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Analyte Chemistry for SPE

The first step in developing a solid phase extraction (SPE) method is understanding your analyte(s) chemistry.  Analyte chemistry dictates physical parameters such as solubility.  It also dictates the interaction of the analyte with both the sorbent and solvents used in the SPE method, as well as any matrix components (e.g. proteins) which could interfere with the extraction method.

Analyte chemistry centers around the functional groups contained within the molecule.  There are 4 types of functional group which will direct the chemistry and physical properties of the analyte.

In SPE we want the analyte (or interferences) to strongly adsorb to the sorbent in order to obtain the cleanest extracts, hence, we need to understand the possible interactions based on functional group chemistry of both the analyte and sorbent.

Non-polar groups are fundamentally carbon in nature and for the most part do not contain any heteroatoms (N, O, S etc., Figure 1).  Any molecular interactions between these types of groups are via van der Waals forces, also known as hydrophobic interactions.  Termed hydrophobic groups, molecules which consist mainly of carbon atoms prefer a non-aqueous environment and will be soluble in non-polar (or less polar) organic solvents which also contain non-polar function groups (e.g. hexane, chloroform).

In terms of SPE sorbents non-polar, hydrophobic analytes will interact strongly with non-polar sorbents containing similar non-polar groups, e.g. C18, C8, phenyl, cyclohexyl.  Therefore, to retain non-polar analytes a non-polar sorbent would be selected.  Hydrophobic analyte/sorbent interactions are facilitated by solvent environments of low hydrophobicity, for example, water, buffers, and aqueous mixtures with low water-miscible organic solvent content - these solvents will repel the analyte into the sorbent increasing the analyte/sorbent interaction.  Solvents with significant hydrophobic character will disrupt the analyte/sorbent interactions.  

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  non-polar solvents

Figure 1: Hydrophobic non-polar groups (left) and non-polar solvents (right, hydrophobic, non-polar regions highlighted).

Polar groups contain dipoles; molecular bonds where the electrons in the bond are shared unequally due to a greater electronegativity on one of the atoms (Figure 2).  Most commonly polar groups contain at least one heteroatom such as oxygen, nitrogen, sulfur, or phosphorous, often bonded to a carbon atom.  Other, weaker common dipoles include unsaturated carbon-carbon bonds (double, triple bonds), aromatic rings, and halogen carbon bonds. 

Polar compounds will be soluble in polar solvents (also known as hydrophilic solvents), highly aqueous solvents, including pure water, most buffers, and solutions containing water or buffers in combination with water-miscible organic solvents at relatively low concentrations. 

The type of interaction between polar analytes, solvents, and sorbents is via a range of polar interactions including dipole-dipole, hydrogen bonding, π- π interactions etc. (Figure 3).  Again, polar molecules will interact with sorbents containing polar groups (e.g. unbonded silica, diol, aminopropyl, cyano).  Analyte sorbent interactions are aided by low polarity solvents such as pure organic solvents with minimal polar character (acetonitrile, THF, ethyl acetate etc.).  Polar interactions can be disrupted by application of a highly polar solvent.  Most alcohols will disrupt polar interactions due to the presence of the highly polar hydroxyl group on the alcohol.  

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  non-polar solvents
Figure 2: Polar groups (left) and polar solvents (right, hydrophilic, polar regions highlighted).
Polar interactions

Figure 3: Polar interactions.

Ionic groups are those which are capable of donating a proton (acids) or accepting a proton (bases, Figure 4).  Ionic groups can exist in either a neutral or ionized form.  The pH at which the group becomes neutral or charged is a function of the specific characteristics of the functional group.

Ionic functional groups exist in conjunction with an ion of the opposite charge, this is referred to as the counter ion, and it can have significant implications for sample preparation, in particular SPE.  For example, an ionized acid (negative charge) may have an ammonium or potassium counter ion (positive charge).  Conversely, an ionized base (positive charge) may have a chloride or acetate counter ion (negative charge).

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Figure 4: Ionic groups.

