Sample Preparation
Solid Phase Extraction – Mechanisms
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Aims and Objectives
Aims and Objectives
Aims


To highlight the various steps in the Protocol for various Modes (Sorbents) for
conventional solid phase extraction procedures including:
o Non-polar SPE
o Polar SPE
o Cation-Exchane SPE
o Anion-Exchange SPE
o Mixed mode SPE
For each mode the various portocol steps will be examined in terms of the solvents
and solutions used, sorbents used, solvent additives, pH and ionic strength
manipulation
Objectives
At the end of this Section you should be able to:





Identify the correct SPE mode to optimise the selectivity of the procedure for the
analyte of interest
Choose the correct sorbent for various modes of SPE
Optimse analyte retention through correct sorbent equilibration, conditioning and
sample loading by controlling sorbent activity, sample and sorbent ionic strength,
analyte and sorbent degree of ionisation
Maximise extract cleanliness by optimising the wash solution solvent strength and
ionic strength and pH where appropriate
Maximise analyte recovery and selectivity by optimising the elution solvent and
ionic strength and pH where appropriate
Content
Non-polar SPE
Overview
Sample Pretreatment
Equilibration and Sample Loading
Washing and Analyte Elution
Polar SPE
Overview
Sample Pretreatment
Sorbent Conditioning, Equilibration and Sample Loading
Sorbent Washing and Analyte Elution
Cation-Exchange SPE
Overview
Sample Pretreatment
Sorbent Conditioning, Equilibration and Sample Loading
Washing and Analyte Elution
Anion-Exchange SPE
Overview
Sample Pretreatment
Sorbent Conditioning, Equilibration and Sample Loading
Washing and Analyte Elution
Minimum pKa Differential
Mixed-Mode SPE
Overview
Sample Pretreatment
Sorbent Conditioning, Equilibration and Sample Loading
Washing and Analyte Elution
Summary
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Non-polar SPE
Overview
Non-polar SPE is the extraction process where molecules with non-polar functional
groups are extracted from predominantly polar matrices onto sorbents exhibiting non-polar
surface properties.
In non-polar SPE, the retention mechanism is the interaction of non-polar groups on the
analytes of interest and the non-polar functional groups on the sorbent, via Van der Waals
forces. This interaction is disrupted (causing elution of the analytes) by solvents with
significant non-polar character. This interaction is facilitated (allowing retention of the
analytes) by solvents having very little non-polar character; or, in other words, very polar
solvents.
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Loading: Column washed with a polar solvent
Since water is the most polar solvent of all, non-polar SPE is an ideal technique for
extraction of aqueous samples. Since most pharmaceutical samples encountered in
bioanalysis are aqueous, non-polar SPE is widely used.
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Elution: Column washed with a non polar solvent
Examples of common sorbents used in non-polar SPE include C18, C8, C6, C4, C2,
phenyl, cyclohexyl, and propylcyano. These surface chemistries are most often bonded to
a base of silica gel. In addition, many commercial polymers also exist that are
fundamentally non-polar in nature.
Sorbents
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Sample Pretreatment
There are six basic steps in non-polar SPE, as in most SPE procedures. These steps are
sample pretreatment, sorbent conditioning, sorbent equilibration, sample loading,
washing, and analyte elution.
Sample pretreatment
Sample pretreatment of aqueous samples for non-polar SPE typically involves sample
dilution, pH adjustment/control, disruption of protein binding, and possibly filtration if the
sample contains particulate matter.
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In general, aqueous samples such as plasma or serum should be diluted at least one-toone with an aqueous buffer. Dilution reduces the viscosity of the aqueous sample,
improving chromatographic mass transfer, and thus improving the efficiency of the
extraction.
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Dilution of plasma sample
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Poor analyte retention
pH adjustment may be necessary for certain ionizable species. For ionizable analytes
whose polarity changes dramatically as a function of their charge, the sample pH should
be adjusted to neutralize the analytes, thus increasing their hydrophobicity, and improving
retention on a non-polar sorbent.
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Good analyte retention
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Protein binding, if active, must be disrupted so the analytes are free in solution to partition
onto the sorbent surface. This disruption may be implemented by addition of an organic
solvent (such as methanol), addition of chaotropic agents like urea or guanidine, or a pH
adjustment.
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Protein binding
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Protein binding disruption by organic solvent addition
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Finally if large amounts of particulates are present, it may be necessary to filter the
samples. For smaller amounts of particulates, the SPE column frit may do an adequate
job of filtration.
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Filtration process
Equilibration and Sample Loading
Many non-polar SPE sorbents are not water-wettable. For such sorbents, analytes
applied in an aqueous sample will not partition onto the sorbent, due to lack of a proper
phase interface between the dry sorbent surface and the sample. Conditioning with a
water-miscible organic solvent such as methanol, acetonitrile or tetrahydrofuran wets the
sorbent surface and allows the analytes to break through the surface tension of the water
and move into the sorbent.
