Entering the Sample Preparation Acronym Maze

The Column, The Column-06-04-2021, Volume 17, Issue 06
Pages: 14–20

Incognito explores the overwhelming array of sample preparation techniques (and acronyms).

I was recently researching sample preparation techniques we are developing in the laboratory, and, as is typical these days, I disappeared down somewhat of an Internet rabbit hole. Whilst I thought my knowledge of sample preparation techniques was relatively comprehensive, I quickly found that this wasn’t the case. This exercise also reminded me of how much we love an acronym—and oh boy is this particular area rich with these! When I started my career, I can clearly remember being bamboozled by the language of analytical sciences, and I often wonder how younger scientists feel when faced with such a huge array of acronyms in the literature, let alone how they gather an understanding of which techniques are applicable in which situations.

I wonder how many readers would recognize all of the acronyms shown in Table 1. Have a go and see how many you get (you may need to scroll up a little in order to hide the answers within the text below). I’ll provide the answers and some further details on each technique in the body of the article.

I know that during my research there were certainly techniques that I had not previously encountered and I wanted to share some of this in the hope that it provides a useful overview of sample preparation techniques you might not have previously considered for your applications. The degree of selectivity achieved by a sample preparation technique is often a primary reason for selection and I’ve also included some information on the relative selectivity of each technique. As my own application involved high analytical throughput, I’ve also included some information on whether there is currently an automated solution for the technique available on the market.

Increasingly stringent regulatory requirements across a range of industries as well as the analytical requirements within our own businesses drive our sensitivity to the very limits of instrument capability. Therefore, I’ve also included information on the ability of each technique to enrich the analytes from sample to final prepared extract.

LLE: Liquid–Liquid Extraction

The acceptor phase (typically an immiscible organic) and donor phase liquids are shaken or mixed to transfer analytes to the acceptor phase based on partition co-efficient. The nature of the acceptor solvent and donor phase pH and ionic strength can be adjusted to favour the transfer of analytes. Widely applicable to liquid samples, especially in the aqueous phase, and simple to implement.

SALLE: Support-Assisted Liquid–Liquid Extraction (also Solid-Liquid Extraction or Supported-Liquid Extraction [SLE])

The donor phase is percolated into and supported within a bed of purified, highly polar solid substrate (diatomaceous earth is typical) with high surface area. The acceptor phase (typically an immiscible organic) is slowly passed through the substrate bed and analyte transfer occurs between the thin layer of donor phase coating the substrate particles, containing the enriched analyte, and the slowly moving acceptor phase. The donor or acceptor phase may be buffered or pH adjusted to promote analyte transfer. Widely used for biological, food, and environmental applications.

CPE: Cloud-Point Extraction

Non-ionic or zwitterionic surfactants are used above the critical micelle concentration to form micelles that contain the target analytes. When the temperature is raised above the cloud point, the so-called coacervative phase undergoes phase separation from the bulk aqueous phase and can be removed via decantation or aspiration. CPE is different from the two closely related techniques of coacervative extraction (CAE) and aqueous surfactant two-phase (ASTP) extraction in which ionic surfactants may be used and phase separation is induced using pH or ionic strength. Whilst CPE was initially focused on the extraction of inorganics, the technique has recently found applications in the extraction of biologics, as well as priority pollutants from water.

UAE: Ultrasonic-Assisted Extraction

The use of ultrasound to enhance extraction efficiency. Whilst the technique is used widely to ensure extraction from complex and intractable matrices, such as natural products, many liquid-based extraction techniques can benefit from the enhanced extraction efficiency when using ultrasound. Typically, the use of ultrasound will enable the volume of the donor solvent to be reduced and the technique is often promoted as a greener alternative to traditional liquid extraction techniques. Widely used in the extraction of biologics and natural products.

