SPE phase & solvent selection

SPE phase selection overview

Choosing the best SPE stationary phase to use depends on the physical/chemical properties (such as pH, pKa and Solubility log P) of your analytes. There are a variety of SPE stationary phases to choose from including: reverse phase, normal phase, ion-exchange, and mixed mode, with mixed mode being a mixture of both normal and reversed phases. This page helps you decide which phase to use when.

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How to choose your SPE phase

Table 1 shows the basic mechanism of separation for each of these phases, while Figure 1 shows that phase selection starts with determining if your sample is soluble in water or organic solvent, your analyte’s characteristics, and which SPE stationary phase to use for each. Figure 1 also shows typical SPE stationary phases used for different applications. Figure 1 is an SPE Phase Selection Flow Chart that shows which Thermo Scientific SPE phase to use depending on your analytes’ solubility and charge.

Table 1: Mechanisms of SPE phase separation
  Reverse Phase Normal Phase Ion Exchange
Retention Mechanism
  • Non-polar/ hydrophobic interactions
  • Van der Waals or dispersion forces
  • Polar interactions
  • Hydrogen bonding, pi-pi, dipole-dipole, and induced dipole-dipole
  • Electrostatic attraction
  • Attraction between charged analyte functional groups with oppositely charged sorbent functional groups
Analyte Characteristics
  • Non-polar functionalities
  • Most organic analytes
  • Alkyl, aromatic, alicyclic functional groups
  • Polar functionalities
  • Hydroxyl groups, carbonyls, amines, alkenes
  • Heteroatoms
  • functionalities with resonance
  • Acids (use anion exchange)
  • Bases (use cation exchange)
Sample Matrices
  • Aqueous-based
  • Biological samples (serum, plasma, urine)
  • Tissue extracts
  • Environmental water samples
  • Non-polar samples
  • Organic extracts
  • Very non-polar solvents
  • Fatty oils, hydrocarbons
  • Aqueous or organic samples (low salt concentration)
  • Biological fluids
  • Solution phase synthesis
Elution Considerations Disrupt interaction with solvents with adequate non-polar character Disrupt interaction with solvents with a more polar character Disrupt interactions by:
  • pH modification
  • Increase salt concentration
Common Sorbents
  • Bonded silica (C8, C18)
  • Polymeric
  • Carbon
Bare silica, Alumina, Bonded silica (aminopropyl, diol) Florisil Bonded silica (SAX, WAX; SCX, WAX)
SPE Stationary Phase Selection

Figure 1. shows the phase (sorbent) depending on if your sample is soluble in a polar, moderately polar, or non-polar organic solvent polar or if your sample is soluble in water soluble if it is ionic or non-ionic.


SPE phases in depth – functionality and typical applications

Polymerics Reversed Phase Silica Phases Normal Phase Silica Phases Ion Exchange Phases
  • Reversed phase non-polar (hydrophobic) phases
  • Mixed-mode Phases
  • Reversed phase hydrophobic phases
  • Normal phase hydrophilic phases
  • Ion-exchange phases
  • Mixed-mode phases
SPE Phase Functionality Surface area Particle size Pore size Analyte Typical Applications
SOLA & SOLAµ HRP Polystyrene divinylbenzene material surface functionalized with pyrolidone 700 to 900m2/g 19 to 25µm 30 to 110Å Retention of polar and non-polar analytes
  • Bioanalytical workflows
Extraction of:
  • Extraction of polar and non-polar analytes
  • Drugs and metabolites in biological matrices
  • Desalting of peptides in serum, plasma, or biological fluids
HyperSep Retain PEP Polystyrene Divinylbenzene material surface modified with urea groups 550 – 750m2 /g 40 – 60μm 55 – 90Å Retention of polar and non-polar analytes
  • Drugs and metabolites in biological matrices
  • Environmental samples
  • Desalting of peptides in serum, plasma, or biological fluids

Reversed Phase Silica Phases:

Separates using non-polar interactions: Van der Waals or dispersion force

SPE Phase Functionality Surface area Particle size Pore size Analyte Typical Applications
HyperSep C18 Highly retentive alkyl-bonded silica phase 470 – 530m2 /g 40 – 60μm 60Å Retains non-polar to moderately polar compounds
  • Drugs and their metabolites in biological matrices
  • Trace organics in environmental water samples
  • Toxins in food samples
HyperSep C8 Less retentive alternative to C18 470 – 530m2 /g 40 – 60μm 60Å Retains non-polar to moderately polar compounds
  • hydrophobic compounds which tend to be retentive on C18 columns
  • HyperSep Phenyl silica-based material with alternative selectivity for basic compounds 470 – 530m2 /g 40 – 60μm 60Å aromatic compounds with a benzene ring in its structure
    • benzodiazepines in biological matrices
    • aromatic compounds

    Mixed-Mode Phases

    These comprise of two functional groups: either non-polar and ion exchange, or hydrophobic and ionic retention. They are ideal for samples with complex structures.

    SPE Phase Functionality Surface area Particle size Pore size Analyte Typical Applications
    SOLA & SOLAµ SCX (mixed mode) Next generation Polystyrene divinylbenzene material surface functionalized with sulphonate groups 550 – 750m2 /g 19 to 25µm   30 to 110Å a versatile polymeric material for the enhanced retention of weak bases
    • Bioanalytical workflows
    Extraction of:
    • Weak bases
    • Drugs and metabolites in biological matrices
    Desalting of peptides in serum, plasma, or biological fluids
    SOLA & SOLAµ SAX (mixed mode) Next generation Polystyrene divinylbenzene material surface functionalized with quaternary groups   550 – 750m2 /g 40 – 60μm 55 – 90Å Polymeric material for the retention of weak acids  
    • Bioanalytical workflows
    Extraction of:
    • Weak Acids
    • Drugs and metabolites in biological matrices
    Desalting of peptides in serum, plasma, or biological fluids
    SOLA & SOLAµ WCX (mixed mode) Next generation Polystyrene divinylbenzene material surface functionalized with carboxcylic acid groups 550 – 750m2 /g 19 to 25µm   30 to 110Å a versatile polymeric material for the enhanced retention of strong bases
    • Bioanalytical workflows
    Extraction of:
    • Weak bases
    • Drugs and metabolites in biological matrices
    • Desalting of peptides in serum, plasma, or biological fluids
    SOLA & SOLAµ WAX (mixed mode) Next generation Polystyrene divinylbenzene material surface functionalized with tertiary amine groups 550 – 750m2 /g 19 to 25µm   30 to 110Å a versatile polymeric material for the enhanced retention of strong acids
    • Bioanalytical workflows
    Extraction of:
    • Weak bases
    • Drugs and metabolites in biological matrices
    • Desalting of peptides in serum, plasma, or biological fluids
    HyperSep Retain-CX Versatile polymeric material for retention of basic compounds 550 – 750m2 /g 40 – 60μm 55 to 90Å a versatile polymeric material for the enhanced retention of basic compounds a wide range of drugs of abuse from biological matrices
    HyperSep Retain-AX Versatile polymeric material for retention of acidic compounds 550 – 750m2 /g 40 – 60μm 55 – 90Å a versatile polymeric material for the enhanced retention of acidic compounds acidic drugs of abuse from biological matrices, such as THC and its metabolites
    HyperSep Hypercarb Unique material for retention of highly polar compounds
    • 100% porous graphitic carbon material
    • Retention properties allow low bed
    Retention of extremely polar compounds Ideal for problem analytes
    SPE Phase Functionality Surface area Particle size Pore size Analyte Typical Applications
    HyperSep Silica A polar sorbent primarily used to retain analytes from non-polar matrices 530m2 /g 40 – 60μm 60Å extract analytes from non-polar solvents such as hydrocarbon, less polar esters and ethers
    • Amines
    • Pesticides
    • Herbicides
    • Carotenoids
    • Fat soluble vitamins
    • Aflatoxins
    • Fatty acids
    • Phospholipids
    HyperSep Florisil magnesia-loaded silica gel 289m2 /g 40 – 60μm 60Å isolation of polar compounds from non-polar matrices
    • Pesticides using AOAC and EPA methods
    • Polychlorinated biphenyls (PCBs) in transformer oil
    HyperSep Cyano silica-based material of low hydrophobicity that is retentive than either silica or diol. 470 – 530m2 /g 40 – 60μm 60Å Retention of polar compounds from non-polar matrices
    • Polar compounds from hexane and oil
    HyperSep Diol silica-based material for extraction of polar compounds 470 – 530m2 /g 40 – 60μm 60Å of polar compounds normal phase extraction and purification of polar compounds
    HyperSep Aminopropyl silica-based material used as both a polar sorbent and a weak anion exchanger 470 – 530m2 /g 40 – 60μm 60Å   petroleum fractionation, saccharides, drugs and drug metabolites