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Poor wetting of surface –little or no analyte retention
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Column conditioning
Just prior to applying the aqueous sample, the conditioning solvent should be displaced
from the sorbent using an equilibration solvent. The equilibration solvent also prepares
the sorbent surface to receive the sample. In order that the chemistry of the sorbent
remains constant throughout the sample loading step, the equilibration solvent should be
as similar to the composition of the sample as possible. For aqueous samples, in most
cases, this means equilibrating the sorbent with the same buffer as used to dilute the
sample.
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Column equilibration
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Good surface wetting and equilibrium – analyte retention
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During sample loading, the single most important consideration is the speed with which
the sample is applied. Too rapid an application will not allow sufficient time for the
analytes to partition into the sorbent surface, resulting in analyte breakthrough and poor
recovery.
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Analyte velocity too high to interact with sorbent functional groups
As a reference, for a 100 milligram bed mass, a reasonable sample loading flow rate for
non-polar SPE is no faster than a few milliliters per minute.
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Optimum analyte velocity enables interaction with sorbent functional groups
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Washing and Analyte Elution
The non-polar retention mechanism is facilitated by polar solvents (for example, water)
and disrupted by solvents containing some non-polar character (methanol, acetonitrile,
isopropanol, tetrahydrofuran, and even more non-polar organics).
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Solvent washing and analyte elution
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Therefore, in order to elute first interferences, then analytes from the surface, it is
necessary to apply solvents of increasing hydrophobicity to the sorbent bed. This is
accomplished by adding the above organics to the buffer in ever higher concentrations.
Important:
Organic solvents are added to the buffer system to alter nonpolar retention. A common selection of organic solvents is:
1.
2.
3.
4.
5.
6.
Water
Methanol
Acetonitrile
Isopropanol
Tetrahydrofuran (THF)
Chloroform
The optimum wash solvent to use for non-polar extraction is a solvent that is non-polar
enough to elute as many interferences as possible, but not sufficiently non-polar to elute
the analytes. The best way to identify such a solvent is to wash the surface starting with
the equilibration buffer, then use progressively more non-polar solvents; for example, first
100% buffer, then 90% buffer / 10% organic, then 80% buffer / 20% organic, etc. Each
wash step should be collected and analyzed for presence of analytes. The wash
containing the highest percentage organic but no analytes will provide the cleanest final
extract.
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Buffer composition
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For elution, it is best to use the weakest elution solvent that has just enough
hydrophobicity to give quantitative analyte elution. Such a solvent will leave behind on the
sorbent surface the maximum number of contaminants. Too strong an elution solvent will
bring these additional contaminants off with the analytes. This solvent may be identified in
the same experiment used to identify the optimum wash solvent.
Polar SPE
Overview
Polar SPE is the extraction process where molecules with polar functional groups are
extracted from predominantly non-polar matrices onto sorbents exhibiting polar surface
properties.
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Polar interactions in a non-polar matrix
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Polar interactions disrupted by a polar solvent
In polar SPE, the retention mechanism is the interaction of polar groups on the analytes of
interest and polar functional groups on the sorbent, typically via dipole-dipole or hydrogenbonding interactions. These interactions are disrupted (causing elution of the analytes) by
solvents with significant polar character (i.e., containing dipoles and/or heteroatoms,
especially polar oxygen or nitrogen atoms). This interaction is facilitated (allowing
retention of the analytes) by solvents having very little polar character; or, in other words,
very non-polar solvents.
Most pharmaceutical biological samples are aqueous in nature. Water is a very poor
solvent in which to begin a polar SPE protocol, since by definition water is extremely
polar, making it a very strong elution solvent for polar extractions. Therefore, analytes
applied to polar SPE columns directly from aqueous matrices tend to breakthrough the
sorbent bed. An ideal initial step for polar SPE of biological samples is a liquid/liquid
extraction into an appropriate non-polar solvent.
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Sample applied in aqueous matrix
-analyte breakthrough occurs
Sample applied in organic matrix
-good analyte retention on sorbent
Aqueous versus organic matrix retention
Examples of common sorbents used in polar SPE include diol, aminopropyl, propylcyano,
unbonded silica, alumina and Florisil™. Less common but nevertheless quite useful are
ion-exchangers, which do not function as ionic sorbents in non-polar solvent
environments, but often have considerable polar character in a non-polar organic solvent.
Other than alumina and Florisil™, most phases used for polar extractions are silica-based
— polymer-based polar sorbents are rare.
Common sorbents
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Sample Pretreatment
There are six basic steps in polar SPE, as in most SPE procedures. These steps are
sample pretreatment, sorbent conditioning, sorbent equilibration, sample loading,
washing, and analyte elution. However, the sorbent conditioning and sorbent equilibration
steps in polar SPE are often combined into a single step.
Sample pretreatment of aqueous samples for polar SPE typically involves transferring the
analytes into a non-polar organic solvent, via a liquid/liquid extraction. This is necessary
since the original aqueous sample matrix will generally cause analyte breakthrough if
applied directly to the polar SPE column.
As in any liquid/liquid extraction, it may be necessary to adjust the pH of the original
sample to facilitate transfer into the organic solvent. For example, if the analytes are
ionic, the pH of the aqueous sample should be adjusted to a value that will convert the
analytes to the neutral (uncharged) form, as this will enhance solubility in the non-polar
organic.
