Reproducing analysis conditions is crucial to achieving consistent, accurate results in gas chromatography–mass spectrometry (GC–MS). Valid reproduction demands appropriate application of technique, solid method design, reliable and accurate equipment, and a dedicated team of well-practiced technicians and researchers. But even when all these conditions are met, users can be held back by the more subtle elements in GC–MS operations, such as cutting or changing a column, or setting up the same experiment on different equipment. Even getting the parameters of a test organized so that it can be reproduced elsewhere — in a laboratory across the hall, the country, or the world — can be daunting. Consistent GC–MS results depend upon retention-time reproducibility.

Any number of factors can affect retention times, including routine maintenance — such as cutting or changing a column, switching to a different column lot, or even moving an analysis to another system. There are several ways to account for the retention-time changes created by these types of system alterations. Some of these methods are based upon the Kovats retention index (RI) system. Historically, these adjustments were made manually, but the processes often were cumbersome and left room for human error. The net was inconsistent results.

Today's changing laboratory environments demand new approaches to solving recurring issues in reproducibility. This article discusses some current approaches to solving the problems encountered in reproducing experimental conditions and evaluates the effectiveness of those approaches.

Kovats Retention Indices

The Kovats RI system overcomes the challenges in cross-system compatibility and results validation. First discovered by Dr. E. Kovats in 1958, RI is a linear scale based upon thermodynamic properties and observation of chromatographic data trends that show a distinct advantage in predicting retention time. The system uses n-alkanes as an index against which retention times of other compounds can be measured. The retention index is information unique to a compound for a given analytical column phase. It is based upon a thermodynamic principle, and it is correlated to chemical structure. The calculation formula is

n-Alkanes are saturated hydrocarbons that consist of straight-chain formations as opposed to molecular structures that involve branching of carbon atoms. n-Alkanes are used as an index because they are nonpolar, chemically inert, and soluble in most stationary phases. These properties make the n-alkane group ideal for a retention-time scale that can be used for measurement of a variety of analytes on a given column phase.

Under linear temperature-programmed oven conditions, n-alkanes show a linear relationship between carbon number and retention time, regardless of column type, dimensions, or flow rate. The RI is defined as the number of carbon atoms in the n-alkane times 100. Thus, the retention index for C₉ would be 900 and C₁₀ would be 1000. A linear plot of these retention index values versus the actual retention time of the C₉ and C₁₀ can then be constructed. From this plot, RI values can be determined for target analytes based upon their retention times from analysis on the same column under identical conditions.

A single chromatographic run can easily determine the retention times of n-alkanes for a given system. From that information and the retention index data from target analytes, it is easy to predict with a high degree of accuracy where specific target analytes will elute on that same system.

The 2005 NIST Mass Spectral Library contains retention indices for numerous compounds. These data can be useful to analysts in several stages of research. First, the retention times for many analyte compounds can be predicted accurately if their retention indices are known and the retention times of n-alkanes are determined experimentally. Second, RI values for a large number of compounds in a method can be calibrated easily. Third, unknown compound isomers with similar mass spectra can be distinguished using mass spectral libraries and retention indices.