    Ion Exchange Phases

    SPE Phase Functionality Surface area Particle size Pore size Analyte Typical Applications
    HyperSep SAX (Strong Anion Exchanger) Strong anion exchange sorbent for extraction of weak acids 470-530m2 /g 40 – 60μm 60Å
    • negatively charged compounds from both aqueous and non-aqueous matrices
    • weak acids such as carboxylic acid
    removal of
    • acidic food pigments
    • removal of phenolic compounds, nucleic acids, and surfactants
    HyperSep SCX (Strong Cation Exchanger) Strong cation exchange sorbent for extraction of charged basic compounds 470-530m2 /g 40 – 60μm 60Å extraction of positively charged compounds from both aqueous and non-aqueous matrices Extraction of antibiotics, drugs, organic bases, amino acids, catecholamines and herbicides

    Mixed-Mode Phases

    SPE Phase Functionality Surface area Particle size Pore size Analyte Typical Applications
    HyperSep Verify-CX Reversed phase C8 group and a strong cation exchanger 470-530m2 /g 40 – 60μm 60Å Non-polar and anionic characteristics for improved analysis of basic drugs of abuse Range of basic drugs of abuse from biological matrices
    HyperSep Verify-AX a reversed phase C8 group and a strong anion exchanger 470-530m2 /g 40 – 60μm 60Å analysis of acidic drugs of abuse A range of acidic drugs of abuse from biological matrices including THC and its metabolites

    Choosing the SPE format and bed weight

    The stationary phase, differing in particle size and distribution, can be packed in a cartridge, 96-well plates, and even pipette tips.

    SPE 96-well plates

    • SPE 96-well plates are ideal when simultaneously processing a large number of small-volume samples in high throughput environments. Is there a strategy for using these that the cartridges don’t have?

    SPE cartridges

    • Plastic SPE cartridges are the most common format used for SPE. They vary in size (e.g., 1mL, 3mL, 6mL, 12mL) and amount of stationary phase (e.g., what?). 3mL cartridges are a popular size and a good starting point for many applications.
    • Smaller cartridges contain smaller amounts of sorbent and require less solvent for elution. This can make a more efficient separation which is useful if evaporation is required.
    • Larger cartridges can be used when larger volumes of solvent are required. A larger sorbent amount can be helpful when analytes are difficult to retain on a column.
    • As a general rule, the cartridge size should
      • Closely match the volume of wash solvent use.
      • Be appropriate for your sample load: Typically, one can load a sample that is approximately 5-10% of the sorbent weight.
    • Take extraction speed and sample capacity into consideration. For example, a faster extraction speed or a higher sample capacity requirement will need a larger cartridge.

    Table 3. Typical sorbent mass, sample size, and elution volumes used for different SPE cartridge sizes

    Cartridge Volume Sorbent Mass Sample Size Minimum Elution Volume
    1mL 50-100mg 2.5-10mg 100-200mL
    3mL 500mg 25-100mg 1-3mL
    6mL 500-1000mg 25-100mg 2-6mL
    12mL 2000mg 100-200mg 10-12mL

    How to select the SPE elution solvent

    The choice of solvent is dependent upon the sample matrix and the retention mechanism used. The table below shows the differing polarities of solvents commonly used in SPE.

    Recommended SPE elution solvents

    The choice of solvent to extract the analyte is dependent on the analyte properties.

    • Nonpolar solvents such as ethyl acetate are strong solvents in reversed phase applications.
    • Polar solvents such as methanol and water are strong solvents in normal phase interactions.
    Recommended Solvents for SPE
    CMD SchemaApp code