, this would require the use of a higher TMOS loading than used in the present study,

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and it has been found to be very difficult to control the domain size when the amount of TMOS exceeds

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6 mL per 10 mL of 0.01 M acetic acid.

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Given the above, the near constant retention factors shown in Table 1 can now be seen as an indication

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the relative volume of the silica skeleton is, within the experimental error, the same for all combinations

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shown in Fig. 1 and Table S-1. Since the external porosity ε is the complement of the relative skeleton

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volume, this also implies the external porosity ε of the produced monolithic structures is the same for all

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geometries shown in Fig. 1. From the results of a series of size exclusion chromatography experiments in

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a previous study on 100 µm i.d. TMOS-based monolithic silica capillary columns, we can estimate the

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external porosity to be on the order of ε = 80%.33,34 From this value, and considering that monolithic

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silica columns after octadescylsilyation typically provide the value of silica skeleton internal porosity, εint

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= 0.4−0.5,34−36 we can estimate the total porosity εt to lie around 88 to 90%. 5 ACS Paragon Plus Environment

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Combining the fact that all geometries in Fig. 1 have the same volumetric fraction of silica, with the fact

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that the number of skeleton branches clearly increases when going from condition A to C, this can only be

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explained by assuming that the skeleton branches themselves become thinner when the amount of PEG is

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increased.25,30

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To quantify the effect of the PEG amount on the resulting structure, we tried to estimate a value for the

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domain size from the available SEM pictures. For this purpose, a very basic 2-D approach was used,

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counting the number of through-pores per cross-sectional area and then dividing both number to arrive

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at an average domain size area.37 This area was then transformed into that of an equivalent equilateral

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hexagon, from which we took the apothem as the characteristic measure. In parallel, we also considered

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a circle with the same equivalent area, and used the corresponding diameter as the characterized

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domain size. To alleviate the experimentalist’s subjectivity, domains were counted by two individual

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people and two different methods were used (resp. drawing hexagonal tiles to indicate the individual

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domains or putting crosses in the through-pore centers). Domain counting was done at three different

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cross-sections (see Figs. S-2a−b of the SI for individual SEMs and for the hexagons and crosses actually

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used for counting the domains). The resulting average number of domains and average domain area are

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given in Table 2, together with the derived size measures (lines 4 and 5 in Table 2). Since there is no

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theoretical ground to prefer the circle-based over the hexagon-based domain size, and given both values

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typically also only differ by 5%, all domain sizes used in the present study relate to the average of both

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measures (line 6 in Table 2). The values in Table 2 provide a quantitative estimate of how the domain

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size of the structures prepared with recipes A, B, C decreases with increasing PEG amount (Fig. 1), an

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observation which is in full agreement with earlier observations made in literature.25,29,30,32

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3.2 Efficiency measurements

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Fig. 2 shows the chromatograms with the C440, C460 and C480 peaks observed in the different monolith

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geometries at u0 = 0.63−0.64 mm/s, i.e., close to the optimal linear velocity of the van Deemter curve (all

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geometries have similar optimal velocities, see Fig. 3 further on). Fig. S-3 of the SI shows chromatograms

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obtained in a second series of 5 µm capillaries. Comparison with Fig. 2 shows a good repeatability of the

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retention factors (max. 5% difference, 2% difference for sol-gel mixture C), and an excellent repeatability

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of the plate height (H) values (only 2% difference).

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Fig. 3 shows the global result of the efficiency measurements in the form of a traditional van Deemter

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plot. For the sake of comparison, we also added the van Deemter curves obtained in two PLOT-columns

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produced with a similar TMOS concentration, one in a capillary with an i.d. of 5 µm and one with an i.d.

