Solid Phase Extraction: Normal Phase Methodology

Non-polar Sample Matrix/ Mobile Phase Environment

In order for polar retention to occur between the sorbent and the sample, the analyte must be introduced to the SPE device in a non-polar sample or mobile phase environment. Therefore, typical sample matrices that can be employed in normal-phase SPE include hydrocarbon or fatty oils diluted in an organic solvent, hexane, isooctane, chlorinated solvents, THF, diethyl ether, and ethyl acetate.

Most organic analytes exhibit some polar functionalities that can be exploited for normal-phase separation. Because many molecules exhibit polar functionality, each interaction can provide different levels of selectivity offering highly selective separations of compounds very similar in structure.


Normal-Phase SPE
Retention Mechanism: Polar Interactions
  • Hydrogen bonding, π-π, dipole-dipole, and induced dipole-dipole
Sample Matrix: Non-polar samples
  • Organic extracts of solids
  • Very non-polar solvents
  • Fatty oils, hydrocarbons
Analyte Characteristics: Analytes exhibiting polar functionalities
  • Hydroxyl groups, carbonyls, amines, double bonds
  • Hetero atoms (O, N, S, P)
  • Functional groups with resonance properties
Elution Scheme: Polar interactions disrupted with a more polar solvent or solution
  • Acetonitrile, methanol, isopropanol
  • Combinations of buffer/solvent or solvent/solvent mixtures
Common Applications:
  • Cleanup of organic extracts of soils and sludge
  • Fractionation of petroleum hydrocarbons
  • PCBs in transformer oil
  • Isolation of compounds in cosmetics

Basic Steps

  1. Sample Pre-treatment Liquid samples should be initially extracted or diluted with a non-polar solvent such as hexane or a chlorinated solvent. Soil, sediment, and other solid samples are initially extracted (Soxhlet or sonication) with a non-polar solvent, and concentrated prior to SPE cleanup. Aqueous residues in the sample can reduce normal-phase retention. It may be necessary to further dry the organic extract with sodium sulfate or magnesium sulfate prior to SPE.

  2. Conditioning/Equilibration Condition and equilibriate with 2-3 tube volumes of a non-polar solvent similar or identical to sample matrix resulting from sample pre-treatment.

  3. Sample Load Apply sample (from step 1) at a consistent and reduced flow rate of ~1-2 drops/second to ensure optimal retention. The compounds should be a non-polar solvent (e.g., hexane) for optimal retention. Note that methanol and acetonitrile are often used as elution solvents in normal-phase SPE, and will often not promote compound retention during sample load.

  4. Wash Sample interferences are often co-retained with compounds of interest during sample load. A wash step is necessary to elute interferences without prematurely eluting compounds of interest. In normal-phase SPE, 1-2 tube volumes of the solvent used in sample pre-treatment and conditioning can be used during wash.

  5. Elution Disrupt polar interactions with a solvent or solvent/buffer mixture more polar than both the sample and wash solutions. Typical elution solvents include water miscible organic solvents such as acetone, acetonitrile, methanol, and isopropanol. Eluting with increasingly polar solvents (or solvent mixtures) in succession can also fractionate multiple compound classes. See “Common Normal-Phase Solvents” table for assistance.

  6. Eluate Post-treatment Normal-phase SPE is often followed by GC analysis, and therefore requires a volatile sample matrix prior to injection. Use sodium sulfate or magnesium sample to remove residual moisture. Further SPE eluate concentration may also be necessary prior to analysis.

Common Normal-Phase Solvents

Solvent Elutropic (e°) or
elution strength
on silica
Retention / Elution
Hexane 0.00 Retention
Isooctane 0.00
Carbon tetrachloride 0.14
Toluene 0.22
Benzene 0.27
Tert-butyl methyl ether 0.29
Chloroform 0.31
Methylene chloride (dichloromethane) 0.32
Diethyl ether 0.29
Ethyl acetate 0.43
Tetrahydrofuran 0.35
Acetone 0.45
Acetonitrile 0.50
40% methanol in acetonitrile 0.67
20% methanol in diethyl ether 0.65
20% methanol in methylene chloride 0.63
Isopropanol 0.63
Methanol 0.73
Water >0.73 Elution
Acetic acid >0.73


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