It may also be appropriate to filter the original sample to remove particulates that are
present. Sometimes the initial liquid/liquid extraction will cause proteins in the sample to
precipitate, and these should be removed prior to sample application to the SPE column.
Protein binding of analytes is less of a concern in this process than in non-polar SPE,
since the initial liquid.
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Filtration process
Note - When performing liquid/liquid extraction as the first step of a protocol prior to polar
SPE, it is not necessary to be greatly concerned as to the quality of the extract. Instead,
the focus should simply be on quantitative transfer of the analyte into the non-polar
organic. Unlike the process whereby liquid/liquid extraction is the primary overall sample
preparation procedure, in this case the polar SPE process will provide the necessary
selectivity in the sample cleanup.
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SPE step provides
high selectivity
Clean quantitative
analyte extraction
Low selective quantitative
analyte extraction
Sorbent Conditioning, Equilibration and Sample Loading
Sorbent conditioning and equilibration for polar SPE is usually quite simple. In most
cases, the sorbent bed is prepared by treatment with a few column volumes of a non-polar
organic solvent; in many cases this is the same solvent into which the analytes have been
extracted from the original aqueous sample. So, for example, if the original sample has
been extracted with hexane, transferring the analytes into the hexane layer, then the
sorbent bed should be conditioned with a few column volumes of pure hexane. In most
cases it is not necessary to equilibrate the sorbent as an additional step prior to applying
the hexane extract of the original sample.
During sample loading, the single most important consideration is the speed with which
the sample is applied. Too rapid an application will not allow sufficient time for the
analytes to partition into the sorbent surface, resulting in analyte breakthrough and poor
recovery.
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Analyte velocity too high to interact with sorbent functional groups
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For polar SPE, the sample application rate should be slower than for a typical non-polar
SPE procedure. This is necessary in order for the analyte polar groups to orient in space
as they pass through the sorbent bed, such that these groups come in close proximity to
polar sorbent groups, thus allowing a retentive interaction to occur.
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Optimum analyte velocity enables interaction with sorbent functional groups
Sorbent Washing and Analyte Elution
The polar retention mechanism is facilitated by non-polar solvents (for example, hexane,
chloroform, MTBE) and disrupted by solvents containing some polar character (THF, ethyl
acetate, isopropanol, acetonitrile, methanol, water and combinations of water-miscible
organics with water). Therefore, in order to elute first interferences, then analytes from
the surface, it is necessary to apply solvents of increasing hydrophilicity to the sorbent
bed. This is accomplished by moving from the first group of solvents to the second group
mentioned above.
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Srong analyte sorbent interaction in non-polar solvent
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Polar interactions disrupted by a polar solvent
The optimum wash solvent to use for polar extraction is a solvent that is polar enough to
elute as many interferences as possible, but not sufficiently polar to elute the analytes.
The best way to identify such a solvent is to wash the surface starting with the solvent,
then use progressively more polar solvents; for example, if the sample extract was in
chloroform, first wash with 100% chloroform, then 90% chloroform/10% methanol, then
80% chloroform/20% methanol, etc. Each wash step should be collected and analyzed
for presence of analytes. The wash containing the highest percentage organic but no
analytes will provide the cleanest final extract.
For elution, it is best to use the weakest elution solvent that has just enough hydrophilicity
to give quantitative analyte elution. Such a solvent will leave behind on the sorbent
surface the maximum number of contaminants. Too strong an elution solvent will bring
these additional contaminants off with the analytes. This solvent may be identified in the
same experiment used to identify the optimum wash solvent.
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Cation-Exchange SPE
Overview
Cation-exchange SPE is the extraction process where molecules with basic (cationic)
functional groups are extracted from predominantly aqueous matrices onto sorbents with
acidic (anionic) surface groups.
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Cationic analyte interacting with an ionic sorbent
In cation-exchange SPE, the retention mechanism is the interaction of charged, cationic
groups on the analytes of interest and charged, anionic functional groups on the sorbent,
via electrostatic (ionic) interactions. These interactions are disrupted (causing elution of
the analytes) by one of three primary methods: a) high ionic strength, in which the
presence of large numbers of ions in the elution buffer creates competition with the
analyte groups for the sorbent groups
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Analyte/sorbent interaction disrupted by high ionic concentration solvent
b) a pH change, in which either the charged analyte groups are neutralized, or the
charged sorbent groups are neutralized
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Charge neutralisation of the sorbent surface causes elution of the analyte
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or c) the use of a buffer containing cationic species with a high affinity for the sorbent
functional groups.
a
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c
b
Introduction of low concentrations of high surface affinity ions causes analyte elution
The cation-exchange interaction is facilitated by a solvent environment that is
simultaneously the converse of each of the conditions “a.” “b.” and “c.” above; that is, a
solvent of low ionic strength, at a pH such that both the analyte groups and the sorbent
groups are charged, and if containing other cations, these cations have a lower affinity for
the sorbent than the analyte functional group cations.