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of 10 µm.18 Comparing the band broadening in the minimal aspect-ratio monolithic columns with the



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PLOT columns, we can infer that adding a silica skeleton to an open-tubular system has two major

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effects:

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- the radial mass transfer distance decreases (positive effect)

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- the disorder increases (negative effect)

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With the present study, we can investigate which of both effects is dominating. Fig. 3 clearly shows that

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the H-values for all produced monolithic columns lie between the 5 and the 10 µm i.d.-PLOT column

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(black-color symbols). This means that, if starting from a 10 µm i.d.-capillary, the addition of a silica

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skeleton as a means to reduce the diffusional distances is beneficial. i.e., in this case the advantage of

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the reduced mass transfer distances outweighs the disadvantage of the increased disorder. For the 5 µm

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i.d.-capillary, the opposite occurs, as in this case all silica monolith structures yield higher H-values than

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the corresponding open-tubular format, implying that here the additional disorder caused by the

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presence of the monolithic skeleton outweighs the advantage of the concomitant reduction of the

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diffusional distances.

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It can hence be concluded that the capillary diameter above which the addition of a monolithic silica

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skeleton (one or more connecting nodes per cross-section) becomes advantageous compared to a PLOT

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column (zero nodes) lies somewhere between 5 and 10 µm i.d., at least for the typical domain sizes (2−3

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µm) that were created in the present study. Smaller domain sizes were not created because this would

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lead us away from the pursued small-aspect ratio structures.

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Another observation that can be made from Fig. 3 is that, for significantly retained compounds, i.e., for

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C460 and C480 (see Figs. 3b−c), the plate heights follow the order HA > HB > HC in the high velocity range

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(for instance, HA = 19.0 µm, HB = 16.9 µm, and HC = 15.6 µm were obtained with the 5 µm i.d.-columns at

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u0 = 5 mm/s). Obviously this is in line with the fact that the domain sizes given in Table 2 also decrease in

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the same direction, with the two A-conditions producing the two largest domain sizes (2.8 µm in the 5

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µm i.d.- and 3.2 µm in the 10 µm i.d.-capillaries) and the two C-conditions producing the smallest

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domain sizes (2.1 µm in both the 5 µm and the 10 µm i.d.-capillaries).

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To analyze these results more in depth, the domain sizes determined in Table 2 were used to calculate

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reduced plate heights (h) and velocities (v0) (see Fig. 4). For the PLOT columns, it is most straightforward

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to use the capillary diameter as the reduction parameter. These reduced parameters have been

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commonly used to assess packing quality (structural homogeneity) of particulate columns, elucidating

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how packing materials are packed well with normalizing difference in particle diameter.38 For the

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present monoliths, this style helps considering how well the skeletons are homogeneously distributed in

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the narrow i.d.-capillaries with normalizing difference in domain size.

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Considering Fig. 4, it is first of all interesting to observe the relatively low values of the curve minima,

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going from hmin < 1.5 for the quasi-unretained component over 1.3 < hmin < 1.9 for C460 (k = 0.6 ) and 1.5 7 ACS Paragon Plus Environment

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< hmin < 2.0 for C480 (k = 1.0). As shown in the zoom-ins presented in the Fig. S-4 of the SI, the minima

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are lowest for the A-recipe and highest for the C-recipe, indicating that the lowest capillary-to-domain

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size aspect ratio leads to the lowest band broadening and vice versa for the one with the highest

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capillary-to-domain size aspect ratio. This is very similar to the observations made by Knox, Kennedy and

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Jorgenson in the packed bed of spheres.1−3 The lower bound values in the above cited intervals, relating

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to the structures with the smallest capillary-to-domain size aspect-ratio, are all considerably smaller

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than any of the values ever cited in literature for monolithic columns and capillaries. The lowest ever

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reported up till now is that in Hara et al. (hmin = 2.2; 4.8 µm plate height/2.2 µm domain size) for 100 µm

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i.d.-second generation capillary columns).29 In millimeter-bore columns, the best ever minimal plate

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heights are even considerably higher, on the order of 3.1 to 3.6. (e.g., hmin = 3.1 (6.7 µm/2.2 µm) in

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Harmann et al.,13 hmin = 3.3 in Cabooter et al.,39 hmin = 3.2 (5.7 µm /1.8 µm) in Ma et al.,40 and hmin = 3.6

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(6.5 µm/1.8 µm) in Gritti et al.36). All these literature data relate to column and capillaries with a much

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wider i.d., and hence also to a much higher capillary-to-domain size aspect-ratios than those considered

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in the current study. Compared to the reference PLOT columns (black data), it is however clear that even

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the lowest aspect ratio structures still produce a significantly higher dispersion than the open-tubular

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format, where there is only one flow path such that there can be no eddy-dispersion.