MAE: Microwave-Assisted Extraction

A non-microwave absorbing acceptor solvent is used with a sample containing a high dielectric constant substance such as water. The sample is rapidly heated using microwave energy and analyte transfer occurs into the acceptor solvent. A variation of the technique uses a microwave absorbing substrate, which is added to the acceptor solvent to induce rapid heating. The technique can use open or closed vessels, the latter being a subgroup of the PFE techniques (see below), provided that the material of construction is non-microwave absorbing. MAE is widely used in the analysis of natural products, polymers, and foods.

ASE: Accelerated Solvent Extraction (see PFE)

A trade name used widely for pressurized fluid extraction.

PFE: Pressurized Fluid Extraction (also Pressurized Solvent Extraction [PSE])

A liquid-solid extraction technique in which the sample and solvent are placed in a sealed container and heated well above the normal boiling point of the extraction solvent. The combination of temperature and pressure results in highly efficient extraction, which is usually completed in a few minutes. Correct selection of the extraction solvent is critical in determining the selectivity of the technique. Smaller volumes of extraction solvent may be required due to the use of increased temperature and pressure and therefore the technique is viewed as a greener alternative. The technique has been used widely in the environmental, food, chemical, petrochemical, and pharmaceutical industries.

SHWE: Superheated Water Extraction

A version of pressurized fluid extraction in which the extraction solvent is water. The superheating of the aqueous solvent changes the dielectric constant and water is said to adopt “solvent-like” properties. The technique is promoted as a greener alternative to traditional PFE using organic solvents.

SFE: Supercritical Fluid Extractio

Supercritical solvents, such as carbon dioxide, are used to extract analytes—typically from solid samples. Supercritical fluids have gas-like diffusivity but liquid-like solvating properties, resulting in enhanced extraction efficiency from intractable matrices. The technique requires specialist equipment that can precisely maintain the extraction solvent above its supercritical point using a combination of back pressure and temperature. The extractants can be collected in a cold-trap, liquid-trap, or onto an absorbent. Modifiers can be added to the supercritical fluid to alter its polarity and hence enable the extraction of more hydrophilic analytes. The technique is a greener alternative to those that use organic solvents. Supercritical fluid extraction has been used extensively and has published applications across a diverse range of industries.

SHE: Static Headspace Extraction

A solid or liquid sample is first heated in a sealed container to release volatile target analytes. The gaseous headspace is then sampled using a gas-tight syringe or loop autosampler. Phase ratio, temperature, extraction time, and agitation can all be used to optimize the extraction efficiency. Static headspace extraction can be used wherever volatile analytes are targeted in solid or liquid samples.

DHE: Dynamic Headspace Extraction

The solid or liquid sample is heated and the volatile target analytes in the headspace vapour (purging) or from within the liquid sample (sparging) are constantly swept from the sample volume using an inert gas. Volatilized analytes are trapped downstream onto an adsorbent material or via cryogenic means, prior to release into the chromatograph via strong heating. Typically, dynamic headspace extraction is used where analyte preconcentration is required.

SPE: Solid-Phase Extraction

An extraction method using a solid substrate, typically coated with a stationary phase of a selected chemistry used to trap, wash, and subsequently elute target analytes. The sorbent is available in tube, cartridge, and well-plate formats. A wide range of substrate chemistries are available and are analogous to the phases used in reversed- or normal-phase high performance liquid chromatography (HPLC), and can also be ionic or mixed-mode for improved analyte specificity. The process typically consists of conditioning the sorbent, applying the sample to trap the analytes of interest, washing to remove interferents, and elution of the target analytes in a small volume using a strong solvent. At each stage, the nature of the solvent, solution pH, and ionic strength can be adjusted to promote analyte adsorption or release and as such the combination of phase choice and chemistry manipulation can lead to a highly selective extraction technique. The highest selectivity is typically obtained when analyte–sorbent interactions are electrostatic. Solid-phase extraction has been extensively employed across a wide of industries and applications.

