Solid phase extraction (SPE) is the process of using solid adsorbents to adsorb target compounds from liquid samples, separating them from the sample matrix and interfering compounds, and then using eluent or heating to desorb, in order to achieve the separation and enrichment of target compounds.

Compared with liquid-liquid extraction, solid-phase extraction has many advantages: solid-phase extraction does not require a large amount of immiscible solvents, and does not produce emulsification during the processing. It uses highly selective adsorbents (stationary phase), which can significantly reduce the amount of solvent used, simplify the sample processing process, and also reduce the required costs. Generally speaking, the time required for solid-phase extraction is half of liquid-liquid extraction, and the cost is one-fifth of liquid-liquid extraction. Its disadvantage is that the recovery rate and precision of the target compound are lower than those of liquid-liquid extraction.

One The Mode and Principle of Solid Phase Extraction

Solid phase extraction is essentially a type of liquid chromatography separation, and its main separation mode is the same as liquid chromatography. It can be divided into normal phase (adsorbent polarity greater than eluent polarity), reverse phase (adsorbent polarity less than eluent polarity), ion exchange, and adsorption. The adsorbent used in solid-phase extraction is also the same as the commonly used stationary phase in liquid chromatography, except for differences in particle size.

The adsorbents used in solid-phase extraction are polar and are used to extract (retain) polar substances. How the target compound is retained on the adsorbent during normal phase extraction depends on the interactions between the polar functional groups of the target compound and the polar functional groups on the adsorbent surface, including hydrogen bonding, π - π bond interactions, dipole dipole interactions, dipole induced dipole interactions, and other polarity polarity interactions. Positive phase solid-phase extraction can adsorb polar compounds from non-polar solvent samples.

The adsorbents used in reverse phase solid-phase extraction are usually non-polar or weakly polar, and the extracted target compounds are usually moderately polar to non-polar compounds. The interaction between the target compound and the adsorbent is hydrophobic interaction, mainly non-polar non-polar interaction, which is van der Waals force or dispersion force.

The adsorbent used in ion exchange solid-phase extraction is a charged ion exchange resin, and the extracted target compound is a charged compound. The interaction between the target compound and the adsorbent is electrostatic attraction.

The selection of adsorbent (stationary phase) in solid-phase extraction is mainly based on the properties of the target compound and the properties of the sample matrix (i.e. the solvent of the sample). When the polarity of the target compound is very similar to that of the adsorbent, optimal retention (adsorption) of the target compound can be achieved. The more similar the polarity of the two, the better the retention (i.e. adsorption), so it is advisable to choose adsorbents with similar polarity to the target compound as much as possible. For example, when extracting hydrocarbons (non-polar), reverse phase solid-phase extraction (using non-polar adsorbents) should be used. When the polarity of the target compound is moderate, both forward and reverse phase solid-phase extraction can be used. The selection of adsorbent is also constrained by the solvent strength (i.e. elution strength) of the sample.

The strength of the sample solvent should be relatively weak compared to the adsorbent, as weak solvents can enhance the retention (adsorption) of the target compound on the adsorbent. The order of solvent strength in forward and reverse solid-phase extraction is different (see Figure 3-13). If the strength of the sample solvent is too strong, the target compound will not be retained (adsorbed) or retained very weakly. For example, when the sample solvent is n-hexane, reverse phase solid-phase extraction is not suitable because n-hexane is a strong solvent for reverse phase solid-phase extraction (see Figure 3-13), and the target compound will not adsorb on the adsorbent; When the sample solvent is water, reverse phase solid-phase extraction can be used because water is a weak solvent for reverse phase solid-phase extraction and does not affect the adsorption of the target compound on the adsorbent.

When selecting the separation mode and adsorbent for solid-phase extraction, the following points should also be considered:

The solubility of the target compound in polar or non-polar solvents mainly involves the selection of the eluent.

2. Whether the target compound is likely to ionize (ionization can be achieved by adjusting the pH value), thus determining whether to use ion exchange solid-phase extraction.

