What is the largest tolerable injection volume

The way you do the sample preparation will result in a strongly diluted sample solution, otherwise you will have to contend with the limit of detection. In any case you would like to directly inject a relatively large sample volume. How large an injection volume can you use without being penalized by noticeable band broadening We assume that the column is not overloaded only a possible peak broadening caused by the injection will be discussed here. 

You can calculate the maximal tolerable injection volume from the variance contributions of all effects to band broadening. This is possible because HPLC consists of stochastic independent processes. However, who has got the time and leisure to do this Therefore, please find in the following paragraph some numbers and recommendations. Without being given additional explanations just believe it  

In order to limit band broadening through injection to less then 1 %, the injection volume should only be up 1/6 of the peak volume. 

Using a flow of 1 ml/min, your peak is 0.6 min broad. The peak volume is 600 pi the acceptable injection volume is therefore 100 pi. If you injected 200 pi, the peak would broaden by 2.5 %. on an older column of 2500 theoretical plates. If you work with a new column with 10 000 theoretical plates, the peak broadening is 9 % compared to a 10 pi injection. 

Case 3 The sample is dissolved in a weaker eluent than the mobile phase 

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