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The fact that the present minimal aspect ratio monoliths produce much smaller hmin-values than silica

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monoliths in broader capillaries and columns cited here above is in full agreement with the observations

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made by Knox and Kennedy and Jorgenson in their aspect-ratio studies of spherical packings.1−3 We can

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hence infer that the unusually low minimal plate height values observed in the present study are also

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due to the fact that the ultra-small capillary diameter simply restricts the radial distance across which

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velocity differences can exist and hence need to be re-equilibrated. The smaller this distance, the

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smaller will be the resulting dispersion. Another reason for the smaller h-values certainly is the fact that

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the limited lateral width inherently restricts the number of parallel flow paths, in turn minimizing the

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number of possible velocity biases and disorder in general.

270

Another interesting observation is that all 5 µm i.d.-capillary columns (closed symbol data) seem to have

271

a deeper minimum and a steeper C-term dominated part of the curve than the 10 µm i.d.-capillary

272

columns (open symbol data). These all seem to have a higher minimum and a flatter C-curve. This is

273

confirmed by the A-, B- , C-values we obtained by fitting the data in Fig. 4 with the simple van Deemter

274

model:

275

h = A+

B + Cν 0 ν0

(1)

276

This relatively simple model was preferred in the present study because it allows for a direct comparison

277

with the Kennedy and Jorgenson studies.2,3 The obtained values for C460 and C480 (retained solutes) are



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278

shown in Table S-5 of the SI, indeed showing that all 5 µm i.d.-capillary columns have C/A-ratio values

279

that are significantly higher (2.5 to 3 times) than for the 10 µm i.d.-capillary columns.

280

To interpret this, it should first be considered that the band broadening arising from radial packing

281

heterogeneities can manifest itself either as a classical A-term contribution (= contribution to h which is

282

independent of velocity) or as a quasi-linear C-term contribution (= contribution to h which varies

283

linearly or quasi-linearly with the velocity), or as some intermediate form.41,42 Whether a velocity bias

284

rather generates an A-term or a C-term type behavior simply depends on the geometric aspect ratio of

285

the zone over which the characteristic velocity bias persists: velocity bias regions that are much wider

286

than long will manifest themselves as a pure A-term contribution, whereas velocity bias regions that are

287

very narrow and persist over a long distance manifest themselves as a pure C-term contribution.

288

Considering the number of flow-through pores cutting across the 10 µm i.d.-capillaries is much higher

289

than in the 5 µm i.d.-capillaries (cf. the numbers in the first row of Table 2), it can be inferred that the

290

number of channel merging points (which can be considered as the main termination points for the

291

velocity biases) is much larger in the 10 µm i.d.-capillaries. Combining the higher number of potential

292

velocity bias termination points with the inevitably larger width of the zones (because of the larger

293

diameter), it is obvious to expect the characteristic velocity bias regions in the 10 µm i.d.-capillaries will

294

be significantly shorter and wider, and will hence display a higher ratio of A-term versus C-term band

295

broadening contribution than the 5 µm i.d.-capillaries, as is indeed found in Table S-5 of the SI.

