Electrospray ionization flow rate limitations

As in HPLC, the coupling of MS detection with CE has provided an excellent opportunity for more selective analysis, but the much reduced flow rates, small injection volumes, limitations in the types of buffers used [since electrospray ionization (ESI) is used in capillary electrophoresis/mass spectrometry (CE/MS)], and need to... 

High-performance liquid chromatography (HPLC) techniques are widely used for separation of phenolic compounds. Both reverse- and normal-phase HPLC methods have been used to separate and quantify PAs but have enjoyed only limited success. In reverse-phase HPLC, PAs smaller than trimers are well separated, while higher oligomers and polymers are co-eluted as a broad unresolved peak [8,13,37]. For our reverse-phase analyses, HPLC separation was achieved using a reverse phase. Cl8, 5 (Jtm 4.6 X 250 mm column (J. T. Baker, http //www.mallbaker.com/). Samples were eluted with a water/acetonitrile gradient, 95 5 to 30 70 in 65 min, at a flow rate of 0.8 mL/min. The water was adjusted with acetic acid to a final concentration of 0.1%. All mass spectra were acquired using a Bruker Esquire LC-MS equipped with an electrospray ionization source in the positive mode. 

Nanospray is a miniaturized version of electrospray. In the original setup of Wilm and Mann (8) it is utilized as an off-line technique using disposable, finely drawn (1 -gm tip), metallized glass capillaries to infuse samples at 10-30 nL/min flow rates. This allows more than 50 min analysis time with just a 1-pT sample. Due to the formation of much smaller droplets and the more effective ionization, there is often no need for LC separation, since the separation is accomplished in m/z or by MS/MS. However, limited reproducibility with respect to quantification and a more complex sample preparation can be seen as drawbacks. An on-line version for hyphenation with capillary and nano-LC as well as CE (slightly modified) is now commercially available. 

Song and Naidong [129] analyzed omeprazole and 5-hydroxyomepra-zole in human plasma using hydrophilic interaction chromatography with tandem mass spectrometry. Omeprazole and its metabolite 5-hydroxy omeprazole and the internal standard desoxyomeprazole were extracted from 0.05 ml of human plasma using 0.5 ml of ethyl acetate in a 96-well plate. A portion (0.1 ml) of the ethyl acetate extract was diluted with 0.4 ml of acetonitrile and 10 /il was injected onto a Betasil silica column (5 cm x 3 mm, 5 /rm) and detected by atmospheric pressure ionization 3000 and 4000 with positive electrospray ionization. Mobile phase with linear gradient elution consists of acetonitrile, water, and formic acid (from 95 5 0.1 to 73.5 26.5 0.1 in 2 min). The flow-rate was 1.5 ml/min with total rim time of 2.75 min. The method was validated for a low limit of quantitation at 2.5 ng/ml for both analytes. The method was also validated for specificity, reproducibility, stability, and recovery. 

The choice of interface is dependent on both the particular analysis and the instrumentation available. Some interfaces require the use of very low flow rates and therefore necessitate the use of either microbore or capillary LC equipment, or a sample splitter if standard-bore equipment is used. Thermospray ionization is the most frequently quoted interface, owing to compatibility with standard-bore instruments. However, the upper molecular weight limit for thermospray ionization is low, and the electrospray interface is becoming popular. The maximum flow rates for different interfaces are listed in Table 3.8.54... 

Electrospray ionization >150,000 Ability to analyze mixtures limited sequence information for pure small peptides adapts easily for LC/MS (NP- and RP-HPLC) most suitable for quadrupoles multiple charging increases upper mass limit well suited to polar or ionic compounds Multiple charging may complicate interpretation of data glycoproteins may not yield useful information limited to flow rates <10 p.l/min... 

Exciting though the potential of electrospray ionization may seem, the use of electrosprays for liquid chromatography/mass spectrometry has been limited in practice by the severe restrictions on solvent composition and volumetric flow-rate that are amenable... 

