• Steen Honore HansenEmail author
Part of the Advances in Delivery Science and Technology book series (ADST)


Separation techniques have become unavoidable in the drug development process. All samples must be considered to consist of several components. It is important to verify that a compound developed in the initial research phase primarily contain the expected compound and not a number of major impurities that may be responsible for effect or side effects, but even a “pure” API do always contain minor impurities which have to be controlled. The formulated drug products contain a lot of excipients and have to be tested for stability where new impurities may be formed. In the clinical trials samples of plasma, urine, tissue etc. also have to be analysed for the drug substance and its metabolites often in very low concentrations. Therefore all these samples must be considered as complex samples and separation is needed in order to determine each component in a selective and reliable way.

Liquid chromatography is most useful for such qualitative and quantitative analysis and is performed using high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography equipment (UHPLC). Using these techniques high efficiency separations are achieved, and in this chapter these techniques are described in detail.


High performance liquid chromatography (HPLC) Ultrahigh performance liquid chromatography (UHPLC) Separation theory Separation selectivity Separation efficiency Detection API purity Drug stability Sample preparation Validation 


  1. Chiang PC, Hu Y (2009) Simultaneous determination of LogD, LogP, and pK(a) of drugs by using a reverse phase HPLC coupled with a 96-well plate auto injector. Comb Chem High Throughput Screen 12:250–257CrossRefPubMedGoogle Scholar
  2. DeStefano JJ, Langlois TJ, Kirkland JJ (2008) Characteristics of superficially-porous silica particles for fast HPLC: some performance comparisons with sub-2-μm particles. J Chromatogr Sci 46:254–260CrossRefPubMedGoogle Scholar
  3. Done JN, Knox JH (1972) Performance of packings in high speed liquid chromatography. II. Zipax. Effect of particle size. J Chromatogr Sci 10:606CrossRefGoogle Scholar
  4. Gilroy JJ, Dolan JW, Snyder LR (2003) Column selectivity in reversed-phase liquid chromatography. IV. Type-B alkyl-silica columns. J Chromatogr A 1000:757–778CrossRefPubMedGoogle Scholar
  5. Huber JFK (1969) High efficiency, high speed liquid chromatography in columns. J Chromatogr Sci 7:85–90CrossRefGoogle Scholar
  6. Huber JFK, Hulsman JARJ (1967) A study of liquid chromatography in columns, the time of separation. Anal Chim Acta 38:305CrossRefPubMedGoogle Scholar
  7. Larsen J, Staerk D, Cornett C, Hansen SH, Jaroszewski JW (2009) Identification of reaction products between drug substances and excipients by HPLC–SPE–NMR: ester and amide formation between citric acid and 5-aminosalicylic acid. J Pharm Biomed Anal 49:839–842CrossRefPubMedGoogle Scholar
  8. Martin AJP, Synge RLM (1941) A new form of chromatogram employing two liquid phases. Biochem J 35(12):1358–1368CrossRefPubMedPubMedCentralGoogle Scholar
  9. The European Pharmacopoeia (2015) Chromatographic separation techniques, 8th edn. 2.2.46, pp 73–79Google Scholar
  10. The United States Pharmacopoeia 38 and National Formulary 33 (2014) 621 Chromatography, pp 424–434Google Scholar
  11. Scott RPW (1992) Liquid chromatography column theory. Wiley, New YorkGoogle Scholar
  12. Snyder LR, Kirkland JJ, Dolan JW (2010) Introduction to modern liquid chromatography, 3rd edn. Wiley, HobokenGoogle Scholar
  13. Van Deemter JJ, Zuiderweg FJ, Klinkenberg A (1956) Longitudinal diffusion and resistance to mass transfer as causes of nonideality in chromatography. Chem Eng Sci 5:271–289CrossRefGoogle Scholar
  14. Wu Y, Levons J, Narang AS, Raghavan K, Rao VM (2011) Reactive impurities in excipients: profiling, identification and mitigation of drug-excipient incompatibility. AAPS Pharm Sci Tech 12:1248–1263CrossRefGoogle Scholar
  15. Yoon SB, Kim J-Y, Kim JH, Park YJ, Yoon KR, Park S-K, Yu J-S (2007) Synthesis of monodisperse spherical silica particles with solid core and mesoporous shell: mesopore channels perpendicular to the surface. J Mater Chem 17:1758–1761CrossRefGoogle Scholar
  16. Yu H, Cornett C, Larsen J, Hansen SH (2010) Reaction between drug substances and pharmaceutical excipients: formation of esters between cetirizine and polyols. J Pharm Biomed Anal 53:745–750CrossRefPubMedGoogle Scholar
  17. Zhang D, Zhu M, Humphreys WG (2007) Drug metabolism in drug design and development. Wiley, New York, pp 263–285CrossRefGoogle Scholar

Copyright information

© Controlled Release Society 2016

Authors and Affiliations

  1. 1.Department of Pharmacy, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations