HPLC / Liquid Chromatography (LC)
History
Historically, chromatography dates back to a Russian chemist, Mikhail Tswett, who gave a lecture in 1903 on the separation of the pigments in green leaves on a chalk column. His first papers were actually published in 1906. Another researcher, David Talbot Day, an American, was simultaneously using chromatography in his work on the separation of hydrocarbons from petroleum. Tswett, however generally receives all the credit because he was able to recognize and understand the chromatographic process.
Tswett filled an open glass column with particles of powdered chalk (calcium carbonate) and alumina. He poured a sample (solvent extract of plant leaves) into the column and allowed it to pass through the particles of powdered chalk. This was followed by pure solvent. As the sample passed down through the column by gravity, different colored bands could be seen separating because some components were moving faster than others. He related these separated, different-colored bands to the different compounds that were originally contained in the sample.
Tswett had created an analytical separation of these compounds based on the differing strength of each compound’s chemical attraction to the particles. He coined the term “chromatography” (from the Greek word chroma meaning color and graphy which means writing – in a sense color writing).
In principle liquid chromatography (LC) and high performance liquid chromatography (HPLC) work the same way except the speed, efficiency, sensitivity and ease of operation of HPLC is vastly superior.
HPLC has been around for about 40 years and is the largest separations technique used. The history of HPLC is summarized below:
- It started as high pressure liquid chromatography in the early 1960’s.
- By the end of the 70’s improvements of column material and instrumentation led to the High Performance Liquid Chromatography.
- Since the 80’s HPLC becomes a major analytical technique.
- Since 2006 new terms came up such as UPLC, RRLC, UFLC.
How does Liquid Chromatography (LC) or High Performance Liquid Chromatography work?
The components of a basic LC or HPLC system are shown in the diagram in Fig. 1:
- Reservoir for mobile phase (liquid)
- Pump
- Injection port (sample inlet)
- Column / HPLC column
- Detector (chromatography detector)
- Computer data station
- Waste container
A reservoir holds the solvent that is the mobile phase. A pump or a high-pressure pump is used to propel the mobile phase at a specified rate through the column (typically ml/min). An injector is able to introduce the sample as a plug into the continuously flowing mobile phase stream that carries the sample into the column. The column contains the chromatographic packing material needed to effect the separation. The packing material is the so called stationary phase. A detector is used to see the separated compound bands as they elute from the column.
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| Fig. 1 : Diagram of a liquid chromatograph |
What are the advantages and limitations of HPLC?
HPLC offers several advantages:
- Speed
- Sensitivity
- Reusable columns
Speed – Analysis times of an hour or less have become common. Less operator time is required comparing to the past.
Resolution – By using selective columns and mobile liquids, liquid chromatography can provide resolution of the millions of nonvolatile compounds that is not possible to analyze by gas chromatography. By using gradient elution even compounds that are strongly retained in columns can be analyzed.
Sensitivity – High sensitivity detectors permit measurement of nanogram (10-9) quantities of material. A few microliters of sample are usually sufficient for a complete analysis.
The small samples used in HPLC can be a disadvantage. If the analytes are collected, the amount of each component available is very small. However, in most cases sufficient material for identification for identification by spectrometric techniques is collected.
Applications of LC and HPLC
LC and HPLC are being used for qualitative and quantitative analysis of:
- Drugs and pharmaceuticals (drug testing)
- Nucleic acids
- Forensic / Urine and other body fluids
- Pesticides, herbicides
- Polymers
- Lipids
- Amino acids
- Carbohydrates
- Surfactants
Relevant Posts
Selection of the Liquid Chromatographic (LC) / HPLC Separation Mode
Selection & Optimization of operating parameters in LC
Liquid Chromatography: Columns in LC/HPLC – Bonded Phase Columns (C18)
Troubleshooting HPLC / Liquid Chromatography Systems – Peak Tailing
Ion-Exchange Chromatography (IEC) - Liquid Chromatography Separation Modes
Selection & Optimization of operating parameters in LC
Liquid Chromatography: Columns in LC/HPLC – Bonded Phase Columns (C18)
Troubleshooting HPLC / Liquid Chromatography Systems – Peak Tailing
Ion-Exchange Chromatography (IEC) - Liquid Chromatography Separation Modes
References
- C.A. Dorschel, J.L. Ekmanis, J.E. Oberholtzer et al. “LC Detectors” Anal. Chem., 61, 951A–968A, 1989
- Nina Hadden et al., “Basic Liquid Chromatography”, Varian Aerograph, 1971
- C. F. Simpson, “Techniques in Liquid Chromatography” Wiley-Hayden: Chichester, England, 1982
- L.R. Snyder. J.J. Kirkland, “Introduction to Modern Liquid Chromatography”, 2nd edition, Wiley, 1979
- A. Weston and P. Brown, “HPLC and CE Principles and Practice”, Academic Press, 1997
- C.F. Poole, S.K. Poole, “Chromatography Today”, Elsevier, New York, 199
Key Terms
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