An important physical parameter of an ionic functional group is the pKa; this is defined as the negative log of the ionization constant (Ka).  By definition, a species in solution at a pH equal to its pKa will exist 50% in the neutral state and 50% in the ionized state.

When working with an ionic species in solution, it is desirable that all molecules of that species exhibit the same charge state since the properties of the neutral and charged states can be radically different.  In order to bring the functional group to either a completely charged or neutral state the pH of the solution should be at least 2 pH units away from the pKa of the species (this is often referred to as the 2 pH rule, Figure 5).

Figure 5: 2 pH rule.

Another parameter relevant to ionic analytes in solution is ionic strength.  This is the concentration of total ionic species in solution.  Ionic strength is particularly important in SPE of ionic species.  Ions in the sample solution compete with the analyte for sorbent binding sites. Poor recovery of the analyte can occur if these counter ions have a greater affinity for the sorbent than the analyte. This occurs in two ways: high counter ion concentrations (ionic strength) and high counter ion chemical affinity for the bonded phase (high counter ion selectivity).  The ionic strength can be altered by adding acids, bases, or neutral salts to the solution.

The interaction of ionic functional groups with either a solvent or sorbent environment will be influenced by the charge state.  Form a solubility perspective; ionized groups tend to favor solubility in highly polar solvents.  Therefore, bases at low pH (ionized) and acids at high pH (ionized) will be more soluble in highly aqueous solvents including pure water, most buffers, and solutions containing water or buffers in combination with water-miscible organic solvents at relatively low concentration.  Conversely, ionic groups in their neutral state tend to favor solubility in non-polar solvents such as acetonitrile, THF, ethyl acetate, chloroform etc. 

When ionic analytes interact with SPE sorbents it is with functional groups on the sorbent surface which possess the opposite charge.  For example, acidic analytes (with a negative charge) will interact with sorbents containing basic functional groups with a positive charge.  For an effective interaction to occur both species must exist in their charged state, therefore, the pH of the solution must be altered to promote this based in the analyte and sorbent pKa.

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Figure 6: Effect of pH on analyte and sorbent ionization state.

Chelating groups are often overlooked when developing a sample preparation strategy.  Chelating groups have a high affinity for transition metals, and are often combinations of polar or ionic groups together.  Some examples of chelating groups include amines, multiple amines, multiple carboxylic acids, or acids in combination with amines (Figure 7).   

Chelating functional groups contribute to the solubility characteristics of the analyte in the same way as the composite of the individual functional groups constituting the chelating group.  For example, a chelating group composed of two amines and a carboxylic acid will affect solubility in the same way as the individual two amines and acid.  Since chelating groups often contain ionizable groups the impact on solubility can also be influenced by pH.

Cheating groups may be used to effect highly selective and unique extractions when performing SPE through in situ modification of commercially available sorbents with certain metals.  Another important implication of chelating groups is their interaction with residual metals in commercial sorbents.  During the manufacturing process it is virtually impossible to create metal free sorbents due to the high cost of this process.  Any chelating groups can interact with these residual metals which may negatively impact on the SPE process.  Due to the unique nature and high energy of the chelating interaction it requires a strong approach for disruption; primarily the addition of competitive chelating agents or the addition of strong acid or bases (Figure 8).

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Figure 7: Chelating groups.

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Figure 8: Interaction of cheating groups with sorbent surface metals.

One term that is often discussed in relation to the physical properties of a molecule and how it affects, for example solubility, is the molecule’s polarity.  The overall polarity of a molecule is a sliding scale; many compounds will contain both polar and non-polar functionalities, as well as ionic groups. 

Polarity can be considered in terms of the polar and non-polar functional groups and how they balance (Figure 9).  For example, acetic acid can be considered very polar as it contains a polar carboxylic acid and a single non-polar CH3 group.  Increasing the non-polar carbon content (e.g. butyric acid) will make the molecule less polar.  On the non-polar end of the scale a compound such as icosanoic acid also contains a polar carboxylic; however, this is outweighed by the number of non-polar carbon groups.  Each strongly polar functional group needs about 6 carbons to balance.

