Nanospray interface

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. 

A chip-based nanospray interface between an HPLC and the MS has been introduced by Advion Biosystems (Ithaca, NY). This instrument aligns a specialized pipette tip with a microfabricated nozzle, set in an arrayed pattern on a silicon wafer. The advantage of this interface is that each sample is sprayed through a new nozzle, thus virtually eliminating cross contamination. 

The mass spectrometer detectors place new demands on the HPLC system. The MS interface requires use of volatile buffers and reagents. Nanospray interfaces especially benefit from low-volume, high-resolution separations. The mass spectrometer is a fast response system and benefits from separation speeds higher than normally supplied by HPLC systems. All of these requirements have provided constraints on new development directions for HPLC systems. 

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. 

Vrouwe, E. X. Gysler, J. Tjaden, U. R. van der Grccf, J. 2000. Chip-based capillary electrophoresis with an electrodeless nanospray interface. Rapid Commun. Mass Spectrom., 14,1682-1688. 

ESI-MS Process Evaluation Setup. The chip glued onto the metal holder depicted in Figure 9.3d was screwed on the Nanospray interface of the mass spectrometer. Cone gas (automatically controlled via the ESI-MS software) was set at a value of 260Lh 1. Nebulizing argon gas was dispensed at various pressures up to about 8 bar. 

Microreactor chips were mounted on a dedicated holder (Figure 9.4a and b), placed on the Nanospray interface of the mass spectrometer using a metal plate. Solutions were introduced on-chip via fused silica capillaries (o.d. = 360 pm) of 100, 40 or 20 pm (i.d.), depending on the microchannel cross-sectional... 

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... 

Different options are available for LC-MS instruments. The vacuum system of a mass spectrometer typically will accept liquid flows in the range of 10-20 p,L min-1. For higher flow-rates it is necessary to modify the vacuum system (TSP interface), to remove the solvent before entry into the ion source (MB interface) or to split the effluent of the column (DLI interface). In the latter case only a small fraction (10-20 iLrnin ) of the total effluent is introduced into the ion source, where the mobile phase provides for chemical ionisation of the sample. The currently available commercial LC-MS systems (Table 7.48) differ widely in characteristics mass spectrometer (QMS, QQQ, QITMS, ToF-MS, B, B-QITMS, QToF-MS), mass range m/z 25000), resolution (up to 5000), mass accuracy (at best <5ppm), scan speed (up to 13000Das-1), interface (usually ESP/ISP and APCI, nanospray, PB, CF-FAB). There is no single LC-MS interface and ionisation mode that is readily suitable for all compounds... 

Hsieh, F. Baronas, E. Mnir, C. Martin, S.A. A Novel Nanospray CE/MS Interface. Rapid Commun. Mass Spectrom. 1999,13, 67-72. 

Several reports concerning the development of stable and rugged sheathless interfaces were proposed. The first sheathless interface was developed by Olivares et al., and two types of sheathless interfaces are currently distinguished. The first one consists of a nanospray needle, which is inserted with a connection unit to the CE capillary. This setup allows changing the spray needle alone independently on the capillary exchange.The second approach involves the use of the end of capillary tip as an emitter with the help of a capillary-outlet conductive coating " or by inserting a conductive wire into the capillary outlet. 

Lord et al. analyzed a mixture of steroids by CEC-ESI/MS and interfaced externally tapered CEC columns in both sheathless and sheath-flow arrangement. Sensitivity was found 20-fold higher in the sheathless configuration. The same conclusion was drawn by Warriner et ah, who evaluated CEC-nanospray/MS vs. CEC-microspray/MS with an ion trap using five corticosteroids. Cahours et al. used CEC-ESI/MS for a drug metabolism study and obtained a simultaneous baseline separation of flunitrazepam and its major metabolites. For CEC-ESI/MS coupling, the commercially available packed-CEC column was connected... 

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. 

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. 

Fig. 8.7. Schematic of a CEC—nanospray ESI-MS interface with conducting vacuum transfer conduit and heated capillary.

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. 

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). 

Simple Chip-based Interfaces for On-line Nanospray Mass Spectrometry... 

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