Classification and Maintenance of HPLC Column

Basic knowledge of HPLC column

HPLC column is a kind of separation and analysis method, separation is the core, so the chromatographic column responsible for separation is the heart of the chromatographic system. You also can choose the C1 HPLC Column, SCX HPLC Column, C8/C18 Standard HPLC Columns, and other HPLC Columns.

The requirements for the HPLC column are high column efficiency, good selectivity, and fast analysis speed. The particle size of various kinds of silica gel used in HPLC column, as well as the bonding phase, alumina, organic polymer microsphere (including ion exchange resin), porous carbon, etc., which are sold on the market, are generally 3, 5, 7, 10um, and the theoretical value of column efficiency can reach 50000-160000 / m.

For general analysis, only 5000 plates are needed for column efficiency; for homologous analysis, only 500 plates are needed; for more difficult separation materials, up to 20000 columns can be used, so the column length of about 10-30cm can meet the needs of complex mixture analysis.

Column efficiency is affected by internal and external factors. To achieve the best efficiency of the column, in addition to the small dead volume outside the column, a reasonable column structure (to reduce the dead volume outside the packed bed as much as possible) and filling technology are also required. Even if the best filling technology is used, the filling condition in the center of the column and along the pipe wall is always different.

The position close to the pipe wall is relatively loose, which makes it easy to produce ditch flow, a fast flow rate, affect the flow pattern of the flushing agent, and widen the spectral band, which is the pipe wall effect. The thickness of the tube wall area is about 30 times the material diameter from the tube wall inward. In a general liquid chromatography system, the effect of the external effect on the column effect is much greater than that of the tube wall effect.

High Purity HPLC Columns

1. HPLC column structure

The chromatographic column is composed of a column tube, pressure cap, ferrule (sealing ring), sieve plate (filter), joint, screw, etc. The column pipe is mostly made of stainless steel. When the pressure is not higher than 70kg / cm2, thick wall glass or quartz pipe can also be used. The inner wall of the pipe requires a high degree of finish. To improve the column efficiency and reduce the pipe wall effect, the inner wall of the stainless steel column is mostly polished. Some people also apply fluoroplastics on the inner wall of the stainless steel column to improve the smoothness of the inner wall.

2. Classification of HPLC Columns

HPLC columns can be classified based on several criteria, including the type of stationary phase, particle size, and application. Here are common classifications:

  1. Based on the Stationary Phase:
    • Reversed-Phase (RP): Hydrophobic stationary phase; commonly used for separating nonpolar and moderately polar compounds.
    • Normal Phase (NP): Hydrophilic stationary phase; suitable for separating polar compounds.
    • Ion-Exchange: Separation based on ion interactions; includes anion-exchange and cation-exchange columns.
    • Size-Exclusion (SEC): Separates molecules based on size; larger molecules elute faster.
  2. Based on Particle Size:
    • Fully Porous: Contain porous particles throughout the column.
    • Partially Porous or Pellicular: Have a solid core with a porous outer layer; often used with smaller particles for increased efficiency.
  3. Based on Application:
    • Pharmaceutical Columns: Designed for pharmaceutical analysis, drug discovery, and quality control.
    • Bioanalytical Columns: Used for analyzing biomolecules such as proteins, peptides, and nucleic acids.
    • Environmental Columns: Suitable for environmental analysis of pollutants and contaminants.
    • Chiral Columns: Designed for enantiomeric separations.
  4. Based on Particle Shape:
    • Spherical: Most common; provides high efficiency.
    • Irregular: Less common; may be used for specific applications.

