How to Optimize Key Variables in GC Analysis: Sample Introduction

An excerpt from LCGC’s e-learning tutorial on GC analysis at CHROMacademy.com

Every analysis can benefit from the best injection possible. This involves selecting the ideal injection technique and optimizing it for every sample. This article covers which injection technique can be used for different sample and analysis types, which parameters should be optimized, and also some of the drawbacks of the inlet.

Split–Splitless Injection

Split injection is conventionally used for analyses in which the sample concentration is high and the user wishes to reduce the amount of analyte reaching the capillary column by performing an “on-instrument” dilution. However, why perform an on-instrument dilution rather than just diluting the sample further before injection? Capillary gas chromatography (GC) columns are limited to the amount of each analyte that can be introduced onto the column before peak shapes begin to deteriorate. Smaller inner diameter columns and thinner stationary-phase films have lower capacity, and analyte concentrations in the order of a few nanograms on column are typical; therefore, we require a reasonably dilute sample. Performing an on-instrument dilution is preferable because it results in sharper peaks. The split ratio is used to control the amount of analyte reaching the column, which ultimately affects peak width and sensitivity. Typical split ratios are in the range 1:20 to 1:400. When using columns with a thick stationary phase film (>0.5 µm) or a large inner diameter (0.533 mm i.d.) the sample capacity increases and lower split ratios are typical, 1:5 to 1:20. With very narrow GC columns (<100 µm i.d.) split ratios can be as high as 1:1000+.

Splitless injection is used for trace analysis as the entire sample is transferred to the analytical column. Analyte transfer from the inlet to the column is slow, which would result in broad analyte peaks; however, two focusing mechanisms occur that mitigate this problem and are a must for optimizing this injection technique. These involve setting the initial oven temperature 20 °C below the sample solvent boiling point, which ensures that condensation and reconcentration of the analyte band takes place in the column. The sample solvent and column polarity should also be matched - for example, by employing nonpolar hexane as the solvent when using a nonpolar poly(dimethylsiloxane) (PDMS) column. The splitless time should be optimized; too short a splitless time and high boiling analytes will be lost, too long a splitless time risks a large solvent peak. Typical splitless times are 20–90 s.

Sample discrimination and sample degradation occur with both split and splitless injection modes. Because of the vaporizing nature of the inlet solvent, backflash can occur, which can cause sample loss, poor resolution, peak shape problems, carry over, and ghost peaks if the injection volume is not optimized based on the particular sample solvent and inlet conditions (temperature, pressure, and liner volume).

Cool-on-Column Injection

Cool-on-column injection is particularly suited to mixtures containing high- and low-volatility analytes and for trace analysis where quantitative reproducibility is important. The sample solvent is deposited directly onto the column, thereby giving high sensitivity. The initial oven and inlet temperature should be set 10–20 °C below the sample-solvent boiling point to allow focusing of the analyte band, which also has the added advantage of reducing sample discrimination and thermal degradation. Matching the column and sample-solvent polarity is also essential to allow a homogeneous solvent film to be formed in the column; if the polarities are different, broad and often split peaks will be observed throughout the entire chromatogram. Because the entire sample is introduced onto the column, the use of a retention gap serves to protect the analytical column from involatile sample components and also allows increased injection volumes (2–5 µL) by providing a large surface area for solvent film formation. Regardless of column internal diameter, a wide-bore (0.53 mm) retention gap must be used. This technique can be prone to sample overload, is difficult to use with columns with inner diameters less than 0.25 mm, and is complex to automate.

Programmed Thermal Vaporizing

This inlet is best suited to the injection of large sample volumes (100 µL is possible, with injections of up to 1 mL having been demonstrated). In solvent vent mode, when carrying out multiple small injections, the time interval should be sufficiently long to allow almost the entire volume of sample solvent to evaporate. The optimum interval time is generally in the range of 2–20 s. For injection volumes below 10 µL use an unpacked baffled liner. For larger volumes use glass wool or beads packed in a straight liner so that the needle just touches the packing to help both solvent evaporation and analyte trapping. Liners with selective adsorption materials increase the range of components that can be trapped (improved trapping of volatiles). The best results are obtained when the boiling point difference between the sample solvent and analytes is at least 150 °C, with lower boiling solvents (<120 °C) being optimum. The initial inlet temperature should be set 30 °C below the solvent boiling point. Normal split ratios are used (50:1 to 200:1). In general, lower temperatures with higher flows are more desirable. Because of the number of parameters that must be optimized, this is the most complex injection technique, which also makes it expensive. If the parameters are not correctly optimized, there can be a loss of volatiles when using solvent vent mode.