Fused Core Particles for HPLC Columns by qov12652


									Fused Core Particles for HPLC
                                                by Joseph J. Kirkland, Timothy J. Langlois,
                                                                   and Joseph J. DeStefano

In recent years, there has been a
strong movement toward the use of
HPLC columns with smaller particles.
While columns with 5-µm particles
have long been the standard, some
users now have turned to columns of
3.5 µm1 or smaller2 to produce faster
separations and increase sample
throughput. The advantages of smaller
particles are well known: faster separa-
tions without sacrificing resolution so
that more samples can be analyzed in
the same time period. Of course, there
are always tradeoffs that must be made
to gain this speed. Smaller particles
decrease the permeability of columns;
thus increased pressures must be used,
resulting in increased stress on pump-      Figure 2     Particle size distribution of 2.7-µm fused core particles.
ing systems and other instrument
components. In addition, columns
with particles of ^3 µm must usually        filtering samples and mobile phases is                tion and the higher density of the fused
be fitted with end frits of 0.5-µm or       often a chore that small-particle users              core particles. The end result is a col-
1.0-µm porosity to ensure that smaller      must endure.                                         umn that produces very fast separations
particles in the particle distribution                                                           for high sample throughput with con-
do not escape to cause detection and        This article describes a 2.7-µm fused                ventional equipment and user-friendly
column life problems. These smaller-        core particle (Advanced Materials                    operating techniques. The thin outer
porosity frits are subject to fouling by    Technology, Inc., Wilmington, DE)                    shell of the particles permits very rapid
microparticulates from samples and          that has been designed to allow very fast            solute mass transfer (optimized kinetics)
mobile phases, eventually causing col-      separations without some of the disad-               so that high mobile phase velocities can
umn failure because of very high back-      vantages of conventional columns with                be used for fast separations without a sig-
pressures. To eliminate this problem,       small, totally porous particles. The char-           nificant loss in column efficiency.
                                            acteristics of these fused core particles
                                            represent a fortunate compromise of
                                            separation speed with modest operating               Materials and methods
                                            pressures. In addition, however, because             The fused core particles were synthe-
                                            of the unique structure of a thin porous             sized in the authors’ laboratory using
                                            shell surrounding a uniform solid silica             proprietary nanoparticle technology.3
                                            core, columns of these materials exhibit             The highly purified Type B silica parti-
                                            unusually high efficiency. While tradi-               cles are 2.7 µm in total diameter with a
                                            tional commercial columns of totally                 0.5-µm-thick outer shell of 90-Å pores,
                                            porous particles rarely show reduced                 resulting in a nitrogen surface area of
                                            plate heights, h (plate height/particle              G150 m2/g. The fused core particles
                                            diameter) of less than 2, columns of the             have been given the trademarked name
                                            fused core particles often exhibit h values          of Halo™, which suggests their physical
                                            of about 1.5 for molecules with molecu-              configuration with the spherical porous
                                            lar weights up to at least 300. This                 shell covering a solid core (Halo fused
                                            unusually high efficiency is believed                core columns available from Mac-Mod
Figure 1       Electron micrograph cross-   by the authors to be a function of the               Analytical, Chadds Ford, PA). A cross-
section of 2.7-µm fused core particle.      extremely narrow particle size distribu-             section electron micrograph of one of

