Materials and Methods

Fully porous 1.7 and 1.9 μm Acquity bridged-ethyl hybrid (BEH) particles were provided by Waters Corporation (Milford, MA), both sizes bonded with C18. Fused silica capillary tubing of 10, 20, 30, 50, and 75 μm inner diameter (i.d.) was purchased from Polymicro Technologies, Inc. (Phoenix, AZ). HPLC grade acetonitrile and acetone and reagent grade trifluoroacetic acid (TFA) were purchased from Fisher Scientific (St. Louis, MO). Deionized water was obtained from a Millipore NANOpure water system (Billerica, MA). Test analytes ascorbic acid,

hydroquinone, resorcinol, catechol, and 4-methyl catechol were purchased from Fisher Scientific (St. Louis, MO). For Kasil frits, potassium silicate (Kasil) from PQ Corporation (Valley Forge, IA) and formamide from Sigma-Aldrich (St. Louis, MO) were both used as received.

2.2.2 Preparation and Analysis of 10-75 μm i.d. Capillary UHPLC Columns

The process of preparing capillary UHPLC columns has been reported previously.1,19-22 In most instances, outlet frits were placed in capillary column blanks by pushing a 1-2 mm plug of 2.5 μm bare nonporous silica particles (Bangs Laboratories, Fishers, IN) 0.5 mm into the capillary using a tungsten wire to allow for the insertion of a carbon microfiber electrode. For columns packed with 1.9 μm BEH particles in the slurry concentration study, outlet frits were found to be more stable when prepared by the Kasil method.23 In the Kasil method, the column blank is gently pushed on a glass microfiber filter (Reeve Angel, Clifton, NJ) that was previously wetted with a 1:1 (v:v) ratio of Kasil and formamide. The column is then placed in an oven at 85°C for at least two hours.

For slurry packing, BEH particles were suspended in acetone at various concentrations (3-100 mg/mL) and the slurry was then sonicated for 10 minutes using a Cole Parmer Ultrasonic Cleaner 8891 (Vernon Hills, IL). The slurry was placed in a packing reservoir and a fritted capillary column blank was then secured into the reservoir using a UHPLC fitting. With acetone used as a pushing solvent, 200 bar was applied to the fritted capillary column from a DSHF-300 Haskel pump (Burbank, CA). The pressure was then gradually increased as the column bed formed until a maximum pressure of 2000 bar was reached. Once the desired bed length had been achieved (~20 cm for all columns), column pressure was slowly leaked until atmospheric pressure was obtained. The column was then placed into a UHPLC injection apparatus

connected to a DSXHF-903 Haskel pump (Burbank, CA) and flushed at 2800 bar with at least 15 column volumes of the mobile phase to be used for column characterization. The pressure was then slowly released and re-initiated at 700 bar where a temporary inlet frit was formed by using

To test column efficiency, a UHPLC injection apparatus was used to inject 200 μM of an isocratic test mixture containing L-ascorbic acid (dead time marker), hydroquinone, resorcinol, catechol, and 4-methyl catechol. The mobile phase used for evaluation was 50/50 (v/v)

water/acetonitrile with 0.1% TFA (except for the poorer performing 30 μm i.d. column from the diameter study which was evaluated using 70/30 (v/v) water/acetonitrile with 0.1% TFA to improve the resolution for peak analysis). Amperometric detection was achieved by amplifying the current generated from an 8 μm diameter (200 μm in length) carbon fiber microelectrode placed at the outlet of the packed capillary and held at +1.1 V vs. Ag/AgCl reference electrode.24 Current-to-voltage conversion was achieved by using a model SR750 current amplifier (Stanford Research Systems, Sunnyvale, CA) with a 109 V/A gain and a 3 dB low pass bandwidth filter set at 3 Hz. A 16-bit A/D converter was set at 21 Hz data acquisition rate and connected to an Intel Core 2 Duo desktop computer. Data was collected using a custom-written recorder program written with LabView 6.0 (National Instruments, Austin, TX).25

Reduced parameter plots (h-v) were constructed by separating the test mixture at a variety of mobile phase velocities. Chromatograms were frequency filtered digitally to remove high frequency noise while low frequency baseline drift was removed by background subtraction. Using Igor 6.0 (Wavemetrics, Inc., Lake Oswego, OR), an iterative statistical moments algorithm with ±3σ integration limits was applied to determine the theoretical plate count and retention time for each peak.26,27 Extra-column band broadening effects from the injector and detector were found to be negligible (~1% total), therefore plate heights were calculated with no attempt to correct for these effects. For the calculation of reduced parameters (h and v, Equations 1-18 and 1-19), the number-averaged particle size and pressure-dependent diffusion coefficient28 for each compound were used.

2.2.3 Microscopic Imaging and Bed Reconstruction of Capillary UHPLC Columns The general process for imaging and reconstructing packed beds is summarized above and has been further explained in the literature9,18, so only key details relevant to the studies described here will be discussed. In previous studies, V450 fluorescent dye had been covalently bonded to amine-modified bare silica. To effectively image reversed-phase particles, this method was modified to allow for the adsorption of a lipophilic dye (Bodipy 493/503, D-3922, Invitrogen, Karlsruhe, Germany) to the stationary phase surface. An initial trial of CLSM imaging applied to C18-bonded particles (2.6 μm Kinetex, Phenomenex, Torrance, CA) using this dye is shown in Figure 2-4. The fluorophore was excited with a 488 nm Argon laser with emission detected in the range of 491-515 nm. These conditions give approximate resolutions of 169 nm and 470 nm in the lateral and axial dimensions, respectively.18 The image size was selected to ensure that the entire column diameter was visible in each lateral image. To ensure sufficient resolution, image slices were taken with 126 nm steps in the axial direction. This means that for a 10 μm i.d. capillary only 119 slices were required while 457 slices were required for a 50 μm i.d. capillary. To calibrate the image stack in the axial direction, a cylindrical

confinement is used to ensure symmetric distribution around the column axis that may be skewed due to capillary drift during imaging. CLSM images of a 30 μm i.d. column in both the capillary and optical axes are found in Figure 2-5.

Once the images have been acquired, they are processed through a series of image filters and masks to determine particle centers (see Figure 2-6). This process is iterated with smaller and smaller filters until nearly all particle centers are determined, with remaining particles fitted manually. To reconstruct the packed bed, spheres are grown out from each of these particle

only the upper half of the packed bed was used to reduce the number of particles reconstructed while still including both wall and bulk packing regions.