When not in use, HPLC columns should be kept filled with solvent, air-tight and free from vibration. Water is generally regarded as an unsuitable conservation medium in view of potential fungal growth. Salt solutions (buffers) may crystallize and cause clogging.
Do not forget to label the column with the type of mobile phase.
After a long period of use or insufficient precautions, the column may become fouled by a build-up of adsorbed materials, especially in the upper packing zones, impairing the overall performance. Before discarding the column, an attempt can be made to regenerate it (albeit with no guarantee of success!). Guard columns must be removed before any such trial begins. It may be a good idea to reverse the column.
Silica Columns
The following solvents are pumped through at a rate of 1–3 ml min1: 75 ml of tetrahydrofuran;
75 ml of methanol;
75 ml of 1–5% aqueous acetic acid if basic impurities are present;
75 ml of 1–5% aqueous pyridine if acidic impurities are present;
75 ml of tetrahydrofuran;
75 ml of tert-butylmethyl ether;
75 ml of hexane (if hexane is subsequently used for chromatography, otherwise stop at ether).
If the separation performance should decrease as a result of the silica adsorbing too much water, the latter can be removed by chemical means22the reagent either being self-formulated or bought as a ready-made version (Alltech Associates, Deerfield, Illinois, USA).
Octadecyl, Octyl, Phenyl and Nitrile Columns23
The following solutions should be pumped through at a rate of 0.5–2 ml min1: 75 ml of water þ 4100ml of dimethyl sulphoxide injected;
75 ml of methanol
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22R. A. Bredeweg, L. D. Rothman and C. D. Pfeiffer,Anal. Chem.,51, 2061 (1979).
23R. E. Majors,LC GC Eur.,16, 404 (2003) orLC GC North Am.,21, 19 (2003).
75 ml of chloroform;
75 ml of methanol.
Water!0.1 M sulphuric acid!water is a further possibility.
If a column has lost its chemically bonded phase, then silane compounds may be used in an attempt to bond it to the silica again.24
Anion-Exchange Columns
The following solvents should be pumped through at a rate of 0.5–2 ml min1: 75 ml of water;
75 ml of methanol;
75 ml of chloroform;
methanol!water.
Cation-Exchange Columns
The following solvents should be pumped through at a rate of 0.5–2 ml min1: 75 ml of water þ 4200ml of dimethyl sulphoxide injected;
75 ml of tetrahydrofuran;
water.
The following sequence works well for anion and cation exchangers:
75 ml of water
75 ml of a 0.1–0.5 M solution of buffer used previously (to increase the ionic strength):
75 ml of water;
75 ml of 0.1 M sulphuric acid;
75 ml of water;
75 ml of acetone;
75 ml of water;
75 ml of 0.1 M EDTA sodium salt;
75 ml of water;
For styrene–divinylbenzene-based cation exchangers: 0.2 N sodium hydroxide is pumped through at 70 C overnight (this removes bacteria which could be present at the surface of the particles).
Styrene–Divinylbenzene Columns
The following solvents should be pumped through at a rate of 0.5–2 ml min1: 40 ml of toluene or peroxide-free (< 50 ppm) tetrahydrofuran;
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24C. T. Mant and R. S. Hodges,J. Chromatogr.,409, 155 (1987).
200 ml of 1% mercaptoacetic acid in tetrahydrofuran or toluene, if the column is blocked by stryrene–butadiene rubber or natural or synthetic rubber.
Special regeneration procedures are recommended for columns used for the separation of biopolymers.25
The column may also be opened to inspect the chromatographic bed. If the packing has collapsed (Fig. 7.18A), then the column is repaired in the following way: (B) the excess packing is removed with a fine spatula and the bed smoothed over; (C) dead volumes are filled with glass beads of about 35mm or suitable porous layer beads and the column is resealed. The frit is replaced by a new one.
Obviously, if enough care is taken (pulse damping, correct preparation of the mobile phase and sample, prevention of precipitation inside the column, etc.), then regeneration and repair work may not be necessary. See the excellent papers by Rabel and by Nugent and Dolan on the subject.26
The cavity left by the collapsed packing in a cartridge with an axially compressed chromatographic bed can be removed by simply drawing up the end fittings.
Column emptying
Column contents should not be removed with a spatula, wire, etc., otherwise the internal walls may become scratched and the packing material itself damaged.
The best way is to remove the end frit and fitting and use the pump to force the contents out.
Direction of Flow
It is best to use a HPLC column always in the same directionorto know why the direction of flow has been changed. Many columns have an arrow which
Fig. 7.18 Repair of columns with collapsed packing.
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25C. T. Wehr and R. E. Majors,LC GC Int.,1(4), 10 (1988) orLC GC Mag.,5, 942 (1987).
26F. M. Rabel,J. Chromatogr. Sci.,18, 394 (1980); K. D. Nugent and J. W. Dolan,J. Chromatogr., 544, 3 (1991).
indicates the direction; in this case it is not clear if it is possible to reverse the column. If the frits at both ends are identical in porosity it is no technical problem to run the column in the other direction, however, frits may differ in pore width and the wide-pore frit would be at the entrance. Flow reversal could not be recommended under these circumstances. If a column has no arrow it is good advice to draw one oneself. The column is then always used in the same direction, therefore fines and non-eluted compounds are concentrated at the inlet. The column is only turned for the purpose of regeneration.
8 HPLC Column Tests