Introduction to HPLC

What is High-Performance Liquid Chromatography (HPLC)?

High-Performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in a mixture.  Chromatography refers to the measurement method, chromatogram refers to the measurement results, and chromatograph refers to the instrument. Chromatography can be used for qualitative or quantitative analysis. Qualitative analysis refers to “what components are in the mixture” while quantitative analysis refers to “how much of each component is present in the mixture” (Fig. 1).

Separating mixtures into their components
Fig. 1 Separating mixtures into its components

The Beginning of Chromatography

Methods for separating components of a mixture include filtration, distillation, and extraction. Chromatography was invented by the Russian botanist Mikhail Semenovich Tswett. In the early 1900s, Tswett packed calcium carbonate in a standing tube, placed pigments extracted from plants on top, and then flushed the tube with petroleum ether as a solvent (Fig. 2).

Tswett’s chromatograph
Fig. 2 Tswett’s chromatograph

As the extracted pigments began to separate on the chromatograph, each individual pigment appeared as a different band of color as if the light was divided into seven colors by a prism in the tube. For this reason, Tswett named this new separation method chromatography using the Greek words chroma, which means color, and graph, which means record. HPLC is a type of separation method commonly referred to as column chromatography and has been developed to enable separation and analysis in a short period of time by increasing pressure compared to atmospheric pressure.

Separation Mechanism in Chromatography

A mixture is placed in a stream of liquid (petroleum ether in Fig. 3) called the mobile phase and moved through a solid medium (calcium carbonate powder in Fig. 3) called the stationary phase. The components in the mixture move with the flow of the mobile phase and interact with the stationary phase. The speed of movement depends on the strength of the interaction between each component and the stationary phase. That is, components that interact strongly with the stationary phase move slowly, whereas components that interact weakly move quickly, so allowing the components to be separated.

Chromatogram (middle), separation mechanism (top)
Figure 3. Chromatography separation mechanism (top) and resulting chromatogram (bottom)

The separated components can be analyzed using different types of detectors. A UV detector, for example, can detect components based on UV absorption. The chromatogram is obtained by measuring the elution time on the X axis and the intensity of the UV signal on the Y-axis. If the measurement conditions are the same, the elution time (peak position) for the standard sample whose components are known and that for the unknown sample can be compared to identify the components for qualitative analysis. In addition, since the absorption intensity is proportional to the concentration, a calibration curve can be prepared using a standard sample, and the component concentration can be determined by measuring the peak area or height for quantitative analysis.

HPLC Detectors

UV/Vis Detector

A UV/Visible detector detects components that absorb light between 190 and 900 nm. For example, aromatics, pigments, proteins, and drugs can all be measured. By selecting the measurement wavelength, it is possible to measure the sample while suppressing the influence of interfering components (Fig. 4). In addition, the sensitivity can be improved by performing measurements at the wavelength where the maximum absorption occurs. By measuring the UV/Visible absorption spectrum at the elution peak and searching a library, it is possible to predict the components that are present. The purity can also be checked from the absorption spectrum.

Figure 4. UV/Visible measurements of crude drug at different wavelengths

Fluorescence Detector

A fluorescence detector detects components that emit light between 220 and 900 nm. Fluorescence detectors are more selective than UV/Visible detectors since the excitation and emission wavelengths are both specific for a particular substance. The excitation and emission wavelengths can be switched programmatically, making it is possible to simultaneously detect fluorescent substances with different wavelengths if the elution time is different (Fig. 5).

Figure 5. Fluorescence measurement using fixed-wavelength

Refractive Index (RI) Detector

A Refractive Index (RI) detector detects components based on their refractive index relative to the mobile phase’s refractive index. Most compounds have a different refractive index to that of the mobile phase, so any component can be detected. However, refractive index variations also occur due to changes in temperature and solvent composition, so it is necessary to perform measurements at constant temperature and in isocratic solvent ratio mode.

Comparison of Detectors

Table 3 summarizes the characteristics of UV/Visible, fluorescence, and RI detectors. UV/Vis and fluorescence detectors are both highly sensitive as well as selective. A gradient elution method can be used with either of these detectors since they are less sensitive to temperature. A RI detector has the advantage of being able to detect a wide range of components; however, it is sensitive to temperature, so the gradient elution method cannot be used. A RI detector has lower sensitivity compared to a UV detector but can detect components that an UV detector cannot. Although they are somewhat expensive, Evaporative Light Scattering Detectors (ELSD) are available that can detect a variety of components with high sensitivity in gradient elution mode.

DetectorSensitivitySelectivityTemperature EffectGradient Elution Method
UV-VisibleNanograms (ng)HighLowApplicable
FluorescencePicograms (pg)Very HighLowApplicable
Refractive IndexMicrograms (μg)N/AHighN/A

Figure 6 shows the lower sensitivity of the RI detector, but that detects components that the UV detector cannot.

Figure 6. Comparison between RI measurement (upper) and UV/Visible measurement (lower)

 

Qualitative and Quantitative Analysis

In most cases, the identification of a target component in an unknown sample is performed by comparing the retention time of the target component to that of a standard sample. If a complex chromatogram with many peaks is obtained or if the retention time of the target component differs from the standard sample, the target component can be identified by adding the standard sample to the unknown sample (Fig. 7).

Figure 7. Identification by comparing retention times in a standard sample (upper) and an unknown sample (lower)

Quantitative Analysis

There are two methods for quantification: external and internal standard methods, both of which are performed using a calibration curve. The external standard method creates a calibration curve by plotting standard concentration versus peak area. Unknown samples are quantified by extrapolating their concentration from their peak area based on the standard calibration curve. The internal standard method creates a calibration curve by adding a fixed amount of internal standard to different concentrations of standard and plotting standard concentration versus the ratio of the peak areas (peak area of standard divided by peak area of internal standard). The same fixed amount of internal standard is also added to the unknown samples which are quantified by extrapolating their concentration from the ratio of peak areas (peak area of the unknown sample divided by peak area of internal standard).

When using the internal standard method, the internal standard is required to be a component not included in the unknown samples, to produce peaks that are completely separable from those for any contamination components, to elute at a retention time close to the quantitative target component, to be chemically and physically stable, and to be highly pure. The advantage of the internal standard method is that it improves the precision of quantitative analysis by reducing error in the injection volume or those caused by evaporation of the solvent.

Figure 8. Quantification by the internal standard method, calibration curve (left), and chromatogram (right)

 

Procedure for Establishing Analysis Conditions

Step 1. Clarify the purpose of the analysis and investigate the components to be analyzed:

Molecular weight, molecular structure, and functional groups of components

Properties (solubility, stability, UV/Visible or fluorescence spectrum, etc.) of components

Sample state (content, concentration, impurities)

Preliminary examination of analysis conditions and pretreatment based on data collections, literature, etc.

Step 2. Consider analysis conditions (column type, mobile phase composition, temperature, separation method, and detection method):

Confirmation of component peaks in standard samples using relatively high concentrations and examination of separation conditions

Measurement at the required concentration, determination of detection method Examination of pretreatment of unknown samples

Confirmation of separation of target components and contaminants in unknown samples

Step 3. Determination of analysis conditions for routine measurements:

Determination of calibration curve linearity and calibration curve type
Quantitative reproducibility including pretreatment
Confirmation of components that are strongly retained in the column and components that are not detected by the detector
Correlation with other quantitative methods

Step 4. Routine quantitative analysis:

Clarification of measurement procedure and documentation
System and column maintenance
Column lifetime
Running cost

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Further Topics on High-Performance Liquid Chromatography (HPLC)