Most pharmaceutical biological samples are aqueous in nature. A protic environment is a
necessity for ion-exchange to occur, and water is highly protic, offering a good starting
point for cation-exchange SPE. However, some biological samples (especially urine and
certain cell-growth buffers) exhibit very high salt (very high ionic strength) and at the very
least require significant dilution to reduce the ionic strength prior to application to an SPE
column.
The most common sorbents used in cation-exchange SPE have either carboxylic acid or
sulfonic acid groups on the surface. These groups can be ionized via a suitable pH
adjustment, thus allowing positively charged cationic analytes to retain. These sorbents
are typically available in both silica-based and polymer-based chemistries. Typical strong
cation exchange sorbents such as sulfonic acid phases are ionised over the whole pH
range. Weaker sorbents such as the carboxylic acid derived phases can have their
degree of ionization (and hence retention strength) altered over a wide pH by
manipulating the solution pH.
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At high pH values, all
sorbent groups are nonionised
At medium pH values,
some sorbent groups are
ionised
At high pH values, all
sorbent groups are
ionised
All silica-based sorbents, including unbonded silica itself, also are capable of being
employed as weak cation-exchange surfaces, since residual silanol groups present on the
surface are weakly acidic. If bonded silicas are to be used, extractions typically work best
with sorbent having short bonded groups (such as propylcyano), since these shorter
chains provide more access to the silanol groups as compared to long chains (such as
C18) which sterically block most of the residual silanols.
At high pH values, all
sorbent groups are nonnon-ionised
At medium pH values,
some sorbent groups
are ionised
At high pH values, all
sorbent groups are
ionised
At high pH values, all sorbent groups are ionised
Acidic alumina may also be used quite effectively as a cation-exchange sorbent. However,
the user should pay careful attention to the quality of the alumina since these materials
often vary widely in properties batch- to-batch.
All silica-based sorbents, including unbonded silica itself, also are capable of being
employed as weak cation-exchange surfaces, since residual silanol groups present on the
surface are weakly acidic. If bonded silicas are to be used, extractions typically work best
with sorbent having short bonded groups (such as propylcyano), since these shorter
chains provide more access to the silanol groups as compared to long chains (such as
C18) which sterically block most of the residual silanols.
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Sample Pretreatment
In addition to sample filtration, if required, and disruption of protein binding, it is necessary
prior to cation-exchange extraction to prepare a sample such that it exhibits the following
two primary qualities: a) the lowest ionic strength possible,and b) a pH such that the
analytes in the sample exist in a charged state.
If the sample is serum or plasma, which have relatively low ionic strengths to start, simple
dilution of the sample with water or a very low ionic strength buffer may suffice to reduce
the original sample’s ionic strength sufficiently for retention. In some cases, such as with
urine samples, the ionic strength is so high that it may be necessary to use a non-polar
SPE procedure to remove excess ions prior to the cation-exchange procedure. This is
often a very simple approach, since inorganic ions are readily washed off of non-polar
phases with simple buffers, in most cases leaving the analytes retained. After analyte
elution from the non-polar sorbent, the low-ionic strength extract may be diluted with water
or a buffer, and applied to the cation-exchange sorbent, where the analytes are again
retained and reconcentrated.
No retention – analyte in uncharged form
Appropriate pH adjustment of the sample to charge the ionic groups on the sample is
necessary to get quality retention on the cation-exchange sorbent. Basic or anionic groups
on the analytes, which are the typical targets for a cation-exchange extraction, are ionized
by dropping the sample pH to a minimum of two pH units below the pKa of the relevant
group. It is recommended to perform this pH adjustment with the strongest acid or the
most powerful buffer possible so as to minimize the increase in ionic strength of the
sample. For example, it is likely to take a much smaller quantity of hydrochloric acid to
drop the pH of a typical amine than of a weaker acid such as acetic acid.
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Good retention – analyte in charged form
Sorbent Conditioning, Equilibration and Sample Loading
Sorbent conditioning for cation-exchange SPE is similar to that used for non-polar SPE. In
most cases, the sorbent bed is first treated with a few bed volumes of a water-miscible
organic solvent, such as methanol, acetonitrile, or THF. This is typically followed by a few
bed volumes of pure water, to prepare the sorbent for the equilibration step.
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Sorbent activated (b) using a non-polar organic solvent
Sorbent equilibration in cation-exchange SPE must accomplish two main purposes: a) to
convert the sorbent counter-ion to one that is easily displaced by the analytes, and b) to
adjust the sorbent pH to an appropriate value so that the sorbent groups are charged.
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At high pH values, all
sorbent groups are nonionised
At medium pH values,
some sorbent groups are
ionised
At high pH values, all
sorbent groups are
ionised
To facilitate analyte retention on the cation-exchange sorbent, it is necessary to
equilibrate with a buffer or a salt solution containing a cation of lower affinity for the
sorbent than the analyte group. This counter-ion will be most readily displaced by the
analyte cation group. The lowest affinity cation available is hydrogen ion (H+), and many
cation-exchange sorbents may be purchased in the hydrogen form. If the native counterion is not hydrogen and must be converted, this is best achieved by washing with an acid,
such as hydrochloric, acetic, or formic. However, care should be taken in the use of very
strong acids such as hydrochloric, since these acids may degrade the integrity of a silicabased sorbent.
a
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b
Counter ion too retentive – complete analyte breakthrough
For pH adjustment of an acidic sorbent, it is necessary to raise the pH of the sorbent to a
minimum of two pH units above the pKa of the sorbent in order to ionize the sorbent
groups. The pKa values for most commercial sorbents are available from the respective
sorbent manufacturer.
