Solid Phase Extraction Common Questions and Answers

Hydrophilic lipophilic balance

“Hydrophilic-Lipophilic Balance (HLB), was originally used to mean “the equilibrium relationship between hydrophilic groups and lipophilic groups in surfactant molecules.” At the beginning of the 21st century, the concept of “hydrophilic lipophilic balance” was Introduced into SPE, the filler matrix is a polymer (such as polystyrene), the matrix is lipophilic, similar to C18, with reversed-phase retention; then bonded to the polymer matrix, such as pyrrolidone, with Hydrophilic, the group can enhance the retention ability of the filler to the polar compound, and maintain the water wettability of the filler to prevent the bed from drying out and improve the stability of the result;

Dima Technology’s ProElut PLS combines a hydrophilic group (pyrrolidone group) and a hydrophobic group (divinylbenzene), which retains both polar and non-polar compounds and is hydrophilic and lipophilic. Balanced characteristics. The same small column is Waters Oasis HLB.

The hydrophilic lipophilic balance column generally has the following characteristics:
A: True versatility: balanced retention of hydrophilic and lipophilic substances, covering non-polar, weakly polar compounds, overcoming the disadvantages of poor retention of polar compounds by C18 adsorbents;
B: Higher stability: water wettability, after the filler is activated, even if the bed is dry, the retention of the target by the adsorbent will not change;
C: Wide pH range of application: The matrix of PLS is organic polymer rather than silica gel, which is stable in the range of pH 0-14, while the silica-bonded adsorbent is stable only in the range of 2-7.5;
D: Higher adsorption capacity: It can retain more targets, effectively prevent the occurrence of “penetration phenomenon” and improve reproducibility;
E: There is no secondary interaction: there is unbonded silanol on the surface of the silica-bonded adsorbent, and the retention of the basic compound is strong. The basic compound is treated with a silica-bonded adsorbent, and the recovery rate is usually low; PLS is an adsorbent for an organic polymer matrix, and there is no secondary interaction. It can be used for basic compounds to obtain satisfactory results.

Target is not recycled or has low recovery

In a complete SPE cartridge operation, the target compound may be present in the sample solution effluent, eluent, eluent, or adsorbent. When the “target is not recovered or the recovery rate is low”, the sample solution may be applied to the sample solution. Add the standard solution, then perform the complete SPE operation and collect and analyze the sample effluent, eluent, and eluate separately, first determine which part of the target compound is present, and then determine which problem from the following:
1. Insufficient retention of the target on the adsorbent;
2. The target is not completely eluted;
3. Other links

Here are some improvements to the specific reasons

1. When more than 5% of the target compound is present in the sample effluent or eluent, it can be concluded that “the target compound is not sufficiently retained on the adsorbent”.
2. When the target compound does not appear in the sample solution effluent and eluent and there is only a small amount or no appearance in the eluate, it can be judged that “the target is not completely eluted”.
3. If the target compound normally appears in the eluent, the problem of insufficient recovery should come from other aspects of sample preparation.

Recyclability is poorly reproducible

Possible cause
Before the sample is loaded, the bed is dry and reactivated, and balanced.
The adsorbent is loosely packed, and the solution is short-circuited. Replace the product, or squeeze the bed with a small stick.
The sample fluid flow rate is too fast. The flow rate is reduced. Generally, when the flow rate is lower than 5 mL/min, the result is stable.

purification effect is not ideal

A: purification mode improvement

In general, the mode of retaining the target compound is better than the mode of retaining impurities. If the item being analyzed is analyzed for a certain compound or a certain type of compound, the purification mode of the target compound is retained; for a simple example, the same is the analysis of carbendazim and thiabendazole (a class of alkaline pesticides) in vegetables. The cation exchange column can be used to specifically retain these pesticides so that they are completely separated from the interferents, and the normal phase adsorbent or graphite is used. The operation of retaining the interference by the carbon black can only remove the main interferent, and there are still many interferents remaining.

In addition, if more than one SPE column can be used for the purification of the target compound, a selective adsorbent should be selected; in terms of selectivity, ion exchange > normal phase > reverse phase.

B: Optimization of eluent and eluent

For the reversed phase mode and the normal phase mode, the elution strength of the eluent should be increased and the elution strength of the eluent should be reduced without affecting the recovery rate.

For the reverse phase mode, the aqueous solution of the target compound can be loaded, and then 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, aqueous methanol solution is sequentially used. Elution, separately collect the eluent analysis, establish an elution curve, and find the solvent system when the target compound is eluted. The methanol content in the eluent should be slightly lower than the solvent system when the target compound is just eluted. The content of methanol in the eluent should be slightly higher than the solvent system when the target compound is completely eluted; when pure methanol cannot elute the target compound, the elution curve of the methanol-methyl tert-butyl ether mixed solution should be tested; For some ionic compounds which are neither soluble in water nor soluble in methanol, an acid or a base may be added to the solvent system.

For a normal phase system, the target compound can be loaded with a solution of n-hexane, followed by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, The solution was eluted with n-hexane methyl tert-butyl ether solution, and the eluent was collected for analysis. The elution curve was established to find out the solvent system when the target compound was eluted. The content of methyl tert-butyl ether in the eluent should be slightly below the solvent system when the target compound is just eluted, and the methyl tert-butyl ether content in the eluent should be slightly higher than the solvent system when the target compound is completely eluted.

C: Reasonable handling of SPE columns

There may be a small amount of interfering substances in the adsorbent in the SPE column. Effective activation can prevent these interfering substances from entering the eluent. The activation solution should select a solvent system with strong elution intensity throughout the operation; in the reverse phase mode, from the sample solution to The eluent mainly involves 0%-methanol aqueous solution, so the activation solution should be pure methanol. When the target compound needs to be eluted with methanol-methyl tert-butyl ether mixture, the activation solution should be selected as methyl tert-butyl ether; In the reversed-phase mode, the 0%-n-hexane-methyl tert-butyl ether solution is mainly involved from the sample solution to the eluent, so the activation solution should be selected as pure methyl tert-butyl ether.

The flow rate is too low

Possible cause
The sample solution contains more particulate impurities, blocking the sieve plate. Centrifuge or filter the sample solution before loading.
Sample solution viscosity is too high dilute the sample with a suitable solvent
The SPE column bed is too high and the resistance to liquid is too high. Use a solid phase extraction instrument and decompress the liquid outlet.
Pressurize at the liquid inlet