The Conventional Column

Conventional analytical columns are 4.6 or 4 mm i.d., 1/4 in (63.5 mm) o.d. They are manufactured from high quality stainless steel such as 316 to withstand the high pressures that are used in packing (up to 10 000 psi; 69 000 kPa) and in operating the columns (around 2500 psi; 17 000 kPa). Manufacturers take particular care with the inner bores of these tubes; some use tubing 'as-drawn', while others use electropolished tubing. There have been reports of chemical modification with silanes to the inner surface.

The column packing material is held in the column with an end frit, which in turn is held in place by the column end fitting. The frit is normally made from a sintered metal, the porosity being determined by the size of the packing material used. For 5 |im materials, a 2 |im porosity frit is normally used, but for 3 |im and smaller, a 0.5 |m frit is common. The most common type of end fittings are those manufactured by Swagelok, Parker and Valco. Typical end fittings are shown in Figure 2.

Frits are held on the column by compression fittings; although it is possible to remove the end fitting to repair or top up the packed material (or to clean or repair a damaged frit), it is not generally recommended as it is not usually possible to regain good performance. Inserting and removing these classical columns from an HPLC instrument cannot be done without tools. In an attempt to make this into a simple 'no tools operation', and to reduce the amount of hardware used in the tube section of the column, the cartridge column was developed. The first successful commercial cartridge column was designed by Brownlee Labs and is today still known as the Brow-nlee system.

In this system, the column design is very simple in that the tubular column has the packing material held in place by frits that are pushed into the end of the tube. This packed tube is held in the cartridge column

Table 1 Definitions of columns

Column type

Internal diameter (mm)

Analytical

20-3

Microbore

2-0.5

Capillary

<0.5

Figure 2 Typical end fittings.

held by hand-tight end nuts that are part of and free to rotate with the cartridge holder.

Since the Brownlee system was introduced, there have been many copies on this basic design and also developments into the finger-tight removable end fitting column (Figure 3). These columns, as shown in Figure 4, again have the frit pushed into the tube or held in place by a frit cap. This frit cap also contains a seal that allows the end fitting to be screwed down onto the frit. This design has many advantages. It allows easy removal of the frit to replace it or top up the column packing material, but more importantly,

Figure 3 Finger-tight end fitting column showing frit cap.
Figure 4 Removable end fitting column.

it allows columns to be coupled together with very low dead volumes.

Apart from the cartridge and removable end fitting columns, a different design approach was taken by Waters with their radial pack systems. As mentioned previously, high pressures are used to pack particles into column to obtain a well-packed bed. In packing columns, voids in the packing bed are produced if insufficient pressures are used; these voids are commonly along the axial direction. The idea of the radial packed column is to pack the material into a soft-walled tube. This tube is held in a device that can apply radial pressure, and so reduce the radial voids. Some examples are shown in Figure 5. This radial compression system has not been applied down to small i.d. tubes, but has been developed for semipreparative systems. In preparative systems, direct axial compression on to the top of the packing is also used.

Columns down to 3 mm i.d. can be used with what is commonly classed as conventional HPLC equipment, with detector flow cells in the order of 8 |L.

PrepPak Cartridge îiMM X 10CM

Figure 5 (See Colour Plate 21) Radial compression column cartridge.

Figure 5 (See Colour Plate 21) Radial compression column cartridge.

Table 2 Volumetric flow rates required to maintain a linear flow velocity of 1.5 mm s_1 in columns of different diameters

Column diameter (mm)

Solvent consumption (mL h -1)

Reducing the column diameter below 3 mm requires detectors with smaller volumes to reduce detector band broadening. Pumps that can produce lower flow rates are also required. Table 2 gives the volumetric flow rates required to maintain a linear flow velocity of 1.5 mm s_1.

Although microbore columns became commercially available in the mid-1980s, they have not displaced 4.6 mm i.d. columns. Many comparison reviews were written in the 1980s of 1 mm versus 4.6 mm i.d. columns. In general the conclusion was reached that solvent saving and packing material costs did not justify the capital expenditure in new equipment and that mass sensitivity enhancements were difficult to achieve. Since then much has improved, both in the instruments and especially in the column design, particularly in the end fittings used on < 3 mm i.d. columns.

From 1990 onward, further research was conducted and there was widespread discussion of the advantages of microbore columns, including increased resolution, decreased solvent consumption, lower heat capacity, increased mass sensitivity and, most importantly, the easier coupling of these columns to secondary systems and mass spectrometers. Columns with i.d. down to 1 mm are conventionally still manufactured from stainless steel, but as the i.d. is reduced so can the wall thickness be reduced. This allows the use of other materials, as the resulting pressure forces in the small i.d. tubes is also reduced. For 2 and 1 mm columns, glass-lined tubing has been used. This is in an attempt to give a very smooth inner surface, which in turn helps column packing and hence efficiency. Columns are now available manufactured from PEEK (poly(ether ether ketone), an ICI polymer). Columns of this material are produced in diameters down to 0.5 mm.

A conventional PEEK column is shown in Figure 6, and a 0.5 mm flexible PEEK column is shown in Figure 7. These columns have no wetted metal components and, since the 0.5 mm columns are flexible,

Figure 6 PEEK column.

for the first time in HPLC it has become possible to consider a different design of instrument, in that there is no longer any need to have a fixed distance between the injector and detector. Another feature of these columns is that they can be cut to any length. If the

Figure 7 PEEK-o-bore column.

Figure 8 Integral guard column.

inlet blocks or becomes contaminated, then this part of the column can be cut off.

Capillary columns of less than 0.5 mm i.d. are almost exclusively manufactured from fused silica, in some cases with a metal outer case onto which the end fittings can be connected.

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