Electrospray interface nanospray

Two factors are driving the market for precise, very-low-flow HPLC pumping systems extremely limited sample sizes in biotechnology and the electrospray and nanospray interfaces that are concentration and flow-rate dependent. It is very difficult to get precise flow and gradient formation from pumps that have a 5- to 10-/iL plunger displacement, even using 3200-step stepper motor drives. This has forced manufacturers to resurrect a very old concept from the earliest days of HPLC, the syringe pump. 

Nanoflow HPLC—HPLC system with accurately controlled reciprocating and syringe pumps designed to use capillary and small diameter, high-resolution columns as front ends for electrospray and nanospray mass spectrometer interfaces. 

Gatlin, C. L. Kleemann, G. R. Hays, L. G. Link, A. J. Yates, J. R. 1998. Protein identification at the low femtomole level from silver-stained gels using a fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal. Biochem., 263, 93-101. 

Gatlin, C.L. Kleeman, G.R. Hays, L.G. Link, A.J. Yates, J.R. Protein Identification at the Low Femtomole Level from Silver-Stained Gels Using a New Fritless Electrospray Interface for Liquid Chromatography-Microspray and Nanospray Mass Spectrometry, Anal. Biochem. 263,93-101 (1998). 

Miniaturized ESI interfaces (nanospray electrospray ionization, nanoESI) match the dimensions of microfluidic chips. On-line couphng of microchips with ESI can be accomplished using different interface geometries blunt end, comer outlet, external capillary, external emitter, or monolithic emitter [27], some of which resemble the nanoESI emitters used in CE-MS (see also Chapter 6). In fact, on-chip capillary channels are often used as CE or LC separation columns, and directly linked with the nanoESI emitters. Atmospheric pressure chemical ionization (APCI) and photoionization (APPI) have also been subject to miniaturization but they have not attracted as much attention when it comes to hyphenation with microchips [28]. This situation may change when the novel nanoAPCI interfaces [29] are perfected, providing the way to transmit and ionize non-polar analytes at low flow rates. 

The HPLC system comprised a 75 ftm x 15 cm PepMap column with a linear gradient of acetonitrile/0.1% aqueous formic acid (5 to 50% acetonitrile over 45 min) at a flow rate of 250 nlmin . Positive-ion electrospray ionization was employed using a nanospray interface. MS-MS specna were acquired over the range m/z 40 to 2000 at a rate of 1 s per scan. 

Today, the two most common LC/MS interfaces are atmospheric pressure ionization interfaces, electrospray (ESI) and ion spray (ISI). Electrospray (Fig. 15.8) and its subtype, nanospray, are recommended for use with proteins and highly polar or ionized compounds. They are very soft ionization, concentration-dependent techniques that result in very little fragmentation and often produce multiply charged molecular ions. 

Ramsey and Ramseyalso described microchip interfacing to an ion trap mass spectrometer. Microfluidic delivery was realized by electroosmotically induced pressures and electrostatic spray at the channel terminus was achieved by applying a potential between the microchip and a conductor spaced 3-5 mm from the channel terminus. Tetrabutylammonium iodide was tested as a model compound with this device. Later, Ramsey et reported use of a microchip nanoelectrospray tip coupled to a time-of-flight mass spectrometer for subattomole sensitivity detection of peptides and proteins. A fluid delivery rate of 20-30 nL/min was readily obtained by applying an electrospray voltage to the microchip and the nanospray capillary attached at the end of the microfabricated channel without any pressure assistance. 

A third strategy for microfluidic-nanospray interfaces, microfahricated, tapered electrospray tips [8-12] is the most promising that has been reported. In fact, several devices with this configuration are now available commercially (for example, from Advion Biosciences and Agilent Laboratories). Several authors have fabricated devices capable of sustaining a stable spray with no dead volume between the channel and tip. For example. Fig. 2a shows a micro-milled electrospray nozzle in poly(methyl... 

One of the major attractions of ESI is its ability to serve as an interface between liquid chromatography and mass spectrometry. There are currently a number of low-flow HPLC systems on the market that are compatible with electrospray, microspray, and nanospray sources. Capillary HPLC systems are interfaced with electrospray conducted in the pL/min flow regime, while nanoflow systems can accommodate nL/min flow rates. When electrospray is coupled with conventional HPLC, it is necessary to accommodate a higher sample flow rate ( 0.1—2mL/min) than normal electrospray can tolerate. To facilitate operation at these higher flow rates, a technique called pneumatically assisted electrospray or ion spray is employed, in which sample nebulization by aflow of gas is used to stimulate a more... 

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