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The CHROMacademy Essential Guide Tutorial
7 Key Secrets to Understanding Solid Phase Extraction


Tony Taylor (Technical Director, Crawford Scientific), gives us his 7 Key Secrets to Understanding Solid Phase Extraction.

It is impossible in this short newsletter to give a comprehensive overview of the technique of SPE!  However, what follows is a highly concentrated Primer that will help you to understand all of the key stages in SPE protocol development.  This understanding is crucial for improving your SPE technique and ultimately to developing better solid phase extraction methods.


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1: Know your analyte(s) and sorbents   jump to section »
2: Know your SPE Protocol Steps - Sorbent Conditioning   jump to section »
3: Know your SPE Protocol Steps - Sorbent Equilibration   jump to section »
4: Know your SPE Protocol Steps – Sample Loading   jump to section »
5: Know your SPE Protocol Steps – Interference Washing   jump to section »
6: Know your SPE Protocol Steps – Analyte Elution   jump to section »
7: The expert tips on Facile Sorbent Processing   jump to section »
 
Secret 1: Know your analyte(s) and sorbents :    
     

To get the best from a solid phase extraction protocol we need to maximize the interaction between the analyte and the sorbent within the SPE device, which allows us to use stronger wash solvents (better selectivity) and less elution solvent (better efficiency).

To carry out an assessment of the molecular properties of analyte we would typically consider:

Log P (or Log D if the analyte is ionisable): this is the analyte octanol:water partition coefficient and gives an indication of the hydrophobicity of the analyte. If the value is positive, the analyte is more hydrophobic and therefore we would consider a more hydrophobic sorbent (C8, C18 etc.), if negative we should consider a less hydrophobic sorbent. These values also give an indication of the protocol solvent strengths that will be used for sorbent (sample) washing and elution.

Figure 1:  Log P analyte assessment
Vitamin D3 LogP = (approx.) +9.5 indicating the need for a more hydrophobic sorbent to gain good analyte retention (C8, C18 etc.).

Glucosamine LogP = (approx.) -2.4 indicating the need for a less hydrophobic sorbent (Phenyl, C4, Nitrile, Non ionised acids or amines etc.). Note: although polar, the analyte would not be fully ionised in the useful pH range (1-12) and therefore ion exchange sorbents would not be advantageous. However, a mixed mode sorbent containing both reversed phase and an ion exchange function would be a very attractive choice for a molecule of this type.

Figure 1.LogP

 

 

 

pKa:  This parameter describes the extent of analyte ionization and defines (sic.!)  the solution pH at which half of the analyte molecules in solution are charged and half are uncharged.  This parameter indicates the possibility of using a charged sorbent to maximize analyte interaction and will indicate, along with an assessment of the analyte functional groups, whether we need a  strong or weak ion exchange sorbent.  The pKa value is also VERY useful in designing protocol solvents especially with regard to solvent pH required to gain maximum analyte retention or elution.

 

 

 

 

 

 

 

Movie 1:  LogP determination
The analyte is shaken in equal volumes of octanol (top layer) and water (bottom layer) and the concentration in each layer measured prior to calculation of:

 

 

 

Movie 2:  Analyte pKa
Ionised and non-ionised analytes partition differenty – and therefore show different solubility characteristics in mixtures of organic and aqueous solvents. For further explanation of pKa and the effect of solution pH on the analyte watch the movies opposite
  • pKa movie 2 shows the dissociation of an acid in various solution pHs
  • pKa movie 3 shows the dissociation of a base
 

 

Movie 3:  Sorbent Selection
Knowing analyte pKa will aid sorbent selection.
The solution pH should be adjusted so that both the analyte and sorbent are ionised during the sample loading and washing steps and then adjusted to nuetralise either the analyte or sorbent for the elution step.


When analytes contain acidic or basic functional groups with low (<1.5) or high (>9.5) pKa values, then weak anion or cation exchange are required as it will be difficult to alter the analyte charge state using solution pH without damaging the sorbent substrate.  The charge state of weak ion exchange sorbents can be more easily altered by solution pH and therefore the sorbent – analyte ionic (strong) interactions can be promoted or disrupted using protocol solvent pH.

