SOLID PHASE EXTRACTION

INTRODUCTION
The conventional process of solid-phase extraction involves passing a liquid sample over a
solid surface to separate target analytes from the sample matrix. The attractive interactions of
the analyte with the solid surface must be greater than the attractive interactions of the
analyte with the sample matrix for retention and extraction to occur. The technique is based
on the same principles as chromatographic separations (partitioning, adsorption, or ion
exchange). This section intends to serve as a practical guide to SPE sorbent and device
selection, extraction steps, and expected performance.

SPE is a powerful method for sample preparation and purification in many fields, including
pharmacology.

PRINCIPLES:

SPE is based on the same principles as chromatographic separations, such as partitioning,

adsorption, or ion exchange.

1.1.Partitioning: This is the process where analytes distribute themselves between

two immiscible phases (usually a solid and a liquid in the case of SPE). The
analytes partition based on their relative affinities for each phase. The goal is to
have the analytes of interest preferentially partition into the solid phase (the SPE
sorbent), while unwanted components remain in the liquid phase (the sample
matrix).
1.2.Adsorption: This is the process where analytes adhere to the surface of the solid
phase (the SPE sorbent). Adsorption can be physical (due to Van der Waals
forces) or chemical (due to covalent bonding). The extent of adsorption depends
on the nature of the analyte, the nature of the sorbent, and the conditions of the
extraction (such as pH and temperature).
1.3.Ion Exchange: This is a special type of adsorption where the analytes are ions
and the sorbent has charged functional groups. The analytes are retained on the
sorbent through electrostatic interactions. The strength of these interactions
depends on the charge of the ion, the charge of the functional group, and the ionic
strength of the solution.

REQUIREMENT

1. Sample Matrix: The first considerations are for the target analyte and the sample
matrix. if the analyte is a polar compound in a water sample, a polar sorbent and a
polar solvent are used for the extraction. If the analyte is a non-polar compound in an
oil sample, you might choose a non-polar sorbent and use a non-polar solvent for the
extraction. Gaseous and liquid samples requiring no chemical modification are
generally the easiest to process because they can be directly introduced into the SPE
system. Solid samples, on the other hand, often need to be prepared or processed in
some way (like being dissolved or suspended in a liquid) before they can be
introduced into the SPE system.
2. Sorbent and Device Selection:
 3.1.Sorbent Selection: The sorbent is the material in the SPE device that selectively
retains the analytes of interest. The choice of sorbent depends on the properties of
the analytes and the sample matrix.

 If the analytes are polar, a polar sorbent might be used because polar
compounds tend to interact strongly with other polar compounds (like
dissolves like).
 If the analytes are non-polar, a non-polar sorbent might be used for the same
reason.

The solubility of the analytes in different solvents is also a key factor. For example, if
the analytes are soluble in polar solvents but not in non-polar solvents, a polar sorbent
might be chosen to retain the analytes while a non-polar solvent is used to wash away
other components of the sample.

3.2.Device Selection: The type of SPE device (like a cartridge or disk) is chosen
based on the nature of the sample matrix and the specific requirements of the
extraction procedure.

 For example, if the sample matrix is a liquid and the volume is small, a cartridge
might be suitable.
 If the sample matrix is a large volume of water, a disk might be more suitable
because it has a larger capacity and allows for faster flow rates.

The mode of interaction refers to the mechanism by which the analytes are retained
on the sorbent. This could be partitioning (where the analytes distribute themselves
between the sorbent and the solvent), adsorption (where the analytes adhere to the
surface of the sorbent), or ion exchange (where the analytes are ions and are retained
by electrostatic interactions with charged groups on the sorbent).

4. Conditioning: Conditioning is required to wet the surface of the sorbent and to remove
any impurities that may be present on the unused tube or disk. Larger amounts of
conditioning solvent will generally not affect the performance but can increase the
performance in some poorly packed cartridges by redistributing the packing into a more
uniform bed.

