FDSC 315 Lecture 3 : Solid-Phase Extraction

What is Kd?

a partition ratio

Describe the principles of SPE.

solid phase extraction is the process of which an analyte is suspended or dissolved in a liquid and extraction with a sorbent material.

What is the sorbent material in SPE made of?

it is often times a powder, but it can also be a surface or an SPE cartridge.

How does one choose the ideal SPE sorbent?

the sorbent material depends on the mechanism of interaction between the sorbent and analyte.

What are the three major applications of SPE?

1. isolation of the analyte from teh matrix and possible interfering molecules

2. preconcentration of the analyte: concentration of analyte is low in initial sample—> concentrate sample on the SPE phase ( basically remove to concentrate sample)

Describe sample cleanup.

cleanup describes isolation of the analyte and removal of interfering molecules. e.g. weakly bound impurities are washed out and the desired analyte (s) is eluted in a cleaner form.

Describe preconcentration of the analyte.

(enrichment): the concentration of the analyte is too low in the initial sample; large amounts of the sample will be concentrated on the SPE phase; the extract is then eluted and concentrated in a small solvent volume.

Give 8 examples of SPE design methods.

Cartridges, disks, 96-wells, pipette tips, solid-phase microextraction or SPME, on-line SPE

dispersive solid-phase extraction, and molecular imprinted polymers.

Describe the cartridge method.

the cartridge resemble a syringe with a filter. A tank is placed underneath the syringes, which is connected to a vacuum pump. This forces the sample through the cartridge. Without a vacuum, gravity is used to pull the solute, it takes longer.

What type of liquid is not ideal for the cartridge method?

samples with a lot of interference, cloudiness, pulp, etc.

Describe the SPE extraction disk method.

the particles of solvent are integrated into a thin disk. ??

Describe the 96-well plate method for SPE.

samples are pipetted into a 96-well plate, and the SPE powder is pipetted directly with the samples. This is good for very small volumes. It is not a very common method.

Describe the interactions that take place during SPE.

interactions that take place include (from weakest to strongest) : Dispersion/london forces, dipole-induced, ion-induced, dipole-dipole, H-bonds, and ion-dipole.

True or false: silica surface is polar.

true. The surface contains hydrogen attached to silicone and oxygen

True or false: surfaces made of C18 chains bonded to silica are rather polar.

false. Hydrocarbons are nonpolar, therefore this renders the silica surface nonpolar. A long hydrocarbon chain will mask the polar properties of the silica.

Why is phenyl used for in nonpolar SPEs?

phenyl is nonpolar, but it is used to extract aromatic compounds.

What is the difference between reversed phase and normal phase SPE?

normal phase chromatography has a very polar stationary phase and a non-polar mobile phase whereas reverse phase chromatography has a non-polar stationary phase and a polar mobile phase.

Describe anion-exchange resins.

a resin with an amine attaches to the compounds (i.e. chlorine). The interaction is based on the charge of the molecule.

Describe the hydrophilic-lipophilic balance (HLB).

The hydrophilic-lipophilic balance of a surfactant is a measure of the degree to which it is hydrophilic or lipophilic, determined by calculating values for the different regions of the molecule

Describe conditioning of the material.

it is the first step before pushing the sample through the cartridge.This activates and/or wets the pores of the stationary phase (improve contact between sample and SPE phase).

Give an example of conditioning of a sample using methylhydroxide.

​​C18 is hydrophobic. The aqueous sample does not come in contact with the C18 phase. MeOH is used to "wet" the phase. First, elute 10 mL methanol through the SPE cartridge. Wait for 5 minutes. Then, elute 10mL of UP water (same pH as the sample).

What happens if the SPE cartridge goes dry?

Do not let the SPE cartridge go DRY, or else you have to restart the entire process.

Describe loading of the sample.

this is the second step. the liquid sample is passed through the SPE phase, and the analyte is retained on the stationary phase. The unretained sample matrix is discarded.

What is the driving force pushing the sample through the cartridge?

gravity, pressure, and vacuum.

Give an example of sample loading.

The sample is loaded onto the cartridge at a rate of 10 mL/minute under vacuum.

What are two reason low analyte recovery may occur?

Low analyte recovery may happen if: Flow rate too fast, or the capacity of the SPE phase is too small for the sample size (overload)

Describe the washing step.

it is the third step. some weakly bound interferences are washed off the cartridge. A weak solvent is used for this purpose.

Give an example of the washing step.

After sample loading, the cartridge is washed with 20 mL of UP water a rate of 10 mL/minute under vacuum.

Describe the drying step.

this is a mini step that may occur after washing. Air is flushed through the cartridge. This makes the next step (elution) more efficient.

Describe elution.

a strong solvent is eluted to recover the analyte(s) of interest from the SPE phase. The fraction(s) containing the analytes are collected.

Give an example of elution.

After extraction on C18 cartridges, the analytes are recovered using elution with methanol.

True or false: you can reuse the cartridge for future extractions.

false; they are disposable.

A sample is known to contain 3.5mg of analyte X. After elution, 2.8mg of X is recovered. What is the recovery of the SPE extraction step?

2.8/3.5 = 80%.

Describe online SPE.

?

You want to analyze saccharides in milk, but issues are interfering. How would you separate these compounds?

Describe 5 benefits of SPE compared to liquid extraction.

Selectivity: a wide range of SPE phases available. Relatively fast: no reflux, shaking or phase separation steps. Low solvent consumption. Preconcentration of the analyte. Can be automated.

What are two disadvantages to SPE.

Needs optimization to prevent breakthrough/overload. Incomplete removal of interferences.

Describe SPME.

SPME is solid-phase microextraction. A polymer-coated fused silica fiber is used for extraction of analytes via directly in the liquid by immersion or from a solid or liquid by headspace extraction (headspace SPME)

In SPME, what occurs after extraction?

After extraction, the analytes are desorbed from the fiber by exposing the fiber in the injection port of a GC or in the desorption chamber of an SPME/HPLC interface.

What is the quantity of the analyte extract proportional to?

The quantity of analyte extracted by the sorbent is proportional to its concentration in the sample medium in the case of equilibrium (and in some specific case under the kinetic regime)

Describe Headspace SPME.

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What are two ways to increase the reaction when separating volatile compounds in HSPME?

adding heat and increasing salt content.

What is the benefit to increasing salinity by adding NaCl to a system for HSPME?

you increase the speed of the reaction because this increases the partition between the air and the liquid.

True or false; when doing SPME in vivo, the animal must always be sacrificed.

false often times they are, however it can be done using a needle in the nonlethal technique.