4.37: Chromatography III
- Page ID
- 38701
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An HPLC assay is being developed to measure the serum concentration of an active drug metabolite. The following information is known about this metabolite: Its molecular weight is 531 daltons; it, like the parent compound, has an aromatic hydroxyl group; and it has a hydroxyl group that the parent does not have. The compound strongly absorbs light at 254 nm. Since the hydroxyl group is found at a single position on the aromatic ring, there are no known structural isomers. Its therapeutic range is approximately 25-100 mg/L.
QUESTION
Which type of chromatography (adsorption, partition, ion-exchange, or molecular exclusion) should be used?
Questions to Consider
- What are the principles governing each of the major classes of chromatography?
- How do the low molecular weight of the compound and the lack of structural isomers affect the choice of a chromatographic mode?
- Would ion exchange chromatography be useful?
- What is the physicochemical basis of polarity?
- What is the relationship between polarity and solvent strength in a normal phase chromatography system? In a reversed-phase chromatography system?
- Answer
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Therefore, the most appropriate chromatographic system would be reversed phase.
Answers to Questions to Consider
- Adsorption chromatography in HPLC makes use of the interaction between polar functional groups on a compound and the polar groups on the solid surfaces of silica or alumina (p. 120).
Partition chromatography makes use of differences in the solubility of a compound in two liquid phases: the mobile phase and the stationary phase (p. 122). The liquid in the stationary phase can be coated or chemically bonded to the stationary phase. It can be either apolar or polar in nature.
Ion exchange (p 123) can only be used when the analyte contains an ionizable group. Separation depends upon the ionic attractions that exist between the counterions on the stationary phase and the analyte, and between the analyte and the mobile phase. - Using Fig. 6.5 (p. 136), one can make the following decisions. Since the molecular weight of the analyte is <1000 daltons, steric exclusion chromatography can be eliminated as a practical approach. The molecule cannot forn hydrogen bonds (phenol group) and is not known to have structural isomers. This allows one to rule out adsorption chromatography.
- There is no ionizable group on this compound and therefore ion exchange chromatography would not be useful.
- Polarity is a concept used to describe the overall interactions between molecules; in liquid chromatography, between solute and solvent molecules and between solute and stationary phase. The four major forces, described in Fig. 5-11 (p. 118), that are the basis of polar interactions all involve the attraction of charges, either induced or permanent. See internet sites: www.cem.msu.edu/~cem333/Week16.pdf, www.laballiance.com/la_info/support/hplc3.htm
- Nomal phase chromatography employs a polar stationary phase. In this system, a strong solvent is one that can remove the most retained, i.e. the most polar, compounds. Therefore, solvent strength in normal phase chromatography increases with the degree of polarity of the mobile phase. In reversed-phase chromatography, the opposite is true. With very apolar stationary phases, the most non-polar compounds are retained, and solvent strength increases as the stationary phase becomes more nonpolar. In other words, solvent strength of the mobile phase is directly related to the polarity of the stationary phase (see pp. 120-121, 135-142). See the internet site in the previous
- Adsorption chromatography in HPLC makes use of the interaction between polar functional groups on a compound and the polar groups on the solid surfaces of silica or alumina (p. 120).