If we increase or decrease the injection volume in HPLC will it make any difference in the retention time of the peak?
Yes, total injection volume can effect retention time. For analytical scale analysis, we prefer to not overload the column with sample so keep the injection volume under 4% of the column's void volume. In general, as you increase the injection volume you increase the diffusion process time it takes to load the sample onto the column (goal: you want to load the sample as a highly concentrated slug onto the head of the column before you start to separate it). Large injection volumes translate to wider peaks. Wider peaks will take a bit longer to elute. So if you compare two different injections of the same sample (one very large, the other very small) the retention time (peak apex) of the larger injection (volume) should be a bit later. *This is a generalization as we have not established any actual values or methods, but the concept is true.
**BTW: The choice of injection solution used also can effect the retention time too (as it influences how the sample is loaded onto the column head and that can be discussed some other time). An understanding of these basic principles are fundamental to understanding the HPLC method development process.
Yes, total injection volume can effect retention time. For analytical scale analysis, we prefer to not overload the column with sample so keep the injection volume under 4% of the column's void volume. In general, as you increase the injection volume you increase the diffusion process time it takes to load the sample onto the column (goal: you want to load the sample as a highly concentrated slug onto the head of the column before you start to separate it). Large injection volumes translate to wider peaks. Wider peaks will take a bit longer to elute. So if you compare two different injections of the same sample (one very large, the other very small) the retention time (peak apex) of the larger injection (volume) should be a bit later. *This is a generalization as we have not established any actual values or methods, but the concept is true.
**BTW: The choice of injection solution used also can effect the retention time too (as it influences how the sample is loaded onto the column head and that can be discussed some other time). An understanding of these basic principles are fundamental to understanding the HPLC method development process.
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