The below-mentioned point explains how injection volume affects several factors in chromatography.
The relation between injection volume and peak height is a type of linear relationship. The appropriate injection volume of the sample in HPLC is associated with the volume. The volume of samples in high-performance liquid chromatography is usually fixed or in autosamplers, it may be variable. The Rheodyne sample loop is commonly used in HPLC to inject samples without disturbing the flow rate of the mobile phase and the pressure of the HPLC system.
The mass overload happens when the chromatographer injects too many analytes and column overload happens when the excess liquid sample is injected into the column. Column and mass overload can reduce column performance due to peak shape and retention time problems.
Here some effects of injection volume in HPLC are mentioned.
- The column is overloaded if too much sample is injected into the column.
- If injection volume increased then the height of the peak will increases.
- The peak width increases by an increase in injection volume.
- The peak broadening is due to volume overloads.
- Increasing injection volume will decrease the resolution between the separated compounds.
- When a very large volume of sample is injected into the HPLC column, the peaks begin to tail and the retention time (RT) may increase.
The size of the column limits injection volume as well as optimal flow rates of the system. If the sample compound and mobile phase are in the same solvent, the ideal injection volume should be as follows:
The most typical liquid chromatography column is a 25 mm x 4.6 mm ID column with a particle size of 5 to 10 microns. The typical ideal sample load for these characteristics or a sample concentration of 1 mg/ml is 10 to 50 microliters.
In HPLC analysis, the optimum injection volume is 10 to 20 μl however, the 10 μl is a default injection volume for most instruments. Since a larger sample injection volume would enhance band spreading and contribute to lower resolution or asymmetric peaks.
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