Starting up a HYDROCELL column:

Whether from storage or fresh from the manufacturer, the baseline can drift when you first use a Hydrocell Ion Exchange or Hydrophobic Interaction Column. The column needs to be activated. The procedure of activating a 4.6 mm Hydrocell column is as follows:

1. Flush with 100% water to remove any salts from your HPLC system, then 80% methanol in water at a flow rate of 3 mL / min. for 10 minutes. 2. Flush the HPLC system with 100% water, and then flush the column with 20% methanol in 1 M sodium chloride solution at flow rate of 3 mL / min for 10 minutes. 3. Flush with your B Buffer of choice at flow rate of 3 mL / min for 10 minutes. 4. Equilibrate with your starting buffer of choice at a flow rate of 2 mL / min. 5. Run a 10 minute blank gradient from 0 -100 % B buffer at flow rate of 2 mL / min.

Hydrocell Column Cleaning Protocol

If the peaks have broadened or the baseline drifts when you use a Hydrocell Ion Exchange or Hydrophobic Interaction Column, the column needs to be reactivated and regenerated. The procedure of reactivating and regenerating a 4.6 mm Hydrocell ion exchange column is as follows:

1. Flush with 80% methanol in water at flow rate of 3 mL / min. for 10 minutes. 2. Flush with water, and then 20% methanol in 1 M sodium chloride solution at flow rate of 3 mL / min for 10 minutes. 3. Flush with B Buffer at flow rate of 3 mL / min for 10 minutes. 4. Equilibrate with the starting buffer at flow rate of 2 mL / min. 5. Run a 10 minute blank gradient from 0 -100 % B buffer at flow rate of 2 mL / min.

Repeat this process, if necessary, until the baseline has stabilized. Adjust the flow rates and volumes for other I.D. columns proportionately.
 

2. An alternative procedure: Elute column with 0.5 M NaOH aqueous solution continuously, at a flow rate of 1 mL / min for 1 hour or leave 0.5 M NaOH inside the column overnight. Flush thoroughly with HCl or HOAc, 1 M, to remove all traces of NaOH from the tubing.

Hydrocell columns with a proprietary hydrophilic oligomeric coating are very hydrophilic and do not have non-specific adsorption and binding. These columns do not need detergents, surfactants or organic solvents in the buffers to circumvent non-specific binding. If you have the problem of retention of your samples on Hydrocell columns, please check the preparations of your samples, buffers, and the equilibrium time with the sample buffer or buffer A first before injection of your samples.

(I) Sample Preparation:
 

1. Solid samples: The samples are prepared by dissolving about 2 mg of sample in 1 ml of the first buffer. If necessary, likewise in the hydrophobic interactive chromatography, a small volume of the secondary buffer can be added to increase the solubility of the samples.

2. Liquid samples: Detergents, surfactants, and solvents that will affect the retention of the samples in the separation should be removed.  Adjust the pH value and salt concentration as close as possible to the first buffer. The concentration of samples should be high enough to be detected by the detector.

3. Preparative samples: In the preparative separation, the size of the sample loop should be increased to increase the loading capacity.  Using a dilute solution repeatedly load the sample instead of using a high concentration of sample.

(II) Buffer Preparation:

Salt concentration and pH values are two factors that greatly affect the separation of biomolecules. A slight change of pH value of your buffer might significantly affect the retention of biomolecules on the column. The pH value of buffers needs to be adjusted to fit the specific separation during methods development.  Variation in retention time might be a result of inconsistent buffer preparation.
 

Hydrocell columns are very hydrophilic and have significant capacity. If the equilibrium time with the first buffer is not long enough, the samples might not have any retention or the retention time might be too short. The equilibrium volume must be at least 10 times the column volume. For example, for a column dimension of 150 x 4.6 mm, there is a column volume of 2.45 ml. The equilibrium volume of the column needs to be at least 24.5 ml.

If the column is equilibrated at 1ml/min, it takes 24.5 min to equilibrate the column. However, Hydrocell columns are produced from rigid polymeric supports and have high mechanic strength. These columns can be equilibrated at high flow rates. The maximum flow rate for a 150 x 4.6 mm analytical column is 6 ml/min. Hence, if the column is equilibrated at 3 ml/min, it will take only 8.16 minutes to equilibrate the column.

C. High Back Pressure:

When Hydrocell columns show back pressures higher than 1000 psi at a normal flow rate, the columns are clogged. The columns need to be cleaned using the column cleaning protocol or eluting with 0.5M NaOH aqueous solution. Flush thoroughly with HCl or HOAc, 1 M, to remove all traces of NaOH from the tubing.

After column cleaning, if the problem still exists, then the inlet frit of column needs to be replaced. It is always recommended to use a guard column or precolumn filter to protect agaist clogging