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SPE Mechanism Selection

Last month we looked at analyte assessment for solid phase extraction (SPE), which is the first step in developing an SPE protocol.1 This involved understanding the analyte chemistry based on the functional groups contained in the molecule - this will affect many of the physical properties of the molecule including solubility and molecular interactions between the analyte, sorbent, and any other matrix components in the sample. 

The next step is to select the correct retention mechanism which will be the focus of this article (Figure 1). 

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Figure 1: Available SPE retention mechanisms.

First consider the analyte and ask a simple question; are there ionizable groups?  If the answer is yes then try to utilize an ion exchange mechanism.  If there are no ionizable groups the sample matrix must be considered as this will determine the separation mechanism, this is due to certain sample matrices promoting analyte retention better by one retention mechanism than another.

If the sample matrix is aqueous or a water miscible organic solvent then a non-polar (reversed phase) mechanism will be appropriate.  If the matrix is water immiscible a polar (normal phase) mechanism will be used.  Following this initial selection of a retention mechanism the analyte chemistry should be considered in greater detail to maximize the retention mechanism(s) available and to provide information that will apply to development of the other SPE protocol steps (e.g. wash solvent and elution solvent strength).

Parameter Example Retention Mechanism
Hydrophobic, non-polar, alkyl, aromatic Non-polar
Polar, hydroxyls. amines, dipoles Polar
Basic groups, cations Cation exchange
Acidic groups, anions Anion exchange

Table 1: Analyte functional groups in relation to retention mechanism.

Sample Matrix Example Retention Mechanism
Aqueous, low ionic strength River water, biological fluids Non-polar, ion-exchange
Aqueous, high ionic strength Sea water, biological fluids (plasma or urine) Non-polar
Non-polar Hexane or olive oil Polar

Table 2: Sample matric properties in relation to retention mechanism.

Figure 2: Retention mechanism selection flow chart based on sample matrix properties.

As well as the stationary phase chemistry, the type of stationary phase, i.e. silica or polymeric, must also be selected.  There are pros and cons of both types of matrix materials, and this decision should be made based on analytical requirements.

Support Material Pros Cons
Silica Wide variety of modified silica sorbents Fines are more prevalent with irregularly shaped particles
Inexpensive Sensitive to some solvents and pH conditions (below pH 2 and above pH 8 the sorbent rapidly deteriorates)
Many validated methods  
Polymer No surface silanol groups which can contribute to unwanted secondary interactions, (particularly advantageous when analyzing basic compounds which are prone to secondary interactions with the silica surface) Fewer modified resins
Higher surface area; hence, they have a higher loading capacity compared to traditional silica-based sorbents  
Stable at pH extremes (pH 0-14)  
Do not have the flow-rate dependence of more traditional silica-based materials  

Table 3: Pros and cons of silica vs. polymer support materials.

 

Example 1 - Testosterone in plasma
Testosterone has no ionizable groups and is moderately hydrophobic (log P 3.37, Figure 3); hence we must consider the sample matrix to select the retention mechanism.  The sample matrix is plasma which is aqueous; therefore, a non-polar retention mechanism would be selected.   Following this the analyte functional groups could be considered in more detail to maximize the retention mechanism.

Figure 3: Testosterone.

Example 2 - Triazines in soil
Triazines are a class of herbicides; there are three main species which are all structurally similar (Figure 4).  These compounds need to be extracted from different matrix types, including soil, muscle tissue, and corn oil.

Figure 4: Atrazines; pKa values and atrazine ionization.1-2

 

All three of the triazine species contain ionizable functional groups with the basic pKas being accessible under standard pH values.  Reducing the pH to 2 units below the basic pKa (pH ~2.0) will result in the fully protonated species, therefore, a cation exchange SPE mechanism can be utilized.  Strong ion exchange media are recommended for initial method development.  With regards to the sample matrix, soil is a solid; therefore the analytes will have to be extracted prior to SPE, atrazines are insoluble in water therefore an organic solvent will be used.  Acetonitrile, which is water miscible, has been used for this extraction procedure.

There are many common situations whereby the matrix nature prevents facile use of a particular mechanism — for example, when working with a urine sample, even if the analyte is ionizable, ion-exchange may be an unreliable mechanism choice, because urine may contain high and variable salt content, which can compromise analyte retention through competition.

Another example occurs when working with adipose tissue extracts — in this case there is a very high lipid content which tends to saturate all available active sites on a non-polar surface, thus inhibiting retention of an analyte via a non-polar mechanism.

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Figure 5: Effect of sample matrix on retention mechanism.

 

Summary
In summary, analyte and matrix properties must be taken into account when considering an appropriate retention mechanism.  The following table (Table 4) and flow chart (Figure 6) summarize retention selection related to analyte and matrix properties.

Retention Mechanism Typical Phases Matrix Properties Analyte Properties Load Solvent Wash Solvent Elution Solvent
Polar Diol, aminopropyl, cyanopropyl, silica Organic (i.e. organic phase from a LLE Water soluble, moderately to strongly polar Small ε0, non-polar (hexane, MTBE) Small ε0, non -polar (hexane, MTBE) Large ε0, polar (H2O, MeOH, EtOH, MeCN)
Non-polar C18, C8 (silica)PS-DVB, DVB (polymer) Aqueous (foods, biological fluids) Organic soluble, hydrophobic (strongly non-polar) High P’, polar (H2O, MeOH/H2O, MeCN/H2O) Aqueous, aqueous/organic Organic, intermediate P’ (MeOH, MeCN)
Cyclohexyl, phenyl, diphenyl Aqueous (foods, biological fluids) Organic soluble, hydrophobic (moderately non-polar) High P’, polar (H2O, MeOH/H2O, MeCN/H2O) Aqueous, aqueous/organic Organic, intermediate P’ (MeOH, MeCN)
C6, C4, C2 Aqueous (foods, biological fluids) Organic soluble, hydrophobic (slightly polar to moderately non-polar) High P’, polar (H2O) Aqueous, aqueous/organic Organic, intermediate P’ (MeOH, MeCN)
Mixed mode Mixed silica
(e.g. C8/SCX) or polymer.  Most commonly a combination of an hydrophobic and ion exchange functional group
Aqueous (foods, biological fluids) Polar, hydrophilic, or hydrophobic Pre-treated sample (diluted, pH adjusted) Aqueous and organic Organic/acid or base
Cation exchange Weak
Carboxylic acid
Aqueous (foods, biological fluids) Basic (cationic) Water or buffer (pH = pKa - 2) Aqueous High ionic strength, pH above analyte pKa, competition with a cation (e.g. Ca+) with greater affinity for the sulfonic acid
Strong
Alkyl sulfonic acid, aromatic sulfonic acid
Anion exchange Weak
Amino, 10, 20-amino
Aqueous (foods, biological fluids) Acidic (anionic) Water or buffer (pH = pKa + 2) Aqueous High ionic strength, pH below analyte pKa, competition with an anion (e.g. SO3-) with greater affinity for the positively charged amine
Strong
Quaternary amine

Table 4: Retention mechanism based on analyte and matrix properties. ε0 = solvent strength, P’ = polarity.

Figure 6: SPE sorbent selection flow chart.

References

  1. https://chemicalize.com
  2. https://www.agilent.com/cs/library/eseminars/Public/Secrets%20of%20SPE.pdf

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Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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