296 297

3.2 Permeability measurements

298

Compared to the open-tubular columns, the addition of a silica skeleton inevitably leads to a

299

significantly decreased permeability. This can readily be seen from Fig. S-6 of the SI, as well as from the

300

permeability data that can be derived from the slopes of the lines in Fig. S-6 and given in Table 1. In

301

agreement with the fact the C-mixture leads to the smallest domain size, the two recipe C-cases have

302

the lowest permeability (steepest ∆P versus u0-relationship). Similarly, it is also straightforward to

303

understand why the two recipe A-cases lead to the lowest permeability and why the two recipe B-cases

304

have some intermediate behavior. Using the estimated domain sizes given in Table 2, the Kv0-

305

permeabilities can be transformed into dimensionless flow resistances φ0 (see also Table 1). The data

306

show the monoliths with the lowest number of nodes (A-recipe) produces a significantly lower flow

307

resistance (φ0 = 30−40) than for the two other recipes leading to a higher number of node points (order

308

φ0 = 60−75). Comparing the flow resistances of the monolithic columns with those of the PLOT columns

309

is not relevant, because of the different characteristic size used to calculate φ0. The flow resistances for

310

the A, B, C-mixtures are significantly smaller than the φ0-values for many other monolithic columns

311

(typically φ0 = 100−250),29,36,39,40,43 and certainly much smaller than the flow resistance of a packed bed

312

column (typically φ0 = 600−800).28,44 The values we measure here are in good agreement with the value 9 ACS Paragon Plus Environment

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313

(φ0 = 60 when ε = 80%) that can be obtained via a quadratic interpolated from the flow resistance (φ0)

314

versus external porosity (ε) data obtained by Gzil et al. for the case of a perfectly ordered and idealized

315

tetrahedral skeleton monolith.45 This agreement hence links the low flow resistance of the presently

316

synthesized structures directly to their high external porosity (ε = 80%).

317 318

3.3 Kinetic performance

319

Fig. 5 combines the information on efficiency and permeability into a kinetic plot of the separation

320

impedance E = H^2/Kv0 versus the ratio of the corresponding Nopt/N-value (see the literature of Billen et

321

al. for physical meaning of plot; Kv0 is shown in Table 1; Nopt/N = u.H/(uopt.Hmin)).46 The N in the Nopt/N-

322

ratio on the x-axis is the efficiency that would be obtained at a given velocity if the column would be

323

made exactly long enough to achieve the imposed ∆Pmax (we selected ∆Pmax = 300 bar in the present

324

study as this was the pressure limit of the Rheodyne injector, while the monolithic silica structure can be

325

assumed to be able to withstand much larger pressures).47 Nopt is the corresponding value at the velocity

326

corresponding to the minimum of the van Deemter curve (also corresponding to the minimum of the E-

327

curves). Originally introduced by Golay, and later applied to LC by Knox, the separation impedance can

328

be considered as a shape quality factor,7 with the lowest impedance value corresponding to the shape

329

that is most suited to produce a given number of plates in the shortest possible time. Please also note

330

the position of the curves in the E vs Nopt/N-curves does not depend on the selection of any

331

characteristic size and is hence a fully geometry-independent measure.

332

Mutually comparing the monolithic silica capillaries in Fig. 5, we see that the curves more or less follow

333

the same pattern as the permeability measurements, with the 5(C) and 10(C)-capillaries (green data,

334

highest aspect-ratio) clearly performing least well and the 5(A) and 5(B)-capillaries (solid red and blue)

335

producing the lowest curves. The 5(A)-capillary owes its low impedance especially to its very low h-

336

value, while the 5(B)-capillary rather owes its low impedance especially to its very low flow resistance

337

(compare the slope of the 5(B)-curve with that of the 10(B)-structure in Fig. S-6). Especially when looking

338

at the more complete set of SEM pictures shown in the SI, it is clear that the two structures with the

339

lowest impedance are also the structures with the lowest number of domains, i.e., with the lowest

340

capillary-to-domain size aspect-ratio. The other 4 geometries clearly have a higher separation

341

impedance, as well as a higher number of domains per cross-section. The E vs Nopt/N-plots for the other

342

analytes provides very similar conclusions (see Fig. S-7 of the SI).

