Capillary volume, micro

Using capillary LC/microelectrospray no additional sample cleanup was necessary before triple-quadrapole mass spectrometric detection [12]. The small volumes of dialysate injected did not affect the sensitivity of the mass spectrometer. The development of microelectrospray and nanoelectrospray interfaces has made the coupling of micro and capillary LC to mass spectrometry much more feasible. Thus, the low flow rates associated with capillary and micro LC make ESI well suited for this approach. 

LC-NMR hyphenation consists of a liquid chromatograph (autosampler, pump, column and oven) and a classical HPLC detector. The flow of the detector is brought via an interface to the flow-cell NMR probe. Using commercial NMR flow-cells with volumes between 40 and 180 p,L, in connection with microbore columns or packed capillaries, complete spectra have been provided from 1 nmol of sample. These micro-cells allow expensive deuterated solvents to be used, and thus eliminate solvent interference without excessive cost. The HPLC eluent can be split in order to allow simultaneous MS detection. 

Furthermore, the micro-capillary-shaped sensor has an excellent sensor-surface-area to sample-volume ratio, which enables quick and efficient capture of biomolecules. 

Figure 14.3 shows a typical capillary gas chromatograph with the major components labeled. This gas chromatograph set up includes compressed gas tanks for the carrier gas (mobile phase) and any necessary detector gases, an auto-injector that employs a micro-syringe for delivering the necessary small sample volumes and an inlet capable of the... 

Since capillaries have very small diameters, the injection volumes in CE are extremely small. Injection volumes in the order of 10-50 nL are commonly applied (a fog droplet is + lOnL). Several approaches have been applied for the injection of such small volumes of sample into the capillary. These included the use of rotary-, split- and micro-injectors, electrokinetic and hydrodynamic injection. Although all these injection techniques have shown to be quite appropriate, electrokinetic and hydrodynamic injection are mostly applied. All the recent commercial... 

When the hydrolysis is complete carefully wash out the condenser with 10-12 c.c. of water. Then distil up to 5 c.c. of liquid into a small conical quartz flask. Use the condenser in the downward position and if necessary add a few boiling capillaries to the liquid in the flask. Repeat the distillation three more times, each time adding 7 c.c. of water. Test the distillate (volume about 20 c.c.) with some barium chloride for sulphuric acid (none should be present), boil for seven to eight seconds and titrate at once with 0-033 N sodium hydroxide solution 1 from a micro-burette having 0-02 c.c. scale-divisions. Use phenolphthalein as indicator and continue the titration until the colour becomes just pink and remains so for a few seconds. For the second titration distil 2 x 7 or 3 x 7 c.c. and for the third and fourth titrations only about 7 c.c. on each occasion. 

Rapp, E., and Tallarek, U. (2003). Liquid flow in capillary electrochromatography generation and control of micro- and nanoliter volumes.. Sep. Set. 26, 453-470. 

Vastly reduced solvent consumption for micro-separation techniques has advantages in that it gives superior solvent suppression when protonated solvents are used [88]. Reduced solvent volumes also make the use of fully deuterated solvents more attractive, eliminating the need for solvent suppression [87]. A low-volume capillary probe with a 7 pi cell volume (1.5 pi active) is commercially available and its application to metabolite identification has been reported [89]. 

The effect of the dwell volume on the retention times of analytes increases with decreasing retention factor at the start of gradient elution and with increasing ratio VpIV, and becomes very significant in the instrumental setup with the dwell volume comparable to or larger than the column hold-up volume, which is more likely to occur in micro- or in capillary LC than in conventional analytical LC (see Figure 5.4) [12]. 

Although this section provides a brief description of most commonly nsed detectors for HPLC, most of the focus is on a few detection modes. Optical absorbance detectors remain the most widely nsed for HPLC, and are discnssed in some detail. We also focns on flnorescence, condnctivity, and electrochemical detection, as these methods were not widely nsed for HPLC in the past, bnt are especially well suited to micro- and nano-flow instrnments becanse of their high sensitivity in small sample volumes. Mass spectrometry has also come into wide and rontine nse in the last decade, but as it is the subject of another chapter, it will not be fnrther discnssed here. Miniaturization has been particularly important for capillary and chip-based electrophoresis, which often employs sub-nanoliter detection volnmes [36,37]. 

EC detection is a promising alternative for capillary electrophoresis microchips due to its inherent characteristics, allowing a proper miniaturisation of the devices and compatibility with the fabrication processes, in case of an integrated detection. Moreover, the low cost associated permit the employment of disposable elements. As the EC event occurs on the surface of electrodes and the decrease in size usually results in new advantages (see Chapter 32), the possibilities of incorporating EC detectors are broad. The simplicity of the required instrumentation, portable in many cases, suit well with the scaling-down trend. Moreover, as the sample volume in conventional micro-channel devices is less than 1 nL, a very highly sensitive detector should be constructed to analyse even modest concentrations of sample solutions. Since sensitivity is one of the accepted characteristics of EC detection EC-CE microchips approach to the ideal analytical devices. 

The micro flow cell Golden Gate [125] can tolerate up to 66 bar at temperatures up to 200 °C. The cell volume amounts to 28 ml and fluidic interfaces are 1/16 in Swagelok capillary fittings. While the dimensions of the actual flow cell are only a few centimeters, the actual optical interface to a spectrometer consist of a macroscale table-top device with a clamping mechanism which positions the flow cell in the optical pathway. 

Sample analysis was performed by using an Applied Biosystems (Foster City, CA) API 3000 triple quadrupole mass spectrometer equipped with a TurboIonSpray source and an Agilent 1100 capillary HPLC system (Palo Alto, CA). The capillary HPLC system included a binary capillary pump with an active micro flow rate control system, an online degasser, and a microplate autosampler. The analytical column was a 300 pm I.D.x 150 mm Zorbax C18 Stablebond capillary column (pore size 100 A and particle size 3.5 pm). The injection volume was 5 pL, and a needle ejection rate of 40 pL/min was used. The pLC flow rate was 6 pL/min. In order to minimize dead volume before the column, the autosampler was programmed to bypass the 8 pL sample loop 1.5 min after injection. The mobile phase consisted of (A) 2 mM ammonium acetate (adjusted to pH 3.2 with formic acid) in 10 90 acetonitrile-water, and (B) 2 mM ammonium acetate in 90 10 acetonitrile-water. The percentage of mobile phase B was held at 32 % for the first minute, increased to 80 % over 8 min, and then increased tol00% over the following 1 min. 

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