the particles is shown in Figure 1. Fused                                                                 Results and discussion
core Halo particles are synthesized with
an extremely narrow particle size distri-                                                                     The unusually high efficiency of
bution, as indicated in Figure 2.                                                                             fused core Halo columns is illus-
                                                                                                              trated by the data in Figure 3. Here,
Densely bonded monofunctional                                                                                 the reduced plate height minimum
dimethyl-C8 and dimethyl-C18 station-                                                                         for the small molecule, naphthalene
ary phases were prepared on the fused                                                                         (MW 128.2), is about 1.5, represent-
core particles by conventional reactions4                                                                     ing about 12,000 theoretical plates
with silanes obtained from Gelest, Inc.                                                                       for this column. Very little increase
(Morrisville, PA). These bonded phase                                                                         in plate height occurs for this small
materials were subsequently endcapped                                                                         molecule when the mobile phase
with trimethylsilane groups. Carbon/                                                                          velocity is more than doubled. This
hydrogen analyses were by Micro-                                                                              effect is the result of the favorable
Analysis, Inc. (Wilmington, DE). The            Figure 3       Comparison of reduced plate height versus      kinetics of the thin porous shell on
level of bonded stationary phase was sim-       mobile phase velocity plots for naphthalene and loraz-        the fused core Halo particles. The
ilar to that for totally porous particles and   epam. Column: 50 × 4.6 mm, Halo C8, 2.7 µm. Sol-              time for solute diffusion in and out
in keeping with that expected for densely       utes: naphthalene (MW = 128.2), mobile phase: 60%             of the thin porous shell containing
bonded materials, where additional silane       acetonitrile/40% water; lorazepam (MW = 321.2),               the stationary phase is minimized
groups cannot be further reacted because        mobile phase: 30% acetonitrile/70% 20 mM sodium               so that band broadening is also
of steric limitations. For example, the         phosphate buffer, pH 3.5. Temperature: 24 °C.                 minimized as the mobile phase
bonded phase level on one sample of                                                                           is increased. Note also in Figure 3
Halo C18 was 3.29 µeq/m2 prior to end-                                                                        that the reduced plate height for the
capping. Particle size measurements were        were performed using 1-µL injections                   drug lorazepam (MW 321.2) at the plate
conducted on a Gemini V instrument              from a model 8125 sampling valve                       height minimum is also about h ~1.5 for
(Micromeritics, Norcross, GA) and elec-         ( Rheodyne, Cotati, CA). The bed                       column efficiency equivalent to that for
tron microphotographs were obtained             stability study was performed with                     the smaller molecule, naphthalene. As
from Micron, Inc. (Wilmington, DE).             the model 1100 instrument using a                      the mobile phase velocity is increased
Surface areas were conducted with a             model 1100 series automatic sampler                    for lorazepam, the plate height increases
Multisizer 3 Coulter Counter (Beckman           (Agilent Technologies). The 5-µm                       slowly as mobile phase velocity increases
Coulter, Fullerton, CA).                        particle Ace column was from Mac-                      as a result of the slower diffusion of the
                                                Mod Analytical, the 3.5-µm particle                    much larger molecule. Yet, at the highest
Chromatographic data were                       Zorbax XDB-C18 column was from                         mobile phase velocity (almost 8 mm/sec
obtained on a model 1100 instru-                Agilent Technologies (Wilming-                         at 328 bar), lorazepam shows a reduced
ment ( Agilent Technologies, Palo               ton, DE), and the 2.5-µm XBridge                       plate height of about 2.5, indicating
Alto, CA) or on a model SPD-6A                  C18 column was from Waters Corp.                       that the 50-mm column is still operat-
liquid chromatograph (Shimadzu                  (Milford, MA). Plate heights were                      ing with about 7000 theoretical plates.
Scientific Instruments, Tokyo,                  calculated with the data systems                       It is believed that the unusually high effi-
Japan) using UV detection with a                using the peak half-height/width                       ciency of fused core Halo columns is the
2-µL cell. Data were recorded from              method.6 Data for the reduced plate                    result of the extremely narrow particle
the model 1100 ChemStation (Agi-                height versus mobile phase velocity                    size distribution and the 30–50% higher
lent) or from an in-house-designed              plots in this paper were fitted to the                  density for the fused core particles (rela-
computer system using a model 900               Knox equation:                                         tive to totally porous particles). These
interface (PE Nelson, Cupertino,                                                                       properties apparently allow the forma-
CA). All data were acquired with                        h = Au1/3 + B/u + Cu            (1)            tion of a more homogeneous packed bed,
a detector response time of 0.1 sec,                                                                   which results in efficiencies higher than
in addition to a data sampling rate             where h is the reduced plate height                    for conventional totally porous packings
of at least 20 points/sec so that               (plate height h/particle size dp); A, B,               (reduced A term, Eq. [1]). While reduced
a minimum of 20 points would be                 and C are column coefficients; and u                    plate heights of 2 for columns is generally
obtained on the very sharp, low-vol-            is mobile phase velocity.6                             accepted as a practical limit, the possi-
ume peaks obtained with the fused                                                                      bility of increased efficiency for packed
core Halo columns. Stainless steel              Solvents for mobile phases were from                   HPLC columns as a result of optimized
column hardware and column frits                Mallinckrodt Baker (Phillipsburg, NJ).                 particles was predicted by Knox.7 In
with 2-µm porosity were from Isola-             Test solutes were from Sigma-Aldrich                   the Knox study, reduced plate heights
tion Technologies, Inc. (Hopedale,              (St. Louis, MO). Lorazepam and van-                    of less than 2 (some as low as 0.6) were
MA). The 4.6 × 50 mm and 2.1 ×                  tin were obtained by dissolving single                 obtained with large, solid glass beads and
50 mm columns used in this study                commercial pills in the appropriate                    superficially porous particles. The higher
were prepared by slurry packing                 mobile phase. No effect from inactive                  density and narrow particle size distribu-
techniques using in-house-designed              excipients in these pills was observed                 tion of the fused core particles appear to
hardware. 5 Chromatographic tests               in the authors’ studies.                               be the characteristics that allow reduced

                                                                                                         AMERICAN LABORATORY • APRIL 2007       19
FUSED CORE continued

                                                            plate heights of less than 2 to
                                                            be a common feature.