Where analyte pKa values fall into the range 2 – 8, either strong or weak ion exchange sorbents may be used and it may be possible to affect charge state changes in both species to alter the ionic interaction using SPE protocol solvent pH.
The most effective interaction types in modern solid phase extraction are known as mixed mode interactions and make use of both hydrophobic interactions and ionic (point-to-point) interactions.  These sorbents may be physical mixtures of C18 and an ion exchange resin or combine both hydrophobic ligands and an ion exchange ligands onto a single substrate.  Polymeric sorbents which contain a hydrophobic backbone with polar or ionisable functional moieties are very popular in modern SPE and are typically used with Generic SPE Protocols as starting points for SPE method development.


 

Movie 4:  SPE Sorbents
Use the tool opposite to see examples of various SPE sorbent surfaces and functional bonded groups. Some mixed mode polymeric sorbents are shown in the roll over tabs at the bottom of the screen.


It’s important to remember that we may have to use Sample Pre-Treatment techniques in order to facilitate good retention on the SPE sorbent.  This typically might involve adjusting the pH of the sample solution to promote (when using ion-exchange sorbents) or suppress (when using more hydrophobic sorbents) ionization.  This stage can make or break an SPE protocol and is often overlooked!

 
Secret 2: Know your SPE Protocol Steps - Sorbent Conditioning :   back to top »
 

The first step in a typical extraction protocol is referred to as column conditioning. The function of this step is to “activate” or “wet” the chromatographic sorbent to allow proper interaction with the sample.

Many extraction sorbents are extremely hydrophobic, and will not wet to an aqueous sample. In this situation, the most common conditioning involves application of a water-miscible organic solvent to the column – typically methanol.

Sorbents most likely to require a conditioning step are either very hydrophobic bonded silicas, such as C18, or very hydrophobic polymer resin sorbents, such as styrene divinyl benzene.

If an extraction sorbent is water-wettable, a column conditioning step may not be necessary, however omission of this step may seriously affect analyte retention.

 


Movie 5:  SPE Sorbent Conditioning
Proper sorbent conditioning can be crucial for good analyte retention (recovery).
These movies demonstrate the need for conditioning and the ‘surfactant’ like properties of methanol in facilitating the retention of analytes from aqueous samples when using highly hydrophobic sorbents.

 
Secret 3: Know your SPE Protocol Steps - Sorbent Equilibration :   back to top »
 

This step is performed just prior to application of the sample and creates a sorbent chemistry environment as similar to the sample as possible. This is required so that the sample chemistry does not change the extraction environment during sample application. If the sorbent is not equilibrated properly, irreproducible results and poor analyte recoveries are often seen. 

pH is important to the effectiveness of the extraction chemistry (i.e. if the analyte, sorbent or a major interferent is ionisable), the sample pH should be adjusted properly in both the sample pretreatment step and the column equilibration step. If a sample is pretreated to a pH of 3.5, and the column is equilibrated to pH 7.5, during the course of the sample application to the column, the pH of the column environment will gradually shift from 7.5 to 3.5. Therefore, the initial portion of the applied sample will experience a different extraction environment than the latter portion of the sample. This can result in irreproducible results and poor analyte recoveries.

The organic and ionic strength of the conditioning solvent should also be matched to the sample solution.  Failure to do so can also lead to poor analyte capture (recovery) and the need to use larger volumes of elution solvent (poor efficiency).

 


Movie 6:  SPE Sorbent Equilibration
Column pH, solvent strength and ionic strength should ALL be adjusted to match the applied sample or analyte breakthrough or poor capture efficiency will result.
The result of an inefficiently captured sample is usually the need for greater volume of elution solvent – resulting in a more dilute extract.

 
Secret 4: Know your SPE Protocol Steps – Sample Loading :   back to top »
 

The single most important element of the sample loading step is the linear velocity of the sample as it passes through the column.

The linear velocity of the sample application is a function of two individual elements – the flow rate at which the sample is applied, and the diameter of the column to which the sample is being applied. For a given flow rate, a wider column will offer a lower linear velocity than a narrow column.

The importance of an adequately low linear velocity relates to the residence time of the analytes in the extraction sorbent. This residence time must be sufficient for the necessary chromatographic interaction to occur. Since this interaction is spatially dependent (in other words, active functional groups on the analyte must orient properly relative to the functional groups on the extraction sorbent), sample application with too small a residence time may result in analyte breakthrough.

The speed of sample application also is a function of the particular extraction mechanism employed. This is especially true when using ion exchange as part of the retention mechanism when the electrostatic point-to-point interactions necessary for analyte retention require longer analyte residence time to achieve the necessary analyte orientation relative to the sorbent surface.