 
Figure 9: Effect of functional groups on polarity.

It would be expected that polar compounds (or compounds containing large numbers of polar functional groups) are soluble in polar solvents; however, even compounds with highly polar groups can be insoluble if balanced by a large hydrocarbon function.  With very large molecules it is often difficult to predict solubility.


Glossary

Hydrophobicity - “Water fearing”.  Hydrophobicity is the association of non-polar groups or molecules in an aqueous environment which arises from the tendency of water to exclude non-polar molecules.  In terms of HPLC hydrophobic stationary phases will not be compatible with water and hydrophobic molecules will be insoluble in water.  Hydrophobic molecules have few polar functional groups and most have high hydrocarbon content (aliphatic and aromatic). 

Hydrophilicity - “Water loving”.  Hydrophilicity is the tendency of a molecule to be solvated by water.  In terms of HPLC hydrophilic stationary phases will be fully compatible with water and hydrophilic molecules will be soluble in water.  Hydrophilic molecules will contain polar functional groups. 

Polarity - Bonds within a molecule will be polar due to a difference in electronegativity of the two atoms.  The difference in charge produces a dipole (i.e. in HCl).  Non-polar bonds will have an electronegativity difference < 0.4 and polar bonds will have an electronegativity difference between 0.4 and 1.7. 

Molecular polarity is a result of the shape and the charge distribution in a molecule.  If the arrangement of atoms within the molecule is symmetric the charges will be balanced, therefore, the molecule is non-polar.  If there is an asymmetric arrangement of bonds within the molecule the charges will not be balanced and the molecule is polar. 

     

Polar molecules are generally soluble in water because of the polar nature of water (like dissolves like). 

Electronegativity - Electronegativity was a concept introduced by L. Pauling and describes the tendency of an atom or group to attract electrons (or electron density) towards itself.  There are several definitions and values for electronegativity in use, therefore, different values may be seen depending on the scale used.  According to Mulliken it is the average of the ionization energy and electron affinity of an atom.  The Pauling scale is more commonly used where the dimensionless relative electronegativity differences are defined based on the bond dissociation energies.

Hydrogen bond - A hydrogen bond is the electrostatic attraction between two polar groups that occurs when a hydrogen atom, covalently bound to a highly electronegative atom such as nitrogen, oxygen, or fluorine, experiences the electrostatic field of another highly electronegative atom nearby.  Hydrogen bonds can occur between molecules (intermolecular) or within different parts of a single molecule (intramolecular).  Depending on the nature of the donor and acceptor atoms which constitute the bond, their geometry, and environment, the energy of a hydrogen bond can vary between 1 and 40 kcal/mol.  This makes them somewhat stronger than a van der Waals interaction, and weaker than covalent or ionic bonds. This type of bond can occur in inorganic molecules such as water and in organic molecules like DNA and proteins.

Dipole-dipole interaction - Dipole-dipole interactions are electrostatic interactions between molecules which have permanent dipole(s). These interactions tend to align the molecules to increase attraction (reducing potential energy).  Polar molecules have a net attraction between them. Examples of polar molecules include hydrogen chloride (HCl) and chloroform (CHCl3).  Often molecules contain dipolar groups, but have no overall dipole moment. This occurs if there is symmetry within the molecule that causes the dipoles to cancel each other out. This occurs in molecules such as tetrachloromethane and carbon dioxide. The dipole-dipole interaction between two individual atoms is usually zero, since atoms rarely carry a permanent dipole.

Molecules which contain a permanent dipole can induce a dipole in other molecules which are nearby resulting in a dipole-induced dipole interaction.

π-π interaction - π-π interactions are a type of non-covalent interaction that involves π systems (electrons in p-orbitals).  Just like in an electrostatic interaction where a region of negative charge interacts with a positive charge, the electron-rich π system can interact with a metal (cationic or neutral), an anion, another molecule or another π system.

 

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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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