How to Choose a Liquid Chromatography Guard Column
During high-performance liquid chromatography analysis, the column is contaminated by samples and chromatographic systems, which result in poor column durability and reduced lifetime.
Contamination from the chromatographic system mainly refers to solid particles produced by the wear of components in the HPLC instrument system, as well as solid particles that are not completely retained by the mobile phase system. Contamination from the sample mainly means that the sample that is not completely dissolved or the sample that has been completely dissolved enters the chromatographic system, and the precipitated contamination system is precipitated due to the difference in solubility of the sample solvent and the mobile phase solvent in the dissolution of the sample.
Especially for the detection of traditional Chinese medicines, natural products, synthetic intermediates and biological drugs, the contamination of the column will be more serious due to the complexity of the matrix. Contamination can lead to increased pressure in the chromatographic system, collapse of the column, discoloration of the filler, etc., resulting in various effects such as decreasing detection costs and inaccurate test results.
The guard column is an important chromatographic consumable in liquid chromatography. The use of a guard column can greatly reduce the contamination of the column and prolong the life of the column.
The guard column is located between the injector and the analytical column and functions in two main ways:
1. Intercepting insoluble particles
2. Adsorption of impurities in the sample matrix
The guard column is recommended for the following situations:
1. Analysis of samples with complex matrices such as traditional Chinese medicines, natural products and synthetic intermediates – the precipitation of the sample due to the difference in solubility and the contamination of the analytical column is more serious, and the influence of the gradient elution is greater.
2. Biological sample analysis – Due to contamination caused by irreversible adsorption of the sample, the guard column can withstand the contamination prior to the analytical column, thereby extending the life of the analytical column.
Guard columns are important, but choosing the right guard column is more important, and improper selection or use can have the opposite effect.
Protection column classification and selection:
The protection column consists of stainless steel and PEEK (polyetheretherketone) in term of material.
Advantages and disadvantages of PEEK column – it is more suitable to choose PEEK column when the sample is sensitive to metal ions. For example, considering this factor choose PEEK analysis column in some protein analysis.
Advantages and Disadvantages of Stainless Steel column – stainless steel protection columns are commonly used in most of the analysis of small molecular compounds. Especially when the mobile phase system uses some solvents with strong solubility such as tetrahydrofuran (THF) or DMSO, stainless steel must be selected, even the piping for the HPLC system needs to be replaced with stainless steel because THF has a strong corrosive effect on PEEK piping.
The protection column consists of card sleeve (column + column sleeve) and directly filled short column in term of structure.
The advantages and disadvantages of directly filling the short column – the protective column of this structure is essentially a short column, generally the filled packing is exactly the same as the analytical column packing. These type of guard columns are used mostly in biological sample analysis. In addition to filtering some insoluble particles, some impurities that are easily adsorbed on the analytical column can be removed. The disadvantage is that disposable and discards when the pollution is serious.
Advantages and disadvantages of the ferrule guard column – the sleeve of the ferrule guard column can be used repeatedly, the column core can be replaced after contamination. The manufacturer provides a matching protection column core, but need to pay attention when purchasing the core, the column core has the fillers which should be matching the analysis column. At the same time, the core and the column sleeve are supposed to be in the same specification and from the same manufacturer. Otherwise, there will be a difference in matching, resulting in abnormal peak shape or leakage.
The protection column consists of independent and direct connection in term of connection method.
Advantages and disadvantages of the independent protection column
The guard column and the analytical column is connected by a pipe, so no worry about the matching problem with the analytical column. However, when using a pipe connection, the improper connection is likely to result in a dead volume.
Advantages and Disadvantages of Direct-Linked Protection Columns – The guard column interface is threaded and carly connected to the analytical column. The dead volume is small. It is recommended to select the direct-connected guard column for UPLC analysis. Disadvantages: Ensure that the interface matches the analytical column when purchasing. Consider factors for guard column selection: 1. Unless specified, select the guard column with the same packing matrix as the analytical column matrix. 2. Calculate the size of the guard column according to the column volume. The volume of the guard column should be 5%~10% of the volume of the analytical column. For example, the volume of the analytical column of 4.6*250mm is about 4mL, and the volume from 0.2~0.4mL of guard column should be selected. At the same time, the inner diameter of the guard column should be the same or equivalent to the inner diameter of the analytical column. Precautions for use of guard column: 1. Use the connecting pipe as short as possible when the independent guard column is connected to the analytical column, and make the interface cut surface smooth, and minimize the dead volume to reduce the extra-column widening effect. 2. The ferrule guard column is generally not marked with the flow direction. It is best to mark it yourself and use it according to the direction of the mark to avoid contamination of the analytical column after the reversed use. 3. When the column pressure rises or the peak shape deteriorates, the directly filled short protection column could be reverse cleaning without connecting the analytic column. Choose cleaning solvent refers to the analytical column cleaning solvent. 4. For casing type guard column, pay much attention to whether there is a matching between the core and the sleeve, and do not screw too tight to prevent the wire from slipping. 5. Ultrasonic cleaning is not recommended for guard columns or core as it may have an effect on the surface modification of the filler. 6. If a guard column is used in multiple analytical columns, the guard column needs to be cleaned before use and the column is protected by mobile phase for a period of time before being used in the analytical column. The use of guard column and its’ difference with the pre-column filter: The purpose of using the guard column is to extend the service life of the analytical column. When the analytical column is expensive, the value of the guard column is especially reflected. In some cases, when the analytical column itself is cheap, the guard column is not required. Pre-column filters are more suitable. The pre-column filter and the guard column have similar appearances, the difference is, the inner core of the protection column is filled with packing, and the pre-column filter inner core is a replaceable filter (generally a perforated stainless steel sintered plate) Therefore, the difference in usage can be inferred from the structure: Pre-column filter – strong versatility, can be used before any analytical column, because its filter element only plays the role of filtering and retaining impurities. After the filter is contaminated, it can be ultrasonically cleaned and used repeatedly. Guard column – In addition to the role of trapping impurities, the column packing can also remove easily adsorbed impurities in the sample, which is especially important for biological sample analysis.