Dead volume measurement

Alternatively, sodium nitro prusside gives a value, close to that of dispersed silica, and, in practice, could be used to determine the total excluded volume or the kinetic dead volume without incurring serious error. Values for the kinetic dead volume measured in this way could be... 

Alhedai et a also examined the effect of exclusion on dead volume measurement. A mobile phase consisting of n-octane, the same chain length as the bonded phase, was employed to ensure no differential interaction between the solute and the two phases. A range of aliphatic hydrocarbons from, n-hexane to n-hexaiiiacohtane were chromatographed at two temperature 30°C and 50°C. The two temperatures were used to ensure that the retention mechanism was solely exclusion and not partition. If partition was the mechanism promoting retention, then different retention volumes... 

The dead point is the position of the peak maximum of an unretained solute. It is not the initial part of the dead volume peak as this represents a retarded portion of the peak that is caused by dispersion processes. The importance of employing the peak maximum for such measurements as dead volume and retention volume will be discussed in later chapters of the book that deal with peak dispersion. 

Again it is seen that only when second order effects need to be considered does the relationship become more complicated. The dead volume is made up of many components, and they need not be identified and understood, particularly if the thermodynamic properties of a distribution system are to be examined. As a consequence, the subject of the column dead volume and its measurement in chromatography systems will need to be extensively investigated. Initially, however, the retention volume equation will be examined in more detail. 

It is clear that the separation ratio is simply the ratio of the distribution coefficients of the two solutes, which only depend on the operating temperature and the nature of the two phases. More importantly, they are independent of the mobile phase flow rate and the phase ratio of the column. This means, for example, that the same separation ratios will be obtained for two solutes chromatographed on either a packed column or a capillary column, providing the temperature is the same and the same phase system is employed. This does, however, assume that there are no exclusion effects from the support or stationary phase. If the support or stationary phase is porous, as, for example, silica gel or silica gel based materials, and a pair of solutes differ in size, then the stationary phase available to one solute may not be available to the other. In which case, unless both stationary phases have exactly the same pore distribution, if separated on another column, the separation ratios may not be the same, even if the same phase system and temperature are employed. This will become more evident when the measurement of dead volume is discussed and the importance of pore distribution is considered. 

As already mentioned, there are two so called "dead volumes" that are important in both theoretical studies and practical chromatographic measurements, namely, the kinetic dead volume and the thermodynamic dead volume. The kinetic dead volume is used to calculate linear mobUe phase velocities and capacity ratios in studies of peak variance. The thermodynamic dead volume is relevant in the collection of retention data and, in particular, data for constructing vant Hoff curves. 

Equation (38) shows that the measurement of (k ) incorporates the same errors as those met in trying to measure the thermodynamic dead volume. However, providing the solute is well retained, i.e.,... 

The silica dispersion showed the smallest retention volume. It should be noted, however, that the authors reported that the silica dispersion required sonicating for 5 hours before the silica was sufficiently dispersed to be used as "pseudo-solute". The retention volume of the silica dispersion gave the value of the kinetic dead volume, /.e., the volume of the moving portion of the mobile phase. It is clear that the difference between the retention volume of sodium nitroprusside and that of the silica dispersion is very small, and so the sodium nitroprusside can be used to measure the kinetic dead volume of a packed column. From such data, the mean kinetic linear velocity and the kinetic capacity ratio can be calculated for use with the Van Deemter equation [12] or the Golay equation [13]. 

The Waters company recommends a system check of the chromatographic equipment that is used for plate number determination and analyses (2) the columns in the GPC unit used are replaced by a zero dead volume union. Then the test sample is injected under the same conditions such as a plate number determination. The 5a peak width measured on a suitable recorded peak is evaluated this 5or width of a 20-/a1 injection should be lower than 150 /a1. 

The detector cells normally are connected by a capillary. For the interpretation of the detector signals the volume of this connection must be known data of the following detectors have to be shifted for the delay toward the first cell. Usually this dead volume cannot be measured immediately, as it has to be determined indirectly by test analyses, e.g.,... 

The exact nature of the dead volume is complex and, in fact, will vary from solute to solute due to the exclusion properties of the stationary phase, particularly if the stationary phase or support is silica or silica based. Thus, to measure (Vo) accurately, a non-adsorbed solute of the same molecular size as the solute should be used and then the correct retention volume (V r) can be calculated and employed for identification purposes. 

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. 

Measuring dead volumes of the individual chambers, including the sample chamber. 

The flow cell is the most important component of a flow injection manifold for CL measurements since maximum radiation should be generated while the solution is flowing in front of the detector. Other attributes of the flow cell are the small dead volume of the cell to allow fast and effective washing between injections... 

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