343

Another clear observation is that the two open-tubular columns have a separation impedance that is

344

significantly smaller than that of any of the monolithic structures (Emin around = 15−20, a value in close

345

in agreement with the theoretical expectations based on the Golay-Aris theory).48,49 This reconfirms the

346

single open-tubular channel as the best possible chromatographic shape (no parallel competing flow

347

paths with different flow resistance and different local retention factor, minimal flow resistance).

348

However, to make the best possible shape work in a practically relevant range of analysis times, the 10 ACS Paragon Plus Environment


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349

lateral diameter of the open-tube needs to be made sufficiently small (5 µm and less).47 Evidently, the

350

shape factor also only takes the kinetic separation aspects into account, and negates the poor mass

351

loadability of PLOT columns with narrow i.d..

352 353

4. Conclusions

354

Comparing the band broadening obtained with silica monoliths synthesized in capillaries with an i.d. of 5

355

and 10 µm with that of porous layer open-tubular (PLOT) capillaries, it can be concluded that the

356

capillary diameter above which the addition of a monolithic silica skeleton becomes advantageous

357

compared to an unobstructed PLOT column lies somewhere between 5 and 10 µm i.d., at least for the

358

typical domain sizes (2−3 µm) that were created in the present study.

359

Within the limitations of the adopted definition of the domain size, we found that domain-sized based

360

reduced plate heights running as low as hmin = 1.3−1.5 for retained compounds are obtained with the

361

structures having the lowest possible capillary-to-domain size aspect-ratio, i.e., for structures whose

362

cross-section only contains one or two node points. The observed hmin-values are significantly smaller

363

than any of the values ever reported in literature, which all relate to structures with a much larger

364

capillary-to-domain size aspect-ratios. As such, the present study confirms the classic observation that

365

ultra-low aspect ratio columns generate a markedly lower dispersion than columns with a larger aspect

366

ratio made in the past by Knox, Jorgenson and Kennedy for the packed bed of spheres, but now for silica

367

monoliths.

368

Interestingly, it was found that the skeleton structures obtained in the 10 µm i.d.-capillaries display a

369

higher ratio of A-term versus C-term band broadening than the 5 µm i.d.-capillaries, in agreement with

370

the visual observation that the velocity bias regions in the 10 µm i.d.-capillaries are inevitably wider

371

(because of the larger diameter) and shorter (because of the higher number of potential velocity bias

372

termination points) than in the 5 µm i.d.-capillaries.

373

In terms of kinetic performance, where band broadening and flow resistance combine in the ability of a

374

given support structure to produce a given number of theoretical plates in the shortest possible time, it

375

is found that the two structures with the lowest kinetic impedance are also the structures with the

376

lowest number of domains or node points, i.e., with the lowest capillary-to-domain size aspect-ratio and

377

resembling most closely that of an open-tubular column. The minimal impedance values (order of Emin =

378

100) are however significantly larger than the Emin-values for the PLOT columns (order of Emin = 15−20),

379

reconfirming the open-tubular format as the column format with the best kinetic performance.

380 381

5. Acknowledgement 11 ACS Paragon Plus Environment

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382

T.H. gratefully acknowledges the financial support of the Research Foundation Flanders (FWO)

383

(12C5414N). We also appreciate the support for SEM from the SURF group of the Vrije Universteit

384

Brussel.

385 386

References

387

(1) Knox, H. J.; Parcher, F. J. Anal. Chem. 1969, 41(12), 1599–1606.

388

(2) Kennedy, T. R.; Jorgenson, W. J. Anal. Chem. 1989, 61(10), 1128–1135.

389

(3) Showchien, H.; Jorgenson, W. J. Anal. Chem. 1996, 68(7), 1212–1217.

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(4) Burns, S.; Grinias, P. J.; Blue, E. L.; Jorgenson, W. J.; Tallarek, U. Anal. Chem. 2012, 84(10), 4496–4503.

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(5) Billen, J.; Gzil, P.; Desmet, G. Anal. Chem 2006, 78, 6191‒6201.

