Journal of Analytical & Bioanalytical Techniques
Open Access

High-performance liquid chromatography; Analytical chemistry; Instrumentation

Case Presentation

High-performance liquid chromatography (HPLC) is a versatile and widely used analytical technique in the field of chemistry, biochemistry, pharmaceuticals, and other scientific disciplines. It allows for the separation, identification, and quantification [1-4] of complex mixtures of compounds with high precision and accuracy. HPLC has revolutionized analytical chemistry by providing researchers with a robust tool for studying a wide range of samples, from small molecules to large biomolecules.

Principles of HPLC

HPLC is based on the principle of chromatography, which involves the separation of components in a sample based on their differential interactions with a stationary phase and a mobile phase. In HPLC, the stationary phase is typically a packed column containing a solid support material with specific properties such as size, polarity, or charge. The mobile phase, often a liquid solvent or a mixture of solvents, flows through the column under high pressure. The sample is injected into the mobile phase, and as it passes through the column, different components interact differently with the stationary phase, resulting in their separation.

Instrumentation and components

HPLC systems consist of several key components, including a pump to generate high-pressure flow of the mobile phase, an injector [3-5] for sample introduction, a column for separation, a detector to monitor the eluting compounds, and a data analysis system. Modern HPLC instruments are equipped with advanced detectors such as ultraviolet-visible (UV-Vis), fluorescence, mass spectrometry (MS), and electrochemical detectors, allowing for sensitive and selective detection of target analytes.

Applications of HPLC

HPLC finds widespread applications in various fields due to its versatility and ability to separate and quantify diverse compounds. Some common applications of HPLC include: Pharmaceutical analysis: HPLC is extensively used for drug discovery, quality control, and pharmacokinetic studies, enabling the analysis of drug compounds, impurities, and metabolites. Environmental analysis: HPLC plays a crucial role in analyzing environmental samples for pollutants, such as pesticides, herbicides, and heavy metals, aiding in monitoring and ensuring environmental safety. Food and beverage analysis: HPLC is employed for the detection and quantification of food additives, contaminants, vitamins, flavors, and other components, ensuring food quality and safety. Biochemical analysis: HPLC is utilized in the analysis of biomolecules, such as proteins, peptides, nucleic acids, and carbohydrates, facilitating [5-8] protein purification, peptide sequencing, DNA analysis, and carbohydrate profiling. Clinical diagnostics: HPLC is applied in clinical laboratories for the analysis of drugs, metabolites, hormones, and biomarkers in biological fluids, aiding in disease diagnosis and therapeutic monitoring.

Advancements in HPLC

Over the years, HPLC techniques have evolved, leading to advancements such as high-resolution HPLC, ultra-high-performance liquid chromatography (UHPLC), and hyphenated techniques like liquid chromatography-mass spectrometry (LC-MS). These advancements have enhanced separation efficiency, sensitivity, speed, and selectivity, allowing for more complex sample analysis and improved detection limits.

Disadvantages of High-Performance Liquid Chromatography

While High-Performance Liquid Chromatography (HPLC) is a widely used and powerful analytical technique, it does have certain disadvantages that researchers should consider: Cost: HPLC systems and the associated equipment can be expensive to purchase and maintain. The initial investment in instruments, columns, detectors, and other accessories can be substantial. Additionally, regular maintenance, calibration, and replacement of consumables can contribute to ongoing costs. Complexity: HPLC requires specialized knowledge and skills to operate effectively. Method development, optimization, and troubleshooting can be time-consuming and require expertise in column selection, mobile phase composition, and detector settings. Inexperienced users may face challenges in achieving optimal separations and obtaining accurate results. Time-consuming: HPLC separations can be relatively slow compared to other analytical techniques. The analysis time can range from minutes to hours, depending on the complexity of the sample and the separation conditions. This extended analysis time can limit the throughput and efficiency of the technique, especially when handling large sample volumes or when rapid analysis is required. Sample Preparation: HPLC often requires sample preparation steps, such as filtration, extraction, or derivatization, to ensure the compatibility of the sample with the chromatographic system. These additional steps can introduce variability, increase the overall analysis time, and potentially lead to sample loss or degradation. Limited Compatibility: HPLC is primarily suitable for analyzing compounds that are soluble in the chosen mobile phase and thermally stable under the separation conditions. Certain classes of compounds, such as high molecular weight polymers or volatile analytes, may present challenges in achieving effective separation and detection using HPLC.

Restricted Sensitivity: Although HPLC can provide excellent sensitivity for many compounds, it may fall short for analytes present in very low concentrations or trace levels. In such cases, additional sample enrichment techniques or more sensitive detection methods, such as mass spectrometry, may be required. Environmental Impact: HPLC analyses often involve the use of organic solvents, which can have environmental implications. The disposal of used solvents requires proper waste management practices to minimize environmental contamination. Despite these disadvantages, the benefits and versatility of HPLC make it a widely adopted analytical technique for various applications. Researchers should weigh these limitations against their specific analytical needs and consider alternative techniques when necessary. In this example, the Table 1 includes columns for the Sample ID, Retention Time (the time at which the peak elutes from the column), Peak Area (the integrated area under the chromatographic peak), and Peak Height (the maximum height of the chromatographic peak). You can customize the table by adding or removing columns as needed, depending on the specific parameters and data you want to record and analyze from your HPLC experiments.

Table 1: It includes columns for the Sample ID, Retention Time (the time at which the peak elutes from the column), Peak Area (the integrated area under the chromatographic peak), and Peak Height (the maximum height of the chromatographic peak).

Conclusion

High-performance liquid chromatography (HPLC) is a powerful analytical technique that has revolutionized the field of analytical chemistry. Its ability to separate, identify, and quantify a wide range of compounds has made it an indispensable tool in various scientific disciplines. With ongoing advancements and improvements, HPLC continues to contribute significantly to research, development, and quality control processes across industries, enabling scientists to explore and understand the complex nature of chemical and biological samples with great precision and accuracy.

Acknowledgement

The authors are appreciative of the JSPS KAKENHI project’s support (No. 26420267).

Competing Interest

The authors say they have no competing interests.

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