The main influential factors in solid phase extraction
Solid phase extraction is a process where a target compound adsorbs onto a solid phase, followed by desorption/elution. Therefore, factors affecting adsorption, desorption, and elution directly impact the efficiency of extraction. These factors include the type of sorbent, strength of the elution solvent, pH, flow rate, and others.
Sorbent Materials
Sorbent materials are the core of solid phase extraction (SPE) technology. Selecting SPE column sorbents with moderate adsorption capacity for the target compound is essential to ensure accurate detection. While it is possible to choose different column sorbents for the same target compound, it is important to adjust the method accordingly.
Strength of Elution Solvent
In solid phase extraction (SPE), the competition between the stationary phase (sorbent) and the mobile phase (sample loading solvent/elution solvent) determines the adsorption of the target compound. Therefore, during sample loading, it is important to choose a sample solvent with appropriate organic solvent content or pH to prevent the target compound from being lost during the loading process. Similarly, during elution, it is crucial to select an elution solvent with the appropriate strength, such as organic solvent content or pH, to ensure complete desorption of the target compound adsorbed onto the sorbent.
pH
For ion exchange stationary phases, the pKa (isoelectric point) of the analytes and interfering substances differ. By adjusting the pH of the solvent, the stationary phase can carry a charge opposite to that of the analyte, while the interfering substances remain uncharged. Alternatively, the stationary phase can carry a charge opposite to that of the interfering substances, while the analyte remains uncharged
Flow Rate
The flow rate during sample loading and elution can affect the adsorption or desorption/elution efficiency. Generally, the flow rate for sample loading and elution is controlled below 1 mL/min. For the trace enrichment of a large volume of samples, such as organic compound enrichment in environmental water samples, the sample loading flow rate should not exceed 5 mL/min. In addition to the mentioned factors, the completion of certain operational steps, such as the degree of activation and the drying of rinsing steps, can also impact the recovery or reproducibility of the results.
Q&A on Solid Phase Extraction Process
Q1. Why is flow rate emphasized in SPE column operations?
Answer: Most researchers desire both speed and quality in their experiments. In solid phase extraction (SPE), there is often a preference for faster flow rates through the column. However, it is important to be cautious of the drawbacks of excessively high flow rates. Once the sorbent material in the SPE column is tightly packed, forming a stationary bed, rapid solvent flow can lead to two issues. Firstly, for bonded phase sorbents, high flow rates may prevent sufficient wetting and expansion, resulting in reduced retention activity. Secondly, excessive flow rates can cause channeling within the column, where the sorbent is not adequately wetted, diminishing the contact area between the sorbent and the sample. This, in turn, affects the mass transfer process of the target analytes, leading to reduced recovery rates.
2. How does the pH of the solvent environment affect compound retention on a small column? Why do we need to consider the pKa value of compounds? Should we consider these factors in a C18 column?
Answer: First, let’s discuss Ka, which refers to acidity coefficient or acid dissociation constant. It represents the ability of an acid to dissociate H+ ions. The pKa value is defined as -log(Ka). For an acid, the following equilibrium exists under the environmental pH:
The left side of the equation represents the proportion of the ionized form (A-) and the molecular form (HA) in the solvent environment. The right side reflects the conversion between the two forms, which depends on the numerical value of (pH – pKa). In ion exchange columns, maintaining the target compound in an ionized state rather than the molecular state is crucial. This directly affects the success of the ion exchange mechanism in the column. In the case of silica-bonded C18 columns, due to the incomplete “end-capping” of silica (-SiOH), at a pH greater than 4.0, the silica will carry a negative charge. This leads to enhanced secondary interactions (primary being non-polar interactions) between ionizable compounds and the silica. Consequently, the retention of such compounds on the silica-C18 is increased. If only non-polar interactions of C18 are considered, it would result in suboptimal recovery
3. Why is GCB commonly used in purification mode rather than enrichment mode in pesticide residue analysis?
Answer: The purification mode in solid phase extraction (SPE) is often used to adsorb impurities, while the enrichment mode is used to adsorb target compounds. GCB (Graphitized Carbon Black) is a type of graphite-like carbon composed of six carbon atoms arranged in a hexagonal plane that stacks together. It has a strong affinity for aromatic planar molecules and exhibits selective adsorption for aromatic compounds. It also possesses good adsorption properties for other analytes. The adsorption and desorption performance of the sorbent directly influence its application range. In the case of GCB, it has strong adsorption capacity but slightly poorer reversibility during desorption. Therefore, it requires the use of appropriate solvents with suitable strength to release the adsorbed substances, making solvent selection for analysis more challenging. Due to its excellent adsorption properties for pigments and other compounds, GCB is commonly used to adsorb impurities such as pigments in pesticide residue sample processing, rather than as a choice for retaining target compounds.
4. Why is vacuum drying commonly performed after the washing step?
Answer: Vacuum drying is typically performed after the washing step for the following reasons:
- In reverse phase extraction, which is commonly used in compound pretreatment, the washing solution is often an aqueous phase or a buffered solution containing water. Removing water facilitates subsequent drying and concentration steps.
- In certain applications, after the washing step is completed, a certain volume of elution solvent, such as 1 mL of methanol, is directly used. This combines the elution and volumetric steps into one process. The preceding step inevitably affects the quantitative results of the elution step.
- Solvent miscibility is another consideration. When there is a significant difference in polarity between the washing solvent used in the washing step and the elution solvent, it can lead to emulsification, phase separation, increased resistance during elution, and the risk of dilution of the elution solution. These factors can result in variations in the effectiveness of elution and ultimately lead to experimental failure
5. When using chromatographic columns with silica-bonded stationary phases, it is common knowledge in chromatography that the recommended pH range for silica-bonded fillers is 2-8. For solid-phase extraction (SPE) columns using silica-bonded fillers, is it necessary to strictly adhere to this guideline?
Answer: Firstly, we need to understand that the manufacturer’s recommended pH range of 2-8 is to ensure the normal lifespan of the chromatographic column. When the pH value exceeds the acidic range, hydrolysis and loss of the bonded phase may occur. When the pH exceeds the alkaline range, it accelerates the dissolution of the silica. This can damage the column’s efficiency and retention characteristics. However, we should note that the impact of solvents outside this pH tolerance range on the chromatographic column is mainly an accumulative process. Over time, solvent flushing gradually dissolves the column, leading to changes in performance and irreversible damage, shortening its lifespan.
One difference between SPE columns and chromatographic columns is that SPE columns are disposable consumables. Therefore, the temporary exposure to solvents outside the pH tolerance range has minimal impact on the retention characteristics of the small column and does not significantly affect retention. As a result, the pH range for SPE columns is generally broader and can be used within a pH range of 1-10 without significant differences in results.
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