ANALYSIS OF SMALL MOLECULES

Small molecules refer to compounds with low molecular weight, typically less than 900 daltons, and examples include amino acids, lipids, sugars, fatty acids, alkaloids, and more.

Various methods are available to separate small molecules, such as High-Performance Liquid Chromatography (HPLC), Liquid Chromatography (LC), Gas Chromatography (GC), Thin-Layer Chromatography (TLC), and Capillary Electrophoresis (CE). Identifying these molecules can be done using Nuclear Magnetic Resonance Spectroscopy (NMR) or Mass Spectrometry (MS). Recently, Liquid Chromatography coupled with Mass Spectrometry (LC-MS) has become a crucial technique for small molecule identification.

For achieving optimal results in the analysis of small molecules using HPLC, UHPLC, or LC-MS, selecting the most suitable stationary phase and mobile phase conditions is crucial. The chemistry of the analyte plays a key role in determining the appropriate column chemistry, and other factors like speed, sample matrix, and number of compounds help define the best suitable base material for the stationary phase.

HPLC OF SMALL MOLECULES

In HPLC analysis of small molecules, the most common separation mode is reversed-phase. However, hydrophilic interaction chromatography (HILIC) and normal-phase chromatography are also suitable, with HILIC being preferred for polar compounds. For ionic compounds, ion-exchange separation modes are used, while ion chromatography is suitable for inorganic anions or cations.

HPLC columns can be packed with various materials, including fully porous silica particles, superficially porous silica particles, polymeric particles, or monolithic silica rods as the stationary phase. Other materials like alumina oxide, zirconia particles, and carbon particles are also used. The stationary phase material typically has a pore size in the range of 60 Å – 160 Å for small molecule separation. In HPLC, particle sizes for the stationary phase range from 3 µm to 5 µm, while UHPLC uses smaller particle sizes, typically 2 µm or below.

Different column selectivities (modifications) can be attached to the stationary phase, with the C18 alkyl chain being the most commonly used for reversed-phase chromatography. However, other modifications like C30, C8, Phenyl, Pentaflourophenyl, and more polar modifications, as well as those with ion-exchange or chiral properties, allow for the separation of almost all compounds soluble in liquids. The mobile phase for RP-HPLC typically consists of an aqueous buffer or water mixed with water-miscible organic solvents like acetonitrile or methanol.

HPLC SAMPLE PREPARATION

Samples with complex and matrix-rich compositions, such as food, beverages, cosmetics, biological samples, and matrix-rich pharmaceutical formulations (e.g., cream, syrup), necessitate effective sample preparation methods to eliminate unwanted components and selectively extract the desired analyte. This becomes crucial when using a stationary phase with very small particle sizes, such as in UHPLC, where particles of 2 µm or below are utilized. Common sample preparation techniques include Liquid-liquid extraction, Solid phase extraction (SPE), and for biological samples, protein precipitation, alongside filtration.

In addition to selectively eluting the target compound and pre-concentrating it, sample preparation also serves to safeguard the HPLC stationary phase from clogging caused by the sample matrix. Monolithic silica-based HPLC columns can endure a high level of matrix interference and necessitate much less sample preparation compared to particulate columns.

DERIVATIZATION

Certain molecules may need derivatization, which can occur either before (pre-column) or after (post-column) the HPLC separation process. Derivatization involves transforming these molecules into derivatives to enhance sensitivity or improve their chromatographic retention in HPLC. This process relies on using chemical reagents with favorable physical and chemical properties to achieve the necessary derivatization.