A Comprehensive Review on High Performance Liquid Chromatography (HPLC): Introduction, Theory, Instrumentation, Advantages, Limitations and Applications

Chromatography is one of the foundations of the analytical chemistry field, as it allows the separation of complex mixtures into separate components that are identified and quantified. High-performance liquid chromatography is one of the methods of chromatography that has proven to be highly powerful and versatile among the other techniques designed in the last 100 years. The method is an improvement of classical liquid chromatography by technological improvement that enhanced its resolution power by a huge percentage, speed, and sensitivity. HPLC is based on the principle of differential partitioning of analytes between a mobile phase (liquid solvent) and a stationary phase (solid material packed in a column). The development of high-pressure pumps, which could drive mobile phases through closely packed columns of small-diameter particles revolutionized the field, making it possible to perform separations that were previously not possible or too time-consuming. Nowadays, HPLC is a vital instrument in many fields of science. When used in pharmaceutical labs, it ensures the purity of drugs and quantifies active components. The environmental scientists use HPLC in order to identify pollutants in minute amounts. It is used by food chemists to examine nutrients, food additives, and food contaminants. HPLC is essential in clinical laboratories as a method of measuring the concentration of biomarkers and therapeutic drug concentrations in biological samples [1]. This review aims to provide a comprehensive understanding of HPLC through theoretical background explanation, instrumentation, its practical advantages, limitations, and various applications of the instrument. This kind of knowledge is critical not only to the novice analytical chemists but also to the professionals who want to make the best out of their practices.

2. Theoretical Principles of HPLC

2.1 Fundamental Separation Mechanism

The mechanism of separation in HPLC is based on the differential distribution of the analytes in two phases: the mobile phase and the stationary phase. Once a sample mixture is injected into the HPLC system, each of the components show different interaction with these phases depending on the chemical properties such as polarity, size, charge, and hydrophobicity. Components that show stronger affinity to the stationary phase, they move more slowly through the column and components that prefer more the mobile phase, move in a faster manner. This differential migration results in temporal and spatial separation of mixture components, allowing them to elute from the column at varying times, known as the retention times [2].

2.2 Retention Factor and Selectivity

The retention factor (k) measures the time of retention of a given compound in the column compared with that of a compound that is not retained. It is determined as k = (tR - t0)/t0, where tR is the retention time of the analyte and t0 is the void time (time needed by a compound not retained to pass through the column). Selectivity (α) is a property used to explain the ability of the chromatographic system to differentiate between two compounds. It is called the ratio of retention factors of two adjacent peaks: α= k2/k1. Greater values of selectivity indicate better separation between compounds [3].

2.3 Column Efficiency and Peak Shape

Column efficiency is represented by the number of theoretical plates (N) and is the degree to which a column is able to create sharp and narrow peaks. The relationship is stated as N = 16(tR/Wb)2, where Wb being the peak width at baseline. The greater the plate numbers correlate with better resolution and sharper peaks. The Van Deemter equation is an equation that explains the effect of different factors on the efficiency of the column. It relates plate height (H) to mobile phase linear velocity (u): H = A + B/u + Cu, where A is eddy diffusion, B is longitudinal diffusion, and C is mass transfer resistance [2].

2.4 Resolution

The separation between two adjacent peaks is quantitatively expressed in terms of resolution (Rs), which is calculated as Rs = 2 [tR2 - tR1)/(Wb1 + Wb2)], where tR1 and tR2 are retention times of the two peaks and Wb1 and Wb2 are the respective baselines widths of the peaks. A resolution value of 1.5 or above is usually indicates separation of baseline. Resolution is a holistic way of measuring the quality of separation, as it depends on column efficiency, selectivity, and retention [4].

3. Instrumentation

An HPLC system consists of a number of combined parts that are important in achieving successful separations.