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(6) Guiochon, G. J. Chromatogr. A 2007, 1168(1−2), 101‒168.

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(7) Billen, J.; Desmet, G. J. Chromatogr. A 2007, 1168, 73‒99.

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(8) Altmaier, S.; Cabrera, K. J. Sep. Sci. 2008, 31(10−11), 2551–2559.

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(9) Mriziq, S. K.; Abia, A. J.; Lee, Y.; Guiochon, G. J. Chromatogr. A 2008, 1193(31), 97‒103.

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(11) Morisatoa, K.; Miyazaki, S.; Ohira, M.; Furuno, M.; Nyudo, M.; Terashima, H.; Nakanishi, K. J.

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Chromatogr. A, 2009, 1216, 7384−7387.

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(12) Bruns, S.; Hara, T.; Smarsly, M. B.; Tallarek, U. J. Chromatogr. A 2011, 1218(31), 5187‒5194.

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(13) Hormann, K.; Mullner, T.; Bruns, S.; Holtzel, A.; Tallarek, U. J. Chromatogr. A 2012, 1222, 46‒58.

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(14) Aggarwal, P.; Tolley, D. H.; Lee, L. M. J. Chromatogr. A 2012, 1219, 1‒14.

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(15) Kanamori, K.; Nakanishi, K.; Hirao, K.; Jinnai, Langmuir 2003, 19(20), 9101–9103.

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(16) Kanamori, K.; Nakanishi, K.; Hirao, K.; Jinnai, H. Colloids Surfaces A Physicochem. Eng. Asp. 2004,

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241(1–3), 215–224.

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(17) Kanamori, K.; Yonezawa, H.; K.; Hirao; Nakanishi, K.; Jinnai, H. J. Sep. Sci. 2004, 27(10−11), 874–886.

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(18) Hara, T.; Futagami, S.; Eeltink, S.; De Malsche, W.; Baron, G. V.; Desmet, G. Anal. Chem. 2016,

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88(20), 10158–10166.

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(19) Forster, S.; Kolmar, H.; Altmaier, S. J. Chromatogr. A 2012, 1265, 88–94.



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410


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(22) Luo, Q.; Shen, Y.; Hixson, K. K.; Zhao, R.; Yang, F.; Moore, R. J.; Mottaz, H. M.; Smith, R. D. Anal.

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Chem. 2005, 77 (15), 5028–5035.

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(23) Luo, Q.; Page, S. J.; Tang, K.; Smith, R. D. Anal. Chem. 2007, 79(2), 540–545.

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(24) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. J. Chromatogr. A 1997, 762(1–2), 135–

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146.

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(25) Nakanishi, K.; Tanaka, N. Acc. Chem. Res. 2007, 40, 863–873.

417

(26) Unger, K. K.; Tanaka, N.; Machtejevas, E. Monolithic Silicas in Separation Science: Concepts,

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Syntheses, Characterization, Modeling and Applications; Wiley-VCH Verlag GmbH&Co.KGaA:

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Weinheim, 2011.

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(27) Desmet, G.; Clicq, D.; Gzil, P. Anal. Chem. 2005, 77, 4058−4070.

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(28) Broeckhoven, K.; Desmet, G. TrAC Trends Anal. Chem. 2014, 63, 65‒75.

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(29) Hara, T.; Kobayashi, H.; Ikegami, T.; Nakanishi, K.; Tanaka, N. Anal. Chem. 2006, 78(22), 7632–7642.

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(30) Hara, T.; Desmet, G.; Baron, G. V.; Minakuchi, H.; Eeltink, S. J. Chromatogr. A 2016, 1442, 42–52.

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(31) Swart, R.; Kraak, J. C.; Poppe, H. TrAC - Trends Anal. Chem. 1997, 16(6), 332–342.

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(32) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Tanaka, N. J. Chromatogr. A 1997, 797(1–2), 133–

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137.

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(33) Hara, T.; Mascotto, S.; Weidmann, C.; Smarsly, B. J. Chromatogr. A 2011, 1218(23), 3624–3635.

