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HPLC Basic Course
1. Principle and System Configuration of HPLC(1)

 HPLC Basic Course

Principle of chromatography


  Chromatography is a technique by which a mixture sample is separated into components. Although originally intended to separate and recover (isolate and purify) the components of a sample, today, complete chromatography systems are often used to both separate and quantify sample components.
The term, “chromatography" was coined by the Russian botanist, Tswett, who demonstrated that, when a plant extract was carried by petroleum ether through a column consisting of a glass tube packed with calcium carbonate powder, a number of dyes were separated, as shown in Figure 1. He named this analysis method "Chromatographie" after "chroma" and "graphos", which are Greek words meaning "color" and “to draw," respectively.
 

  Diagram showing Tswett's experiment  
  Figure 1. Diagram showing Tswett's experiment  


  "Chromatography" represents a separation technique; whereas a "chromatograph" is a system for performing chromatography. The chart displaying the time-dependent change in signal intensity as a result of the separation is called a "chromatogram”.
As shown in Table 1, gas and liquid chromatography are common classifications that are based upon the mobile and stationary phases utilized for the separation.
 

  Table 1. Type of chromatography  
 
Mobile phase Stationary phase Analysis Sample Types
Gas Solid/Liquid Gas chromatography (GC) Samples that are gaseous at ordinary temperatures and samples that vaporize when heated
·Odorous samples such as petrochemicals, perfumes, and thinner are easier to analyze by GC.
·High molecular weight compounds are measured after pyrolysis.
Liquid Solid/Liquid Liquid chromatography (LC) Liquid samples and solvent-soluble solid samples
·Compared to GC, LC has a wide range of measurement subjects.
·High molecular weight compounds can be analyzed, if soluble in solvent.
 


  Although the intended use of GC and LC are the same (i.e., separation and quantification), the measurement subjects are different, as the sample conditions differ at separation. The stationary phase typically indicates a column (fillers), while the mobile phase, which is referred to as the eluent in LC, indicates a vehicle to pour a sample into the column.  

  How is a sample separated into its components in the column? The speed of a migrating sample component depends on whether the component has an affinity for the stationary or mobile phase. This affinity appears via various actions: adsorption, partition, ion exchange, etc. As shown in Figure 2, components that have a higher affinity for the mobile phase compared with the stationary phase migrate more rapidly, while components that have a higher affinity for the stationary phase are eluted from the column later. The order and resolution of the components emerging from the column depend on the type of selected stationary and mobile phases.
What does HPLC stand for?
HPLC is short for the High Performance LC. HPLC is an analysis method that yields high performance and high speed compared with traditional column chromatography because of the forcibly pumped mobile phase. Recently, ultrafast analysis using a high-pressure-resistant apparatus has been attracting attention. UHPLC (Ultra High Performance LC) is becoming established as an abbreviation for this ultrafast LC method.
 

  Diagram showing separation  
  Figure 2. Diagram showing separation  

  Chromatography is based on the principal that under the same conditions, the time between the injection of a component into the column and the elution of that component is constant. This characteristic is used to perform qualitative or quantitative analysis. Such analyses are explained here using the measurement of aspartame, a synthetic sweetener contained in beverages.  

 The next chapter is "Configuration of an HPLC system".
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