Depending on the usage conditions and sample cleanliness, most columns can undergo potential changes such as chemical contamination, surface changes, and partial Disturbance of the Silica material during use (Void). Such changes can lead to the deterioration of the packing materials in the column, Which reduces the separation selectivity of certain compounds. Such special separation selectivity is often impossible to reproduce. Resulting in the need to develop methods or perform a lot of unnecessary diagnostic work for instrument systems, chromatographic columns, and mobile phases in the future.
Due to the nature of the reversed-phase surface, column performance (resolution, retention) may change slightly during the first few injections as the organic solvent diffuses out of the core of the particles.
For zwitterionic compounds such as polypeptides, this so-called "column conditioning" occurs for most polypeptides larger than 10,000 molecular weight.
A column can be conditioned by repeated injections of a polypeptide until column performance remains constant. This typically requires injection of about 100 μg of the polypeptide for a 4.6 mm i.d. x 250mm column.
Alternatively, column conditioning can be accomplished by injecting a commonly available protein such as ribonuclease followed by elution with a typical acetonitrile gradient with 0.1% TFA.
What is Column Conditioning?
A pre-saturation column, located between the pump and injector, saturates the mobile phase with dissolved silica thereby protecting the analytical column from solvent degradation.
Clean, stable baselines are critical to good liquid chromatography, and properly conditioning the HPLC column is a helpful way to ensure a good baseline. Analysts need to flush out the storage solvent and equilibrate the column to the solvent mixture and may need to use an intermediate solvent to ensure complete mixing and flushing.Â
Every reversed-phase HPLC column should be conditioned for the first time use, following long-term storage, and when significant changes in the mobile phase. The mobile phase utilized in the conditioning was thought to be similar to those used in the consequent chromatography.
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The general procedure of column conditioning is as follows:
- Wash the column with about 3 column volumes of aqueous mobile Phase with a low flow rate.
- Run linear gradient system from 100% aqueous mobile phase to 100% organic mobile phase up to 2-3 column volume at the identical flow rate as above.
- Equilibrate the column on a minimum of 5 column volumes with the organic mobile phase, until the monitor signals get stable.
- If you change the mobile phase system, a blank run should be done to verify any impurity that may emerge in the mobile phase.
How long should the HPLC column be equilibrated?
This is a question commonly asked by HPLC analysts, particularly those relatively new to the technique. The range of currently available column dimensions means that a one size fits all approach is not appropriate. The amount of mobile phase which should be flushed through a column before it is ready to use is usually expressed in terms of the column volume, the amount of mobile phase required to fill the column.
Equilibration time is dependent on the flow rate and dimension of the column. In general, flushing the column with ten up to twenty column volumes is enough for equilibration. Therefore the flow rate has to be kept in mind. It is possible to increase the flow rate during equilibration to shorten the equilibration time. If a column should be equilibrated with a buffered mobile phase or with a mobile phase containing an ion-pair reagent, the equilibration time should be extended.Â
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When a column has just been installed on a reversed-phase HPLC system then it will typically require between 10 and 20 column volumes before it is fully equilibrated and ready to use. However, some applications are likely to require additional column volumes. Examples are methods that include ion-pairing reagents and chiral HPLC methods. In these cases, suitable equilibration should be investigated and documented for future use.Â
A starting point for investigation may be approximately 30 column volumes. When re-equilibrating after a gradient injection has been run (prior to the next injection) 10 column volumes are recommended. Although it refers to the column volume, the volume that is interested in is more correctly called the void volume, Vm. This is the volume of the HPLC column that is not taken up by the stationary phase. This is typically approximately 70% of the total column volume.Â
There are two methods that we can use to calculate the void volume:
Method 1
Use the dimensions of the column and the formula for the volume of a cylinder:
The dimensions of interest on HPLC column are shown below.
The result of the calculation above is the total column volume. To convert this value to the void volume we multiply by 70%, therefore the formula becomes:
The radius, r, is equal to the internal diameter divided by two:
Example
A HPLC column of internal diameter 4.6mm and length 10cm:
Note: express both i.d. and length in cm.
To calculate the required equilibration time simply multiply the calculated void volume (in mL) by the number of required column volumes, e.g. 10, then divide by the flow rate (in mL/min) to determine the total time required.
For a flow rate of 1 mL/min on the column above, this would result in an equilibration time of 11.6 minutes for 10 column volumes.
Method 2
Inject an unretained solute to obtain t0, the column dead time (minutes). Then multiply this by the flow rate to obtain the void volume:
Where F is the flow rate expressed in mL/min.
Example
The flow rate for the method is 1.0 mL/min.
The column dead time, t0, is obtained from the chromatogram in Figure and is equal to1.05 minutes.
Therefore, Vm = 1.0 x 1.05 = 1.05mL
As previously, to calculate the required equilibration time you can multiply the calculated void volume (in mL) by the number of required column volumes, e.g. 10, then divide by the flow rate (in mL/min) to determine the total time required.
However, we can see that it is multiplied by the flow rate and then divided by the flow rate. The purpose of this was to show the principle of the calculation and compare it to method 1.
In fact, the required equilibration time is just t0Â multiplied by the number of column volumes required. In this example, the equilibration time is simply 1.05 x 10 = 10.5 minutes.
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