10 guidelines to increase the lifetime of your column.....

1. Use the columns at the correct flow rates, and generate pressures much lower than the maximum pressure limit of the column. Set this value in the method for the pump. 
2. Always apply the ready‑to‑use guard column. All the non‑eluting components from the sample and the mobile phase will then accumulate in the guard column. 
3. Use low concentration buffers (< 0.1 M) and a pH between the minimum and maximum column limits, provided by the column manufacturer.
4. Use high pressure filters between pump and the sampler. 
5. Never leave a buffered mobile phase inside a non‑running pump, to prevent any crystallization, which will also effect the quality and lifetime of the column. 
6. Turning the injection valve must be done as quickly as possible. If the injection valve is turned too slowly at a high flow, a pressure wave will cause voids in the packing and the accompanying double peaks. 
7. Do not drop the column or any other mechanical shocks: if the column is started up immediately at high flow rates and pressures, you will create a pessure wave, as in point 6. 
8. Only connect the column to the system with the advised type of fittings, Fittings must be in good condition. Different types of columns can be connected to the instrument with 'finger tight' PEEK fittings. 
9. Always use pure, filtered, degassed solvents for the mobile phase. Be sure that the pump is perfectly regulated and damped. 
10. Replace the guard column in time (before the column is generating higher pressures) and regenerate the column with the correct series of flushing solvent mixtures.

HPLC columns packed with 3, 5, 7 or 10 μm particles are made by using the so‑called slurry technique at pressures of 30 to 60 MPa (1 MPa = 10 bar). A newly packed column has therefore a high packing density and can stand rough handling. Nevertheless mechanical shocks should be avoided (e.g. dropping on the floor!). Older columns are even more sensitive.

Pressure shocks (injection, column switching, pump pulsation) may cause instability in the packing. This will cause a decrease in column efficiency, broad peaks or even double peaks. 

The maximum flow rate is reached when the average velocity u of the mobile phase through the column should not exceed 0.4 cm/sec. Above this velocity there is a chance of channelling inside the packed bed which will result in double peaks in the chromatogram. 

Double peaksThe pump should be set with a maximum pressure limit which is set at a pressure of 10% below maximum column limit (check manufacturers manual).

Blockages of columns are the result of a clogging of solid or poorly soluble particles or compounds from the samples or the eluent in the top or frit of the column. The contaminations can come from: 

1. The mobile phase (buffers!)
2. The pump (seals, plungers), injection valve or other parts of the system
3. The sample  

These particles can be present in the mobile phase if it is not properly filtered beforehand. Also bacterial growth can occur inside the eluent reservoir or buffer salts can precipitate.

The sample itself, however, is the main source of column contamination and blockage. This contamination may be either solid particles or components of the sample which do not elute under the chosen chromatographic conditions and remain on top of the packing. This will result in an increasing pressure drop.  

Problems due to impurities from the eluent and pump can be prevented by placing a low pressure filter in the solvent reservoirs or at the inlet of the pump. A high pressure filter behind the pump and the injection valve is also very effective.

Problems resulting from contaminated samples can be avoided by proper clean-up, if possible, of the samples before injection. It is always advised to use a guard column between the injector and the analytical column. Guard column

A guard column:

• Has a small internal volume
• Should be connected with a short capillary, or better directly coupled to the analytical column.
• Should be filled with the same kind of material (e.g. reversed phase or ion exchange) as the analytical column, or with a material of 30-40 micron porous particles to avoid excessive back pressure.

A guard column designed and used in this way has no significant effect on the separation efficiency. 

Serum extract analysis with guard column

Serum extract analysis after many injections

Effect of a ‘guard’ column on efficiency

Important signs of saturation of the guard column are:

• Gradual increase of pressure drop
• Worsening of the peak shape
• Shifting retention times
• Increasing baseline
• Instable baseline
• Ghost peaks (especially in gradient analysis)
• Detectable peaks when injecting a clean solvent

If a column is blocked, one or more of the following actions should be taken: 

1. Replace the inlet frit or screen, if possible
2. Rinse the column and regenerate it (in backflush) 
3. Replace the column 

Contamination from the mobile phase and/or sample can accumulate on top of the packing and giving a brownish colour.

Broadening and tailing of peaks due to an accumulation of non‑eluting components on top of the packing cannot only result in a high back pressure but also in a changing retention behaviour, lower retentions  and less resolution. In that case the column needs to be regenerated with one or more solvents with higher elution strengths than the mobile phase used for the analysis. Make sure that consecutive solvents are completely miscible and are compatible with the packing material.