Gradients can be used with equal ease for either ionization technique. In most cases, cycle time for system reequilibration (determined by the overall system dead volume) provides the practical limitation to their usage. If, for example, a particular HPLC pump/autosampler combination has 1.0 mL of dead volume (or dwell volume, the volume of all plumbing between where the solvents are mixed and the column head) and is operating at a flow rate of 1.0 mL/min (typical for APCI), then the lag time between when the gradient is initiated and when the correct solvent composition reaches the pump head is 1 min (1.0 mL/(1.0 mL/ min)). If the flow rate is only 0.2 mL/min (typical for electrospray), then the lag time will be 5 min. This means that a typical gradient run would require 5 min to initiate reequilibration plus whatever time is required for elution and final reequilibration (usually 10 to 20 column volumes). This is clearly an unacceptable time delay. 

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. 

Most recently (Schneider 2006) an experimental source was used to conduct studies under conditions of total solvent consumption , with pneumatically assisted nebu-lization to stabilize the ESI process, a heated laminar flow chamber to enhance desolvation and ion production, and various atmosphere-to-vacuum aperture diameters to maximize ion transfer. The motivation for these experiments was to investigate the proposal that the reason for the much lower ionization sampling efficiencies at higher flow rates ( o,L.min and above) is that the electrosprayed droplets are much larger in view of the much larger ESI needle tip diameters required to maintain flow rates in this regime, and thus are much less efficiently evaporated down to the Rayleigh and/or ion evaporation limits than the droplets formed from the 1 (tm diameter tips nsed in nano-ESI (Juraschek 1999 Schmidt 2003). 

The combination of the electrospray ion source with HPLC has without a doubt become the LC/MS interface in recent years. It is a particularly powerful combination, since this ionization technique covers a wide range of samples [38] that are commonly separated by HPLC [39] or electrophoresis [40]-[44]. The ESI source exhibits concentration-dependent behavior and thus gives optimal signals at most flow rates. The principle of the ionization process is discussed in Section 20.4.9. The most important feature of this interface is aspray needle which can be connected directly to the separation column, if the flow rates are compatible. Initially the major limitation was that only low flow rates (a few pL/min) could be used, but now flow rates of 1 mL/min or more are possible by using heated sprayers or ultrasonic devices. Splitting of the flow is possible as well, allowing two detectors to be used simultaneously. Since buffers can be used as long as they are volatile and not too concentrated, a sheath flow... 

Mobile phase The assay utilizes an isocratic mobile phase of 45% 10 mM ammonium acetate with 0.1% formic acid (mobile phase A) and 55% methanol (mobile phase B) at a flow rate of 0.4 mL/min. The flow rate may be adjusted if necessitated by the system back pressure or flow limits of the electrospray ionization source of the available equipment. 

Solvent removal and ionization are combined during electrospray in which the HPLC eluate (at flow rates as high as 1 mL/min) is sprayed through a capillary electrode at high potential to form a fine mist of charged droplets at atmospheric pressure. As the solvent evaporates, gas-phase sample ions are formed. The first application of electrospray LC-MS to the analysis of retinoids was reported by van Breemen and Huang (308). Retinoic acid, retinol, retinal, and retinyl acetate were analyzed without derivatization using a C30 reversed-phase HPLC column. Retinoic acid formed abundant deprotonated molecules, [M-H] , with a limit of detection of 23 pg injected on-column. Positive ion electrospray produced an abundant protonated molecule for retinal, and a base peak of m/z 269 was observed for retinol and retinyl acetate, which corresponded to elimination of water or acetic acid, respectively, from their protonated molecules. The limits of detection of retinal, retinol, and retinyl acetate were 1.0 ng, 0.5 ng, and 10 ng, respec-... 

For electrospray ionization, initially, 100 lm i.d. stainless-steel capillaries, i.e. hypodermic needles, were used for sample introduction. With such a device, the flow-rate is limited to 10 pL min i, which is too high for many biochemical applications and too low for effective LC-MS coupling. Therefore, the initial system was modified. 

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