Movie 7:  Sample Loading
The importance of sample linear velocity on analyte retention (recovery)

 
Secret 5: Know your SPE Protocol Steps – Interference Washing :   back to top »
 


Movie 8:  Washing
The most selective extractions, which produce the least convoluted chromatograms, involve the use of the ‘strongest’ wash solvents

This step (or steps!) wash the sorbent to selectively remove undesired contaminant species, while leaving the target analytes retained on the sorbent surface. This step is performed using a solvent with a higher elution strength than the solvent in which the sample was applied to the column, relative to the particular extraction mechanism being employed. This may involve the use of increased solvent strength (% organic), ionic strength or different pH.

In many cases, the wash solvent is a “weaker” version of the elution
solvent – that is, the elution solvent diluted with a weaker eluent. This ensures miscibility of all the solvents in the extraction protocol, resulting in the fastest equilibration of each protocol step with the next, and the greatest reproducibility.

One common error in SPE protocols is the use of wash solvents that are too 'weak'. Philosophically, the concept of a wash solvent is to remove as many of the undesired contaminants as possible. Many SPE users employ very weak elution solvents due to a concern for eluting analytes during wash steps. Unfortunately, this often results in poor quality extracts, since many of the contaminants are left on the column during the wash, and subsequently eluted along with the desired analyte in the elution step.

In general, this issue leads to the following important conclusion – wash steps should be performed with the strongest solvent possible that does not elute the analyte. This ensures removal of the greatest amount of contaminants, resulting in the cleanest possible extract.

With hydrophobic extraction mechanisms, subsequently higher strength aliquots may be used to determine (via the analysis of the wash solvent), the highest eluotropic strength which does not elute the analyte.  Similarly in mechanisms involving ion exchange, the pH of the wash solution can be adjusted to suppress the ionization of the analyte or sorbent with respect to interfering species but NOT the analyte.

With mixed mode mechanisms, the strongest wash solutions possible can be used.  Even if the ionic interactions between the analyte and sorbent surface are interrupted (using pH or ionic strength) during the wash phase, the organic strength can be adjusted so as to retain the analyte by this secondary interaction.  The converse is also true, and so a host of wash solvents may to be used to wash interfering species from the sorbent surface whilst the analyte is ‘flipped’ between the two retention mechanisms.

 

 

Movie 9:  Washing Mixed Mode Extractions
The ability to switch analyte retention mechanisms between electrostatic and hydrophobic interaction leads to the possibility of obtaining the cleanest extracts

 
Secret 6: Know your SPE Protocol Steps – Analyte Elution  :   back to top »
 

The types of solvents used for elution must be capable of disrupting all of the retentive interactions between functional groups on the analytes and sorbent. An elution solvent which disrupts only some of these interactions will bring about only partial elution, resulting in irreproducible results and often poor analyte recoveries.

Many modern SPE procedures employ sorbents specifically designed to exhibit multiple retention mechanisms. Such sorbents are often referred to as “mixed mode” sorbents, and are extremely popular in pharmaceutical applications, for extraction of basic and acidic drugs. Analyte elution from these surfaces often requires especially “strong” elution solvents, to ensure complete recovery of the analytes from the sorbents.

Having said all of the above, one all too common error in SPE protocols is to use elution solvents that are too strong. The elution step is an additional opportunity for sample cleanup by leaving undesired contaminants behind on the sorbent during elution. Use of a very strong elution solvent will tend to elute these contaminants along with the desired analytes.

This situation leads to another important conclusion – elution steps should be performed with the weakest solvent possible that provides complete elution of the analytes. This leaves behind on the sorbent the greatest possible amount of contaminants, resulting in the cleanest possible extract.


Movie 10:  Analyte Elution
Elution requires all analyte interactions to be disrupted via increased eluotropic strength, increased ionic strength, the use of a more selective counter ion or a change in solution pH.

One common error is the use of eluents which are too strong – ultimately resulting in a less selective protocol and more a more convoluted chromatogram.

 
Secret 7: The expert tips on Facile Sorbent Processing    back to top »
 

Use of Soak Steps

Under certain circumstances it may be necessary to include a soak step at various points within the SPE protocol.  During a soak step the solvent applied to the column is allow to remain stationary within the cartridge and the flow rate and linear velocity of the solvent fall to zero.

