In the pharmaceutical industry, related substance test analysis is crucial, especially when the HPLC method is a gradient approach. In gradient method analysis, getting a smooth baseline with no extra peaks is usually a difficulty.
In such instances, a Guard column can be employed to provide a smooth baseline and exclude any extra peaks caused by impurities in the solvent used to prepare the mobile phase and diluents.
"If the method has already been verified, method Equivalency data is necessary before normal usage of the Guard column."
What is a guard column?
A guard column is a column or cartridge that is positioned between the injector and the analytical column to protect it. It prevents contaminants and suspended particles from entering the analytical column. It is typically 2 cm long with an interior diameter of 4.6 mm. To provide a small pressure loss, guard columns are filled with pelicullar particles of roughly 40 m size.
The efficient operation of an HPLC instrument is based on the absence of chemical contaminants or solid suspensions in the mobile phase and sample. Precaution and handling of mobile phase use addresses the precautions that should be taken during the preparation and use of the mobile phase. The importance of cleansed sample injection (Centrifuge or filter with syringe filter) is always beneficial.
HPLC column is a critical component of the HPLC system which requires careful handling and protection. It is expensive to keep replacing columns frequently so your objective should be to maximize the useful life of the column so that every time you get the desired accuracy and consistency of results.
The chromatographic performance of the HPLC column degrades with time due to the progressive buildup of contaminants and suspensions. Particles greater than 2μm in size present in the mobile phase or sample begin to settle on the column's input frit, disrupting flow homogeneity. Smaller particles generate more backpressure, obstructing the flow route in the stationary phase.
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Nature of Contaminants
- In reverse phase separations, highly retained substances such as fatty acids. Irreversibly retained substances, such as residual proteins, that were not entirely eliminated after sample extraction.
- Particulate impurities can be caused by non-filtration of samples or by the wear of system components such as seals in the pump or injector.
- Crystalline deposits formed as a result of the drying of residual buffers inside the column.Â
- After usage, washing columns with HPLC grade water or buffer solutions inhibit the development of salt deposits.
Desirable Features of Guard Columns
- To avoid separation issues, the Guard column should ideally have the same packing as the analytical column.
- To reduce back pressure, the internal ID of the guard column should be comparable to that of the analytical column. It is better to have a shorter guard column length, but it should be long enough to prevent highly held compounds from reaching the main column.
- The Frit facing the injector should be detachable for cleaning, with roughly 2 mm of material removed and replaced with fresh material.
- When compared to refilling guard columns, disposable cartridge guard columns are more convenient and cost-effective.
Guard columns need to be changed on regular basis but intermediate change becomes necessary through observation of changes in chromatographic behavior such as an increase in backpressure, peak broadening, and changes in the retention time of peaks. However, the frequency of change can be decided on the basis of the chemical composition of the sample, the presence of highly retained or irreversibly retained components, injection volume, or the number of injections.
What is the difference between HPLC Guard Columns and Guard Cartridges?
HPLC guard columns and cartridges safeguard analytical, semi-preparative, and preparative HPLC columns. They contribute to column life by eliminating particle pollutants and highly absorptive chemicals from samples.
Guard columns should ideally have the same stationary phase as the analytical column. To avoid back-pressure, guard column internal IDs should be comparable to those of the main column. Smaller is usually preferable, but they must be large enough to successfully prevent highly held chemicals from negatively impacting the major column. Cartridge-type guard columns are also a simple and low-cost alternative to refillable guard columns.
Guard Column Hardware Design vs. Guard Cartridge Hardware Design
Guard Column | Guard Cartridge |
Guard columns are exactly what they sound like: tiny columns. It is similar to an HPLC column, except it is much shorter and loaded with larger resin particles. The bigger particle size of the resin captures particles in the sample and mobile phase without interfering with chromatography or causing significant back pressure in the system. The guard column's casing is composed of 316 stainless steel. | This design is modular for both stainless steel and PEEK housings, and cartridge replacement is straightforward and tool-free. The cartridge-type kit comes with a holder and replaceable cartridges, with additional replacement cartridges supplied separately. The cartridge holder has been modified to reduce dead volume and avoid further column band widening. Cartridge holders are reusable. |
Volume: Analytical: 114 µL Prep: 548 µL | Volume: Analytical (PEEK): 57 µL Analytical (ss): 80 µL Prep (ss): 321 µL |
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