System Dead Volume

The system dead volume must be reduced to an absolute minimum, particularly when using very efficient narrow-bore SEC columns. Extra column dispersion becomes a greater consideration as the column volume is reduced, and dead volume should be minimized in all parts of the system, including injection valves, connecting tubing, and detectors, if the column performance is to be realized. 

A value of 2.5 m for extra-column dispersion does not tell us what our system dead volume needs to be (except that it needs to be very small). Much of the extra-column dispersion in a chromatograph can be considered to occur as the solute passes through tubes, as shown in Fig. 2.3d. This effect is well understood, so that the dispersion produced can be calculated in some cases if the various dimensions are known. 

In terms of volume, V0 is a measure of the system dead volume from the injector to the detector. For a well designed system with low extra-column dispersion, V0 will be roughly equal to the dead volume of the column, that is, the volume of the column not occupied by the packing particles. 

Using nano LC-MS at submicroliter per minute flow rates requires special attention to plumbing, system dead volume, valve switching, large volume sample injection, precolumn methodology, automation, online sample clean-up, and multichannel parallel operation of a single MS. The techniques discussed below are particularly useful for nano LC-MS-MS applications. 

M mass of solute to be separated N number of effective theoretical plates P pressure Q flow rate R resolution S peak capacity Sm specific heat of mobile phase Ss specific heat of adsorbent Sg specific heat of detector cell walls V volume in conventional units Vo system dead volume Vr retention volume V r corrected retention volume Vm volume of mobile phase in the column Vs volume of stationary phase in the column Ve extra column volume... 

Attachment of the Pyrolysis System. The attachment of a pyrolyzer to a GC system should be made so that minimum dead volume remains in the system. Dead volume can be tested for by injection of methane into the GC column a tailing methane peak indicates the existence of dead volume. Such voids drastically reduce resolution and may also trap polar or more volatile fragments. The system should also be tested for contamination from previous runs by firing the pyrolyzer without sample. Generally, such a blank run should be made from time to time to ensure the absence of memory effects. A typical configuration of the so-called on-line approach is presented in Fig. 4.7.4. 

Typical hose-flow characteristics of this system are summarized as follows hose length, 210 m inside radius, 0.48 cm hose volume, 16 L flow rate, 6 L/min flow velocity, 130 cm/s Reynold s number, 11,700 (at 20 °C) time in hose, 160 s predicted and observed hose smear, 1.2 and 2.8 s, respectively and estimated system dead volume (includes manifold in laboratory), 400 cm. After an impulse enters the hose, the predicted hose smear represents the exit interval between signal maximum and one standard deviation (for this experiment a 4.5-L/min flow rate was used). Flow becomes turbulent at about 150 L/min. The clean pump does not behave as a pure dead volume, so the effective volume is only an estimate however, by using the estimates just discussed and considering a gain factor of 0.35 as a threshold, the spatial resolution can be determined for the system of 50 m in the horizontal scale at a 5-m/s (10 knot) ship speed or of 25 m at 2.5 m/s (5 knots). 

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. 

For continuous processes, the lowest possible system dead volume will enable the operation with low average holding times. This may be important in some applications, especially those involving bacteria-laden liquids. Low system dead volume is also desirable for batch or continuous processes to minimize the volumes of cleaning solutions required during a cleaning cycle. 

The major drawbacks in the case of this system have been connected in the past with the unspecific nature and lack of sensitivity of the detectors employed (e.g. refractive index detection, UV monitor, flow calorimeter). These may be overcome by the use of a locating agent after the column as in ion-exchange chromatography or by the analysis of specific derivatives followed by colorimetric or fluorimetric detection. The latter, termed precolumn derivatisation, appears to be the more suitable approach since commercially available HPLC systems may be adopted without modification and the risk of peak broadening with increase in system dead volume are minimised. 

System dead volume, volume of mobile phase in the column... 

These advantages do not come without some cost Several modifications of conventional LC apparatus are necessary to permit use of microbore columns. In particular, when using microbore LC, it is essential to minimize system dead volume. Modifications of conventional LC apparatus necessary for microbore column work are described further in this chapter see Note 1 and Fig. 1). 

The chromatography method, including the manufacturer and model for the LC system and autoinjector, if applicable, should be provided. Ideally, the exact same equipment that was used for method development should be used to validate the method. After the method has been validated, other types of equivalent equipment may be used but, since factors such as system dead volume and carryover may impact the method, additional validation experiments may be required to support these changes. (Section 10.2.10). Similar to what was described above for the preparation of solutions and reagents, a description of all chemicals and solvents used for the preparation of mobile phases should be provided along with examples of how these are prepared and stored when... 

Suitable manifold dead spaces. The system dead volume in theory should be as small as possible but in reality it should be carefully evaluated, especially for static volumetric systems. The volumes of adsorbed gas are calculated by the pressure difference between the experiment (with a reactive gas) and the blank (dead volume calibration with an inert gas) the smaller the dead volume, the higher the difference in pressure and more precise is the adsorbed volume calculation. On the contrary, by decreasing the system dead space the gas dose to be injected should be decreased accordingly to avoid the risk of injecting too much gas that might overtake the necessary amount to form a monolayer or, in the best case, to produce an isotherm with few experimental data points. In fact, when the injection volume is too small it is very difficult to calibrate it with the required precision. 

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