The success of SPE largely depends on the careful selection of the sorbent material, the
conditioning and washing steps, and the elution conditions.

PROCEDURE

The extraction process uses tubes or disks, which is similar to liquid chromatography (LC).
Here are the key points:

1. Sample Introduction: The sample volume can vary from a few microliters to several
litres. Samples are generally processed to become suitable liquid samples and poured
quantitatively onto the top tube frit or disk.
2. Processing Methods: Tubes can be processed individually by simply pouring the
liquid sample on top of the frit or disk and allowing the solvent to drip through the
 packed bed by gravity. Other methods include the use of syringe plungers, single tube
processors, air or nitrogen lines, centrifuge tubes, and vacuum flask apparatus.
3. Retention Capacity: The sample matrix itself can contribute to the loss of retention
capacity by not properly wetting the solid-phase surface. To reduce the risk of sample
breakthrough, small amounts of organic solvent can be added (1-5%) to maintain
properly wetted conditions.
4. Breakthrough Testing: One trick to test for breakthrough is to attach two or more
tubes in series and apply the sample solution. If analytes are detected in more than the
first tube, the breakthrough is occurring.
5. Washing Step: In some cases, it is desirable to wash the sorbent bed after application
of the sample matrix. The washing step typically uses similar volumes as the
conditioning step.
6. Elution: Eluting the analytes of interest from the packing is customarily done with as
small a volume as necessary to completely remove the analytes while leaving behind
any interfering compounds that were retained during the wash step.
APPLICATIONS

Solid Phase Extraction (SPE) is a versatile technique used in various fields due to its ability
to concentrate and purify analytes from different types of samples. Here are some of its
applications:

1. Environmental Analysis: SPE is used for the concentration of trace organic

pollutants from water. It helps in the detection and quantification of pollutants in
environmental samples like air, water, and soil.
2. Clinical and Pharmaceutical Applications: SPE is used for the purification of drugs
of abuse from blood and urine. It’s also used in the analysis of pharmaceuticals,
including the extraction and purification of drugs from biological samples3.
3. Food and Beverage Analysis: SPE is used for the extraction of organic compounds
from food and beverages. It helps in the detection of contaminants or the analysis of
nutritional components.
4. Novel Methodologies: There are also different variants of SPE techniques such as
solid-phase microextraction (SPME), in-tube solid-phase microextraction (IT-SPME),
and magnetic solid-phase extraction (MSPE) that have been developed for specific
applications.
 LIQUID LIQUID EXTRACTION
Liquid-liquid extraction is a versatile and dependable separation technique wherein an aqueous
solution is usually brought into contact with another organic solvent, exclusively immiscible with the
former, to affect a legitimate and actual transfer of either one or more solutes into the latter. The
normal-feasible separations which can thus be achieved are found to be rather easy, fast,
convenient and effective reasonably. Invariably such separations may be performed by shaking the
two liquids in a separatory funnel for a few minutes; and may be extended either to large quantities
of pharmaceutical substances or trace levels.

In short, liquid-liquid extraction has been employed predominantly and effectively not only for the
pre-concentration and isolation of a ‘single’ chemical entity just before its actual estimation but also
for the extraction of classes of organic compounds or groups of metals, just before their usual
estimation either by chromatographic techniques or by atomic-absorption methods.

PRINCIPLE
According to this law, at a constant temperature, a solute distributes itself between two immiscible
solvents (a and b) in contact with each other in such a way that at equilibrium, the ratio of its
concentration in the two layers is constant, irrespective of its total amount. This can be
mathematically expressed as:

The constant (Kp) is also known as the distribution coefficient or the partition coefficient. For dilute
solutions, at a specified constant pressure and temperature, the mole fraction of a solute is directly
proportional to its concentration in molarity or mass per unit volume; which implies that these may
be employed instead of mole-fraction