                                                            Predictably, as shown in Fig-
                                                            ure 4, the smaller (2.7 µm) par-
                                                            ticle size of the Halo particles
                                                            results in significantly higher
                                                            efficiency for lorazepam than
                                                            5-µm and 3.5-µm particle col-
                                                            umns. The less steep increase in
                                                            plate height with mobile phase
                                                            velocity increase for the Halo
                                                            particles is also in keeping with    Figure 7       Separation of test mixture. Column:
Figure 4       Effect of particle size. Columns: 50 × 4.6   the smaller particle size and the    4.6 × 50 mm, 2.7-µm Halo C8. Mobile phase: 75%
mm, Ace 5-C18, 5 µm; Zorbax XDB-C18, 3.5 µm;                superior kinetic properties of       methanol/25% 20 mM potassium phosphate buffer, pH
Halo C18, 2.7 µm. Solute: lorazepam (MW = 321.2).           the fused core structure. The        7.0. Flow rate: 1.5 mL/min. Temperature: 25 °C.
Mobile phase: 30% acetonitrile/70% 20 mM sodium             higher efficiency of the smaller-     Solutes: 1) uracil, 2) butyl paraben, 3) propranolol, 4)
phosphate buffer, pH 3.5. Temperature: 24 °C.               particle fused core column does      naphthalene, 5) acenaphthene, 6) amitripylene.
                                                            come with the price of a higher
                                                            operating pressure. However,
                                                            the operating pressure at the
                                                            highest mobile phase velocity
                                                            used for a very fast separation is
                                                            still within the pressure limits
                                                            of most of the HPLC instru-
                                                            ments currently in use.

                                                            Relative to results for a col-
                                                            umn of 2.5-µm hybrid parti-
                                                            cles, the 2.7-µm particle Halo
                                                            column shows significantly
                                                            better performance for the
                                                            large drug molecule, vantin
Figure 5     Comparative column performance for                                                  Figure 8       Separation of aromatic acids. Column:
                                                            (MW 557.6), as illustrated in
vantin. Columns: 50 × 4.6 mm, 2.5-µm XBridge                                                     4.6 × 50 mm, 2.7-µm Halo C8. Mobile phase: 55%
                                                            Figure 5. Not only is the plate
C18 (k = 9.5), 2.7-µm Halo C8 (k = 8.7). Solute:                                                 methanol/45% 25 mM sodium phosphate buffer, pH
                                                            height about 67% smaller, but
vantin (MW = 557.6). Mobile phase: 35% acetoni-                                                  2.5. Flow rate: 2.20 mL/min. Temperature: 24 °C.
                                                            the increase in plate height
trile/65% 20 mM sodium phosphate buffer, pH 3.5.                                                 Column pressure: 360 bar. Solutes: 1) uracil, 2)
                                                            with mobile phase veloc-
Temperature: 24 °C.                                                                              phthalic acid, 3) 2-fluorobenzoic acid, 4) 3-nitroben-
                                                            ity increase is less, due to
                                                                                                 zoic acid, 5) 3-fluorobenzoic acid, 6) m-toluic acid.
                                                            enhanced kinetic properties
                                                            of the fused core structure.