dSPE: Dispersive Solid-Phase Extraction

A form of SPE in which the extraction media in powder form is dispersed into the sample solution rather than the sample liquid being passed through an immobilized bed of the extraction media. In its simplest form, dispersive SPE is used to retain matrix interferents on the extraction media, prior to it being centrifuged and the resulting sample solution aspirated from the supernatant. dSPE is most often used with intractable matrices or homogenates and is an integral step in the QueChERS workflow. QueChERS (Quick, Cheap, Effective, Reliable Safe—that acronym readers can have for free!) has become well established as an extraction technique following its introduction for the extraction of pesticides from fruits and vegetables. It is a two-step process in which the first step involves a salting-out extraction using buffered or unbuffered solvent (acetonitrile is typical), followed by dispersive SPE of an aliquot from step one to remove interferences and matrix compounds. Whilst the application areas for dSPE are diverse and still evolving, perhaps the most popular applications are in food and environmental analysis.

MIPSE: Molecularly-Imprinted Sorbent Extraction

A further extension to the SPE principle in which silica or polymer stationary phases are manufactured using one or more target analyte molecules as a template to form “lock and key”-type molecular recognition sorbents. This method of manufacture improves the specificity of the sorbent towards the analyte or structurally related analytes used as the original template and can yield very impressive levels of specificity and sensitivity. Whilst MIPSE is applicable to any situation in which SPE can be used, it is perhaps best used when the target analyte functional group chemistry or molecular volume is distinctly different from potential interferents. There has been a recent uptick in interest for MIPS extraction of target biologics from both endogenous and exogenous matrices.

SPME: Solid-Phase Microextraction

A technique in which a small diameter liquid polymer or solid sorbent coated fibre is immersed into a liquid sample or suspended in the headspace above a liquid or solid sample to selectively extract either dissolved or volatile target analytes, respectively. Depending upon the nature of the polymer coating, analytes will diffuse into the coating until equilibrium is reached. The first in our list of microextraction techniques used where a high degree of analyte enrichment is required; in these techniques the acceptor phase has a lower volume than the donor phase or the acceptor phase possesses the ability to concentrate analytes for direct introduction into the separation or detection equipment. Optimization of the SPME extraction technique involves selection of the appropriate fibre coating chemistry, coating film thickness, sample extraction time, extraction temperature, sample agitation, and sample to headspace volume ratio, alongside several other variables. In gas chromatography (GC), the fibre is usually placed into the inlet and the analytes thermally desorbed onto the column. Cryo‑focusing can be used to reduce analyte band widths if required. The technique can also be used with LC by desorbing the fibre with a liquid extractant, although this approach is less popular. A recent development is the so-called SPME Arrow, in which a metal rod of larger diameter than the traditional fibres is used and is coated with a thicker and larger surface area sorbent layer. SPME Arrow is purported to result in a 10-fold increase in sample sensitivity compared to the fibre-based technique. SPME has found applications in a wide range of applications including flavours and fragrance, forensics, toxicology, environmental, and biological analysis.

SBSE: Stir-Bar Sorptive Extraction

A version of SPME in which the liquid polymer or solid sorbent is coated onto a stir bar that is used to agitate the sample whilst target analytes are adsorbed to the stir-bar surface. SBSE is also an equilibrium technique, the advantage being the increase of surface area and therefore capacity of the sorbent compared to SPME. Extraction and equilibration times are in the tens of minutes, similar to those for the SPME technique. In GC, the stir-bar is typically placed into a thermal desorption unit for analyte introduction to the GC column. In LC, analytes are desorbed by washing or immersing into a suitable solvent prior to sample introduction. The SBSE technique is argubably more popular than SPME for LC applications and is particularly suited to the extraction of low- to medium-polarity analytes. Applications include food and beverage, biological samples, environmental matrices, and pharmaceutical products.

HSSE: Headspace Sorptive Extraction

A name given to SPME when sampling the headspace above a liquid or solid sample. Also known as HS-SPME—headspace solid‑phase microextraction.

