How to Achieve Higher Injection Volumes (G7167C, G7137B Hybrid Multisamplers)

Whenever scaling a method down from a larger to a smaller column it is important that the method translation allows for reducing the injection volume in proportion to the volume of the column to maintain the performance of the method. This keeps the volume of the injection at the same percentage volume with respect to the column. This fact is particularly important if the injection solvent is stronger (more eluotropic) than the starting mobile phase. Any increase will affect the separation particularly for early running peaks (low retention factor). Sometimes, it is the cause of peak distortion and the general rule is to keep the injection solvent the same or weaker than the starting gradient composition. This has a bearing on whether, or by how much, the injection volume can be increased. The user should check for signs of increased dispersion (wider or more skewed peaks and reduced peak resolution) when trying to increase the injection size. If an injection is made in a weak solvent, the volume can probably be increased further because the effect will be to concentrate the analyte on the head of the column at the start of the gradient. Conversely if the injection is in a stronger solvent than the starting mobile phase, then increased injection volume will spread the band of analyte down the column ahead of the gradient resulting in peak dispersion and loss of resolution.

Perhaps the main consideration in determining injection volume is the diameter of the column as this has a big impact on peak dispersion. Peak heights can be higher on a narrow column than with a larger injection on a wider column because there is less peak dispersion. With 2.1 mm i.d. columns typical injection volumes might range up to 5 – 10 µL but it is dependent on the chemistry of the analyte and mobile phase, as discussed earlier. In a gradient separation, injection volumes of about 5 % of the column volume might be achieved while maintaining good resolution and peak dispersion. One way to achieve larger injections is to use a trapping column selected by a switching valve to capture and concentrate the injection before switching and injecting it onto an analytical column, see Sample Enrichment. The valve can be conveniently located in the Multicolumn Thermostat.