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(34) Hara T., Study on Preparation and Characterization of Monolithic Silica Capillary Columns for High

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Separation Efficiency in High Performance Liquid Chromatography, PhD thesis in Justus-Liebig-

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Universität Giessen (online available), 2013.

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(35) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. J. Chromatogr. A 1998, 797(1–2), 121–

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(37) Courtois, J.; Szumski, M.; Georgsson, F.; Irgum, K. Anal. Chem. 2007, 79, 335−344.

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(38) Neue, D. U. HPLC Columns: Theory, Thechnology, and Practice; Wiley-VCH: New York, 1997.

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(39) Cabooter, D.; Broeckhoven, K.; Sterken, R.; Vanmessen, A.; Vandendael, I.; Nakanishi, K.; Deridder, S.; Desmet, G. J. Chromatogr. A 2014, 1325, 72‒82. 13 ACS Paragon Plus Environment

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(40) Ma, Y.; Chassy, A.W.; Miyazaki, S.; Motokawa, M.; Morisato, K.; Uzu, H.; Ohira, M.; Furuno, M.; Nakanishi, K.; Minakuchi, H.; Mriziq, K.; Farkas, T.; Fiehn, O.; Tanaka, N. J. Chromatogr. A 2015, 1383, 47‒ 57.

441

(41) Giddings, C. Dynamics of Chromatography: Principles and Theory; Dekker, M., Ed.; CRC Press: New

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York, 1965.

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(42) Desmet, G. J. Chromatogr. A 2013, 1314, 124−137.

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(43) Gritti, F.; Guiochon, G. J. Chromatogr. A 2011, 1218, 5216–5227.

445

(44) Desmet, G.; Clicq, D.; Gzil, P. Anal. Chem. 2005, 77(13), 4058–4070.

446

(45) Gzil, P.; De Smedt, J.; Desmet, G. J. Sep. Sci. 2006, 29, 1675–1685

447

(46) Billen, J.; Guillarme, D.; Rudaz, S.; Veuthey, J.-L.; Ritchie, H.; Grady, B.; Desmet, G. J. Chromatogr. A

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2007, 1161, 224–233

449

(47) Hara, T.; Eeltink, S.; Desmet, G, J. Chromatogr. A 2016, 1446, 164–169.

450

(48) Poppe, H. J. Chromatogr. A 1997, 778, 3–21.

451 452

(49) Causon, T. J.; Shellie, R. A.; Hilder, E. F.; Desmet, G.; Eeltink, S. J. Chromatogr. A 2011, 1218 (46), 8388–8393.

453



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454

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Figure caption

455 456 457 458 459 460

Figure 1. Scanning electron micrographs of the silica monoliths obtained with recipes A, B, C in capillaries with a 5 µm and a 10 µm i.d.. Column: (a) Capillary(5)-A, (b) Capillary(5)-B, (c) Capillary(5)-C, (d) Capillary(10)-D, (e) Capillary(10)-B, and (f) Capillary(10)-C. The numbers in the parentheses show capillary i.d.. Scale bars correspond to 1 µm. The measurements were conducted at 10 000-fold magnification.

461 462 463 464 465 466

Figure 2. Chromatograms obtained for coumarins C440, C460 and C480 with ODS-modified monolithic silica columns. Column: (a) Capillary(5)-A, (b) Capillary(5)-B, (c) Capillary(5)-C, (d) Capillary(10)-D, (e) Capillary(10)-B, and (f) Capillary(10)-C. Mobile phase: 70:30% (v/v) methanol/water. Measurement temperature: 25 °C. Effective length (e.f.) and total length (t.l.) are shown for all the columns. Retention factor (k), theoretical plate number (N), and plate height (H) observed for coumarin 480 are shown.

