Column volume dimensions

In this section, the effect of the extra-column volume on chromatographic performance is discussed for columns and connection tubings of various internal diameters. Relevant theoretical equations are summarized, and the effects of extra-column volume on apparent retention, selectivity, efficiency, and column pressure are discussed. The influence of the dwell volume on separation is also discussed, and recommendations on acceptable ratios of extra-column to column volume dimensions... 

As a practical result, the amount of gel to be prepared for a preparative column must exceed the nominal volume of the final column by 10%. For the packing of production-scale columns the maximum pressure rate of the column has to be considered. The large columns consist mostly of borosilicate glass tubes with similar pressure stabilities. For example, a Superformance column with dimensions of 1000 mm in length and 50 mm in width is pressure stable up to 14 bar. Therefore, Fractogel EMD BioSEC should be packed with a... 

Loading capacities in size exclusion chromatography are very low because all separation occurs within the liquid volume of the column. The small diffusion coefficients of macromolecules also contribute to bandspreading when loads are increased. The mass loading capacities for ovalbumin (MW 45,000) on various sizes of columns can be seen in Table 10.5. The maximum volume that can be injected in size exclusion chromatography before bandspreading occurs is about 2% of the liquid column volume. The maximum injection volumes for columns of different dimensions can also be seen in Table 10.5. 

The extra-column dispersion governs the dimensions of the column that we use. In the calculation above, the dispersion is increased by about 8% by the extra-column effects. If we want the dispersion to be increased by no more than this, then should not be any smaller than the value calculated above. This in turn limits the retention volume, and thus the volume of the column itself. The minimum column volume we can use will depend on the amount of extracolumn dispersion and on what we consider to be an acceptable increase in peak width that is produced by extra-column effects. In practice, this acceptable increase is taken as 10%, based on an unretained solute, and if we take 50 (i as a typical figure for extracolumn dispersion then the minimum column diameter works out at about 4.5 mm for a column 25 cm long. 

FIGURE 13.3 Raw and deconvoluted mass spectra of a yeast ribosomal protein (L16) from a 2DLC(SCX/RP)/MS experiment were obtained, where mass spectral adducts were observed because of insufficient washing of the second-dimension RP column (Panel a, 7 column volumes of wash). Panel b shows mass spectra for the same protein from an experiment with sufficient second-dimension wash volumes (Panel b, 14 column volumes of wash). 

The loading capacity of SEC columns is quite modest compared to interactive modes of chromatography. A rule of thumb dictates that the sample volume capacity is about 2% of the column volume. A typical analytical SEC column with dimensions of 8 x 300 mm has a VM of 10 to 11 ml, providing a sample volume limit of about 200 pi. The mass loading limit for such a column is about 1 to 2 mg. Above these volume and mass limits, resolution will be compromised. Sample capacity will scale in proportion to column volumes for different column lengths and diameters. 

Column volume. X. The total volume of the column which contains the stationary phase. (IUPAC recommends the column dimensions be given as the inner diameter and the height or length of the column occupied by the stationary phase under the specific chromatographic conditions. Dimensions should be given in millimeters or centimeters.)... 

Column Dimensions (mm) Column Volume (mL) Flow rate (mL/min) Equilibration Time (min)... 

The injection is a critical factor in fast LC methods and must be considered to maintain column efficiency. Injection volumes that are too large can cause volume overload of the column, which results in broad, flat-top shaped peaks with low plate counts that are more pronounced for earlier eluting components. As injection volume is increased, peak height should increase however, peak width should remain the same. If peak width increases as well, this is indicative of volume overload. As column dimensions are reduced, the maximum injection volume must be reduced by the ratio of the column volumes [see equation (17-33) in Section 17.7.4], For example, reducing... 

A column with dimensions 1x35 cm is sufficient to separate 1 ml of liposome or the DRV liposome dispersion. The column is pre-calibrated and at the same time saturated with a dispersion of empty liposomes mixed with a quantity of the encapsulated material (in each case). The void volume of such columns should be between 7 and 13 mL and the bed volume between 17 and 21 mL. 

