Chromatography

Efficient SPE (Part 1 of 2)

Efficient SPE – Modern polymer and classical silica phases

2CB-columns300

Solid Phase Extraction (SPE) is an effective method for sample preparation used in the most laboratories today. For nearly 30 years diverse SPE phases, packed in columns, cartridges and 96-well plates or as bulk material, are commercially available and will be expanded with newly developed materials almost every year. While at the beginning silica-based sorbents were predominant, polymers have entered into the market in the last years. More than 50 different types of SPE phases make a decision for the user not easy. Especially for RP phases, which can be used for multiple applications, the question is: “Silica or polymer RP phase?”

The following article will present the main silica and polymer sorbents with their features and advantages regarding an effective application.

Basic Principles

The main purpose of SPE is to eliminate undesirable substances from a sample (clean-up) while at the same time concentrating the component(s) of interest in the sample (enrichment).  The result is a reduced number of components in the sample matrix prior to analysis by techniques such as HPLC, GC, TLC, MS, NMR, ELISA, and so on.

Whether the SPE adsorbent is silica-based or polymer-based, the main steps of the SPE procedure are generally the same: (a) conditioning of the adsorbent, (b) the sample application by vacuum (e.g. with vacuum manifold) or pressure (adsorption), mostly a washing of adsorbent, and (c) the elution (desorption) of the components of interest.

basic principle of spe procedure

 

 

 

 

 

 

 

 

 

 

SPE phases are most commonly categorized by primary interaction mechanism with the analyte of interest:

  • Reversed Phase RP (extraction of hydrophobic analytes from aqueous matrix)
  • Normal Phase NP (extraction of polar analytes from non-polar organic solvents)
  • Ion exchange IC (extraction of charged analytes from aqueous or non-polar organic samples)

Phases with combined multiple interaction mechanisms are called mixed-mode phases. A classification by silica and polymer sorbents is generally not realised. But for a selection of a phase, it is important in regard to a successful and effective application.

Silica Phases

Silica phases with reversed phase mechanisms comprise – inter alia – silica with -C18, -C8, -C4 and -C6H5 surface modification. Their primary interactions to analytes from mostly aqueous samples are of hydrophobic nature. Solvents with lower polarity in comparison with water (MeOH, CH2Cl2, CHCl3, … hexane) are selected for the elution.

Reversed phase silicas

 Phase  Modification  Key feature
 C18 ec  octadecyl, endcapped  highest hydrophobicity
 C18  octadecyl, non-endcapped  secondary interactions with polar groups
 C18 Hydra  octadecyl, polarly adjusted  selectiv for polar analytes (pesticides, phenols etc.)
 C18 PAH  octadecyl, adjusted for PAHs  selectiv for PAHs
 C8  octyl  strong secondary interactions
 C4  butyl  very strong secondary interactions
 C6H5  phenyl  additional π-π-interactions
 CN  cyanopropyl  additional electronical interactions

 

The most common SPE silica phases are C18 modified silicas. Manufacturers differentiate basically 3 types of C18 sorbents: (a) endcapped (C18ec), (b) non-endcapped (C18) and (c) phases, adjusted to polar analytes (e.g. C18 Hydra).

Silica C18 ec is distinguished by the highest hydrophobicity of the silica phases. Thus, it is advantageous for clean-up of samples with large structural variations. High polarity differences effect thereby a separation of the analytes from matrix ingredients. Above all, substances with non-polar structures (e.g.fatty acids) are well-extracted from aqueous matrices with polar ingredients (e.g. blood, urine, rich food) for example.
A generally applicable phase is the non-endcapped C18 phase, which forms additional interactions with polar groups of analytes. Many organic substances from aqueous solutions can be cleaned and enriched well in this way.
C18 phases, specially adjusted to polar analytes are suitable for polar compounds such as pesticides and their metabolites, as well as substances with hydroxy, amine or nitro functional groups. They are distributed under trade names like C18 Polar Plus (Mallinckrodt Baker), C18 OH (Varian) or C18 Hydra (MACHEREY-NAGEL).

C18 PAH sorbent was developed specially for enrichment of PAHs from water in order to achieve higher recovery rates for EPA PAHs as compared to conventional phases.

C8 and C4 phases feature stronger secondary interactions with polar compounds, because shorter alkyl chains do not shield as strong as a C18 modification. These phases are recommended, if compounds are too strongly bonded on C18 – and elution yields only moderate recovery rates (e.g. drugs from plasma).

Phenyl phase C6H5 shows considerably different selectivity for the extraction of aromatic and non-aromatic compounds (e.g. flavours from brandy) in comparison to C18 and C8, due to additional π-π-interactions. Also cyanopropyl silica CN facilitates selective interactions by a high electron density of the CN group. It can be used under RP and NP conditions.

Contrary to polymeric RP phases, whose popularity increased significantly in the last years, silica RP phases feature a broad range of specific selectivities. They are described in many application notes, in literature and in databases (see SORBTECH application database) and are mostly more inexpensive.

Non-modified normal phase silica forms polar interaction, like hydrogen bonds and dipole-dipole interactions. Samples are mostly organic extracts, solvents with a higher polarity as the sample solvent (e.g. hexane) are used for elution (e.g. ether, MTBE, THF, acetone, CH2Cl2, CHCl3).

Normal phase silicas and sorbents

 Phase  Modification  Key feature
 Silica Gel  unmodified  strong affinity to polar compounds (hydrogen bonds)
 OH  diol  besides hydrogen bonds weakly hydrophobic interactions
 NH2  aminopropyl  additional weak anion exchange
 Alumina *)  aluminium oxide  alternative selectivity for SiOH                      *no silica
 Florisil® *)  magnesium silicate  alternative selectivity for SiOH                     *no silica

 

Unmodified silica SiOH has a very strong affinity to polar compounds. It has a high importance for the cleaning of alkane extracts from oils and fatty matrices (e.g. hydrocarbons from diesel, antibiotics or pesticides from fish).

Polar aminopropyl silica NH2 is characterised by additional weak Anion Exchange properties. In aqueous media with pH < 7.8 it acts as Anion Exchanger, whereas in non-polar solvents the hydrogen bonds dominate. It is well-suitable for the extraction and fractionation of lipids.

Further NP materials are silica-based diol phases OH, but also aluminium oxides Alox and magnesium silicate Florisil®.

Bonded Phase silica with Strong Anion and Strong Cation Exchangers are available.  Interactions between charged analytes and the functional groups of the Exchanger are formed, depending on the pH of the aqueous sample. A separation of the ionic bond is effected by a changed pH value of the solvent or a change in ionic strength of the buffer at the elution.

Ion exchanger silicas

 Phase  Modification  Key feature
 SB (SAX)  quaternary amine  strongly basic anion exchanger
 SA (SCX)  benzenesulphonic acid  strongly acidic cation exchanger
 PCA (WCX)  propylcarboxylic acid  weakly acidic cation exchanger
 PSA  propylsulphonic acid  very strong cation exchanger

 

Cation Exchange silicas – bonded phase silica with terminal groups such as benzenesulfonic acid SA (SCX), propylcarboxylic acid PCA (WCX), and propylsulphonic acid PSA modifications are available. They differ by their Exchanger strength. Thus, they are used selectively depending on the target analytes.  For example SA are used to capture amino acids, PCA to capture strong cations, and PSA to capture weak cations.

For special applications in pharmaceutical, environmental and food sector special and combined silica phases are available. These phases are exactly adjusted on special tasks (analyte and matrix) and often described and required by public regulations (e.g. Pharmacopeia, EPA, EN, ISO, DIN). For a summary of SPE phases for special applications please click here!

 

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