Introduction to HPLC

HPLC is an abbreviation for high-performance liquid chromatography. Chromatography refers to the measurement method, chromatogram refers to the measurement results, and chromatograph refers to the instrument. Chromatography separates components in a particular substance and performs qualitative and quantitative analyses on those components. Qualitative analysis refers to “what kind of compound each component is”, and quantitative analysis refers to “how much of each component is present” (Fig. 1).

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

The beginning of chromatography

Methods for separating mixed compounds 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). Individual pigments appeared as color bands as if the light was divided into seven colors by a prism in the tube. For this reason, Tswett named this separation method chromatography using the Greek words chroma (color) and graph (record).

Tswett’s chromatograph
Fig. 2 Tswett’s chromatograph

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 Chromatogram (middle), separation mechanism (top)

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 system configuration

High-performance liquid chromatography (HPLC) is a type of separation method referred to as column chromatography and has been developed to enable separation and analysis in a short time using high pressure. An HPLC system consists of five parts: a pump for liquid delivery, an injector for sample injection, a column for separation, a detector, and a data processor (Fig. 4). The pump is used to deliver the solvent (mobile phase) and sample to the detector. For sample injection, an injector (manual) or an autosampler (automatic) is used to introduce the sample into the mobile phase. To achieve more stable separation, an oven may be used to keep the column at a constant temperature. The detector detects the separated components, which vary depending on the detector type. The data processor displays the detected signal (chromatogram) on a computer and analyzes it. The data processor allows identification and quantification of components.

HPLC configuration
Figure 4 HPLC configuration

HPLC pumps

As shown in Fig. 5, pumps are classified according to their flow rate:

· Nano LC pumps: 1 µL/min or less

· Micro LC pumps: several tens of µL/min

· Semi-micro LC pumps: several hundreds of µL/min

· Analytical pumps: several mL/min

· Preparative pumps: several tens of mL/min or more

The pump flow rate for normal analysis is several mL/min.

Classification of pumps based on flow rate
Figure 5. Classification of pumps based on the flow rate

Manual injection versus automatic sampling

There are two types of sample introduction methods: manual injection in which a syringe is inserted into the injector and the sample is injected manually, and auto sampling, whereby a large number of samples automatically can be injected sequentially.

Column oven

The column is where the separation occurs and the elution time of the sample components depends on the column temperature. To obtain reproducible results, a column oven should be used to stabilize the elution time. For samples that are well separated at low temperatures, a column oven may be used to further improve the separation by lowering the temperature of the sample.

Column Oven Diagram

Types of detector

As shown in Table 1, there is a variety of detectors that can be used depending on the target sample. The detector choice depends on your application needs and samples. For general use, the UV or PDA detectors are the most common covering a wide range of applications and components. When higher sensitivity is required, a fluorescence detector or a mass spectrometer can be used. For a more universal detection for compounds that don’t absorb or fluorescence, an evaporative light-scattering detector, or a differential refractive-index detector is more appropriate.

Detector type Measurement principle
UV/Vis detector Absorbance
PDA detector Absorbance
Differential refractive index detector Refractive index
Fluorescence detector Fluorescence
Electrochemical detector Oxidation / reduction
Electrical conductivity detector Conductivity
Mass spectrometry detector MS
Optical rotation detector Optical rotation
Circular dichroism detector Circular dichroism
Evaporative light scattering detector Light scattering

Separation principle

In HPLC, individual components are separated using a column, based on the difference in the degree of interaction between the sample components and the column. Components with a low degree of interaction with the column are eluted first. These interactions include adsorption, hydrophilic interactions, hydrophobic interactions, electroaffinity, penetration, and exclusion (Fig. 6).

Column types and separation modes

As shown in Table 2, there are various types of columns and separation modes that can be used, and the optimum choice depends on the nature of the sample and the analysis that is required. When an organic solvent is used as the mobile phase, a normal-phase column (mainly silica gel) can separate and analyze samples composed of fat-soluble components based on adsorption. When a water/methanol solvent is used as the mobile phase, separation can be achieved based on hydrophobic interactions in reversed-phase mode. GPC columns separate sample components based on their molecular size using pores. Ion exchange columns separate ion components based on electrical affinity.

Mode Stationary phase Mobile phase Interaction Features
Normal phase Silica gel Organic solvent Adsorption Separation of fat-soluble components
Reversed-phase Silica C18 (ODS) Water / MeOH Hydrophobic The most commonly used method
GPC (non-aqueous) Polymer Organic solvent Gel permeation Molecular weight distribution measurement
GFC (aqueous) Hydrophilic polymer Buffer Gel permeation Biopolymer separation
Ion exchange Ion exchanger Buffer Electric affinity Separation of ionic components

Normal phase vs. reversed-phase

Normal-phase chromatography and reversed-phase chromatography are completely different methods. In normal-phase chromatography, a low-polarity solvent is passed through a high-polarity column and the low-polarity components are eluted first. In reverse-phase chromatography, which is the most commonly employed technique, a more polar solvent is passed through a non-polar (often C18 or similar) column with polar components eluting first.

