HPLC Basic Course

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1. Principle and System Configuration of HPLC (1)

 Principle and System Configuration of HPLC (1)

Principle of Chromatography

Figure 1. Diagram showing Tswett’s experiment

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.

“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.

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.


2. Principle and System Configuration of HPLC (2)

Philosophy of Qualitative analysis and Quantitative analysis by chromatography

Qualitative analysis

A standard sample of aspartame was measured, yielding a peak at 12.5 minutes. This peak corresponds to aspartame, itself.
Next, a sample prepared from a beverage was measured under the same conditions, yielding a number of peaks. The peak appearing at 12.5 minutes can be regarded as that of aspartame (Figure 1).
The following conditions were the same: the type of fillers, column sizes, column temperature, composition of the mobile phase, and flow rate.

Figure 1. Example of aspartame measurement

Quantitative analysis

The height and area of a peak are proportional to the concentration of the corresponding component. A calibration curve is created using the standard sample. The concentration of aspartame in the beverage can be determined from the peak area of the detected aspartame.

Attention should be given to the fact that a qualitative analysis includes many uncertainties. Other components may have been eluted together with aspartame. LC and GC systems are good at determining the content of a certain component in a sample, rather than the types of the components of a sample. Performance of a quantitative analysis requires the preparation of a calibration curve. It is very difficult to perform the qualitative or quantitative analysis of a component for which a standard sample is not available.


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One Reply to “HPLC Basic Course”

  1. I like how you mentioned that separating and quantifying are the popular methods used today in chromatography. The lead researcher in the lab my sister works in is thinking of getting a portable liquid chromatography system because he’s considering performing research at the location of some unspecified substances. It seems like a good idea for the lead researcher to think about getting equipment from a reputable supplier so that he can do proper research out in the field.

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