467 468 469 470 471 472 473

Figure 3. Van Deemter plots obtained for (a) C440, (b) C460, and (c) C480 with ODS-modified monolithic silica columns and PLOT columns. Symbol: Capillary(5)-A (), Capillary(5)-B (), Capillary(5)-C (), Capillary(10)-A ( ), Capillary(10)-B ( ), Capillary(10)-C ( ), PLOT(5)-A ( ), and PLOT(10)-A ( ). Mobile phase: 70:30% (v/v) methanol/water. Measurement temperature: 25 °C. The measurement errors on H-values were within ±0.10 in the range of all the linear velocities. PLOT(5)-A and PLOT(10)-A are same as used in our previous study.18

474 475 476 477

Figure 4. Plots of reduced plate height against reduced mobile phase velocity with ODS-modified monolithic silica columns and PLOT columns. Solute: (a) C440, (b) C460, and (c) C480. Measurement conditions and symbols are same as shown in Fig. 3.

478 479 480

Figure 5. Plots of separation impedance (E) versus Nopt/N for the ODS-modified monolithic silica columns and PLOT columns. Solute: C480. Measurement conditions and symbols are same as shown in Fig. 3.

481 482 483 484 485 486 487 15 ACS Paragon Plus Environment

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488

Tables

489

Table 1. Column properties of fabricated ODS-modified capillary columns Column

kC460a

kC480b

Column permeabilityc (Kv0) (m2)

Flow resistanced (φ0)

Capillary(5)-A

0.62

0.96

2.5 × 10-13

39.2

Capillary(10)-A

0.63

0.98

2.0 × 10-13

30.8

1.04

-13

66.6

-13

58.2

-14

Capillary(5)-B Capillary(10)-B

0.67

1.05

2.1 × 10 1.4 × 10

Capillary(5)-C

0.64

1.00

8.3 × 10

62.4

Capillary(10)-C

0.66

1.02

7.4 × 10-14

75.0

0.87

-13

36.3

-12

33.6

PLOT(5)-A

e

PLOT(10)-A

490 491 492 493

0.67

0.56 e

0.55

0.86

7.2 × 10 2.9 × 10

a,b

Retention factors were obtained for C460 and C480 in 70:30% (v/v) methanol:water. Column permeability (Kv0) was calculated with coumarin 440.5,30 dFlow resistance (φ0) was calculated as φ0 = d2/Kv0 (d: domain size of silica monolith or through-pore size in PLOT columns). ePLOT capillary columns used in our earlier study.18 c



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494

Table 2. Estimated domain size of monolithic silica capillary columns with 5 and 10 µm i.d. Parameters

(5)-A

(5)-B

(5)-C

(10)-A

(10)-B

(10)-C

Diameter (µm)a

5.57

5.31

5.51

10.90

11.28

11.09

3.00

4.15

5.15

9.50

14.17

20.85

8.12

5.34

4.63

10.07

7.10

4.64

3.06

2.48

2.31

3.40

2.86

2.31

3.21

2.61

2.42

3.57

3.00

2.43

3.14

2.55

2.37

3.48

2.93

2.37

b

Domains

c

Domain area

Apothem (µm)

d

Cyl. diameter (µm)

e

Domain size (av.) (µm)

495 496 497 498 499 500 501

f

a

Capillary diameters were determined with SEM pictures in Fig. S-2. bDomain numbers pre cross-sectional area were counted by two individual persons and subsequently the obtained numbers were averaged. cThe values were calculated by dividing the crosssectional area of capillary by domain numbers. dThe values were determined by assuming a through-pore shape is a regular hexagon. eThe values were obtained by assuming a through-pore shape is a circular form. fDomain sizes were given by averaging the values obtained from regular hexagon-model and cylinder-model.

502


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Figure 1 in the main text 44x19mm (600 x 600 DPI)

ACS Paragon Plus Environment


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revised Figure 3 in the main text 94x135mm (300 x 300 DPI)



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revised Figure 4 in the main text 87x111mm (300 x 300 DPI)


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For TOC only 83x46mm (300 x 300 DPI)


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Chromatographic Properties of Minimal Aspect Ratio Monolithic Silica

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