Small Molecule HPLC
Analysis of small molecules involves studying compounds with low molecular weights, typically less than 900 daltons. Common examples of small molecules include amino acids, lipids, sugars, fatty acids, alkaloids, and various other biological and chemical substances. Several techniques are employed for the separation of small molecules, such as High-Performance Liquid Chromatography (HPLC), Liquid Chromatography (LC), Gas Chromatography (GC), Thin-Layer Chromatography (TLC), and Capillary Electrophoresis (CE). For their identification, techniques like Nuclear Magnetic Resonance Spectroscopy (NMR) and Mass Spectrometry (MS) are commonly used. In recent years, Liquid Chromatography coupled with Mass Spectrometry (LC-MS) has become a critical method for identifying small molecules due to its sensitivity and versatility. Achieving optimal results in HPLC, UHPLC, or LC-MS analysis of small molecules relies heavily on the careful selection of the appropriate stationary phase and mobile phase conditions. The chemistry of the analyte is essential in determining the most suitable column chemistry. Factors such as analysis speed, sample matrix, and the number of compounds also play significant roles in choosing the ideal stationary phase base material to ensure accurate and effective analysis.
Overview
HPLC of Small Molecules
The HPLC analysis of small molecules is typically conducted in reversed-phase separation mode. For polar compounds, hydrophilic interaction chromatography (HILIC) and normal-phase chromatography are also viable, with HILIC being the preferred method. Ion-exchange separation modes are employed for ionic compounds, and ion chromatography is used for separating inorganic anions or cations.
The HPLC column is generally packed with materials such as fully porous silica particles, superficially porous silica particles, polymeric particles, or a monolithic silica rod. Other materials like alumina oxide, zirconia particles, and carbon particles are also used. The typical pore size of the stationary phase material for small molecule separation ranges from 60 Å to 160 Å. For traditional HPLC, the stationary phase particle size ranges from 3 µm to 5 µm, while UHPLC typically uses smaller particle sizes (2 µm or below). Column selectivity is determined by modifications attached to the stationary phase. The C18 alkyl chain is the most common column chemistry in reversed-phase (RP) chromatography, though other modifications (e.g., C30, C8, Phenyl, Pentafluorophenyl, or polar modifications) as well as ion-exchange and chiral modifications are used to separate a wide variety of compounds. The mobile phase for RP-HPLC typically consists of an aqueous buffer or water, combined with water-miscible organic solvents like acetonitrile or methanol.
HPLC Sample Preparation
Complex samples, such as food, beverages, cosmetics, biological samples, and matrix-rich pharmaceutical formulations (e.g., creams, syrups), require efficient sample preparation methods to remove unwanted components and selectively extract the target analyte. This is particularly important for UHPLC, where small stationary phase particle sizes (2 µm or smaller) are used. Common sample preparation techniques include liquid-liquid extraction, solid phase extraction (SPE), protein precipitation, and filtration for biological samples. The goal of sample preparation is not only to concentrate and selectively elute the target compound but also to protect the HPLC stationary phase from clogging due to the sample matrix. Monolithic silica columns are more tolerant of matrix interference and require less sample preparation compared to particulate columns.
Derivatization in HPLC
In some cases, derivatization is necessary before (pre-column) or after (post-column) the HPLC separation. Derivatization converts the target molecules into derivatives that improve their sensitivity or chromatographic retention. Chemical reagents with desirable physical and chemical properties are used for the derivatization process to ensure better separation and analysis in HPLC.