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  • 77

    3 Micro-HPLCHeather Kalish and Terry M. Phillips

    3.1 IntroduCtIon

    High-performance liquid chromatography (HPLC) has become a standard separation technique used in both academic and commercial analytical laboratories. However, there are several draw-backs to standard HPLC, including high solvent consumption, large sample quantity, and decreased detection sensitivity. Micro-HPLC (HPLC) is a term that encompasses a broad range of sample volumes and column sizes (as shown in Table 3.1), but Saito and coworkers provided narrower defi-nitions in their review based on the size of the columns.1

    Micro-columns range in size from 0.5 to 1.0 mm internal diameter (i.d.); capillary columns range in size from 0.1 to 0.5 mm i.d., and nano-columns range in size from 0.01 to 0.1 mm i.d., as seen in Figure 3.1. Additionally, the size of the column tends to dictate the materials the col-umns are manufactured from.1 Micro-columns are made from stainless steel tubing, while cap-illary and nano-columns are manufactured from mainly fused silica, glass-lined stainless steel, pressure-resistant plastic (polyetheretherketonePEEK), or fused silicalined PEEK (PEEKSil). Fused silica is advantageous to glass-lined stainless steel because it is flexible and inert, but it can allow for chemical bonding to the inner column wall.1 Commercially available capillary and

    Contents

    3.1 Introduction ............................................................................................................................773.2 HPLC Systems ..................................................................................................................... 783.3 Advantages of HPLC Systems .............................................................................................803.4 Columns .................................................................................................................................. 81

    3.4.1 Open Tubular .............................................................................................................. 813.4.2 Semi-Packed ............................................................................................................... 813.4.3 Packed ......................................................................................................................... 81

    3.5 Stationary Phases .................................................................................................................... 823.5.1 Micro-Particulate ........................................................................................................ 823.5.2 Monolithic ................................................................................................................... 823.5.3 Monoliths Prepared by Porogen Alteration ................................................................ 833.5.4 Monoliths Prepared by Carbon Nanotube Incorporation ........................................... 833.5.5 Monoliths Prepared by Porogen Alteration and Surface Alkylation ..........................843.5.6 Monoliths Prepared by Photo-Initiated Polymerization .............................................843.5.7 Monoliths Prepared by the SolGel Method ..............................................................84

    3.6 Gradient Elution Systems .......................................................................................................843.7 Detectors .................................................................................................................................873.8 Applications of HPLC ..........................................................................................................88

    3.8.1 Pre-Concentration .......................................................................................................883.8.2 Multidimensional Liquid Chromatography ................................................................ 913.8.3 Other Applications ......................................................................................................92

    References ........................................................................................................................................92

  • 78 Handbook of HPLC

    nano-columns are available from a number of companies including the following columns used in the authors laboratory: MicroTech Scientific (Vista, CA), Dionex/LC Packings (Sunnyvale, CA), Eksigent Technologies (Dublin, CA), Waters Corporation (Milford, MA), Agilent Technologies (Santa Clara, CA), and Shimadzu Scientific Instruments (Kyoto, Japan).

    Horvath and coworkers first investigated miniaturization of the HPLC column in the late 1960s in a series of articles examining the separation of nucleotides.24 The comparison of open tubular and stainless steel columns with i.d. of 0.51.0 mm packed with novel pellicular column materials indicated that the packed columns were superior for LC. Over the next decade, numerous research groups made significant advancements in the reduction of the column size, column construction materials, and packing supports. Ishii and coworkers continued to work with 0.5 mm i.d. columns, but investigated columns made of Teflon that were slurry packed with 30 m particle diameter pel-licular particles.510 High speed, efficient separations were demonstrated by Scott and coworkers on 1.0 mm i.d. columns.1115 Novotny and coworkers further reduced the column i.d. to 50200 m, packed with 10100 m particles.16,17 They concluded that with a 70 m i.d. column packed with 30 m particles, good efficiency could be obtained without excessive inlet pressure.17 Over the next three decades, significant advancements have been made in the areas of column composition, detector interface, and hardware design, which are the subject of numerous review articles.1,1823

