Internal Diameter of the Column

Conventionally, analytical SEC columns have been produced with an internal diameter of 7.5 mm and column lengths of 300 and 600 mm. In recent years environmental and safety issues have led to concerns over the reduction of organic solvent consumption, which has resulted in the development of columns for organic SEC that are more solvent efficient (13). By reducing the internal diameter of the column, the volumetric flow rate must be reduced in order to maintain the same linear velocity through the column. This reduction is carried out in the ratio of the cross sectional areas (or internal diameters) of the two columns. Eor example, if a 7.5-mm i.d. column operates at 1.0 ml/min, then in order to maintain the same linear velocity through a 4.6-mm i.d. column the flow rate would be... 

Thus the internal diameter of the column at sill level =... 

An HPLC column is a straight, stainless steel calibrated tube (sometimes coated on the inside with an inert material such as glass or PEEK 5 ) between typically 3 and 25 cm in length. The internal diameter of the column can vary from 0.5 to 5 mm (Fig. 3.6). The stationary phase is held in the column by one porous disc at each extremity. Dead volumes within the column are kept as small as possible. The flow of the mobile phase into the column must not exceed a few ml/min. Some manufacturers offer microcolumns that have an internal diameter in the order of 0.3 mm and are 5 cm in length. Typical mobile phase flow rates for these narrow columns can be as low as a few pl/min. Because complete elution of compounds with these microcolumns requires only a few drops of mobile phase, the stationary phase and pumps have to be adapted accordingly. These narrow columns not only substantially reduce the amount of mobile phase which has to be used but also improve resolution by diminishing the diffusion inside the column. They are well suited for HPLC-MS (cf. Chapter 16). 

A mixture of proteins is separated on a column with a stationary phase of carboxymethylated cellulose. The internal diameter of the column is 0.75 cm and its length is 20 cm. The dead volume is 3 ml. The flow rate of the mobile phase is 1 ml/min. The pH of the mobile phase is adjusted to 4.8. Three peaks appear upon the chromatogram corresponding to the elution volumes V, V2 and V2 at 12 ml, 18 ml and 34 ml respectively. 

Load the sample on an appropriate RP column (internal diameter of the column chosen according to the starting biological material available, 300 A for granulometry, internal diameter from 3.8 to 7 mm, a Ci8 or C8 column is recommended for step 1). It is recommended to equilibrate the column with 2% acetonitrile in acidified water (0.05 % TFA) to remove the hydrophilic molecules and to properly stack the interesting material. 

Elute the peptides with a linear gradient of acetonitrile in acidified water at a flow rate between 0.8 and 1.3 mL/min depending of the internal diameter of the column. Flow rate can be 0.8-1 mL/min if the column has 4.6 mm of internal diameter and increased to 1.3 mL/min for a semi-preparative column (7 mm of... 

In such a situation the internal diameter of the column might also effect the equilibration process but Molander et al. [37] found that even using a temperature gradient, the differences were minimal for columns narrower than 4.6 mm internal diameter. A recent study has found that elevated temperatures, up to 70°C, markedly improved the efficiency and peak shapes of bases with intermediate pH eluents [38]. 

Cut a circle of Whatman No. 54 filter paper the same size as the internal diameter of the column and allow to float onto the settled... 

Column-liquid chromatography (CLC) can be conveniently divided into those systems which use packed columns and those which use open tubes (Figure 3.1). Capillary tubes (<4 < 350 pm) are used in open-tubular chromatography and the stationary phase is coated on the internal surface. Packed-column systems can be sub-divided arbitrarily into capillary columns, microbore columns, analytical columns and preparative columns according to the internal diameter of the column (Figure 3.1). 

To compare or predict the behaviour of capillary columns it is useful to calculate the phase ratio f3 = V- /V (Figure 2.13). Calling the internal diameter of the column and df the film thickness deposited on its inner surface, an approximate calculation leads to ... 

The internal diameter of the column affects several chromatographic aspects. In the beginnings of modern liquid chromatography there was much discussion of the infinite diameter effect (20-22). Due to slow radial mass transfer, for certain combinations of particle diameter and column diameter, solute injected directly onto the center of a column will traverse the length of the column without ever approaching the column walls. For poorly packed columns this significantly increases column efficiency, by eliminating wall effects. However, for well-packed columns the effect is rather small. The practical utilization of this phenomenon also requires specialized injection apparatus and decreased column sample capacity. For these reasons, this concept is now little discussed. 

It is clear that as the internal diameter of the column increases, so does the mobile-phase volume, and then the volume of mobile phase that is necessary for any given chromatographic analysis. This affects the cost of the analysis, both directly in increased usage of chromatographic solvents, and also through increased solvent disposal cost. This cost factor is one of the primary reasons driving the interest in small-diameter columns, which will be discussed in more detail shortly. 

D = internal diameter of the column in feet Fm = adjustment factor for the material of construction Fs = adjustment factor for the tray spacing Ft = adjustment factor for the type of trays... 

In open tubular columns dp is replaced by the internal diameter of the column, dc- Diffusion rates in a gas are much higher than in a liquid, therefore in GC the mobile phase effects. Cm, are much smaller than the corresponding effects in the stationary phase, Cs- In HPLC, Gm and Cs are of comparable significance. 

The efhciency of an HPLC column is determined by the type of stationary phase particle and the quality of the column packing. The internal diameter of the column is not affecting the performance characteristics [10], although it has been deliberated that columns with low aspect ratio, i.e., ratio between internal diameter and particle diameter, have higher separation efficiency. A recent and extensive experimental study did not show convincing evidence for higher column efficiency for columns with an aspect ratio from 7.5 to 30 [11]. 

Height equivalent of a theoretical plate Internal diameter of the column Retention factor (formerly capacity factor)... 

Let us take a typical column of 15 cm length and packed with S-/im particles. This column would have a plate count of about 10,000 plates under normal running conditions. Table 3.1 shows the standard deviation of a peak as a function of the retention factor for several internal diameters of the column. 

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