Ultrahigh pressure liquid chromatography/time-of-flight mass spectrometry for fast separations

Abstract

Recently, ultrahigh pressure liquid chromatography (UHPLC) has been shown to overcome the pressure limitations that small particles impose on conventional pumping systems. High speed separations in UHPLC produce peak widths that range between 100 to 1000 ms, of which many are too narrow to be monitored by scanning mass spectrometers. The only mass spectrometer that is fast enough for such separations is the time‐of‐flight mass spectrometer (TOFMS). State‐of‐the‐art TOFMS instruments for liquid chromatography can record and store complete mass spectra at rates as high as 100 spectra s−1. In this study, high speed separations with high resolution were demonstrated using 13–15 cm×29–100 μm i.d. capillaries packed with 1.5 μm nonporous octadecylsilane‐ and isohexylsilane‐modified silica particles utilizing a home‐built UHPLC system. The UHPLC system was successfully coupled to TOFMS via a liquid‐sheath electrospray interface. Separations of selected combinatorial chemistry samples, pharmaceutical compounds, and herbicides were completed in less than 100 s using UHPLC/TOFMS. Total column efficiencies ranged from 20,000–30,000 plates. The fundamental and practical aspects of UHPLC/TOFMS are discussed. Results are compared with those obtained from typical capillary LC. © 2000 John Wiley & Sons, Inc. J Micro Sep 12: 462–469, 2000

New hyphenated methodologies in high‐sensitivity glycoprotein analysis

“…First, and perhaps most importantly, multidimensional separations have been emphasized in proteomic analyses [18][19][20][21][22][23] for several years. The overall separation efficiencies of chromatographic columns can be significantly enhanced through the combination of very small particles with increased column length, leading to the use of ultrahigh pressures in microcolumn LC [24][25][26][27]. Finally, the properties of monolithic capillary columns, operating in either the electromigration mode (CEC) [28][29][30][31][32][33][34] or a pressure-driven mode [35,36] for the separations of both peptides and oligosaccharides, provide an additional route to solving the problems of peak overlap in proteomic and glycomic studies.…”

Development of Fast HPLC Methods

“…The tremendous improvement in performance that was demonstrated over conventional columns (i.e., stainless steel tubes 3-4.6 mm in diameter packed with particles 3-5 μm in size) generated significant interest in this technique. A number of academic research labs have subsequently conducted research using UHPLC with nonporous particles, notably the laboratories of Milton Lee (Brigham Young University) [22][23][24][25][26][27][28][29] and Luis Colón (SUNY-Buffalo). [30,31] The practical challenges and limitations of the technique have largely limited its use to research environments.…”

“…Low flow rates and narrow peak widths make capillary UHPLC particularly suitable for coupling with mass spectrometry via nanoelectrospray ionization [23,45]. Tolley et al [34] have used very-high pressures of around 1000 bar (15,000 psi) to separate bovine serum albumin digests on columns packed with 1.5-μm nonporous reversed-phase particles by gradient elution, with quadrupole/time-of-flight (Q-TOF) tandem mass spectrometry for detection.…”

“…The retention factor k* for a linear gradient is given by (17)(18)(19)(20)(21)(22) where Δ% is the change is %B from gradient start to end (the gradient range), and S is a constant depending upon the molecular structure of the sample [49]. For molecular weights below 500, S ≅ 5 and the equation reduces to (17)(18)(19)(20)(21)(22)(23) The retention factor k* is a constant for all solutes eluting in a linear gradient. This simple equation provides the basic insight for a starting point for developing or optimizing a method.…”

“…In addition, largest changes in resolution occur at k* = 2-10. Once the method has been optimized for selectivity, equation (17)(18)(19)(20)(21)(22)(23) provides insight on how to optimize for speed. If any single parameter is changed independently, k* will change.…”