                                                            The mechanical strength of the               prepared prior to the final sample
                                                            fused core particles, coupled with           injection to ensure a mobile phase
                                                            a narrow particle size distribution          that was equivalent to the starting
                                                            and higher particle density, also            mobile phase. After these 500 sample
                                                            allows unusually stable column               injections at a flow rate of 1.0 mL/min
                                                            beds to be formed by the slurry              that resulted in 260 bar, the column
                                                            packing method. Figure 6 shows               showed no change in solute retention
                                                            the results of a column stability            or efficiency, within the precision of
                                                            study for a 2.1 × 50 mm fused                making up the mobile phase.
Figure 6       Halo column stability study. Column:         core Halo C8 column. Here, the
2.1 × 50 mm, 2.7-µm Halo C8. Mobile phase: 50%              column was operated for 71 hr                Examples of separations with fused
acetonitrile/50% water (premixed). Flow rate: 1.0           with continuous premixed mobile              core Halo columns are shown in
mL/min. Column pressure: 260 bar. Temperature:              phase flow (over 40,000 column                Figures 7 and 8. The high column effi-
24 °C. Solutes: 1) uracil, 2) phenol, 3) 4-chloro-1-        volumes) with solvent recycle.               ciencies and good peak shapes are the
nitrobenzene, 4) naphthalene. Solid line: first chro-        During this period, 500 sample               result of the favorable particle con-
matogram; dotted line: chromatogram after 71 hr of          injections were made on the col-             figuration, densely bonded surfaces,
continuous flow (>40,000 column volumes) and 500             umn with an automatic sample                 and the very high purity of the Type B
sample injections.                                          injector. Fresh mobile phase was             silica that make up the particles.

Conclusion                                       4.   Snyder, L.R.; Kirkland, J.J. Introduc-     7.   Knox, J.H. J. Chromatogr. A 1999,
                                                      tion to Modern Liquid Chromatography,           831, 3.
Fused core particles for HPLC columns                 2nd ed., John Wiley and Sons: New
have been developed that are well                     York, NY, 1979; Chapter 5.
suited for very fast separations and high                                                        The authors are with Advanced Materials
                                                 5.   Kirkland, J.J.; DeStefano, J.J. J. Chro-
sample throughput at modest column                    matogr. A 2006, 1126, 50.                  Technology, Inc., 3521 Silverside Rd.,
backpressures. The 2.7-µm high-purity            6.   Snyder, L.R.; Kirkland, J.J.; Glajch,      Ste. 1-K, Quillen Bldg., Wilmington, DE
silica particles have a solid core and                J.L. Practical HPLC Method Develop-        19810, U.S.A.; tel.: 302-477-2513;
a 0.5-µm-thick outer shell with 90-Å                  ment, 2nd ed., John Wiley and Sons:        fax: 302-477-2514; e-mail: jkirkland@
pores, providing a surface area of 150                New York, NY, 1997; Chapter 2.             advanced-materials-tech.com.
m2/g. The unusually high efficiency for
columns of these particles is believed to
be a feature of the very narrow particle
size distribution and the higher particle
density. Reduced plate heights, h, of
~1.5 for small molecules represent a
level of efficiency that has previously
not been reported for stable, commer-
cial HPLC columns. The thin outer
porous shell on these fused core par-
ticles allows rapid solute mass transfer
(fast kinetics) so that column efficiency
is degraded very little as mobile phase
velocity (flow rate) is increased. These
particles can be slurry packed into very                                                                    A NEW
stable column beds, and continuous
use of pressures up to at least 400 bar                                                                  DIMENSION
is indicated since the column bed has
been carefully stabilized. Samples and
mobile phases can be treated in the
same manner as for columns of 5-µm                                                                     ICP Analysis
particles since 2-µm frits are used on                       HORIBA Jobin Yvon presents the Master
the inlet and outlet, enhancing column                    of CCD-based ICPs for true multi-line analysis
lifetime compared to columns of ^3-
µm particles.

As for most systems, operation at higher
temperatures (e.g., 40–60 °C) will further
increase column efficiency and decrease                                                           - New proprietary
column backpressure. For best results, the                                                         ICP-based database
high-efficiency columns of the fused core                                                         - Unique interactive
particles should be used in high-quality
instruments with low-volume fittings,                                                              assistance tools
injectors, and low-volume detectors to                                                           - Full benefit of multi-line
minimize extra-column band broaden-                                                                analysis
ing. Data handling systems should be
used that capture at least 20 data points/
sec (using detector response times of 0.1                                                               Bring ICP expertise
sec) to maintain peak integrity for fast,
low-volume (sharp) peaks.                                                                               to your lab with an
                                                                                                      easy-to-use instrument.
References                                                                                       Be confident in the quality of
1.   Kirkland, K.M.; McCombs, D.A.;
                                                                                                         your results.
     Kirkland, J.J. J. Chromatogr. A 1994,
     660, 327.
2.   Kirkland, J.J. J. Chromatogr. Sci.
     2000, 38, 535.
3.   Kirkland, J.J.; Langlois, T.J. Substrates          www.jobinyvon.com
     with Porous Surfaces. Patent applied
     for, Feb. 2006.
                                                                                 Web access AL20.com?3706

                                                                                                      AMERICAN LABORATORY • APRIL 2007   21

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