Today, column material is normally Type 316 stainless steel, once again chosen because it offers the best compromise of cost, workability, and corrosion resistance. Most commercial columns available today have internal diameters of either 2.6-3 mm or 4.6-5 mm. Most leading manufacturers obtain a great amount of interchangeability between column sizes without excessive fitting replacement by supplying columns with a variety of internal diameters, but a uniform ¼ in. outside diameter. Naturally, preparative columns have larger diameters: usual O.D. values are 3/8 - 5/8 in., corresponding to internal diameters of 6.4-12.7 mm, respectively.
Standard HPLC column.
Most modern liquid chromatography plumbing is performed with compression fittings such as Swagelok , Parker-Hannifin , Gyrolok, etc. These offer a great deal of flexibility, can be connected and disconnected frequently without loss of sealing power, are commonly available, and may be used without special seating and flanging tools allowing users to install, maintain, modify, and improvise equipment. Great care should be taken in the choice of end fittings, connectors, and unions however, since inclusion of rapid diameter changes corners, and other unswept areas in a system can destroy a good separation. It is best to obtain the above parts from a reputable liquid chromatograph manufacturer since most of these companies offer fittings which are specifically made for LC.
Many reducing unions and column end fittings incorporate special devices designed to hold packings in place and/or prevent their flowing into the detector or detector tubing. The most frequently used of these consist of fine porous stainless steel fritted filter discs, usually with an average opening of 1 mm. These may be forced into the ends of columns or may be in the connector or end fitting. The important point is that the fritt disc must be tightly fitted to avoid occurrence of large-diameter spaces at the edge where the disc contacts the wall. An additional benefit of the frit is derived from the fact that it fills what otherwise might be a large empty area in conversion zones thus, the sample cannot diffuse, cross mix, or undergo other efficiency-killing effects when passing through these areas.
Connector tubing must be of low cross section to hold separated components in narrow bands as they are transferred from column-to-column, from column-to-detector, or from detector-to-detector when mounted in series. Tubing having an internal diameter in excess of 0.634 mm (0.025 in) can allow peak mixing and band broadening and it is good practice to use tubing no larger than 0.12 mm (0.005 in) in areas where sample is to be transported.
A line filter should be used between the pump and the sample injector to prevent particulates from clogging the column inlet. Porous stainless steel filters having a porosity of about 2 µm are typically used in commercial instruments; however, 0.5-,um porosity filters (e.g., Alltech Associates) are desirable with columns of less than 10-,um particles. To facilitate solvent changeover, the volume of the line filters should be small.
Pressure monitors are used in the LC equipment as diagnostic tools to optimize separation and to indicate system problems (e.g., plugging or leaks). Diaphragm or Bourdon-type gauges are simple, inexpensive, and generally robust. On the other hand, strain-gauge pressure transducers are more precise and have a smaller internal volume, which facilitates the changing of solvents. In addition, straingauge types are available with high- or low-pressure alarms or cut-off circuits, to protect the pump against high-pressure overload by column plugging or the instrument against fire due to solvent leaks (low-pressure alarm).
It often is advantageous to run ion-exchange, size-exclusion and reverse-phase columns at higher temperatures, and to precisely control the temperature of liquid-liquid columns. Therefore, column thermostats are a desirable feature in modern LC instruments. Generally, temperature variation within the LC column should be held within +0.2°C. Maintaining a constant temperature is especially important in quantitative analysis, since changes in temperature can seriously affect peak-size measurement. It is sometimes important to be able to work at higher temperatures for the size-exclusion chromatography of some synthetic polymers because of solubility problems, but precise temperature control is not so important in this case. High-velocity circulating air baths are most convenient in LC (as in GC). These devices usually consist of high-velocity air blowers plus electronically controlled thermostats, with configurations similar to those used in gas chromatographs. Alternatively, LC columns can be jacketed and the temperature controlled by contact heaters or by circulating fluid from a constant-temperature bath. This latter approach is practical for routine analyses, but is less convenient when columns must be changed frequently.
Since separations are accomplished in minutes, fraction collectors are usually not
needed in modern LC. Manual collection is normally used, and many commercial instruments
have convenient sample-collection ports on the outlet of the detector. Fraction collectors
are sometimes used in preparative chromatography and in conventional size-exclusion
chromatography with larger-diameter columns, because separations are usually much slower.