Devices & Accessories

HPLC Columns

We have pre-packed HPLC columns of various sizes and designs in stock. Also column combinations for linear upscaling, for example, 4.6 and 20 mm ID with the same packing are available.


Because of the enormous diversity no current summary of all available HPLC columns is provided in endless lists. We rather recommend contacting us so that we can advise you in the choice of the right HPLC column for your application.

The selection guide helps determining the right column for your specific application. Based on the properties your sample such as solubility and polarity, you can quickly identify the appropriate separation phase.

HPLC Trennsäulen

HPLC Column Selection Guide

We have phases in stock for lipids, organic acids, PAH, peptides, DNA, environmental analysis, metal ion analysis, food and beverage, sugar, pesticides, ion chromatography and many more.

HPLC columns are available with an inner diameter (ID) of up to 50 mm. Of course you can also pack your HPLC columns yourself cost-effectively and quickly, using our column packing station with static or dynamic axial compression.

Tips for Care and Storage of HPLC Columns

The lifetime of a column is not least dependent on the handling and storage. In the following we would like to give you a few suggestions and provide information.

Columns with Silica Gel

Due to the high mechanical stability and good physicochemical surface properties these columns filled with silica gel are currently the most widely used HPLC columns. Furthermore, they are compatible with many organic solvents, which makes them even more interesting. HPLC columns are readily pressure stable up to 400 bar; however pressure surges should be avoided, as the silica gel at the column inlet could be damaged leaving a gap in the packing material which in turn can result in double peaks.

Since HPLC columns resemble a filter, the use of pure solvents is a prerequisite for clean separations. Impurities block adsorption sites, alter the selectivity of the separation column and can show up as so-called ghost peaks in the chromatogram. To avoid such irreversible adsorption at the column inlet, a pre-column should always be installed upstream. It should be replaced at regular intervals or when a predetermined back pressure is exceeded. Pre-columns extend the life time of the separation column substantially. The pre-column also retains smaller particles, such as abrasion of the pump seal, from the injection valve or from the sample. As an alternative to a pre-column, in-line filters may be used.

Before changing the solvent on the column from aqueous to organic solvents, it is crucial to wash the buffer solution from the column, as otherwise buffer salts could precipitate in the organic solvent and block both the column and the capillaries.

Firmly seal the column inlet and outlet for storage of separation columns with silica gel.

  • Overnight - store in the currently used eluent
  • For up to 1 week - wash buffer-free and rinse briefly with methanol
  • For a long time – rinse with methanol, followed by acetonitrile

Equilibration

After a throughput of 15-20 column volumes of eluent the HPLC column ought to be equilibrated. The equilibration time, however, strongly depends on the dimension of the separation column.

Equilibration times ca. Dimensions Column volume Flow rate
10 min ID 2.0 x L 50 mm 0.11 ml 0.25 ml/min
25 min ID 2.0 x L 150 mm 0.33 ml 0.25 ml/min
45 min ID 2.0 x L 250 mm 0.55 ml 0.25 ml/min
30 min ID 4.0 x L 150 mm 1.50 ml 1.00 ml/min
45 min ID 4.0 x L 250 mm 2.20 ml 1.00 ml/min
15 min ID 4.6 x L 50 mm 0.58 ml 1.00 ml/min
35 min ID 4.6 x L 150 mm 1.74 ml 1.00 ml/min
60 min ID 4.6 x L 250 mm 2.91 ml 1.00 ml/min

Equilibration times can be reduced by higher flow, however, increasing operating pressures in the HPLC system have to be monitored consequently.

Regeneration of NP-Columns “Never Use Water!“

For columns with normal phase (silica, diol, nitro and amino phases) use the following washing procedure.

  • Rinse with 20 column volumes heptane or hexane.
  • Rinse with 5 column volumes isopropanol.
  • Rinse with 4 column volumes acetonitrile.
  • Rinse with 5 column volumes isopropanol.
  • Rinse with 20 column volumes heptane or hexane.
  • Now equilibrate the HPLC column.

Regeneration of RP-Columns

For regenerating RP-columns, so-called reverse phases, the following rinsing routine is well established.

  • Rinse with 20 column volumes HPLC water.
  • Rinse with 20 column volumes acetonitrile.
  • Rinse with 5 column volumes isopropanol.
  • Rinse with 20 column volumes heptane or hexane.
  • Rinse with 5 column volumes isopropanol.
  • Wash with 20 column volumes acetonitrile.
  • Now equilibrate the HPLC column.

Columns with Polymer Phase

HPLC columns with polymeric material are more sensitive to mechanically stress than columns with silica gel material. They are also more sensitive to solvent changes and react with macerating or shrinking, which can cause a pressure increase or dead volumes.

As the polymer material exhibits very different properties dependant on the respective producer, no general suggestions for handling the column can be given.

A column with hydrophobic unmodified polystyrene / divinylbenzene copolymer can also be stored with aprotic solvents, for example, acetonitrile, for a long time.

Polymer-based ion exchangers, in contrast, we recommend storing in a 0.01% aqueous sodium azide solution at +6°C to +8°C in the refrigerator to prevent contamination.

Regeneration of Polymer Columns

Double peaks or increases in operating pressure after a certain operating time mostly originate from a dirty column. Usually the HPLC column can be regenerated; however, for each of the different polymer-based column versions another regeneration process is necessary. In case of need send us your inquiry so that we can inform you about the correct procedure.