Columns nanoflow

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. 

In order to reduce peak broadening in the nano-ESI needle attached to the nano-LC column, the use of packed needles has been promoted. Gatiin et al. [58] reports the use of 100-pm-ID fused-silica in-needle packed columns (10-pm particles) with a laser-pulled tip with a diameter of -2 pm. Figeys and Aebersold [59] reported the use of such in-needle RPLC columns for the LC-MS analysis of tryptic digests in combination with a nucrofluidic device to generate a nanoflow solvent gradient via electroosmotic flows. 

Vouros and co-workers recently demonstrated the effectiveness of nanospray to improve both the S/N quality concurrent with a reduction in matrix effects such as ion suppression [102]. Rather than use a nanobore LC column, the authors constructed a unique postcolumn flow that allowed for nanospray flow rates with conventional column formats. A cylindrically symmetric flow splitter was shown to preserve chromatographic peak shape at split ratios as high as 2000 1 thus, effluent from a conventional 2.1-mm Cl8 column at 200 pi. /min was reduced to 100 nL/min. The heart of the flow splitter was a fused-silica needle emitter with an orifice diameter of 5 pm. Tlie nanoflow splitter was applied to the in vitro metabolite analysis of a test... 

The liquid volume of a sample required for analysis depends on the ionization technique, MALDI or ESI, and the introduction technique (see Table 4.1). The following statements assume that we are analyzing a sample near the detection limit of the analyte in a specific mass spectrometer. For MALDI-MS, the researcher typically spots 0.1 to 1 jL onto the MALDI sample plate. Thus, a minimum starting volume of 1 of 5 jL of sample is recommended. For ESI, the required sample volume is primarily dependent on the sample introduction technique. If the researcher uses a nanoflow electrospray technique, capillary EC, or capillary electrophoresis, then typically a l-pL voliune is required. However, larger sample volumes are recommended for ease of handhng. If the voliune is small, then the analysis may be limited to one experiment when additional MS or MS-MS experiments are desired. For higher flow rate ESI sources, the researcher should supply 50 pL or more for direct infusion experiments or for loading 5 to 20 pL onto an analytical EC column. 

Shimadzu has produced the Prominence HPLC, Agilent has developed the 1200 Rapid Resolution HPLC (Figure 3.22), Thermo Scientific has the Accela LC system and Waters has brought out the Acuity UPLC. Jasco s X-LC system allows two systems to fit in the footprint previously occupied by one traditional system. It is also a modular system of detectors and autosamplers, which allows it to be customised. LC Packings has launched the UltiMate 3000, which is a nanoflow LC system for use with columns of 50 im and larger. Modular systems are common now, e.g. Cecil Instruments produces both HPLC and ion chromatography systems and various detectors can be accommodated including UV-Vis, refractive index, conductivity and fluorescence. 

Commercially, Agilent Technologies produces chips for both direct infusion into a mass spectrometer and for HPLC-MS applications. The chips accommodate nanoflow rates with an electrospray ionisation source, are about the size of a credit card and are rensable. The infusion chip is for collecting direct MS or tandem MS data. The protein HPLC chip has both a sample enrichment and CIS separation column on the chip, as well as the connections and spray nozzles for electrospray. There are also a small molecule chip and a glycan chip. The chip being used is placed in a Chip Cube MS interface which positions the sprayer tip perpendicular to the MS inlet (Figure 10.7). 

While the sample is loaded onto the trapping column and desalted by flushing with 0.1% TFA at an increased flow rate, the nano-HPLC branch is equilibrated with 0.1% FA. The UV baseline is set on the FA containing solution. By switching the HPLC valve, the trapping-column loop is then integrated into the nanoflow. Now, the nano-flow pushes the small volume of TFA solution through the... 

Watanabe, T., Nakanishi, K., Ozawa, T., Kawasaki, H., Lite, K., et al. (2010) Semi-online nanoflow liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry of synthetic polymers using an octadecylsilyl-modified monolithic silica capillary column. Rapid Commun. Mass Spectrom., 24, 1835-1841. 

During the initial years of HPLC, the most common internal diameter of conventional analytical columns was 2 mm, except for columns for size exclusion chromatography that often used to be 7-8 mm. Soon, the standard inner diameter changed to 4.6 mm, the standard length was reduced from 60 to 15-25 cm, and the particle size of the column packing materials was reduced from 40-50 to 5-10 pm. The dimensions of the columns were initially related to the commercial availability of stainless steel tubing. Today, the internal diameters of conventional HPLC columns and microbore HPLC columns are in the range of 2-5 and 0.5-1 mm, respectively. Capillary columns typically have 0.1-0.5 mm ID and nanoflow columns have <0.1 mm ID (Table 3.1). 

The columns are commonly made from stainless steel, with the exception of capillary and nanoflow columns that are usually made of fused silica tubing. Some steel columns have a glass inner wall in order to be more inert. The stationary phase particles are held in place by frits at each end of the column (Figure 3.7). 

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