Benzonitrile

HPLC Method for Analysis of Benzonitrile, Toluene, Benzylamine on Primesep A by SIELC Technologies

Separation type: Liquid Chromatography Mixed-mode

1. Benzonitrile: Benzonitrile (C₆H₅CN) is an aromatic organic compound. It is a colorless liquid with an almond-like odor. Benzonitrile is used as a solvent and a chemical intermediate in the production of a variety of compounds, including pharmaceuticals, dyes, and perfumes.
2. Toluene: Toluene (C₇H₈) is a colorless, water-insoluble liquid with the smell associated with paint thinners. It is a mono-substituted benzene derivative, consisting of a CH₃ group attached to a phenyl group. It is an important organic solvent and is also used to produce benzene, and a number of consumer products including certain types of glue, paint thinners, and nail polish removers.
3. Benzylamine: Benzylamine (C₆H₅CH₂NH₂) is a primary amine compound containing a benzyl group attached to the amino group. It is a colorless liquid with a faint almond-like odor. It is used as a chemical intermediate in the synthesis of a variety of substances, including pharmaceuticals, dyes, and polymers.

These chemicals are widely used in organic synthesis. Please handle these substances with care, as they can have hazardous effects. Always refer to the safety data sheets for each chemical and adhere to recommended safety procedures.

These three compounds be separate and analyzed on a reverse-phase Primesep A, 4.6 x 150 mm, 5 µm, 100 A, dual ended column with a mobile phase consisting of water, Acetonitrile (MeCN) and Sulfuric acid as a buffer modifier. This analysis method can be UV detected at 210 nm.

LOD was determined for this combination of instrument, method, and analyte, and it can vary from one laboratory to another even when the same general type of analysis is being performed.

High Performance Liquid Chromatography (HPLC) Method for Analysis of Benzonitrile, Toluene, Benzylamine

Condition

Description

Parabens are common preservatives in pharmaceutical and cosmetic industries. They are esters of p-hydroxybenzoic acid. Method for separation of methyl paraben, propyl paraben, benzonitrile and toluene was developed on a Obelisc R column. All four compounds are neutral and are retained by reverse-phase mechanism. In case of reversed-phase stationary phase, no effect of pH is observed. Retention time for all four compounds changes on an Obelisc R column when pH is changed. pH of the mobile phase affects ionization state of stationary phase. Obelisc R column has C12 carbon chain and carboxylic acid with pKa of 4. At lower pH (pH 2, TFA), carboxylic acid of stationary phase is not ionized and thus adds hydrophobicity to stationary phase. Obelisc R column can be used for analysis of basic, acidic and neutral compounds with suitable detection techniques – UV, ELSD, CAD, LC/MS.

This application shows the effect of mobile phase composition on retention of acidic, basic and neutral compound. Retention of basic and acidic compounds is controlled by buffer pH and concentration, while retention of neutral compound is controlled by the amount of acetonitrile. Buffer pH changes not only ionization state of ionizable basic and acidic compounds, but ionization state of trimodal stationary phase Obelisc R. Available detection techniques include UV, ELSD, LC/MS, and Corona.

Dequalinium, or Dequadin, is an antiseptic and disinfectant. It consists of two quaternary amines connected by C10 hydrophobic chain. It’s a strongly basic compound that is hard to analyze on reverse phase column and obtain good peak shape. Compound shows a significant tailing (symmetry below 0.5 and USP tailing factor of over 2) due to interaction of quaternary groups with residual silanols. In this HPLC application dequalinium is analyzed on Primesep D mixed-mode column with perfect symmetry and high efficiency. Primesep D column has hydrophobic chain and a basic group close to the surface of silica gel. This group provides shielding effect and prevents interaction between acidic silanol groups and quaternary amines of dequalinium. Method can be used for analysis and quantitation of hydrophobic amines when peak shape is not satisfactory on traditional HPLC columns. Relative comparison of several modern LC columns (Zorbax from Agilent, XTerra from Waters and Gemini from Phenomenex) shows that Primesep D mixed-mode column can be successfully used in determination of hydrophobic quaternary amines and diamines. If a compound is retained on C18 column with poor peak shape, switching to mixed-mode column can provide a valuable alternative. Benzonitrile is used to ensure high quality of packing and good peak shape for neutral analytes. Method uses trifluoroacetic acid as an additive to the mobile phase. Other acids and buffers can be used depending on the detection technique.

Separating basic, acidic and zwitterionic compounds in one run in reverse-phase HPLC can be very challenging. The methods might require the use of ion-pairing reagents and complex gradients that can make MS-compatibility difficult. Obelisc R column which has both positive and negative ion-pairs embedded in the stationary phase allows for fine tuning and separation of a wide range of compounds with different ionic properties. Acids, bases, amino acids and neutral compounds were separated isocratically in one run using a simple MS-compatible mobile phase of acetonitrile (ACN) and water with Ammonium Acetate (AmAc) buffer. Can also be UV detected at 250nm.

Primesep 100 separates a mixture of amino acids (tyrosine, phenylalanine), organic acids (benzoic acid, mandelic acid), amines (benzylamine, pyridine), and neutrals (benzonitrile, toluene) in one HPLC run by combining reversed-phase, cation-exchange, and polar interactions. The method is tunable and peak order can be changed significantly by adjusting acetonitrile and trifluoroacetic acid concentrations. The separation method uses a mobile phase mixture of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) and compatible with UV, mass spec (LC/MS) and evaporative light scattering (ELSD) detection.

Primesep B separates acids, bases, and neutrals in one injection. Maleic acid, benzonitrile, and benzylamine are baseline resolved by a combination of reversed-phase, ion-exchange, and ion-exclusion mechanisms. Excellent peak shape results with a mass spec compatible mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.

Primesep A separates acids, bases, and neutrals in one injection. Maleic acid, benzonitrile, and benzylamine are baseline resolved by a combination of reversed-phase, ion-exchange, and ion-exclusion mechanisms. Excellent peak shape results with a mass spec compatible mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.

Primesep 100 retains and separates an acid, base, and neutral in one HPLC injection. Maleic acid, benzylamine, and benzonitrile are resolved by ion-exclusion, ion-exhange and reversed-phase modes. The separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.

Primesep 200 separates acids, bases, and neutrals in one injection. Maleic acid, benzonitrile, and benzylamine are baseline resolved by a combination of reversed-phase, ion-exchange, and ion-exclusion mechanisms. Excellent peak shape results with a mass spec compatible mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.