Liquid Chromatography

Abstract

Liquid chromatography, with various modes, is a technique, which deals with the separation of molecules in samples by using specific solvents or buffers. Liquid chromatography or high‐pressure liquid (HPLC) is a popular quantitative analytical technique applied in many areas of chemical, biomedical and pharmaceutical sciences. HPLC is used for separation, detection and measurement of amino acids, proteins, lipids, carbohydrates, vitamins, trace elements and ions in various types of samples. It is also used for the analysis of polymorphisms in genes coding for specific proteins or enzymes. HPLC is widely used in diagnostic and research purposes in industry, hospitals, educational and research institutions. Liquid chromatography currently accounts for about 60% of separation technology applied in life sciences worldwide. HPLC analytical methods have high assay precision, specificity and accuracy.

Key Concepts

  • HPLC is an important analytical technology used worldwide.
  • HPLC is used for the analysis of micro‐ and macromolecules in human body fluids, water and food items for research and diagnostic purposes.
  • HPLC is also used for the analysis of polymorphisms in genes coding for the enzymes.

Keywords: HPLC; columns; detectors; analysis; solutes; solvents; buffers; adsorbents; counter‐ions

Figure 1. The column and the tubings in an HPLC system.
Figure 2. A typical HPLC system consisting of a degasser, a pump, mobile‐phase delivery system (isocratic or gradient), a detector (UV, fluorescence or electrochemical), auto‐sampler and integrator (computer‐aided). The column may be placed in an oven for constant temperature.
Figure 3. A typical chromatogram of a monobromobimane (MBrB)‐derivatised plasma sample of (a) a healthy control subject, (b) an epilepsy patient on anti epileptic drug and (c) a patient with kidney disease. Samples were analysed according to the method of Mansoor et al. (). Cys, cysteine; CG, cysteinylglycine; HCY, homocysteine. Column, 150 mm × 4.6 mm i.d.; stationary phase, particles ODS 3; mobile phase contains ion‐pairing agent tetrabutylammonium phosphate; column temperature 25°C.
Figure 4. A standard curve for cysteine.
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References

Bishop ML, Fody EP and Schoeff LE (2010) Clinical Chemistry, Techniques, Principles, Correlations, 6th edn. USA: Wolters Kluwer.

Blake CJ (2007) Status of methodology for the determination of fat‐soluble vitamins in foods, dietary supplements, and vitamin premixes. Journal of AOAC International 90: 897–910.

Cabo R, Kozik K, Milanowski M, et al (2014) A simple high‐performance liquid chromatography (HPLC) method for the measurement of pyridoxal‐5‐phosphate and 4‐pyridoxic acid in plasma. Clinica Chimica Acta 4 (33): 150–156.

Cremonesi L, Stenirri S, Fermo I, et al. (2003) Denaturing HPLC analysis of DNA deletions and insertions. Human Mutation 22: 98–102.

Dolan JW (2000a) Starting out right, Part I. Selecting the tools. LC‐GC The Magazine of Separation Science 13: 12–15 [www.lcgcmag.com].

Dolan JW (2000b) Starting out right, Part II. Measuring satisfaction. LC‐GC The Magazine of Separation Science 13: 72–76 [www.lcgcmag.com].

Dorsey JG, Foley JP, Cooper WT, Barford RA and Barth HG (1990) Liquid chromatography: theory and methodology. Analytical Chemistry 62: 324R–356R.

Frueh FW and Noyer‐Weidner M (2003) The use of denaturing high‐performance liquid chromatography (DHPLC) for the analysis of genetic variations: impact for diagnostics and pharmacogenetics. Clinical Chemistry and Laboratory Medicine 41: 452–461.

Kudo M, Moteki T, Sasaki T, et al (2008) Functional characterization of human xanthine oxidase allelic variants. Pharmacogenetics and Genomics 18: 243–251.

Larsen TL, Nilsen V, Andersen DO, et al. (2008) Comparison of high‐pressure liquid chromatography (HPLC) and Griess reagent‐spectroscopic methods for the measurement of nitrate in serum from healthy subjects. Clinical Biochemistry 41: 1474–1481.

Lim CK (1986) HPLC of Small Molecules, a Practical Approach. Oxford: IRL Press.

Lough WJ and Wainer IW (eds) (1996) High Performance Liquid Chromatography, Fundamental Principles and Practice. London: Blackie Academic and Professional.

Mansoor MA, Svardal AM and Ueland PM (1992) Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine and glutathione in human plasma. Analytical Biochemistry 200: 218–229.

Premstaller A and Oefner PJ (2003) Denaturing HPLC of nucleic acids. PharmaGenomics 3: 20–37.

Seljeskog E, Hervig T and Mansoor MA (2006) A novel HPLC method for the measurement of thiobarbituric acid reactive substances (TBARS). A comparison with a commercially available kit. Clinical Biochemistry 39: 282–286.

Snyder LR (2000) HPLC : past and present. Analytical Biochemistry 72: 412A–420A.

Swartz ME and Krull IS (1997) Analytical Method Development and Validation. USA: Marcel Dekker, Inc.

Zacharis CK and Tzanavaras PD (2013) Liquid chromatography coupled to on‐line post columns derivatization for the determination of organic compounds: a review on instrumentation and chemistries. Analytica Chimica Acta 798: 1–24.

Further reading

LaBerge GS, Shelton RJ and Danielson PB (2003) Forensic utility of mitochondrial DNA analysis based on denaturing high‐performance liquid chromatography. Croatian Medical Journal 44: 281–288.

Sanz AM, Ayala JH, González V and Afonso AM (2008) Analytical methods applied to the determination of heterocyclic aromatic amines in foods. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences 862: 15–42.

Skoog DA, Holler FJ and Nieman TA (1998) Principles of Instrumental Analysis, 5th edn. USA: Saunders College Publishing.

Synder LR and Kirkland JJ (2010) Introduction to Modern Liquid Chromatography, 3rd edn. USA: John Wiley & Sons.

Vogeser M and Seger C (2008) A decade of HPLC‐MS/MS in routine clinical laboratory‐goals for further developments. Clinical Biochemistry 41: 649–662.

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How to Cite close
Mansoor, Mohammad Azam(Jun 2015) Liquid Chromatography. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002679.pub3]