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Suitable counter ion – complete analyte retention
Cation-exchange is extremely sensitive to sample loading speed. In particular, it is very
easy to pass the sample through the sorbent bed too quickly, in which case breakthrough
(inadequate analyte retention) is likely. A common reference point for sample application
to a cation-exchange sorbent is no faster than 1 milliliter per minute for a 100 milligram
sorbent bed. This speed limitation is a consequence of the “point-to-point” nature of ionexchange, i.e., a very specific individual group on an analyte molecule must encounter a
very specific group on the sorbent in order for retention to occur.
Washing and Analyte Elution
The cation-exchange retention mechanism is facilitated by a) low-ionic strength buffers, b)
buffers at a pH such that both the analyte and the sorbent remain charged, and c) organic
solvents, which have little or no effect on the ionic retention mechanism. Any of these
reagents make excellent wash solvents for cation-exchange.
Conversely, analyte elution is facilitated by a) high-ionic strength buffers, b) buffers at a
pH such that either the analyte or the sorbent are neutralized, or c) buffers containing a
counter-ion that has a very high affinity for the sorbent (much higher than the analyte
cation group affinity for the sorbent).
When using high-ionic strength as the elution mechanism, it is often important to consider
compatibility of the elution buffer with the final analytical technique to be used. LC/MS
systems, for example, do not cope well with high ionic strength samples, so it may be
necessary to dry down and reconstitute the elution solvent. In this case, a volatile buffer
salt, such as ammonium acetate should be used rather than a non-volatile salt such as
sodium chloride.
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Analyte elution facilitated by high ionic strength buffer
pH adjustment to facilitate analyte elution offers two possibilities: a) neutralization of the
analyte groups, by raising the pH to a minimum of two pH units above the analyte pKa, or
b) neutralization of the sorbent bed, by lowering the pH to a minimum of two pH units
below the sorbent pKa. If possible, neutralization of the analyte groups is desired versus
neutralization of the sorbent, since sorbent neutralization will release all species retained
on the sorbent during the extraction protocol, which may include undesired contaminants.
a
b
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Analyte elution facilitated through analyte neutralisation
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a
b
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Analyte elution facilitated by neutralisation of the sorbent bed
The third approach to analyte elution is to use a buffer containg a counter-ion that has an
extremely high affinity for the sorbent surface groups. These high-affinity cations can
readily displace the analyte cation groups, which may have much lower affinity for the
sorbent, from the surface. Examples of these cations are listed in the reference table of
counter-ions at the end of the course and in the course manual.
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Analyte elution facilitated by counter ion with high affinity for the sorbent
Anion-Exchange SPE
Overview
Anion-exchange SPE is the extraction process where molecules with acidic (anionic)
functional groups are extracted from predominantly aqueous matrices onto sorbents with
basic (cationic) surface groups.
Extraction of acidic molecules
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In anion-exchange SPE, the retention mechanism is the interaction of charged, anionic
groups on the analytes of interest and charged, cationic functional groups on the sorbent,
via electrostatic (ionic) interactions. These interactions are disrupted (causing elution of
the analytes) by one of three primary methods: a) high ionic strength, in which the
presence of large numbers of ions in the elution buffer creates competition with the
analyte groups for the sorbent groups
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Analyte elution facilitated by high ionic strength buffer
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b) a pH change, in which either the charged analyte groups are neutralized, or the
charged sorbent groups are neutralized
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Analyte elution facilitated by neutralisation of the sorbent bed
or c) the use of a buffer containing anionic species with a high affinity for the sorbent
functional groups.
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Analyte elution facilitated by high sorbent affinity counter ions
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The anion-exchange interaction is facilitated by a solvent environment that is
simultaneously the converse of each of the conditions “a.” “b.” and “c.” above; that is, a
solvent of low ionic strength, at a pH such that both the analyte groups and the sorbent
groups are charged, and if containing other anions, these anions have a lower affinity for
the sorbent than the analyte functional group anions.
Most pharmaceutical biological samples are aqueous in nature. A protic environment is a
necessity for ion-exchange to occur, and water is highly protic, offering a good starting
point for anion-exchange SPE. However, some biological samples (especially urine and
certain cell-growth buffers) exhibit very high salt (very high ionic strength) and at the very
least require significant dilution to reduce the ionic strength prior to application to an SPE
column.
The most common sorbents used in anion-exchange SPE have some type of amine group
on the surface; primary, secondary, tertiary, quaternary and multiple amine surfaces are
all available. These groups can be ionized via a suitable pH adjustment, thus allowing
negatively charged anionic analytes to retain. These sorbents are typically available in
both silica-based and polymer-based chemistries.
