Columns Dead volume

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. 

The idea of the effective plate number was introduced and employed by Purnell [4], Desty [5] and others in the late 1950s. Its conception was evoked as a direct result of the introduction of the capillary column or open tubular column. Even in 1960, the open tubular column could be constructed to produce efficiencies of up to a million theoretical plates [6]. However, it became immediately apparent that these high efficiencies were only obtained for solutes eluted at very low (k ) values and, consequently, very close to the column dead volume. More importantly, on the basis of the performance realized from packed columns, the high efficiencies did not... 

The XK column system is a medium-pressure jacketed glass column system designed for operating pressures up to 5 bar (0.5 MPa). Column dead volumes are less than 0.1% of the total column volume. Wetted materials include EPDM, TEFZEL, superpolyoxymethylene, and flurorubber. Columns use nylon nets of 10-)Lim mesh size and may be used with most SEC media with particle diameters >20 /xm. Columns are intended for use with aqueous solutions and... 

Unfortunately, exclusion chromatography has some inherent disadvantages that make its selection as the separation method of choice a little difficult. Although the separation is based on molecular size, which might be considered an ideal rationale, the total separation must be contained in the pore volume of the stationary phase. That is to say all the solutes must be eluted between the excluded volume and the dead volume, which is approximately half the column dead volume. In a 25 cm long, 4.6 mm i.d. column packed with silica gel, this means that all the solutes must be eluted in about 2 ml of mobile phase. It follows, that to achieve a reasonable separation of a multi-component mixture, the peaks must be very narrow and each occupy only a few microliters of mobile phase. Scott and Kucera (9) constructed a column 14 meters long and 1 mm i.d. packed with 5ja... 

All the solutes in exclusion chromatography are eluted between the interstitial column volume and the column dead volume (i.e. the pore volume). Consequently, the column must be large enough to provide... 

A value for the column dead volume is required in most calculations. It is convenient to have one cosponent of the test mixture as an unretained solute. 

Nevertheless it must be pointed out that, in calculating (k ), the value taken in practice is often the ratio of the corrected retention distance (the distance in centimeters on the chart, between the dead point and the peak maximum) to the dead volume distance (the distance in centimeters on the chart, between the injection point to the dead point on the chromatogram). This calculation assumes the extra column dead volume is not significant and, unfortunately, in almost all cases this is not true, (k ) values calculated in this way will be in error and should not be used for solute identification. Where computer data processing is used and no chart is available the distances defined above would be replaced by the corresponding times. 

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 (2). 

The concept of the effective plate number was introduced and employed in the late nineteen fifties by Purnell (7), Desty (8) and others. Its introduction arose directly as a result of the development of the capillary column, which, even in 1960, could be made to produce efficiencies of up to a million theoretical plates (9). It was noted, however, that these high efficiencies were were only realized for solutes eluted close to the column dead volume, that is, at very low k values. Furthermore, they in no way reflected the increase in resolving power that would be expected from such high efficiencies on the basis of the performance of packed columns. This poor performance, relative to the high efficiencies produced, can be shown theoretically ( and Indeed will be, later in this book) to result from the high phase ratio of capillary columns made at that time. That is the ratio of the mobile phase to the stationary phase in the column. The high phase ratio was... 

It is seen from equation (15) that the maximum overload volume is linearly related to the (k ) value of the first eluted solute of the critical pair, the function (a-1) and the column dead volume, Consequently, the larger the column, either in length and/ or radius, the larger the sample volume can be, This assumes that the column is of such a size, that it can be efficiently packed with practical techniques and that the particle size of the packing is chosen such that the pump pressure available can provide the necessary mobile phase flow-rate. 

Volume of the mobile phase in the column (dead volume)... 

COI.ITMNST7F. Analytical reversed phase columns for proteins and peptides range from 50 to 250 mm in length having 4 to 5mm i.d. Columns with 2mm i.d. have also been employed for peptide separations in microsequencing applications. The use of microbore columns with less than 2 mm i.d. is severely hampered by instrumental constraints due to small extra-column dead volumes and difficulties in obtaining accurate... 

FIG. 5 Breakthrough curves as a function of HSA concentration in the eluent on a monoclonal anti-HSA immunoadsorbent. Eluent 10 mM potassium phosphate buffer. pH 7.4, 150 mM NaCI, 20 C flow rate 8.33 mm3-s-1 30 x 4.6 mm column dead volume 0.3 cm3. Solid line experimental breakthrough curve. Dotted line fit of the theoretical model Eqs. (7)-(l 1) with kj = 0], (From Ref. 23, with permission from Academic Press. Inc.)... 

Ion chromatography (1C) allows the separation of substances in the form of ions, chiefly in aqueous solutions. Mobile phases used in the technique contain relatively large amounts of salts stabilizing the pH and determining the sequence of analyte retention. They enable the separation of only cations or only anions the ions that are not separated by a selected phase (cationic or anionic) are eluted in the column dead volume. As the next step, they can be loaded into appropriate ion-exchange columns in the second chromatographic dimension [148]. 

The dead volume will not simply be the total volume of mobile phase in the column system (V ) but will include extra-column dead volumes (V ) comprising volumes involved in the sample valve, connecting tubes, and detector. If these volumes are significant, then they must be taken into account when measuring the dead volume. 

---