How to Clean HPLC Columns Properly?

High-Performance Liquid Chromatography Columns

There are three basic types of liquid chromatography columns: liquid-liquid, liquid-solid, and ion exchange. Liquid-liquid columns are less popular because of their limited stability and inconvenience. In liquid-solid columns, the stationary phase is solid, and the analytes are absorbed into the stationary phase, thus analyzing the composition of the mixture. Typically, HPLC is preceded by a guard column to protect and extend the life of the analytical column. The function of the protective column is to remove irreversible particles, contaminants, molecules, etc. from the column. You also can choose C18 Alkaline HPLC Columns, Reversed Phase C4 HPLC Column, Phenyl-Ether HPLC Columns, C18 Standard HPLC Columns, and other HPLC-Columns using.

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Commonly used HPLC columns are made of stainless steel, but can also be made of thick glass, polymers (e.g. polyether ketone), a combination of stainless steel and glass, or a combination of stainless steel and polymers. There are many advantages to using stainless steel, for example, stainless steel is more resistant to corrosion, high pressure, and wear and tear than glass, which also reduces the cost of use. Typical HPLC columns are 3 to 25 cm long and 1 to 5 mm in diameter, unlike GC columns, which are usually straight. The typical diameter of the particles that fill the column is between 3 and 5 µm. The efficiency of a liquid chromatography column will increase when the diameter of the particles that accumulate inside the column is reduced.

Peaks or peak broadening can lead to a reduction in column efficiency. The ideal situation will be that sharp peaks are resolved. The longer the substance stays in the column, the wider the peak will be. HAWACH columns have high flow rates, low back pressure, cumulative column efficiency, high column efficiency, good separation, short equilibration times, short analysis times, and solvent savings. HAWACH columns are available in a variety of tube sizes such as 4.6*150, 4.6*200, 4.6*250, 4.6*350, 6.0*150, 6.0*250, 6.5*150, 6.5*250, etc. particle size: 3μm, 5μm are available.

Cleaning HPLC columns

We all know that HPLC columns have a finite life cycle and even after good cleaning and storage, they will reach the end of their life cycle. However, there are certain measures you can take to maximize the life of your columns. Once you are ready to use your HPLC column, you can help maintain its integrity by installing in-line filters, trap columns, and a protective column. These will help prevent contaminants from damaging your HPLC column.

After using the column, it is always recommended to clean the column prior to storage. The cleaning procedure usually involves an isocratic/gradient wash using the closest solvent system to the last solvent on the column but using HPLC-grade water instead of buffer. This will ensure that the buffer component is removed from the column. You can then increase the percentage of organics in the wash to further remove any hydrophobic impurities.

The reversed-phase HPLC columns may be repeatedly injected into a sample containing a strongly retained material, and in particular, the sorbent components such as large molecular weight or hydrophobic biological flow proteins and strong alkaline substances may be adsorbed on the silica medium.

In addition, some mobile phase additives such as ion-pairing test reagents and reactants can adsorb on the filling surface and change their properties. Contaminated columns can cause peak shape differences, poor retention of repeatability, high back pressure, and baseline drift. Typically, these columns can be cleaned using organic solvents or agents capable of destroying contaminants and binder phases or silica surfaces.

The users should be careful when using reagents that are corrosive or destructive to the binding phase. Additionally, the use of sample preparation minimizes contact between the HPLC columns and undesired materials to reduce contamination. We suggest using the HPLC protect column as pre-filter. Use Pre-filter, HPLC protect column, can reduce pollution from contamination and possibly store the analytical HPLC columns.

Backwashing the HPLC column

If you notice a deterioration in peak shape accompanied by an increase in backpressure, it is recommended that the column is backflushed. Before backflushing, it is important to ensure that your mobile phase or in-column solvent is miscible with your cleaning solvent and that your flow rate does not exceed half the typical recommended flow rate for the column. For detailed procedures, follow our instructional column maintenance guide. You can determine the column volume using the following formula: V = pr2L where V is the column volume in ml, r is the column radius in cm and L is the column length in cm.

To properly clean a reversed phase HPLC column after use, first, replace your mobile phase with 95% HPLC grade water and 5% acetonitrile and then rinse through the HPLC column at approximately 10 times the column volume at half the flow rate. This will remove any buffer left in the HPLC column. You can then gradually move as necessary to increase the organic content to approximately 95% acetonitrile and 5% water, and after backwashing, connect the column forward and to the regular mobile phase before use. Please note that when backwashing the column, disconnect the column from the detector. Learn how to properly backwash your HPLC column using these tips.

HPLC column storage

It is also important to store your columns properly after use, especially for long periods of time. It is important not to store your HPLC column while it still contains any buffers or ion-pair reagents. To ensure this, flush five column volumes s without buffers of the mobile phase through the HPLC column. For columns with ion-pairing reagents, it may be necessary to extend the wash time to completely remove them from the column. After washing, place the column backward in acetonitrile/water (65:35 v/v), using methanol instead of acetonitrile, 100% hexane or IPA for normal phase columns, 100% methanol for ion exchange columns, and acetonitrile/water (80:20 v/v) for HILIC columns.