Low pH values, all basic
functional groups are
ionised
High pH values, all basic
functional groups are nonionised
Basic alumina may also be used quite effectively as a cation-exchange sorbent. However,
the user should pay careful attention to the quality of the alumina since these materials
often vary widely in properties batch-to-batch.
Sample Pretreatment
In addition to sample filtration, if required, and disruption of protein binding, it is necessary
prior to anion-exchange extraction to prepare a sample such that it exhibits the following
two primary qualities: a) the lowest ionic strength possible,and b) a pH such that the
analytes in the sample exist in a charged state.
If the sample is serum or plasma, which have relatively low ionic strengths to start, simple
dilution of the sample with water or a very low ionic strength buffer may suffice to reduce
the original sample’s ionic strength sufficiently for retention. In some cases, such as with
urine samples, the ionic strength is so high that it may be necessary to use a non-polar
SPE procedure to remove excess ions prior to the cation-exchange procedure. This is
often a very simple approach, since inorganic ions are readily washed off of non-polar
phases with simple buffers, in most cases leaving the analytes retained. After analyte
elution from the non-polar sorbent, the low-ionic strength extract may be diluted with water
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or a buffer, and applied to the anion-exchange sorbent, where the analytes are again
retained and reconcentrated.
Appropriate pH adjustment of the sample to charge the ionic groups on the sample is
necessary to get quality retention on the anion-exchange sorbent. Acidic or cationic
groups on the analytes, which are the typical targets for an anion-exchange extraction, are
ionized by raising the sample pH to a minimum of two pH units above the pKa of the
relevant group. It is recommended to perform this pH adjustment with the strongest base
or the most powerful buffer possible so as to minimize the increase in ionic strength of the
sample. For example, it is likely to take a much smaller quantity of ammonium hydroxide
to raise the pH of a typical acid than of a weaker base such as triethyl amine.
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Poor analyte retention as solution pH dictates analyte is unchanged
Maximum analyte retention as solution pH dictates both analyte and sorbent are charged
Sorbent Conditioning, Equilibration and Sample Loading
Sorbent conditioning for anion-exchange SPE is similar to that used for non-polar SPE. In
most cases, the sorbent bed is first treated with a few bed volumes of a water-miscible
organic solvent, such as methanol, acetonitrile, or THF. This is typically followed by a few
bed volumes of pure water, to prepare the sorbent for the equilibration step.
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Sorbent equilibration in anion-exchange SPE must accomplish two main purposes: a) to
convert the sorbent counter-ion to one that is easily displaced by the analytes, and b) to
adjust the sorbent pH to an appropriate value so that the sorbent groups are charged.
To facilitate analyte retention on the anion-exchange sorbent, it is necessary to equilibrate
with a buffer or a salt solution containing an anion of lower affinity for the sorbent than the
analyte group. This counter-ion will be most readily displaced by the analyte anion group.
The lowest affinity anions available are fluoride and hydroxide ions. Most anion-exchange
sorbent are not available in these forms, so the counter-ions must be converted. This is
best achieved by washing with a suitable salt at high ionic strength; for example, if
conversion to fluoride is desired the sorbent may be equilibrated with high molarity sodium
fluoride.
For pH adjustment of a basic sorbent, it is necessary to lower the pH of the sorbent to a
minimum of two pH units below the pKa of the sorbent in order to ionize the sorbent
groups. The pKa values for most commercial sorbents are available from the respective
sorbent manufacturer.
Anion-exchange is extremely sensitive to sample loading speed. In particular, it is very
easy to pass the sample through the sorbent bed too quickly, in which case breakthrough
(inadequate analyte retention) is likely. A common reference point for sample application
to a cation-exchange sorbent is no faster than 1 milliliter per minute for a 100 milligram
sorbent bed. This speed limitation is a consequence of the “point-to-point” nature of ionexchange, i.e., a very specific individual group on an analyte molecule must encounter a
very specific group on the sorbent in order for retention to occur.
Washing and Analyte Elution
The anion-exchange retention mechanism is facilitated by a) low-ionic strength buffers, b)
buffers at a pH such that both the analyte and the sorbent remain charged, and c) organic
solvents, which have little or no effect on the ionic retention mechanism. Any of these
reagents make excellent wash solvents for anion-exchange.
Low ionic strength buffer – no analyte elution
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Conversely, analyte elution is facilitated by a) high-ionic strength buffers, b) buffers at a
pH such that either the analyte or the sorbent are neutralized, or c) buffers containing a
counter-ion that has a very high affinity for the sorbent (much higher than the analyte
anion group affinity for the sorbent).
When using high-ionic strength as the elution mechanism, it is often important to consider
compatibility of the elution buffer with the final analytical technique to be used. LC/MS
systems, for example, do not cope well with high ionic strength samples, so it may be
necessary to dry down and reconstitute the elution solvent. In this case, a volatile buffer
salt, such as ammonium acetate should be used rather than a non-volatile salt such as
sodium chloride.
a
b
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Analyte elution facilitated by high ionic strength buffer
pH adjustment to facilitate analyte elution offers two possibilities: a) neutralization of the
analyte groups, by lowering the pH to a minimum of two pH units below the analyte pKa,
or b) neutralization of the sorbent bed, by raising the pH to a minimum of two pH units
above the sorbent pKa. If possible, neutralization of the analyte groups is desired versus
neutralization of the sorbent, since sorbent neutralization will release all species retained
on the sorbent during the extraction protocol, which may include undesired contaminants.