ISSE: In-Sample Sorptive Extraction

A name given to SPME when the extraction takes place within the sample. This name can also be applied to a newer development of the SPME technique in which a metal “blade” of larger surface area is used for liquid samples or to penetrate a solid sample and allowed to equilibrate, primarily in the fields of bioanalytical chemistry and in vivo diagnostics. The larger surface area of the sampling device is combined with ultra-thin films to allow rapid equilibration and a significant enrichment factor of the target analytes. The blade may be wetted with solvent and can act as an electrospray ionization emitter source when a high voltage is applied or the blade can also be used for ambient or direct sampling into the mass spectrometer.

DI-SDME (SDME): Direct-Immersion Single-Drop Microextraction

A technique in which a small amount of immiscible solvent (acceptor phase, typically 1–3 μL) is suspended (typically from the end of a syringe) within a liquid sample to extract target analytes. The same syringe is then used to introduce the solvent and extracted analytes into the chromatography system. As the acceptor phase–donor phase ratio is very small, the degree of enrichment is high and good analyte sensitivity can be achieved. The technique can be optimized by altering the chemical nature and volume of the acceptor solvent, temperature and degree of agitation, and pH and ionic strength of the donor solvent. Of course, the degree of agitation should be limited to a rate that does not cause the hanging drop to be stripped from the syringe tip. A further implementation of this technique, also known as liquid‑liquid‑liquid or three-phase microextraction, involves an intermediary immiscible solvent of lower density and volume that is placed on top of the donor solvent. The hanging drop is then immersed within it. Typically, the donor phase will be aqueous, the intermediary phase an organic solvent, and the hanging drop aqueous—the analytes first partitioning into the organic intermediary and then back extracting from the intermediary solvent into the acceptor solvent. This approach allows extraction of ionogenic analytes through the manipulation of pH and ionic strength. A dynamic version of this technique has also been developed in which an organic plug within a microsyringe barrel is withdrawn into the syringe as the aqueous sample is aspirated. A thin film of acceptor solvent remains on the syringe barrel inner wall whilst the remainder of the syringe barrel is filled with donor solvent. The high interfacial area provides good extraction efficiency and this operation is repeated several times to increase the enrichment factor of the technique. SDME techniques have been used in environmental analysis and for the analysis of food and beverage for BTEX (benzene, toluene, ethylbenzene, and xylene) and priority pollutants, as well as flavour and fragrance applications. SDME has also been investigated for the analysis of contaminants and pollutants in pharmaceutical formulations.

HS-SDME: Headspace Single-Drop Microextraction

The headspace version of DI-SDME in which the acceptor phase is suspended above a solid or liquid sample to extract and enrich the volatile species that are evolved from the sample under ambient or heated/agitated conditions. The sample manipulation options for SHE such as pH, ionic strength, heating, and agitation are all also available in HS‑SDME.

HF-LPME: Hollow-Fibre Liquid-Phase Microextraction

A hollow microporous fibre of typically 1.5 cm is suspended from the end of a microsyringe and the acceptor phase is drawn into the lumen (channels) and the wall pores of the fibre. The technique then proceeds in the same fashion as HS-SDME. The high interfacial area and relatively low acceptor phase volume result in good sample enrichment factors. The advantage of the hollow fibre technique is that the acceptor phase is more mechanically stable, allowing a greater degree of sample agitation and the possibility to re-immerse the fibre into a liquid sample in the dynamic form of the technique.

CFME: Continuous-Flow Microextraction

A version of the single-drop microextraction technique in which a single drop of acceptor solvent is immersed into a flowing stream of donor solvent, usually driven by an HPLC or peristaltic pump, within a microextraction chamber.

DLLME: Dispersive Liquid–Liquid Microextraction

An extraction technique based on a ternary solvent system in which the donor and acceptor solvents are immiscible and the third solvent (often called a dispersing or consulate solvent) is fully soluble in both phases. The acceptor and dispersing solvents are rapidly injected into the donor solvent and a fine mist of acceptor solvent droplets is formed, which presents a very high interfacial area for analyte extraction. The ternary mixture is then centrifuged to separate the two immiscible phases and the acceptor solvent harvested by aspiration using a microsyringe. The acceptor–donor solvent ratio is typically very low to promote high analyte enrichment factors and this often requires the use of a conical vial to enable efficient harvesting of the acceptor solvent from below or above the donor solvent layer post centrifugation. If a dispersing solvent is not used, then the acceptor solvent can be dispersed using ultrasonic assistance and the technique is then abbreviated to UA-DLLME. The technique has applications in water and environmental, pharmaceutical and clinical, industrial chemistry, and the food, flavour, and fragrance industries.

