Columns are usually made of heavy-wall glass (withstands pressures up to 200 bar) or stainless steel (up to 800 bar or larger ) tubing.

Column dimensions

The outer diameter of larger I.D. columns is typically 1/4 inch, irrespective of I.D.

The development of HPLC continues, not just in terms of HPLC techniques, but also stationary phases and column technology. Column hardware and the packing material have improved significantly over the years.

The three column parameters tabulated here (particle size d_p, length and internal diameter) play a vital role in the development of HPLC. In particular, the miniaturization of these three parameters is the result of efforts that continue to this day. In addition since manufacturing processes have improved substantially, new stationary phases (e.g. spherical particles of narrow size distributions) can be produced routinely now.

• Column length L
• Internal diameter I.D. of the column
• Particle size (d_p) of the packing material
• Flow / linear velocity of the mobile phase

The relation between column dimensions and HPLC instrument’s contribution to the total band broadening is also discussed in this chapter.

Column dimensions affect the:

• Separation. The separation power of a column increases with the square root of the column length.
• Analysis time. Under fixed analysis conditions, the analysis time is proportional to the length of the column. The shorter the column, the faster the analysis.
• Pressure. For a given eluent and temperature, the pressure drop across a column is proportional to the length of the column.    
• Mobile phase consumption. For a given eluent velocity, the mobile phase consumption is proportional to the square of the column diameter. 
• Injection volume. The maximum injection volume depends on the stationary phase properties, the ionic state of the sample and on the column's diameter.

The packing procedure is essential for the quality, stability and longevity of HPLC columns. In brief, the quality of HPLC columns is mainly determined by:

• Quality of the liquid suspension of a packing material (2-10%v/v)
• The applied column filling pressure (400 to 1500 bar)
• Final rinsing and conditioning steps

Since smaller particles provide larger number of plates per unit column length, modern columns employ 5, 3, and 1.8 µm stationary phase particles. Given these very small particle diameters, it is not possible to dry-pack HPLC columns efficiently. Therefore, wet- fill or slurry packing methods have been developed to manufacture high quality HPLC columns:

• First, the particles are suspended at 2-10% v/v in a suitable solvent.
• Next, the column exit is closed with a fine-meshed stainless steel frit or screen and the suspension is forced into the analytical column at high pressures.
• After the high pressure filling process, the column inlet is closed by another frit. 
• Finally the column is rinsed to remove the suspension solvents used during the packing process. 

After the packing procedure, the analytical column is tested and several relevant column parameters are determined. The column plate number, retention factors, selectivity and peak asymmetry are important parameters and are used to monitor the quality of the packing process.

If the column is stored for a short period, storage in mobile phase is suitable as long as the mobile phase is not aggressive, does not contain a buffer and has a neutral to weakly acidic pH.

If the column is not to be used overnight, the mobile phase should be kept running at a slow rate, especially if the mobile phase contains buffers that can precipitate in the column. 

Long-term storage If a buffer solution is used, the column is initially flushed with buffer-free water with 10% or more modifier to prevent salt precipitation. After that the column is finally flushed with the pure organic solvent. In all cases the column manual should be consulted.

Buffers may promote bacterial growth in HPLC columns, which may result in clogging of a column. As a result, RPLC columns are properly stored filled with 100% methanol or acetonitrile, which effectively preclude bacterial growth. If the column was used with a buffer, however, it must be flushed in a stepwise manner with water/modifier mixtures of increasing modifier content to remove all traces of buffer. If any traces of buffer salt remained, they would precipitate as the column is flushed with pure organic solvent for storage.

In the event that the RPLC column must be used with a buffered eluent after storage, it must be reconditioned by a stepwise increase in the water/buffer concentration in the eluent prior to use. In all cases and also for other column types, please refer to the user's guide of that column.  

If columns have not been used for a period of time time, they may be somewhat dried out. To restore the column performance it should be flushed with pure modifier at a low flow rate.

Conventional columns are available with standard connection fittings of different types.

Columns can also be produced as glass or stainless steel cartridges that fit into special holders.  This has the advantage that the cartridge columns can be replaced rapidly while the connecting capillaries remain in place. No spanners are used once the holders have been connected to the capillaries.

The tubing material must be seamless and smooth. Steel columns are made of polished tubing while glass columns are naturally sufficiently smooth inside.

Dirty samples can quickly raise system pressures and can contribute to shifts in retention, poor peak shape, and loss of separation. The proteins in biological samples, fats in foodstuffs, polymers in industrial samples, and biological residues in environmental analyses are frequent culprits. In these cases, the use of a guard column is highly recommended. 

The guard column prevents minute particles from the sample or from the mobile phase from clogging the top of the expensive analytical column. A sudden or even a steady increase in the pressure in an HPLC system usually indicates clogging of the column or other parts of the system, such as the injection valve or connecting capillaries.

Protecting the analytical column with a guard column is always recommended, especially for routine analyses. A guard column typically has dimensions of 10 – 50 mm (length) and an I.D. of 1 - 3 mm.  These columns are inserted between the injection valve and the top of the analytical column. Due to the small dimensions of the guard column, they have only a minor effect on the peak shape and resolution.

The guard column can be incorporated into the cartridge system of the analytical column. For conventional columns, it is possible to connect a guard column directly to the inlet fitting of the analytical column. In this way the two columns can be connected without dead volume.

Guard columns can enhance separation efficiency
A well-designed guard column will only generate a negligible reduction in efficiency, peak symmetry or separation.

The frequency of replacement of the guard column is a function of the sample composition, pretreatment and injection volume. There is no fixed rule indicating when to replace the guard column.

Indications of a saturated guard column include:

• Increased pressure drop across the column
• Shifts in retention times
• Poor peak shape
• Loss of resolution
• Drift of the baseline
• Unstable baseline
• Appearance of ghost peaks, especially with gradient elution analyses

It is important to replace the guard column before these effects begin to appear in chromatograms. This frequency must be determined experimentally and may vary from application to application.

The following are important considerations in choosing a guard column:

• Length smaller than 1/10 of the analytical column
• Internal diameter
• Direct coupling to analytical column is most favourable
• Particle size: 5 < d_p < 30 micron 

In order to minimize the loss of efficiency caused by a guard column, the internal volume of the guard column should be small. In practice this means that the length of a guard column is equal to or less than 10% of the length of the separatory column. 

In practice, a guard column of 3.0 mm ID is typical for an analytical column of 4.6 mm I.D., while a 3.0 mm I.D. analytical column is combined with a guard column of 2 mm ID.

In terms of packing material, there are two options:

• The same particle size as the analytical column, e.g. 5 µm. This results in the best overall efficiency, but these guard columns are relatively expensive and are easily blocked by dirty samples.
• Large particles of 30 – 40 µm. Advantages include low pressure drop, easy (re)filling and low cost. Some loss in efficiency may occur.

Both types of guard columns are commonly used in analytical laboratories.