Monday, March 29, 2021

HPLC column care and maintenance

The column is such an essential part for the separation in high-performance liquid chromatography, hence the proper care and maintenance of the column are needed for better performance and longer life. Every chromatographer knows the importance of columns in HPLC analysis, since the resolution, theoretical plate number, peak symmetry, tailing factor, and system suitability depend on column performance.

Column conditioning:

Every reversed-phase HPLC column should be conditioned for the first time use, following long-term storage, and when the significant changes in the mobile phase. The mobile phase utilized in the conditioning ought to be similar to those used in the consequent chromatography.
The general procedure of column conditioning is as follows:
1. Wash the column with about 3 column volumes of aqueous mobile Phase with a low flow rate.
2. 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.
3. Equilibrate the column on a minimum of 5 column volumes with the organic mobile phase, until the monitor signals get stable.
4. If you change the mobile phase system, a blank run should be done to verify any impurity that may emerge in the mobile phase.


Table of contents

 

Column conditioning

Column cleaning

Column storage

Regeneration of the reversed-phase column

Important points

Column cleaning:

Regularly cleaning the HPLC column is essential, particularly when dealing with samples that have any column foul contaminants and particulate matter. Increased back pressures, fluctuating baseline, and loss of resolution are all signs that the column may require to be cleaned. In silica-based reversed-phase media, the peak has loss resolution because of peak branding and it may also be caused by dissolution of the matrix during regular use of mobile phase or buffer of above pH 7.
A typical procedure for cleaning an HPLC column is as follows.

  • All the solvent using in the analysis should be HPLC grade and filtered through the 0.45-micron nylon filter membrane and degassing is also necessary.
  • Equilibrate the column at a low flow rate with several column volumes of mobile phase containing HPLC grade water.
  • 100% - 20 column volumes of water
  • 50:50% - 20 column volumes of water: acetonitrile or methanol
  • 100% - 20 column volumes of acetonitrile or methanol.The buffer solutions are very harmful to the performance and lifespan of the column, hence wash the column with an appropriate volume of water, so that salt does not get accumulated in the column and system.

Column storage:

Reverse phase columns are based on silica gel so they should not be stored in an aqueous solution. This is because the silica has inherent instability under aqueous conditions. Silica-based reversed-phase columns are generally stored in pure organic solvents, for example, methanol, which should be free from additives or TFA.

Regeneration of the reversed-phase column:

The RP-HPLC columns include C8, C18, CN C1, C30, C4, and phenyl stationary phases. To wash and generate the column, flush it with the following solvents.

  • 20 column volumes of 95:5% of water: acetonitrile (v/v)
  • 20 column volumes of 100 % of acetonitrile
  • 5 column volumes 100 % of isopropanol
  • 20 column volumes 100 % of hexane
  • 5 column volumes 100 % of isopropanol
  • 20 column volumes 100 % of acetonitrile
  • 20 column volumes of water: acetonitrile (95:5 v/v)
After washing with this sequence of solvents, the column needs to re-equilibrate with your mobile phase composition.

Important points:

Since almost 90% of the separation is working using reverse phase chromatography (RP-HPLC), some important points to consider in protecting and improving the life and performance of HPLC columns are listed below.
  • Properly install the column when you change or replace it.
  • Confirm the efficiency of the new column supplied by the vendor.
  • The column should be protected from knocks, bumps, and mechanical shock.
  • To protect against impurities and other foreign particulates, always use a guard column.
  • Always use HPLC grade solvents (methanol, Acetonitrile, and water, etc.) for analysis.
  • To exclude impurities and foreign particles, filter out the mobile phase using 0 .45 μ and before injecting the sample filter the sample using a 0 .20 μ membrane filter.
  • Degas all mobile phases and samples before use for the analysis.
  • Always use freshly prepared buffer solution (phosphate, acetate, and formate, etc.) for analysis.
  • Before injecting the sample, saturate the column with the mobile phase.
  • Do not overload the HPLC column resulting in broad peaks of analytes.
  • If a column oven is available then maintain column temperature, and also maintain the room temperature.
  • Always maintain the pH of the buffer/mobile phase in the range of 2.0 to 9.5 pH.
  • Avoid using a viscous buffer. Phosphate buffer concentrations should be less than 20 mM.
  • Avoid injecting the highly acidic and basic sample into the sample loop, always neutralize the sample.
  • Do not inject biological samples directly into the HPLC injector.
  • Avoid any sudden pressure fluctuation during the analysis.
  • Do not work with high flow rates, it creates pressure
  • Periodically check the performance of the column with the standards and do not operate HPLC system at back pressure greater than 4000 psi.
  • After each use, thoroughly clean the column with HPLC grade water and a suitable solvent.
  • Avoid storing the column with buffers, store it with the necessary solvents (methanol/acetonitrile), and do not allow it to dry. 

 

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