Regenerating the column can be done in backflush mode but this is not essential. Use approximately 20 times the column volume for each solvent. Check the column manual first before starting the regeneration procedure! 

PAKs standard before column regeneration

PAKs standard after column regenerationImportant rules for column regeneration:

1. Always use HPLC‑grade solvents.
2. With silica columns (normal phase chromatography) always use dry solvents.
3. Not every solvent flushing step is necessary. Depending on the original elution strength of the mobile phase 1 or 2 solvents are sufficient.
4. Never use acetone or other ketones in combination with ‑NH₂ polar bonded phases
5. Reversed phase columns can be regenerated with acetonitrile or THF instead of methanol. However, it is more expensive and more hazardous. Also iso-propanol is used in some cases (consult your supplier).

"Voids" often occur in the top of the column. Further down the column minute channels can develop which are not visible, but will result in double peaks in the chromatograms. Small channels or dead volumes can sometimes be removed by backflushing the column.

Poor column quality

Column performance after regeneration

“Double” chromatogram

Performance after “backflush” regeneration

Unstable column?

Pulsation of the pump and pressure changes due to the operating of the sample valve are two other forms of mechanical load on the column. To observe the pulsation of the pump a proper pressure read‑out device is necessary. A pressure pulse can also be caused by air bubbles or inefficient check valves and seals.

Switching the flow on and off frequently and changing mobile phases with different properties will reduce the column lifetime.

Modern pumps have the capability to built up pressure in a short period of time thus putting the column under a high pressure in a relatively short time. It is therefore better to start up the column at a low flow rate and then increase it gradually.

Changing the eluent?
When dealing with QC analyses it is often better to use one column and one mobile phase for a particular analysis and not to change the eluent frequently.

• It nearly always takes a relatively long time before the column reaches equilibrium with the new mobile phase. In some cases it is almost impossible to use the column for other purposes after flushing it with buffers or ionpair reagents. These reagents are irreversibly adsorbed onto the packing material.
• Gradient elution is another technique in which the equilibrium between column and mobile phase is important. Before a new injection is made be sure that the eluent has returned to its original composition and the column is properly equilibrated.

If salts are used in the mobile phase, the pump should run continuously during the night. A flow rate of 0.3 ‑ 0.5 ml/min is usually sufficient to prevent any buffers from crystallizing inside the column or other parts of the instrument (pump, injector, detector). If it is necessary to run during the night, the eluent from the detector can be returned directly to the eluent reservoir in which the eluent is gently stirred. If the eluent is not contaminated during the above procedure the column is ready for use the next morning.

If the column has been contaminated strongly it can be cleaned during the night by a regeneration procedure, preferably with a stronger eluent. Obviously the column will then not be immediately ready for use next morning.

Leaks can be the result of incorrect treatment of column connections and fittings as well as the wrong combination of (non compatible) couplings.

The most widely used fittings in HPLC are of the compression type.

• Most fittings are standardized only on the type of thread used (10/32 UNF).
• The other specifications such as the angle of the conic the ferrule has to seal, the shape and length of the ferrule and the insert depth of the capillaries vary.
• To connect a ferrule to a 1/16" capillary it is sufficient to hand tighten the nut in the fitting and afterwards tighten it further for 3/4 of a turn with the correct spanner (inch‑size!).
• A once connected ferrule usually needs only a 1/4 of a turn to make a leak free connection.

Stainless steel nuts and ferrules have the disadvantage that once fitted they cannot be removed from the capillary.

PEEK connections are a good alternative.

• They are made in one piece (nut + ferrule) of this strong polymer.
• Those fittings can be hand‑tightened and can still withstand a pressure up to 25.0 MPa. The ferrule does not clamp itself into the capillary so it can be used several times on couplings that differ in insert depth.
• If these are not tightened too much they can be used several times and can always be removed from the capillary whenever necessary.

If the column is not used for one or more days it is best to leave it in the HPLC instrument with the mobile phase inside. This does not apply when the mobile phase contains a concentrated buffer.
For longer periods of time (e.g. more than a few days), buffers etc. should be flushed out of the column with water / modifier mixtures and replaced by a suitable organic solvent. Consult the column manual for proper flushing and storage directions. 

After long‑term storage the column should be started up with a moderate flow rate and pressure, with solvents which are completely miscible with those used for storage.