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Analyte elution through analyte neutralisation
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Analyte elution through surface neutralisation
The third approach to analyte elution is to use a buffer containing a counter-ion that has
an extremely high affinity for the sorbent surface groups. These high-affinity anions can
readily displace the analyte anion groups, which may have much lower affinity for the
sorbent, from the surface. This effect is dramatically more pronounced in anion-exchange
than in cation-exchange, and therefore can be highly useful. Examples of these anions
are listed in the reference table of counter-ions at the end of the course and in the course
manual.
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Analyte elution using strong counter ion
Minimum pKa Differential
The issue of first sample pH adjustment, then sorbent bed pH adjustment, brings out a
very important issue when performing ion-exchange SPE, whether cation or anion
exchange — that of minimum pKa differential. As discussed earlier in the course, for an
ionic group to be substantially in either the charged or neutral form, the pH of the
surrounding environment must be a minimum of two pH units away from the pKa of the
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group. For a cation to be charged, the pH must be two units above the pKa, and for an
anion the pH must be two pH units below the pKa.
This requirement may create difficulties if not considered carefully. For example, assume
that it is desired to perform cation exchange on a molecule that has a pKa of 6.7, using an
anionic (cation-exchange) sorbent with a pKa of 4.5. According to our two-pH unit
premise, for the analyte to be charged, we must be at pH 4.5 or below, but for the sorbent
to be charged we must be at pH 6.5 or above. Clearly these two conditions cannot be met
simultaneously.
The corollary to this is that to perform ion-exchange properly, we need a) a four pH unit
differential between the pKa value of our anion and cation, and b) the cation must have a
pKa higher than the anion. This situation may well dictate which sorbents are chosen for
the specific analytes to be extracted, based on the known pKa of the analyte and the pKa
of the sorbent.
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Effect of pH in analyte retention
Mixed-Mode SPE
Overview
Mixed-mode SPE is an extraction approach involving sorbents which are designed to
exhibit two or more primary interactions for analyte retention. The use of multiple
retention mechanisms offers unique advantages in terms of selectivity and breadth of
applicability to many different chemical structures.
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Commercial mixed-mode sorbents may be silica or polymer-based, and are typically
produced in one of two ways — either by bonding the sorbent concurrently with two
different functional group chemistries, or by blending discrete sorbent chemistries in
appropriate ratios to give the combination of retentive properties desired. Although the two
approaches result in materials of similar properties, it is generally recognized that the
blending approach is preferred for two reasons: a) it is easier to bond to a surface or
synthesize a single functional group chemistry reproducibly than to do so with multiple
functional groups, and b) if different retentive properties are required, it is much more
flexible to simply blend single functional group sorbents in different ratios than to create an
entire new synthesis to achieve the same effect with multiple functional groups on a given
surface.
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The most common mixed-mode sorbents have a hydrophobic functional group in
combination with an ion-exchange functional group. Hydrophobic groups may vary from
short chain, less retentive — thus more selective - materials (such as a C2 surface) to
highly retentive surfaces, such as C18. Ion-exchangers may be either cation or anion
exchangers, or even both. Hydrophobic chemistries pair well with ion-exchange
chemistries, since both allow effective loading of analytes in similar matrices; for example,
highly aqueous samples. This is an ideal condition for pharmaceutical biological samples
such as serum, plasma, blood, urine, CSF or various buffers.
The standard approach in employing a mixed-mode sorbent is to use the sorbent to
extract an analyte that exhibits functional group characteristics capable of interacting with
both of the active modes in the sorbent. For example, a mixed mode sorbent containing a
cation exchanger and a hydrophobic chemistry works well with a basic analyte that also
has some hydrophobicity. Thus when the analyte is loaded, the cation exchanger
interacts with the basic functional group on the analyte, and the hydrophobic sorbent
group interacts with the hydrophobic analyte groups. In fact, this particular combination
(i.e., hydrophobic and cation-exchange) is by far the most popular sorbent used in
pharmaceutical extraction of basic drugs. For the steps described to follow, we will
assume this is the chemistry combination with which we are dealing.
In order to effectively elute an analyte that is being retained on a mixed-mode surface via
two separate mechanisms, it is necessary to use an elution solvent that simultaneously
disrupts both of the retention mechanisms. This concept is key to the strength of a mixedmode sorbent in providing sample extracts of extraordinary cleanliness.
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Sample Pretreatment
One of the advantages of mixed-mode SPE is that it is more forgiving than single-mode
techniques in terms of raw sample quality. Multiple retentive mechanisms require only
that a sample is optimized initially to accommodate one of the active sorbent mechanisms.