For gas chromatographic studies, a Chrom-5 chromatograph with a flame-ionization detector was used employing a sampling column of dimensions 100 cm x 0.3 cm and 250 cm X 0.3 cm. After filling with the respective adsorbents of 0.20-0.25 mm radius fraction, the columns were conditioned for 6 h at 70-170°C in a flow of the carrier gas (nitrogen). The liquid adsorbate samples (volume not exceeded 0.2 pi) were introduced... 

Method transfer from one laboratory to another one (from development to routine, from manufacturer to customer and so on) can be difficult because HPLC separations are influenced by many parameters. At the new place the resolution of a critical peak parr can be worse than required or the whole chromatogram looks different. In order to prevent such surprises, whenever possible, it is necessary to describe every detail of the method column dimensions, stationary phase (maybe even the batch number), preparation of the mobile phase (the order the individual components are mixed can be critical), temperature, volume flow rate, extra-column volumes of the instrument, the dwell volume in the case of gradient separations (see Section 4.3) as well as detection and integration parameters. It can be useful to designate alternative stationary phases, i.e. materials which are located close to each other in representations such as Figure 10.9. The true temperature in a column oven must be verified because it can differ from the requested one Method transfer also includes the detailed description of sampling, storage and sample preparation. 

Insufficient conditioning of the column with mobile phase will result in retention time drift (see Figure 10.6). If the amount of time allowed for the conditioning process is too short, there will be a shift in retention time over the course of a large number of samples as the column continues to equilibrate. The column equilibration time will be dependent on the dimensions of the column. It is usual to condition the column with between 10 and 20 column volumes of mobile phase (see Table 10.1). 

The dimensions of a separation column are defined by the column length L and the internal diameter d. Both determine the column volume V, the length having a... 

The column volvime is defined as about one-half the volume calculated from the actual dimensions of the column. The column volume may, of course, be measured experimentally in each case if the interfaces can be clearly seen. 

In terms of column volumes (CVs), the ideal FC mobile phase elutes the desired component in 3-6 CVs and will separate two components if their CVs differ by 1 or more. A CV is defined as the volume of solvent filling the sorbent pores and spaces between particles in a column of any dimensions, and it is related to Rf by the equation CV = 1/Rf. As examples, an Rf of 0.90 indicates that 1.1 CV will be required to elute the compound from the column, Rf 0.50 requires 2.0 CV, and Rf 0.10 requires 10 CV. 

Before a numerical solution of transport equations is attempted, these were transferred into a dimension-less form to assess more conveniently the relative influence of the various transport steps on the overall transport rate. With normed concentrations in the interparticle column volume, eq. (46), of sorbent particles, eq. (47), and in the micropores of the latter, eq.(48). 

Another example of a highly complex protein mixture is the separation of proteins obtained from the lysate of Escherichia coli cells, which is typically carried out by multidimensional chromatography. In top-down proteomics, the use of a high-capacity ion exchanger in the first dimension is the very common first step toward the identification of individual proteins. Figure 3.288 shows such separation on a 50 mm X1 mm i.d. ProSwift WAX-IS. Its small column volume in combination with its relatively high capacity favors this column for use as a first dimension in multidimensional chromatography. 

Prepare a 25cmx 1.2 cm bed of Amberlite XAD-2 resin in a glass column. The dimensions are not critical. Wash the resin with 5-6 bed-volumes of water. 

This concerns columns with i.d. > 500 tm. For columns with i.d. < 500 tim and capillaries with i.d. <75 pm, this rule no longer applies. Since band broadening is not a problem in capillaries < 75 pm, such capillaries are optimized with regard to the time required to flush the capillary volume and the danger of blocking. It is possible to run a column of dimensions 100 mm x 75 pm with an output capillary of dimensions 250 mm x 50 pm however, the dwell time of the sample in the capillary is larger than that on the colurrm (for non-retained compounds). 

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