3. Is it possible for the target compound to form covalent bonds with the adsorbent? If covalent bonds are formed, there may be difficulties during elution.

4. The degree of competition between non target compounds and target compounds on the adsorption sites of the adsorbent is related to whether the target compound can be well separated from interfering compounds.

II Common adsorbents (stationary phase) for solid-phase extraction

Given that solid-phase extraction is essentially a separation method in liquid chromatography, in principle, materials that can be used as packing materials for liquid chromatography columns can be used for solid-phase extraction. However, due to the high column pressure and the requirement for high column efficiency in liquid chromatography, the particle size of the fillers is strictly required. In the past, 10 μ m particle size fillers were commonly used, but now columns often use 5 μ m fillers, and even 3 μ m fillers (as the HPLC pump pressure increases, the particle size of the fillers gradually decreases). The requirement for the particle size distribution of the filler is also very narrow. The pressure applied to the solid phase extraction column is generally not high, and the separation purpose is only to separate the target compound from the interfering compound and the matrix. The column efficiency requirement is generally not high, so the packing used as a solid phase extraction adsorbent is relatively coarse, generally at 40 μ m, and the particle size distribution requirement is not strict, which can greatly reduce the cost of the solid phase extraction column. The types and uses of adsorbents commonly used in solid-phase extraction are shown in Table 3-4.

III Solid phase extraction equipment and operating procedures

A simpler solid-phase extraction device is a small column with a diameter of several millimeters (Figure 3-14), which can be made of glass, plastic such as polypropylene, polyethylene, polytetrafluoroethylene, or stainless steel. There is a sintered sieve plate with a pore size of 20 μ m at the lower end of the small column to support the adsorbent. If there is no suitable sintered sieve plate for the self-made solid-phase extraction column, glass wool can be added instead of the sieve plate to support the solid adsorbent and allow the liquid to flow through. Fill a certain amount of adsorbent (100 ㎎~1000 ㎎, depending on the needs) on the sieve plate, and then add another sieve plate on top of the adsorbent to prevent damage to the column bed when adding samples (glass wool can also be used as a substitute if there is no sieve plate). There are currently various specifications of solid-phase extraction columns equipped with various adsorbents available for sale, which are very convenient to use (Figure 3-15).

The general operating procedure for solid-phase extraction is as follows:

1. Activated adsorbent: Before extracting the sample, rinse the solid-phase extraction column with an appropriate solvent to keep the adsorbent moist and able to adsorb target compounds or interfering compounds. Different modes of solid-phase extraction require different solvents for column activation:

(1) The weakly polar or non-polar adsorbents used in reverse phase solid-phase extraction are usually rinsed with water-soluble organic solvents such as methanol, followed by rinsing with water or buffer solutions. It is also possible to rinse with a strong solvent (such as hexane) before rinsing with methanol to eliminate impurities adsorbed on the adsorbent and their interference with the target compound.

(2) The polar adsorbent used in solid-phase extraction is usually rinsed with the organic solvent (sample matrix) containing the target compound.

(3) The adsorbent used in ion exchange solid-phase extraction can be rinsed with the sample solvent when used for samples in non-polar organic solvents; When used for samples in polar solvents, water-soluble organic solvents can be used for rinsing, followed by rinsing with an appropriate pH of an aqueous solution containing certain organic solvents and salts.

In order to keep the adsorbent in the solid-phase extraction column moist from activation to sample addition, approximately 1ml of the solvent used for activation treatment should be maintained on top of the adsorbent after activation treatment.

2. Sample loading: Pour the liquid or dissolved solid sample into the activated solid-phase extraction column, and then use vacuum (Figure 3-16), pressure (Figure 3-17), or centrifugation (Figure 3-18) to introduce the sample into the adsorbent.

