FAQ Analysis and Solutions About HPLC Columns

HPLC analysis, which is supposed to have broader potential applications, is not limited by environment temperature and the boiling point of samples. After more than 30-year rapid development, the theory of HPLC has been more and more mature in the basic theory, the instruments, the chromatography columns, etc. And now HPLC has become one of the most advantageous separation and analysis methods in many fields, such as the chemical industry, environment, pharmacy, food, and so on.

According to the different separation mechanisms, liquid chromatography can be divided into five kinds: liquid-solid adsorption chromatography, liquid-liquid distribution chromatography, ion-exchange chromatography, ion-pair chromatography, and molecular exclusion chromatography, which can be also called gel permeation chromatography.

For instance, in the process of liquid-solid adsorption chromatography, the mobile phase is liquid and the stationary phase is solid adsorbent. The substances are separated according to the different adsorption effects. And when doing liquid-liquid chromatography, the mobile phase and stationary phase are both liquid chromatography, which uses the distribution of sample components between two immiscible liquid phases for separation.

FAQ analysis and solutions about HPLC columns

1. The column pressure is too high, after a period of use.
Solution: First check whether the HPLC system is the cause. After eliminating the system cause, the reason is basically due to the accumulation of impurities in the column during the experiment. Try to improve the operating conditions. After the sample is finished, it should be rinsed off in time. For example, the mobile phase of the buffer salt must be washed with a high proportion of aqueous solution (such as 90%) and then stored with the organic phase.

2. Sieve plate blockage and column head collapse.
Solution: If it is determined that the filter screen of the column head is contaminated, the column can be flushed to normal pressure in the opposite direction with methanol, or the column head should be removed, the sieve plate should be carefully removed, and the solution should be sonicated for about 20 minutes with a 5% or so nitric acid solution. Then, it is sonicated with pure water for about 20 minutes and reloaded into the column.

3. The packing of the column head is contaminated by the sample.
Solution: If it is determined that the packing of the column head is contaminated, remove the stud screws, dig out the contaminated packing in the front part of the column, and refill with the same column packing. And after careful repair, reinstall the stud screws.

4. The salt in the buffer in the column encounters a high concentration of methanol or other organic solvent to form crystals and precipitate.
Solution: If it is determined to be salt crystals, rinse the column with 10% methanol/water to dissolve all the salt in the column, and then change the high concentration of methanol.

5. What is an HPLC column, and how does it work?

An HPLC column is a critical component of High-Performance Liquid Chromatography (HPLC) systems. It consists of a cylindrical tube packed with stationary phase particles. During analysis, the sample mixture is injected into the column, and the mobile phase (liquid solvent) carries the sample components through the stationary phase. As the sample interacts with the stationary phase, its components are separated based on their various physicochemical properties, leading to distinct peaks in the chromatogram.

6. What are the different types of HPLC columns available?

There are several types of HPLC columns, including:

• Reverse-phase columns: For hydrophobic compounds. Such as Reversed Phase C4 HPLC Column.
• Normal-phase columns: For polar compounds. Such as CN HPLC Column, Polar Columns in HPLC, C18 Alkaline HPLC Column.
• Ion-exchange columns: For charged compounds.
• Size-exclusion columns: For separating molecules based on size.
• Chiral columns: For enantiomeric separations.
• HILIC columns: For polar compounds using a hydrophilic stationary phase.

7. What factors can affect HPLC column performance?

Several factors can impact column performance, including:

• Mobile phase composition and flow rate.
• Column temperature.
• Sample preparation and injection volume.
• Column age and condition.
• Sample matrix and pH.
• Column particle size and pore size.
• Solvent compatibility with the column material.

8. How do I choose the right HPLC column for my application?

Selecting the right column depends on your sample’s nature and the required separation. Consider factors like compound polarity, molecular weight, analyte stability, and sample complexity. Consult the column manufacturer’s technical guides, applications support, and chromatographic method development resources.

9. How often should I replace my HPLC column?

The column’s lifespan depends on various factors, including the type of column, the frequency of use, and the sample matrix. Regular column maintenance and proper usage can extend its life. As a general guideline, consider replacing the column after 150 to 300 injections or when you notice a decrease in resolution and peak shape.

10. How do I store and maintain my HPLC column properly?

• Store columns in a clean, dry, and cool environment to prevent degradation.
• Avoid exposure to direct sunlight and extreme temperatures.
• Keep the column in an appropriate solvent or mobile phase when not in use to prevent the phase from drying out.
• Flush the column with an appropriate solvent after each use.
• Regularly perform column conditioning and equilibration before analysis.

11. Why am I experiencing poor peak shapes or resolution in my chromatograms?

Several factors can contribute to poor peak shapes and resolution, such as:

• Incorrect mobile phase composition or flow rate.
• Sample overloading.
• Column contamination or degradation.
• Air bubbles in the mobile phase.
• Incorrect column equilibration.
• Incompatible sample solvent.

Carefully review your chromatographic conditions and optimize them accordingly.

12. What can I do if my HPLC column becomes blocked or clogged?

• Try flushing the column with an appropriate solvent to remove debris.
• Use HPLC guard columns to protect the main column.
• If the blockage persists, consider using a suitable column regeneration kit or contact the manufacturer for guidance.

Notes of HPLC column

1. Whenever possible, the column bed must be kept dry, especially during the injection test and column flushing process, the mobile phase should not be left empty for more than 30 minutes. Otherwise, the column will dry up and can be discharged into a large amount of non-ejectable bubbles which even cause partial collapse or central cracking of the bed.
2. For use and storage, it is strictly forbidden to use force to absolutely prevent accidental violent impact or falling at high places. Otherwise, it will easily cause mechanical damage to the column bed, then there will be no regeneration.
3. When the column and the chromatograph are coupled, the valve or pipe must be cleaned.