PharmD Info

High-performance liquid chromatography (HPLC) is a widely used analytical technique in the field of chemistry, biology, and pharmaceutical sciences. HPLC allows scientists and researchers to separate, identify, and quantify different compounds in a sample. There are several types of HPLC techniques available, each with its own advantages and limitations. In this article, we will explore the most common types of HPLC used in scientific research, and provide insights into their applications, benefits, and limitations.

Reverse-phase HPLC (RP-HPLC)

Reverse-phase HPLC (RP-HPLC) is the most commonly used HPLC technique. It separates compounds based on their hydrophobicity or lipophilicity. In RP-HPLC, a non-polar stationary phase, such as C18, is used with a polar mobile phase, such as water or methanol. The more hydrophobic or lipophilic a compound is, the longer it will interact with the stationary phase and the longer it will take to elute. RP-HPLC is highly versatile and can be used to analyze a wide range of compounds, including small molecules, peptides, and proteins.

Normal-phase HPLC (NP-HPLC)

Normal-phase HPLC (NP-HPLC) separates compounds based on their polarity. In NP-HPLC, a polar stationary phase, such as silica gel, is used with a non-polar mobile phase, such as hexane. The more polar a compound is, the longer it will interact with the stationary phase and the longer it will take to elute. NP-HPLC is often used to separate and analyze polar compounds, such as carbohydrates, nucleic acids, and steroids.

Ion-exchange chromatography (IEC)

Ion-exchange chromatography (IEC) separates compounds based on their charge. In IEC, a stationary phase with charged functional groups, such as a resin with sulfonic acid groups, is used with a mobile phase containing ions of opposite charge. The charged compounds in the sample will bind to the stationary phase, and the degree of binding will depend on the strength of the charge. IEC is commonly used to separate and analyze charged molecules, such as amino acids, peptides, and proteins.

Size-exclusion chromatography (SEC)

Size-exclusion chromatography (SEC) separates compounds based on their size. In SEC, a stationary phase with pores of a specific size is used with a mobile phase. The larger the molecule, the less it can enter the pores of the stationary phase, and the faster it will elute. SEC is often used to separate and analyze large molecules, such as proteins, nucleic acids, and polysaccharides.

Affinity chromatography (AC)

Affinity chromatography (AC) separates compounds based on their specific interaction with a ligand immobilized on the stationary phase. The ligand can be an antibody, enzyme, or receptor. The compound of interest will bind to the ligand, and the non-specifically bound molecules will elute first. AC is highly specific and can be used to isolate and purify a specific protein, enzyme, or other biomolecule.

Hydrophilic interaction chromatography (HILIC)

Hydrophilic interaction chromatography (HILIC) separates compounds based on their hydrophilicity. In HILIC, a polar stationary phase, such as silica or amino, is used with a polar mobile phase, such as acetonitrile or methanol. The more hydrophilic a compound is, the longer it will interact with the stationary phase and the longer it will take to elute. HILIC is often used to separate and analyze polar and hydrophilic compounds, such as carbohydrates, nucleotides, and peptides.

Chiral chromatography

Chiral chromatography separates enantiomers, which are mirror images of each other. In chiral chromatography, a stationary phase containing chiral selectors, such as cyclodextrin or chiral ligands, is used with a mobile phase. The enantiomers will interact differently with the chiral selector and will have different retention times. Chiral chromatography is commonly used in pharmaceutical sciences to separate and analyze chiral drugs and natural products.

Multi-dimensional chromatography

Multi-dimensional chromatography is a powerful technique that combines two or more types of chromatography to achieve high-resolution separations. For example, a sample can be first separated by RP-HPLC, and then the eluted fractions can be further separated by size-exclusion chromatography. Multi-dimensional chromatography is often used to analyze complex samples, such as proteins, peptides, and natural products.

In conclusion, HPLC is a versatile and powerful technique that can be used to separate and analyze a wide range of compounds in different fields of research. By understanding the principles and applications of different types of HPLC, scientists and researchers can select the most appropriate technique for their analytical needs.