Isocratic elution vs. gradient elution

In reversed-phase chromatography and ion-exchange chromatography, gradient elution may be used to improve the separation and reduce the measurement time. As an example, chlorogenic acid and rutin can be separated using an ODS column and a methanol/1% acetic acid solution. First, let’s look at an analysis example that was performed without changing the composition ratio of the solvent. In the case of a methanol/1% acetic acid ratio of 40/60, component A could not be separated well (Fig. 7, upper left). In the case of a methanol/1% acetic acid ratio of 30/70, the separation was completed but it took a long time (Fig. 7, lower left). However, by applying a gradient from a methanol/1% acetic acid ratio of =30/70 to 45/55 (changing the concentration), the retention of the column was strengthened, and component A was reliably separated and eluted in a short time (Fig. 7, right).

UV/Vis detector

A UV/Vis detector detects components that absorb light between 190 and 900 nm. For example, aromatics, pigments, proteins, and drugs can be measured. By selecting the measurement wavelength, it is possible to measure the sample while suppressing the influence of interfering components (Fig. 8). In addition, the sensitivity can be improved by performing measurements at the wavelength where the maximum absorption occurs. By measuring the UV/Vis absorption spectrum at the elution peak and searching for a library, it is possible to predict the components that are present. The purity can also be checked from the absorption spectrum.

Fluorescence detector

A fluorescence detector can measure fluorescence in the wavelength range of 220-900 nm. Since the fluorescence wavelength depends on the excitation wavelength, the results are more selective than can be obtained using a UV/Vis detector. Since the excitation and fluorescence wavelengths can be switched programmatically, it is possible to simultaneously detect fluorescent substances with different wavelengths if the elution time is different (Fig. 9).

(Upper: fixed-wavelength mode Left: Ex = 275 nm, Em = 400 nm Right: Ex = 450 nm, Em = 525 nm, Lower: using wavelength programming mode)

RI detector

The RI (refractive index) detector detects the differences in the refractive index of materials. Most compounds have a different refractive index to that of the solvent, 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 (Fig. 10).

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

Comparison of detectors

Table 3 summarizes the characteristics of UV/Vis, fluorescence, and RI detectors. UV/Vis and fluorescence detectors are highly sensitive and they can measure the sample selectively. The gradient elution method can be used because the detectors are less sensitive to temperature. The 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. Although they are somewhat expensive, evaporative light scattering detectors are available that can detect a large variety of components with high sensitivity and can be used with gradient elution.

Detector Sensitivity Selectivity Temperature effect Gradient elution method
UV/Vis – ng High Low Applicable
Fluorescence – pg Very high Low Applicable
RI – μg N/A High N/A

Qualitative analysis

In most cases, identification of a sample component is performed by comparing its retention time with that in a standard sample. If a complex chromatogram with many peaks is obtained or if the retention time of the target component differs between the standard and the actual sample, the target component is identified by adding the standard sample to the unknown sample (Fig. 11). Analyzing the HPLC-collected components by IR or mass spectroscopy enables reliable qualitative analysis.

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

Quantitative analysis using HPLC

There are two methods for quantification: the external and internal standard method, both of which are performed using a calibration curve. The external standard method creates a calibration curve for a standard sample and then the unknown samples are quantified using that pre-established calibration. In the internal standard method, a fixed amount of an internal standard substance is added to an unknown sample when creating a calibration curve using a standard sample, and a calibration curve is created with the concentration ratio vs. peak area ratio for quantification. As shown in Fig. 12, the internal standard substance is required to be a component not included in the actual sample, 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 prevents errors in the injection volume or those caused by evaporation of the solvent.

The procedure for establishing the analysis conditions is summarized below.

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

1. Molecular weight, molecular structure and functional groups of components

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

3. Sample state (content, concentration, impurities)

4. Preliminary examination of analysis conditions and pretreatment based on datasheet and literature

Step 2: Consider analysis conditions for the column, mobile phase conditions, temperature, separation method, and detection method

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

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

3. Confirmation of separation of target components and contaminants in unknown samples

Step 3: Determination of analysis conditions that can be used for routine measurements

1. Determination of calibration curve linearity and calibration curve type

2. Quantitative reproducibility including pretreatment

3. Confirmation of components that are strongly retained in the column and components that are not detected by the detector

4. Correlation with other quantitative methods

Step 4: Routine quantitative analysis

1. Column lifetime

2. Running cost

3. Clarification of measurement procedure and documentation

4. System and column maintenance

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