The soak step allows time for chemical and physico-chemical equilibrium to be established between the solvent and sorbent bed and can greatly improve retention and elution efficiency and reduce the effects of variable flow rate through the cartridge.

During sorbent conditioning a soak step can be used to improve the sorbent surface activation if the solvent is allowed to soak for (30 secs. to 2mins. for a typical 100mg sorbent bed mass), prior to the equilibration step.  This approach is often used with non-polar sorbents that are highly hydrophobic such as C18 (especially the end-capped phases) and for some C8 end-capped phases.

During analyte elution, the volume of elution solvent, the number of solvent aliquots required and the rate at which the elution solvent is applied for successful analyte recovery can all be affected if a soak step is employed.

By allowing a time period where the analyte species can diffuse into the extraction solvent and allow equilibrium to be reached, whilst the solvent is stationary, can be advantageous.  The soak step will ensure maximum analyte recovery in the elution solvent, may reduce the volume of extraction solvent required and will render the elution step much less flow rate dependent.


Movie 11:  Soak Steps
Stopping the flow of protocol solvent and allowing equilibration of solvents with sorbents and analyte can have drastic effects on improving the quality and repeatability of extraction methods.

 

 

Use of Drying Steps

Often a drying step may be included after the interference removal / wash steps.

This can be particularly important when an aqueous sample has been loaded and the elution step of the protocol involves water immiscible solvents.  In this case, any water that remains on the surface of the sorbent may exclude the immiscible elution solvent and cause low recovery of the analyte.  Polar sorbents generally require longer drying times than hydrophobic phases due to their higher wettability.

The drying step may be significantly reduced or even eliminated when water miscible solvents are used for the elution step. However, the presence of small amounts of water in the final eluate solution can significantly increase the time required for solvent evaporation (where analyte pre-concentration is required) or solvent elimination prior to residue reconstitution into a solvent more suitable for the separative stage of the analysis.

Even small traces of water in the eluate solution mean that to evaporate the solution to dryness will required extended heating at higher temperatures.  This can be highly unsatisfactory when the analytes are thermally or chemically labile and analyte degradation must be considered.


Movie 12:  Drying Steps
The use of a drying step is often critical when using water immiscible protocol solvents or solvents which do not wet the sorbent surface well, even in the presence of trace amounts of water

 

The following subjects are covered in full multi-media at CHROMacademy.com

The Theory Of HPLC
Introduction (1.5hrs)
Chromatographic Parameters (3hrs)
Band Broadening (3hrs)
Column chemistry (4hrs)
Reverse phase (partition) chromatography (6hrs)
Ion-Pair Chromatography (3hrs)
Normal phase (absorption) chromatography (3hrs)
Gradient HPLC (3hrs)
Quantitative and Qualitative HPLC (3hrs)
FAST HPLC (4.5hrs)

Theory and Instrumentation of GC
Introduction (1.5hrs)
Chromatographic Parameters (3hrs)
Band Broadening (3hrs)
Gas Supply and Pressure Control (2hrs)
Sampling Techniques (4.5hrs)
Sample Introduction (5hrs)
GC Columns (5.5hrs)
GC Temperature Programming (3hrs)
GC Detectors (2.5hrs)

Instrumentation of HPLC
Mobile Phase Considerations (3.5hrs)
Solvent Pumping Systems (4hrs)
Autosamplers (4.5hrs)
Detectors (4.5hrs)

Solid Phase Extraction
Molecular Properties (4hrs)
SPE Overview (3.5hrs)
SPE Mechanisms (4.5hrs)
Method Development (5.0hrs)
Primary Sample Preparation Techniques (2hrs)
Liquid / Liquid Extraction Techniques (1.5hrs) Approaches to Automation for SPE (1.5hrs)

Fundamental GC-MS
Introduction (1.5hrs)
GC Considerations (4.5hrs)
GC -MS Interfaces (2.5hrs)

Fundamental LC-MS
Introduction (1.5hrs)
Electrospray Ionisation Theory (6.5hrs)
Electrospray Ionisation Instrumentation (4hrs)
Mass Analyzers (9.5hrs)
Atmospheric Pressure Chemical Ionisation (3.5hrs)
Atmospheric Pressure Photoionisation (3hrs)
Solvents, Buffers and Additives (3.5hrs)
Vacuum Systems (3hrs)
Flow Rates and Flow Splitting (3hrs)

MS Interpretation
General Interpretation Strategies (11hrs)

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