HLLME: Homogeneous Liquid–Liquid Microextraction

An extraction technique similar to DLLME, but the three-phase system containing the donor, acceptor (immiscible), and dispersing solvents are fully mixed (homogenized) at the beginning of the experiment using agitation or sonication. Extraction occurs across the very high interfacial contact area as the immiscible phases are separated by adding salt or an ion-pair reagent, a surfactant, or changing temperature or pH value. The phase separation is often assisted by centrifugation. Typically, the acceptor–donor solvent ratio in HLLME is higher and hence enrichment factors lower than in the DLLME technique.

MASE: Membrane-Assisted Solvent Extraction

A small-scale liquid–liquid extraction technique in which the donor phase is separated from a lower volume acceptor phase through the use of a semi‑permeable membrane (typically low-density polyethylene). The analytes pass from the donor phase through the semi-permeable membrane into the immiscible acceptor phase, which is usually held within a cone of membrane suspended in the donor phase. The technique is seen as a convenient way of reducing the amounts of solvent used in traditional liquid–liquid extraction and the donor phase pH and ionic strength may be manipulated to promote extraction. Any application suitable for liquid–liquid extraction can be adapted for use with the MASE technique.

The acronym MASE is also used for matrix solid-phase extraction in which a bonded phase particulate sorbent is used as an abrasive to disrupt a solid sample matrix and is blended to facilitate extraction from complex solid samples. The sample and sorbent are transferred to a column and analytes eluted using appropriate solvents.

MESI: Membrane Extraction with Sorbent Interface

An adapted version of the dynamic headspace technique in which a hollow fibre or flat sheet polymer membrane is placed in the headspace environment of the sample. An inert gas is passed through the membrane and analytes permeable to the membrane flow from the headspace through to an adsorbent trap. Once accumulated, the adsorbed analytes are thermally desorbed into the chromatographic system.


Phew… that’s a lot of sample extraction options! I’m also convinced that I’ve missed some of the techniques and I haven’t included many of the emerging techniques used to extract analytes such as peptides, proteins, and amino acids—the article would simply have been too long. Whilst researching the options for my application development, I came across the following acronym CA-HS‑HF-LPME-GC-FID (cooling assisted headspace hollow fibre liquid phase microextraction gas chromatography flame ionization detection), which in my 35+ years of chromatography experience had me scratching my head! With all of these techniques and their sub-variants I wonder how on earth any of us ever feel empowered to make sample extraction choices beyond the realms of those with which we are already familiar. I wanted to produce this article because there was no good reference that gave a reasonable summary of all major techniques with an indication of the various pros and cons with respect to selectivity, enrichment factor, and automation capability. I’ve discovered so many new methods or adaptations and extensions of those with which I was already familiar, and it is striking how many new methods (and acronyms) have been introduced since I began my chromatography career, when liquid–liquid extraction was by far the norm and solid‑phase extraction was the emerging new sample preparation technique.

Hopefully this summary will serve the purpose of helping less experienced folk to make informed sample extraction choices when developing analytical methods. I leave you with the thoughts of the psychologist Barry Schwartz from his book, The Paradox of Choice, “when people have no choice, life is almost unbearable…But as the number of choices keeps growing, negative aspects of having multiple options begin to appear....the negatives escalate until we become overloaded. At this point choice no longer liberates, but debilitates.”


  1. B. Schwartz, The Paradox of Choice (Harper Perennial; New York, USA, 2004).

Contact Author: Incognito
Contact the Editor: kjones@mjhlifesciences.com

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