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Graph of Retention Volume of a Series of Ions against their
Ion Volume (Cubic Angstroms)
The effect of solvent composition on the retention of a series of solutes, commonly used to measure column dead volumes, was also investigated by these authors. They employed mixtures of methanol and water as the mobile phase and measured the retention volume of the same salts together with a silica gel 'dispersion' (containing particles 0.002 micron in diameter). They also measured the retention volume of the components of the mobile phase methanol, and water. The silica 'dispersion' was chosen to simulate a solute of very large molecular size. The results they obtained are shown in figure
The results indicate that there is little effect of mobile phase composition on the retention volume of the solutes employed, or the silica 'dispersion'. It should be pointed out, however, that there are no values included for methanol between 0% and 10% methanol where adsorption of the methanol on the reverse phase surface significantly changes the value of its retention volume.
It is seen that the lowest retention volume is obtained for the silica 'dispersion'. This material, however, is difficult to use as it requires sonication for at least 5 hours before use. This was necessary, to obtain complete dispersion of the particles and, thus, ensure symmetrical peaks.
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
Graph of Retention Volume of a Number of Different Solutes against Composition of the Mobile Phase
è silica smoke
¦ Sod Nitrate î Sod. Sulphite
¦ Methanol (D) î water
Î 20 40 60 80 100
used to determine the linear mobile phase velocity and capacity factors for use in dispersion equations such as those of Van Deemter( 12) or Golay (13). The maximum dead volume recorded was that of water or methanol at concentrations above 10% v/v of methanol. This would appear to represent the thermodynamic dead volume for small molecules. It should be emphasized, however, that the thermodynamic dead volume of the column for larger molecules will be significantly less due to the exclusion characteristics of the silica support.
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 n um. irhexane iu rrhexati iacufilane 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
would be obtained at the different temperatures for each solute. The results obtained are shown in figure (3)
Graph of Retention Volume of n-Alkanes against Carbon Number
It is seen that an approximately linear relationship exists between the retention volume of each alkane and its carbon number and that the smaller molecule exhibits the greatest retention. This is a direct result of the exclusion properties of the silica gel support. The fact that the data, taken at the two different temperatures, fall on the same straight line confirms that little or no partition is taking place and that the difference in retention between the individual solutes is entirely related to their molecular size.
It follows that retention measurements on silica based stationary phases for the purpose of obtaining thermodynamic data is fraught with difficulties. Data from solutes of different molecular size cannot be compared or related to other interacting variables ideally, thermodynamic measurements should be made on columns that contain stationary phases that exhibit no exclusion properties. However, the only column system that might meet this requirement is the capillary column which, unfortunately introduces other complications which win be discussed later.
The best compromise for silica based stationary phases is to use corrected retention volume data for solutes eluted at à (Þ of greater than 5 and only
compare chromatographic data for solutes of approximately the same molecular size. A summary of the data for the Zorbax column obtained by Alhedai et al is shown in Table 2.
Table 2 Summary of the Physical and Chemical Properties of the Zorbax column
Column Length 25cm
Column Radius 2.3mm
Column Packing Zorbax C8 Reverse Phase
Carbon Content of Reverse Phase lO.OBw/w
Equivalent Decane Content (dimethyl Octane) 11,88w/w
Total Column Volume 4.15ml
Total Volume of Silica in the Column 0.96ml