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    [Sample Prep] 시료 전처리 방법 -SPE (Solid Phase Extraction) 고체상 추출법

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    이매스

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SPE(Solid Phase Extraction) : 고체상 추출법, 혼합물로부터 목적으로 하는 화합물과 불순물을 물리·화학적 성질에 근거해 분리하는 방법으로 크로마토그래피를 하기전 샘플을 농축 시키거나 깨끗하게 하기 위해 사용하는 일반적인 전처리 기술이다. 액체액체 추출법 (Liqiud-Liqiud Extraction : LLE)에 비해서 인체에 유해한 유기용매를 대량으로 사용하지 않는다는 특징을 가지고 있고 또 효율이 높다는 장점이 있다.


SPE 방법 5단계

1. Sample pretreatment – e.g. pH adjustment, protein precipitation, filtration

2. Column conditioning – the column is made ready for the sample being loaded

3. Sample loading – the sample is loaded onto the cartridge

4. Column wash – interferences are washed from the column

5. Sample elution – the sample is eluted by a suitable solvent and collected – interference left behind on cartridge


고체상 추출법 카트리지의 종류

1. Normal phase SPE procedure

typical solid phase extraction involves five basic steps. First, the cartridge is equilibrated with a non-polar or slightly polar solvent, which wets the surface and penetrates the bonded phase. Then water, or buffer of the same composition as the sample, is typically washed through the column to wet the silica surface. The sample is then added to the cartridge. As the sample passes through the stationary phase, the polar analytes in the sample will interact and retain on the polar sorbent while the solvent, and other non-polar impurities pass through the cartridge. After the sample is loaded, the cartridge is washed with a non-polar solvent to remove further impurities. Then, the analyte is eluted with a polar solvent or a buffer of the appropriate pH.A stationary phase of polar functionally bonded silicas with short carbons chains frequently makes up the solid phase. This stationary phase will adsorb polar molecules which can be collected with a more polar solvent


2. Reversed phase SPE

Reversed phase SPE separates analytes based on their polarity. The stationary phase of a reversed phase SPE cartridge is derivatized with hydrocarbon chains, which retain compounds of mid to low polarity due to the hydrophobic effect. The analyte can be eluted by washing the cartridge with a non-polar solvent, which disrupts the interaction of the analyte and the stationary phase. A stationary phase of silicon with carbon chains is commonly used. Relying on mainly non-polar, hydrophobic interactions, only non-polar or very weakly polar compounds will adsorb to the surface.



3. Ion exchange SPE

Ion exchange sorbents separate analytes based on electrostatic interactions between the analyte of interest and the positively or negatively charged groups on the stationary phase. For ion exchange to occur, both the stationary phase and sample must be at a pH where both are charged.

- Anion exchange

Anion exchange sorbents are derivatized with positively charged functional groups that interact and retain negatively charged anions, such as acids. Strong anion exchange sorbents contain quaternary ammonium groups that have a permanent positive charge in aqueous solutions, and weak anion exchange sorbents use amine groups which are charged when the pH is below about 9. Strong anion exchange sorbents are useful because any strongly acidic impurities in the sample will bind to the sorbent and usually will not be eluted with the analyte of interest; to recover a strong acid a weak anion exchange cartridge should be used. To elute the analyte from either the strong or weak sorbent, the stationary phase is washed with a solvent that neutralizes the charge of either the analyte, the stationary phase, or both. Once the charge is neutralized, the electrostatic interaction between the analyte and the stationary phase no longer exists and the analyte will elute from the cartridge.

- Cation exchange

Cation exchange sorbents are derivatized with functional groups that interact and retain positively charged cations, such as bases. Strong cation exchange sorbents contain aliphatic sulfonic acid groups that are always negatively charged in aqueous solution, and weak cation exchange sorbents contain aliphatic carboxylic acids, which are charged when the pH is above about 5. Strong cation exchange sorbents are useful because any strongly basic impurities in the sample will bind to the sorbent and usually will not be eluted with the analyte of interest; to recover a strong base a weak cation exchange cartridge should be used. To elute the analyte from either the strong or weak sorbent, the stationary phase is washed with a solvent that neutralizes ionic interaction between the analyte and the stationary phase.








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