| | Reversed Phase | Normal Phase | Ion Exchange |
| Analyte | Moderate to low polarity
| Low to high polarity/neutral
| Charged or Ionizable |
| Separation Mechanism | Separation based on hydrophobicity | Separation based on polarity | Separation based on charge |
| Sample Matrix | Aqueous | Non-polar organic solvent | Aqueous/ Low ionic strength |
| Condition/ Equilibrate SPE Sorbent | 1. Solvate with polar organic 2. Water | Non-polar organic | Low ionic strength buffer |
| Preliminary Wash Step | Aqueous/buffer | Non-polar organic | Low ionic strength buffer |
| Elution Steps | Increase polar organic content | Increase eluotropic strength of organic solvent mixture | Stronger buffers - ionic strength or pH to neutralize the charge |
| | | | AX
[Anion Exchange] | CX
[Cation Exchange] |
| Sorbent Functionality | C18, tC18, C8, tC2, CN, NH2, HLB, RDX, Rxn RP | Silica, Alumina, Florisil, Diol, CN, NH2 | Accell Plus QMA, NH2, SAX, MAX, WAX | Accell Plus CM, SCX, MCX, WCX, Rxn CX |
| Sorbent Surface Polarity | Low to Medium | High to Medium | High | High |
| Typical Solvent Polarity Range | High to Medium | Low to Medium | High | High |
| Typical Sample Loading Solvent | Water, low strength buffer | Hexane, chloroform, methylene chloride | Water, low strength buffer | Water, low strength buffer |
| Typical Elution Solvent | CH3OH/water, CH3CN/water | Ethyl acetate, acetone, CH3CN | Buffers, salts with high ionic strength, increase pH | Buffers, salts with high ionic strength, decrease pH |
| Sample Elution Order | Most polar sample components first | Least polar sample components first | Most weakly ionized sample component first | Most weakly ionized sample component first |
| Mobile Phase Solvent Change Required to Elute Compounds | Decrease solvent polarity | Increase solvent polarity | Increase ionic strength or increase pH | Increase ionic strength, or lower pH |
This has been a brief introduction to sample enrichment and purification using solid-phase extraction [SPE]. The best way to start using SPE is to first learn what others have done with analytes and/or matrices similar to those of interest to you. You will find > 7,700 references to the use of SPE in the Resource Library on waters.com. Fill in the blank with a partial compound or matrix name in the following search phrase:
“Sep-Pak” OR “Oasis” AND ______*
NOTE: Rather than risk a spelling error, use an asterisk [*] with a root name for best results. Using this same search string, even more references [> 60,000] may be found on GOOGLE Scholar.