3. Washing and elution: After the sample enters the adsorbent and the target compound is adsorbed, the weakly retained interfering compound can be washed off with a weaker solvent first, and then the target compound can be washed off with a stronger solvent and collected. Rinsing and elution, as described above, can be carried out by vacuum, pressure, or centrifugation to allow the eluent or elution solution to flow through the adsorbent.

If the adsorbent is chosen to have weak or no adsorption on the target compound, but strong adsorption on the interfering compound, the target compound can be washed down and collected first, while the interfering compound is retained (adsorbed) on the adsorbent, and the two can be separated. Figure 3-19 shows schematic diagrams of two methods. In most cases, the target compound is retained on the adsorbent and subsequently eluted with a strong solvent, which is more conducive to sample purification. Figure 3-20 shows solid-phase extraction

The general program schematic diagram used.

In order to facilitate the use of solid-phase extraction, many manufacturers have not only produced various specifications and models of solid-phase extraction columns, but also developed many solid-phase extraction devices to make solid-phase extraction more convenient and simple to use. Supelco provides a single tube processing plug (Figure 3-21) for pressurizing a single solid-phase extraction column, which can be easily used in conjunction with the solid-phase extraction column. For example, in order to enable multiple solid-phase extraction columns to be evacuated simultaneously, Supelco provides vacuum multi manifold devices with 12 and 24 apertures (Figure 3-22), which can simultaneously process multiple solid-phase extraction columns. The Dalian Institute of Chemical Physics, Chinese Academy of Sciences, and the National Center for Chromatography Research and Analysis have also developed a vacuum solid-phase extraction device.

Figure 3-23 shows the flowchart of how to select the solid-phase extraction mode based on the properties of the sample matrix (solvent), target compound, and interfering compound

4 Solid phase Micro Extraction (SPME)

Solid phase microextraction is a new extraction and separation technology developed on the basis of solid phase extraction. Compared with liquid-liquid extraction and solid phase extraction, it has the advantages of short operation time, small sample size, no need for extraction solvents, suitable for analyzing volatile and non-volatile substances, and good reproducibility. Many research results have shown that adding appropriate internal standards to samples for quantitative analysis results in excellent reproducibility and precision. The solid-phase microextraction device is shaped like a microsampler, consisting of a handle and an extraction head or fiber head. The extraction head is a 1cm long molten fiber coated with different adsorbents, connected to a stainless steel wire and wrapped in a thin stainless steel tube (to protect the quartz fiber from breakage). The fiber head can be extended or retracted inside the steel tube, and the thin stainless steel tube can penetrate rubber or plastic gaskets for sampling or injection. The handle is used to install or fix the extraction head and can be used forever (Figure 3-24)

The key to solid-phase microextraction is to choose a coating (adsorbent) on non quartz fibers, so that the target compound can adsorb on the coating while interfering compounds and solvents do not adsorb. Generally, when the target compound is non-polar, a non-polar coating is selected; Choose a polar coating when the target compound is polar.

The sampling method of solid-phase microextraction is to insert the solid-phase microextraction needle tube (stainless steel sleeve) through the sealing gasket of the sample bottle and into the sample bottle. Then push out the extraction head and immerse it into the sample (immersion method) or place it in the upper space of the sample (headspace method) for extraction. The extraction time is approximately 2-30 minutes to achieve adsorption equilibrium of the target compound. Retract the extraction head and pull out the needle (Figure 3-25).

Solid phase microextraction can be used for gas chromatography or liquid chromatography (Figure 3-25). When used for GC, the solid-phase microextraction needle (stainless steel sleeve) is inserted into the GC injection port, the handle is pushed, the fiber head is extended, and the target compound is thermally desorbed using the high-temperature pyrolysis of the injection port. After desorption, it is carried into the chromatographic column by the carrier gas. When used for HPLC, the solid-phase microextraction needle tube (stainless steel sleeve) is inserted into the solid-phase microextraction/HPLC interface desorption cell, and then the target compound is eluted through the desorption cell using the HPLC mobile phase, and brought into the chromatographic column.