The History and Principles of HPLC

Ion Chromatography is high-performance liquid chromatography (HPLC) and is a liquid chromatography method for the analysis of anions and cations.

In a narrow sense, ion chromatography is a chromatographic method in which a low exchange capacity ion exchange resin is used as a fixed relative ionic substance for separation and a conductivity detector is used to continuously detect changes in effluent conductance. Ion chromatography is defined in the principles and applications of ion chromatography: liquid chromatography for separation and detection of the ionicity of a substance.

Development history

In 1975, Small et al. successfully solved the problem of continuously detecting column effluent with a conductivity detector, that is, using an anion or cation exchange column with low exchange capacity, separating the inorganic ions with a strong electrolyte as the mobile phase, and the effluent passed through a scale. To inhibit the column of the ion exchange resin that is oppositely charged to the separation column packing. In this way, the counter ions of the ions to be measured in the mobile phase are removed, and the background phase conductance of the mobile phase is lowered, thereby obtaining high detection sensitivity. Since then, with true ion chromatography (IC), IC has also been isolated from liquid chromatography as a chromatographic separation technique.

In 1979, Gjerde et al. used a weak electrolyte as the mobile phase. Since the mobile phase itself has a low conductivity, it can be directly detected by a conductivity detector without using a suppression column. Ion chromatography using a suppression column is called double column IC or suppressed IC, and ion chromatography without a suppression column is called single column ion chromatography ( single column IC) or nonsuppressed IC (nonsuppressed IC).

HPLC is the abbreviation of high-performance liquid chromatography. It is some kind of chromatographic analysis technology used to separate the mixtures to confirm and quantize the proportion of each composition. Basically, HPLC depends on the pump to pressure the samples and makes them go through the HPLC columns that are filled with adsorbing materials. As a result, each component of the samples can be separated. In general, HPLC is mostly applied in biological chemistry and analytical chemistry.

In the 1960s, gas chromatography technology had limitations on the analysis of organics with high boiling points. In order to separate the macromolecular substances that are difficult to be gasified, such as protein and nucleic acid, classical liquid chromatography was introduced to the theories and methods of gas chromatography. At the end of the 1960s, Kirkland and other scientists invented the first HPLC instrument in the world and thus opened the era of HPLC.

As a whole, HPLC uses stationary phases with smaller grain sizes to fill in the HPLC columns, increases the numbers of the column plates, and drives the stationary phases with high pressure, which can make the separation finish within only hours or even dozens of minutes.

The Principles of Using HPLC Columns

In general, HPLC technology uses all kinds of interaction forces to separate the mixtures. These interaction forces are mostly some kind of non-covalent properties between the analytes and the HPLC columns. When using the HPLC columns, the liquid samples that are to be detected will be injected into the HPLC columns at different times. Through pressure moving in the stationary phases, as the interaction forces between different substances in the samples and the stationary phases are different, the substances will leave the HPLC columns in sequence. The laboratory workers will get different peak signals by using the detectors, and each peak point represents one kind of chemical compound. Finally, by analyzing and comparing the signals, we can judge the substances that are contained in the samples to be detected.

Furthermore, the chromatographic instrument that is designed with the liquid as the mobile phase is called liquid chromatography. In comparison, liquid chromatography that is equipped with a high-pressure infusion pump, high-efficiency stationary phase, and a high-pressure sensitive detector is called high-performance liquid chromatography. There is a great variety of HPLC which are used in laboratories. However, no matter what kind of HPLC, it has four basic parts: a high-pressure infusion system, a sample loading system, a separation system, and a detection system.

1. Stationary Phase and Mobile Phase:
   • Stationary Phase: In HPLC, separation occurs on a stationary phase packed into a column. This phase can be composed of various materials, such as silica, polymer, or metal, and is chemically modified to provide specific separation properties.
   • Mobile Phase: It is a liquid solvent or mixture of solvents that carries the sample through the column. The choice of mobile phase is critical for achieving effective separation.
2. Sample Injection:
   • The sample is introduced into the mobile phase stream in a small volume. This can be done manually or using an autosampler.
3. Column:
   • The column is a crucial component. It’s packed with the stationary phase and provides the platform for the separation process. The column’s properties, including length, diameter, and stationary phase, are tailored to the specific separation needs.
4. Separation Mechanisms:
   • HPLC separates compounds based on their differential interactions with the stationary phase and mobile phase. Common mechanisms include:
     • Adsorption Chromatography: Based on surface interactions with the stationary phase.
     • Partition Chromatography: Relies on differential solubility in the mobile and stationary phases.
     • Ion-Exchange Chromatography: Separation is based on ionic interactions.
     • Size Exclusion Chromatography (SEC): Separates based on molecular size.
5. Detection:
   • After separation, the eluting compounds are detected. Common detectors include UV-Visible spectrophotometers, fluorescence detectors, and mass spectrometers.
6. Data Analysis:
   • The data collected from the detector is processed to generate a chromatogram, which represents the intensity of the detected compounds over time.
7. Applications:
   • HPLC is widely used in various fields, including pharmaceuticals, environmental analysis, food and beverages, forensics, biochemistry, and more.

Good Separation Effect

Diol HPLC column is mainly used for the separation of peptides, proteins, and polar drug molecules. This column has ultra-pure fully porous spherical silica gel, which greatly improves the stability of chromatographic column separation and has a good symmetrical peak shape and high column effect; besides, it ensures the inert, homogeneous, and reproducible surface of silica gel with a unique surface modification method. Moreover, Diol HPLC columns are mainly used for sample analysis, especially for mobile phase and sample filtration in chromatographic analysis, which has a good effect on protecting the chromatographic column system and injection valve from contamination and can be widely used in gravimetric analysis, microanalysis, colloid separation, and aseptic experiments.

Technological Superiority

Compared with the conventional HPLC column, the Diol HPLC column is a much more stable and reproducible ortho phase column than the silica gel HPLC column and amino Acid HPLC column. It is modified on the basis of pure silica gel, and some compounds can provide better selectivity and chromatographic peak shape. And it adopts unique bonding technology to ensure a stable stationary phase structure and excellent batch and batch reproducibility.