FACTORS AFFECTING LIQUID-LIQUID EXTRACTION

a) Effect of temperature and inert solutes
b) Effect of pH on extraction
c) Effect of ion-pair formation
d) Effect of synergistic extraction.
 a. Effect of temperature and inert solutes
Partition coefficients are typically not sensitive to temperature when the two solvents are more or
less immiscible and the concentrations are relatively low in both phases. This allows the effect of
temperature on the partition coefficient to be conveniently estimated from its effect on the
solubilities of the substance in the two respective solvents

The use of inert solutes such as calcium chloride, magnesium chloride, and sucrose can be effectively
employed in the development of solutions to challenging extraction problems. These solutes allow
for efficient extractions from water into solvents like acetone, ethanol, and methanol, which are
completely miscible with water in the absence of salt.

b. Effect of pH on extraction

Generally, it has been found that the organic acids and bases do exist in aqueous solution as
equilibrium mixtures of their respective neutral as well as ionic forms. Thus, these neutral and ionic
forms may not have the same partition coefficients in a second solvent; therefore, the quantity of a
substance being extracted solely depends upon the position of the acid-base equilibrium and
ultimately upon the pH of the resulting solution. Hence, extraction coefficient (E) may be defined as
the ratio of the concentrations of the substance in all its forms in the two respective phases in the
presence of equilibria; and it can be expressed as follows :

c. Effect of Ion-pair formation

Ion-pair formation needs its due recognition because it very often gives rise to unexpected
extractions. In a true sense, ion-pair may be regarded as a close association of an anion and cation,
and therefore, it usually takes place either in a polar or a non-polar solvent. In reality, the ion pairs
are invariably formed by the union between comparatively large organic anions and (much smaller)
cations. Interestingly, the resulting ion pairs have been found to show their appreciable solubility in
polar solvents; hence, these species may be extracted conveniently under such experimental
parameters where neither individual component ion could.

A few vital criteria towards the formation of an improved aqueous extractable ionic species are,
namely :

 Formation of a neutral metal-chelate complex or by ion association

 Creation of larger and more hydrophobic molecular species
Effect of Synergistic Interaction

METHODOLOGY

1. Preparation of Phases: The process begins with the preparation of two immiscible
phases. One of these is usually water, and the other is typically an organic solvent.

2. Mixing: The two phases are then placed into a device known as a separatory funnel which
has a stopcock attached to the bottom end. The compounds in the system will be distributed
between these two phases.

3. Extraction: During the extraction phase, an organic compound that is dissolved in an

aqueous phase is transferred to an organic solvent. This process is dependent on the
partition coefficient of the compound.

4. Separation: After the extraction, the two phases are allowed to separate. The phase that is
enriched in solute(s) is referred to as the extract, while the feed solution that is depleted in
solute(s) is called the raffinate.

5. Repetition: To increase the efficiency of the extraction, the extraction process is often
repeated several times.

6. Estimation: After the process is completed the layer of the phase that is at the bottom of the
separatory funnel ( phase having more density) is collected using the stopcock. The two
phases are then titrated to estimate the content of the compound with respect to the phase.

The methodology can vary depending on the nature of the compounds being extracted. For instance:

 Extraction of Neutral Compounds: If the desired organic compound is neutral (i.e., is neither
acidic nor basic), the extraction sequence usually involves simply extracting with an organic
solvent several times.
  Extraction of Acidic Compounds: If the desired compound is acidic, we can selectively
deprotonate that compound by using an aqueous base. This will pull the deprotonated
compound into the aqueous phase.

 Extraction of Basic Compounds: If the product is basic, we can perform a sequence very
similar to the acidic compound above. We can protonate the basic compound by using an
aqueous acid, pulling the protonated compound into the aqueous phase.

The typical apparatus used in an extraction is the separatory funnel. These come in many different
sizes, but the typical size is 100 ml.

This technique is widely used in various fields, including the production of fine organic compounds,
the processing of perfumes, the production of vegetable oils and biodiesel, and more.

SUPPLEMENTARY MATERIALS (NOT EXACTLY IN SYLLABUS BUT TAUGHT IN CLASS)