3. Maintenance of HPLC Columns:

Proper maintenance is crucial for the optimal performance and longevity of HPLC columns. Here are key maintenance practices:

  1. Initial Conditioning:
    • Solvent Conditioning: Condition the column with the mobile phase to remove air bubbles and ensure proper wetting of the stationary phase.
  2. Mobile Phase Compatibility:
    • Choose Compatible Solvents: Select mobile phase solvents that are compatible with the stationary phase material to prevent damage.
  3. Column Installation:
    • Proper Installation: Install the column correctly, ensuring proper connections and minimal dead volume.
  4. Use of Guard Columns:
    • Guard Columns: Use guard columns to protect the analytical column from particulate matter and contaminants.
  5. Avoiding Extreme pH Conditions:
    • pH Range: Stay within the recommended pH range for the column material to prevent degradation.
  6. Column Cleaning:
    • Routine Cleaning: Clean the column regularly using appropriate procedures and solvents to remove contaminants.
  7. Avoiding Strong Solvents:
    • Avoid Harsh Solvents: Avoid using solvents that can damage the column material; refer to the manufacturer’s guidelines.
  8. Avoiding Extreme Temperatures:
    • Temperature Control: Keep the column at a stable temperature, and avoid extreme temperature fluctuations.
  9. Proper Storage:
    • Storage Conditions: Store columns in a cool, dry place, and follow the manufacturer’s recommendations.
  10. Regular Checks:
    • Pressure Checks: Monitor backpressure regularly to detect changes in column performance.
  11. Optimizing Mobile Phase Flow Rates:
    • Flow Rate: Operate the column within the recommended flow rate to prevent column damage and maintain efficiency.
  12. Column Regeneration:
    • Regeneration: If applicable, follow recommended regeneration procedures to extend column life.
  13. Document Usage:
    • Record Keeping: Keep records of column usage, including the number of injections, to schedule replacements proactively.
  14. Verify Column Integrity:
    • Performance Checks: Periodically check column performance using test mixtures or standard compounds.
  15. Retirement Criteria:
    • Replace Timely: Retire columns based on recommended usage limits or signs of performance decline.

4. The effect is the same as that of polishing.

The column joints at both ends of the chromatographic column are equipped with sieve plates, which are sintered stainless steel or titanium alloy with a pore diameter of 0.2-20um (5-10um), depending on the particle size of the packing. The purpose is to prevent the packing from leaking out. The inner diameter of the column is generally determined according to the length of the column, the particle size of the packing, and the reduced flow rate, so as to avoid the pipe wall effect.

5. The development direction of the HPLC column

Due to the emphasis on the analysis speed, the short column has been developed. The length of the column is 3-10cm, and the particle size of the filler is 2-3um. To improve the analytical sensitivity and connect with MS, a narrow-diameter column, capillary column, and micro-diameter column with an inner diameter of less than 0.2mm were developed.

The advantages of small-diameter columns are ① saving mobile phase; ② increasing sensitivity; ③ less sample; ④ using the long column to achieve high resolution; ⑤ easy to control column temperature; ⑥ easy to realize LC-MS joint use.

However, as the volume of the HPLC column becomes smaller and smaller, the effect of of column effect becomes more significant. It needs a smaller cell volume detector (even on-column detection), smaller dead volume column joints, and connecting parts. The matching equipment shall have the following performance: the infusion pump can accurately output a low flow rate of 1-100ul / min, and the injection valve can accurately and repeatedly inject small-volume samples.

Because of the small amount of sample and the requirement of a high sensitivity detector, electrochemical detection assist mass spectrometer has outstanding advantages in this respect. The performance of the HPLC column is not only related to the performance of the stationary phase but also related to the filling technology. Under normal conditions, when the particle size of the filler is more than 20um, the dry method is more suitable for the preparation of the column; when the particle size is less than 20um, the wet method is ideal.

Generally, there are four filling methods: ① high-pressure homogenization method, which is mostly used for the filling of analytical columns and small-scale preparation columns; ② radial compression method, waters patent; ③ axial compression method, which is mainly used for filling large-diameter columns; ④ dry method, which is highly technical, most laboratories use filled commercial columns.

It must be pointed out that the filling technology is an important link in obtaining HPLC columns, but the fundamental problem lies in the performance of the packing itself and the reasonable structure of the matching chromatograph system.