Towards an accurate method for column void volume determination using liquid chromatography-mass spectrometry

Area of Science:

• Analytical Chemistry
• Chromatography

Background:

• Accurate prediction of analyte retention times in chromatography relies on knowing the column void volume.
• Current methods for measuring column void volume are inconsistent and often rely on experimental estimations.

Purpose of the Study:

• To investigate the utility of deuterated acetonitrile as an isotopically labeled mobile phase component for determining column void volume.
• To compare void volume measurements obtained using deuterated acetonitrile with those from conventional void volume markers.

Main Methods:

• Investigated deuterated acetonitrile elution behavior in binary water-acetonitrile mixtures across various compositions, stationary phases (C8, C18), and temperatures (30, 40°C) using LC-MS.
• Compared elution data with common void volume markers: uracil, phloroglucinol, and N,N-dimethylformamide.
• Utilized three-way ANOVA to assess the significance of mobile phase composition and temperature on elution behavior.

Main Results:

• Higher temperatures led to lower elution times for deuterated acetonitrile.
• Mobile phase composition significantly impacted the elution of deuterated acetonitrile and conventional markers, indicating they exhibit retention-like behavior.
• Void volumes determined using deuterated acetonitrile were 24-36% higher than those from conventional markers.

Conclusions:

• Deuterated acetonitrile serves as a superior isotopically labeled marker for accurate column void volume determination in water-acetonitrile mobile phases.
• The minor disturbance method with deuterated acetonitrile provides a more integral average void volume, resolving discrepancies in chromatographic predictions.
• This method offers a more reliable approach to measuring the elusive column void volume, improving retention time predictions.