Separation and Extraction

Separation and extraction

Extraction is one method of sample preparation that commonly takes place before processes in analytic chemistry. The point of extraction is to enrich the analytes and remove interfering substances from the sample. Extraction leverages the fact that different substances (and substance classes) have different degrees of solubility. That is why extraction is different from other separation methods, such as filtration, in which substances are separated based on their particle size.

Sample separation or extraction – Is there a difference?

Extraction is a separation method that serves to pull one or more substances from a mixture of substances. In sample preparation, this step is used to remove undesired substances from a sample. These substances can distort the analysis or interfere with the analyzers themselves. Extraction can also serve to enrich the analyte. This in turn allows for a higher level of detection accuracy. Sample preparation with extraction thus increases the quality of the results and of the process of chemical analysis itself.

An extraction agent (solvent) is used to separate the relevant substances from each other. Each substance's characteristic solubility is the determinant in the separation process. This underlying principle distinguishes extraction from many other separation methods, such as:

  • Filtration (particle size)
  • Decanting (density)
  • Centrifuging (density and resulting centrifugal force)
  • Liquid-liquid extraction (density)
  • Distillation (boiling temperature)

Thus, extraction is only one of many sample separation methods. In practice, other solvent-free separation methods are also referred to as extraction, but this is somewhat of a misnomer.

Sample extraction methods

In the context of sample preparation, extraction can fall into one of many categories based on the state of matter of the sample and the solvent.

Liquid-liquid extraction (LLE) uses a liquid solvent to separate a dissolved substance from a liquid sample. One simple example is the separation of fat from milk, which can be done with petroleum ether, for instance. LLE is sometimes known as solvent extraction.

Solid phase extraction (SPE) is used to separate the analytes from a sample and isolate them via a solid phase. This type of extraction is very common in analytical laboratories. The basic principle of solid phase extraction is identical to that of column chromatography. It consists of flowing a sample solution through a stationary phase (sorbent) that has specific interactions with the analytes, solvent and sample matrix. There are two variants: Either the analyte becomes enriched on the stationary phase (analyte retention) or passes through it (retention of interfering substances). SPE is especially versatile, since many phases are available for a wide range of analytes and matrices.

And although solid phase microextraction (SPME) functions without a solvent, it also deserves a mention. SPME is an innovative type of extraction that uses a coated fiber as a phase. This is coated with a liquid or a solid. When the sample comes into contact with the fiber, the analytes from the sample spread out on the fiber. After the extraction period has passed, this fiber can be placed directly into a chromatograph to perform the analysis. Its strong suit in terms of application lies in gas chromatography (GC).

Procedure of the SPE method

This variant of solid phase extraction is widespread in sample preparation. Compared to liquid-liquid extraction, it has some critical advantages thanks to its lower solvent consumption, shorter extraction time, and potential for automation. It is perhaps no surprise that SPE is a mainstay in the pharmaceutical industry, conservation and foodstuffs chemistry. Laboratories in these fields in particular benefit from efficient SPE. Lastly, sample preparation tends to be an especially time-intensive step in the analytic process.

Steps in the process

The SPE method can be broken down into four steps.

  1. Conditioning the sorbent
    The solid phase is conditioned by wetting it with a solvent. In the case of an aqueous sample matrix, this solvent is water. The selection of substances for conditioning depends on the SPE method. The aim is to create an environment that enables maximum interaction between the sorbent and the sample.1
  2. Loading the sample
    The sample is flowed through the SPE column, either under positive or negative pressure. Depending on the SPE variant, either the analyte will be retained, or the impurities in the sample will be retained.
  3. Washing the sorbent
    Washing the sorbent is only necessary in SPE methods where the analyte enriches on the sorbent. In this case, the phase is washed in order to remove any impurities that may have accumulated.
  4. Elution
    If the analyte was retained, the final step is the elution of the analyte from the sorbent. A suitable solvent is used for this purpose. If the extraction involved retention of the impurities, then the analyte is fed into the column, flows through the sorbent without interacting with it, and exits the bottom of the column. The separate washing step is then not necessary.


