Kinesis Solutions Techtips

Splitless injection is suitable for trace level determinations i.e. in situations where the analytes may be in the concentration range of low ppm, µg/mL. This allows much lower detection limits (higher sensitivity) because most of the sample will be transferred onto the column rather than out the split vent. The split vent valve remains closed during splitless injection (See Figure 1) and is opened about 30-60 sec after injection. If the vent were left closed for the duration of the analysis, a tailing solvent peak would obscure early eluting component peaks. Peak widths tend to be wider (especially for earlier eluting peaks) than for split injection. Specific guidelines need to be followed with splitless injections to obtain acceptable peak shapes and widths for many compounds.

This article will give some insights into peak focusing methods necessary for splitless injection and the GC conditions required for sharp peaks.

One thing to remember about splitless injection is that the gas flow through the liner is equal to the column flow. So the flow through the liner could be as low as 0.5mL/min. This means there is a longer time to flush the sample into the column before the start of the temperature program. In fact the sample / solvent could take up to ten minutes to be totally flushed from the liner. For this reason the split vent is always turned back on after about one minute to quickly remove any residue left in the liner to produce a sharp solvent peak (See Figure 2).

Getting narrow peaks - solvent effect
It is extremely important to get the sample into the column in the narrowest band possible (See Figure 3). The broadness of the band will affect peak resolution. If narrow bands are not achieved in the first part of the column, the separation following is a lost cause i.e. non-recoverable. Unfortunately, splitless injection transfers the sample very slowly from the inlet liner to the column resulting in broad bands. This is because the flow through the liner is the same as the flow through the column (which is of the order of 1.0 mL/min for narrow bore capillary columns) and thus the transfer rate is slow. For splitless injection, for a splitless time of 1 minute and a one mL liner volume, the initial peak width will be approximately one minute wide. Hence, there must be a band / peak focusing mechanism to sharpen the broad peaks back into sharp peaks so that good resolution is achieved.

The most common method of peak focusing in splitless injection is known as the solvent effect. Solvent focusing involves injection of the sample solvent at an oven temperature at least 10-20°C below the boiling point of the solvent. Since the column temperature is below the solvent boiling point, the sample solvent condenses in the front part of the capillary column. The less volatile components will follow the solvent band into the column and the solvent film traps these solute molecules (See Top Figure 4). The analytes will have a greater affinity for the solvent than for the gas phase. Solvent focusing occurs by evaporation first of the solvent at the injector end of the column and the analytes become more and more concentrated in the ever decreasing solvent band as the oven heats up. Eventually all solvent will evaporate and at this stage, the analytes will have been focused as a narrow band on the column (See Bottom Figure 4). This process is called the solvent effect. A chromatogram showing the effectiveness of the solvent effect can be seen in Figure 5. Note the sharp early-eluting peaks after solvent focusing.

Splitless Time
In Figure 6, the top chromatogram shows an example of what happens when the splitless time is too short. You can see that C22 has not had enough time to vaporise completely. The sample has spent a little over10 seconds in the injection port and has not gained enough energy from the liner to completely vaporise the hydrocarbons. There is a lot of high boiling point discrimination.

The bottom chromatogram (See Figure 6) shows the same sample under the same conditions except the split flow turns on one minute after the start of the run. The first thing you will notice between the two chromatograms is the difference in response of the heavier compounds. The high boiling point compounds have now been able to absorb enough energy from the liner to vaporise completely. The relationship between the split time and boiling point (shown through increasing carbon number of aliphatic hydrocarbons) can be see in Figure 7.

Getting narrow peaks – cold trapping
If a solute boiling point is approximately 150°C or above the initial column temperature, that solute will focus at the front of the column without the solvent effect. As the solute molecules are transferred from the inlet liner to the capillary column, the compounds condense in the capillary column and thermal focusing counteracts the slow transferral process. Thus, what would otherwise result in a wide band has now become a narrow band (See Figure 8). This process is called "cold trapping". Either the solvent effect or cold trapping has to occur to obtain good peak widths or shapes for splitless injections.

Getting narrow peaks – liner inner diameter
The liner inner diameter can make a dramatic difference in peak shape (Figure 9), especially of the early-eluting peaks. A smaller inlet liner diameter results in higher velocities through the liner and thus faster transfer of components into the capillary column.