With a single mechanism only, the sample must be optimized to accommodate that
particular mechanism. The problem with this may best be exemplified by a hydrophobic,
basic analyte from urine. Urine typically carries a relatively high ionic strength. If this
sample is applied directly to a cation-exchange sorbent, the analyte is likely to experience
breakthrough due to competing matrix ions. Sample dilution may help but may be
insufficient to reduce the ionic strength adequately.
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However, if the same sample is applied to a mixed-mode cation-exchanger/hydrophobic
sorbent, the analyte may be retained by the hydrophobic mechanism without regard to the
ionic character of the sample. This is analogous to applying the sample to a sorbent with
hydrophobic properties only, but the mixed-mode will contribute further benefits in
subsequent steps.
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Regarding the specific sample preparation steps, as with all samples, it may first be
necessary to address the issue of protein binding or filtration removal of particulates.
As far as altering the chemistry of the sample, we must prepare the sample according to
the mechanism we wish to use. So, for example, if we are using a combination ionexchanger/hydrophobic mixed-mode sorbent, and we wish to use the ion-exchange
mechanism, the sample must be prepared as if we were using an ion-exchange-only
sorbent. This might involve sample dilution and pH adjustment.
Sorbent Conditioning Equilibration and Sample Loading
Sorbent conditioning for mixed-mode SPE is a combination of that used for both
hydrophobic sorbents and ion-exchange sorbents.
Typically the surface is first
conditioned with methanol, acetonitrile or THF, to wet the sorbent.
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Sample conditioning
Then the excess organic solvent is removed using a suitable buffer. In this case the buffer
may also be of a pH to ionize the ion-exchange groups, but this is only necessary during
equilibration if the ion-exchange mechanism is desired to be active when the sample is
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first applied. Also, if a counter-ion change is desired for the ion-exchanger, this should be
performed.
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Equilibration buffer used to adjust solution pH ensuring cation exchanger is fully ionised
The requirements for the sample loading step, as with the previous steps, is dependent on
which mechanism is to be employed as the primary retentive mechanism. If ion-exchange
is to be used, the sample should be applied at a rate comparable to that used for ionexchange alone, that is , relatively slowly. If the primary initial retentive mechanism is the
hydrophobic mechanism, then the sample may be loaded much faster.
Washing and Analyte Elution
Washing and elution are two steps in mixed mode which exemplify the strength of the
technique in providing very clean extracts. The principle here is straightforward — since
our target analytes are retained (presumably) by both retention mechanisms, we can
selectively wash off interferences that are retained only by a single mechanism.
Therefore, in a mixed-mode extraction we typically employ two separate wash steps. For
example, again assuming that our analyte is both basic and hydrophobic, and is retained
on a mixed mode sorbent containing both cation-exchange and hydrophobic chemistries,
we can first use a wash step which removes only matrix components that have ionic
character. An example of this might be salts from a urine matrix. This step might use, for
example, a high ionic strength buffer. Such a wash step would have no influence on the
retention of the analytes via a hydrophobic mechanism. Then, as a second wash, we
might use a pure organic solvent, which will remove matrix components with only
hydrophobic character (no ionic retention), without affecting the ionic retention of the
analytes. Using this double-wash approach very effectively removes many interferences
from the sorbent without affecting the analytes.
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For effective analyte elution, we must employ a solvent that disrupts both retention
mechanisms simultaneously.
For mixed-mode sorbents with ion-exchangers and
hydrophobic groups, this is typically an organic solvent (such as methanol, acetonitrile, or
THF), modified with a strong acid or base to negate the ionic retention. So, for example, a
typical elution solvent might be methanol containing a few percent of ammonium
hydroxide or hydrochloric acid.
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As with other retention mechanisms, maximum extract purity is achieved by a) washing
with the strongest solvent possible that does not elute the analytes, and b) eluting with the
weakest solvent possible that gives high analyte recovery, yet leaves behind on the
sorbent as many remaining interferences as possible.
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Summary
Mixed-mode extraction is often somewhat confusing to users, yet can be understood quite
simply by recalling the following guidelines:
Be certain to identify which sorbent mechanisms are represented by any given
commercial product. Although there are many different combinations of sorbent
chemistries available, by far the most common are a hydrophobic chemistry paired with
either an anion or a cation-exchange chemistry.
Consider the retentive mechanisms as individual chemistries acting together, and treat the
sorbent appropriately. For example, conditioning steps to prepare the cation-exchange
element of a mixed mode sorbent are the same as those prescribed for a cation-exchange
single mechanism sorbent. All of the pH, ionic strength, and counter-ion principles apply
equally. The hydrophobic element should also be treated in a similar, analogous manner
to a standalone hydrophobic sorbent.
Bear in mind that the two retentive mechanisms may help to retain an analyte from a
matrix environment that would be incompatible with a single mechanism sorbent. Again,
the principle here is to use the favoured mechanism to overcome matrix limitations until
the analyte is retained and the uncooperative interferences removed through washing.
Use aggressive wash solvents that only intercede in one of the two retention mechanisms,
so as to preserve analyte retention via the other mechanism. For maximum elution
effectiveness, be sure to use a solvent that simultaneously disrupts both retention
mechanisms.
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