The time and solvent required for extraction can have a significant impact on profitability, especially in laboratories with a high throughput. Higher-performance extraction allows for more precise results by removing impurities from the sample.2 From this, it is possible to derive some requirements for SPE.

Solid phase extraction further helps reduce analysis time because it attains a much higher degree of separation than liquid-liquid extraction. It requires only a few steps, which are easy to automate. For example, all SPE steps proceed with the same column without the necessity for additional handling. Pressure-assisted sample loading is another contributing factor. Multiple samples can be prepared in parallel with one SPE machine.

SPE machines consume much less solvent compared to liquid-liquid extraction. At the same time, the analyte becomes significantly more concentrated, which makes it easier to later quantify the analyte in a measuring instrument.  Moreover, (semi-)automatic SPE processes ensure that extraction proceeds reproducibly, free of contamination and without losing any analyte.3

Interference factors

SPE is often used as a sample preparation method when the goal is to remove impurities from the sample before the actual analysis takes place. These impurities mainly affect the quality of the results.

More complex sample matrices make it significantly harder to analyze substances of interest. The more compounds are present in the sample, the harder it is to qualitatively and quantitatively detect the analytes. If impurities are removed from the sample, fewer contaminants reach the analyzer, which often improves the service life of certain wear parts in the instrument. That is why, with SPE, particular care must be taken to use a phase that guarantees optimal separation of the substances present. Like selecting the eluent, this is part of what it takes to develop an SPE method.

When separating or fractionating into multiple substance classes, it is also possible to use multiple eluents in succession. In this case, solvents with increasing or decreasing polarity are used.

Separation methods in various industries

SPE has opened up a wide range of applications as a separation process. Thanks to the availability of many different solid phases and procedures for elution, this method can be specifically adapted to different analytes and matrices. The main areas of application are environmental analysis, pharmaceutical and biochemical analysis, and food analysis.

Environmental analysis

The sum parameter AOX (adsorbable organic halides) is used in wastewater monitoring. Halides occur in technical processes and often take a long time to degrade in the environment.  Therefore, they concentrate in food webs and can be harmful to people.

One process for measuring AOX is described in DIN EN ISO 9562. It describes interference factors that can distort the measurement of AOX. For example, a high concentration of organic substances in the sample can cause AOX readings to come out too low. False high readings, meanwhile, can be caused by a higher presence of chlorides. Diluting the sample can generally minimize interference factors, but sample dilution simultaneously raises the quantitation limit for this parameter.

In the event of high chloride concentrations, sample dilution can make it impossible to detect AOX at all. This is why a sample preparation method was developed which enables the measurement of AOX in samples with high chloride content and/or organic substances: the so-called SPE-AOX method. Measurement of SPE-AOX is described in Annex A of ISO 9562. Using solid phase extraction, the sample is purified of interfering chloride ions, and the analyte concentrates on the solid phase. Following elution with methanol and dilution with water, the sample is then adsorbed on activated carbon and measured in a traditional AOX analyzer.

When preparing samples for the SPE-AOX method, it is possible to use the fully automatic APU 28 system. This system can prepare up to 28 samples for analysis. The APU sim represents another semi-automatic system for SPE-AOX sample preparation, allowing the operator to process up to 6 samples at a time.

Pharmaceutical analysis

Like all methods in forensic toxicology, pharmaceutical analysis must meet special requirements for the validity of results. In this context, manual SPE enrichment represents a risk factor because the procedure is prone to errors.

For example, to detect THC, the active substance in cannabis, institutions use gas chromatography with a mass spectrometer as a detector (GC/MS). Sample preparation, meanwhile, is done with SPE. Previously manual SPE can be migrated to an automatic sample preparation system to increase efficiency. This ensures no obstacles to validity.4

Foodstuffs analysis

The roasting of coffee inevitably produces the carcinogen acrylamide. Because this process cannot be abated, production must feature continuous monitoring of the acrylamide content in order to ensure food safety.5

Matrix reduction also plays an important role in the analysis of coffee samples. Solid phase extraction has proven a suitable method for this. The actual analysis is then done with HPLC-MS, for example. This method achieves an excellent recovery rate of acrylamide. Especially with the high sample volume in food analysis, the ability to automate SPE represents a massive potential efficiency boost in the laboratory.6