Technical data

Agilent 1290 Infinity LC System Manual and Quick Reference 51

Optimization of the Agilent 1290 Infinity LC System

How to Achieve Higher Injection Volumes

The standard configuration of the Agilent 1290 Infinity Autosampler includes

a variable volume sample loop for up to 20 µl injections. The metering device

can inject a maximum volume of 40 µl and the sample loop cartridge can be

exchanged to allow this (refer to the 1290 Infinity Autosampler manual for

details). The system delay volume due to the autosampler will increase

accordingly.

Whenever a method is scaled down from a larger column to a smaller column

it is important that the method translation makes an allowance for reducing

the injection volume in proportion to the volume of the column to maintain

the performance of the method. This is to keep the volume of the injection at

the same percentage volume with respect to the column. This is particular

important if the injection solvent is stronger (more eluotropic) than the

starting mobile phase and any increase will affect the separation particularly

for early running peaks (low retention factor). In some cases 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 and the user

should check for signs of increased dispersion (wider or more skewed peaks

and reduced peak resolution) in trying to increase the injection size. If an

injection is made in a weak solvent then 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 will have 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 to10 µl but it is very dependent

on the chemistry of the analyte and mobile phase as discussed above. A rough

guide to maximum injection volume can be gained by looking at the volume of

the column (see Table 7 on page 44) – in a gradient separation injection

volumes of about 5 % of the column volume might be achieved whilst

maintaining good resolution and peak dispersion.