    3.2 hPlC systeMsToday nearly all of the major HPLC companies offer a HPLC system or at least the possibility to modify a standard instrument to accept micro-bore columns. In our laboratory, we routinely use the HPLC systems Ultimate from Dionex/LC Packing (Figure 3.2), the Extreme Simple 4-D

    (A)(B)

    (C)

    (D)

    FIGure 3.1 A comparison of columns used in routine and HPLC. (A) A laboratory built PEEK nano-flow HPLC column measuring 100 m i.d. 5 cm long, (B) A commercial C-18 PEEK capillary HPLC column measuring 75 m i.d. 25 cm long, (C) A commercial C-8 stainless steel HPLC column measuring 1 mm i.d. by 10 cm long, (D) a routine commercial reversed-phase stainless steel column measuring 4.6 mm i.d. 25 cm long, and (E) A commercial HPLC size exclusion preparative column measuring 7.5 mm i.d. by 30 cm.

    taBle 3.1units of length and Volume used in hPlCsymbol Prefix use in Volume (l) use in length (m)

    c Centi 102 102

    m Milli 103 103

    Micro 106 106

    n Nano 109 109

    p Pico 1012 1012

    f Femto 1015 1015

  • Micro-HPLC 79

    system from Micro-Tech Scientific (Figure 3.3), and the 2-D system from Eksigent Technologies (Figure 3.4). Although the majority of these instruments are used for proteomics research, Sajonz et al.24 used the Eksigent Express eight-channel HPLC system to perform multiparallel, fast normal phase chiral separations, providing near real-time separations. Using a panel of test racemates, these investigators demonstrated rapid analyses, which were comparable to those obtained by conventional, but much slower HPLC procedures.

    In addition to the commercially available systems, several authors have described labora-tory-built systems using commercially available components from companies such as Upchurch Scientific (Oak Harbor, WA). One of the first reported laboratory-built micro-bore HPLC systems was described by Simpson and Brown,25 which was a simple adaptation of a standard HPLC system to accept micro-bore columns built from guard columns. A complete system has been described based on dual microdialysis syringe pumps (CMA Microdialysis, Chelmsford, MA) or dual syringe pumps (Harvard Apparatus, Inc., Holliston, MA), a microinjection port, and a micro-column; the latter components being obtained from Upchurch scientific (Figure 3.5). This system was coupled with a laser-induced fluorescence (LIF) detector and used to measure neuropeptides in sub- microliter samples.26 A further modification of this system was built to perform immunoaffinity isolations of biomedically important analytes from clinical samples.27

    The advent of microfabrication greatly improved HPLC design and will eventually provide the ultimate lab-on-a-chip. Shintani et al.28 built a multichanneled HPLC for the separation of

    (A) (B)

    FIGure 3.2 (A) The Ultimate HPLC system by Dionex/LC Packings, used in our laboratory for proteom-ics analysis. (B) A close-up of the injection port, columns, and switching valves on the Ultimate system.

    (A) (B)

    FIGure 3.3 (A) The Extreme Simple 4-D system by Micro-Tech Scientific, used in our laboratory for HPLC work. The configuration shows the instrument setup as an eight-pump system (B) A close-up of the injection port, columns, switching valves, and four of the eight pumps on the Extreme Simple system.

  • 80 Handbook of HPLC

    multiple analytes within the same sample. This system employed an array of monolithic columns driven by a single HPLC pump and a chip-based microinjection device. Detection was achieved with a multichannel ultraviolet (UV) detector based on fiber optics. Further, Yin et al.29 developed an entire HPLC system on a microfabricated chip made from laminated polyimide layers. Following chromatographic separation on reversed-phase particles, the separated analytes were detected using an ion-trap mass spectrometer, a custom-built interface, and an integrated nanospray tip. A similar chip-based system has been described by Lazar and colleagues.30 The HPLC sy