Improve the resolution of the chromatographic peak

Question

Hi, I wanted to ask what factors I can change in order to improve the resolution of the chromatographic peak, which is now very broad, bearing in mind that I am using CIMac r-Protein L Analytical Column(and I don't want to change), with: bed volume (CV) : 0.1 ml Flow rate : 1 mL / min Injection volume : 50 &micro;L Analysis time : 8 min Temperature : 22 &deg;C. DAD: wavelenght 280 mAU and bandwing 4 nm without reference 
 Thanks :)

Wolney Vasconcelos · Answer

Okay, a few things to check: 
 1- My best bet is this is an old column. Considering nothing has changed and it has gotten worse overtime, it is now time to replace the column. One good indication of a bad column is peak broadening. 
 2- If you are creating a new method from scratch, then what is the solvent and the gradient? Usually a stronger organic solvent at the time the peak shows up helps with peak broadening 
 3- My first instinct was to say that 50ul is too much but given the response for the smaller peak, I don't think that`s an issue anymore. 
 4- Has the retention time shifted significantly? That would help understand the influence of several factors, specially solvent strength, temperature and pH.

Improve the resolution of the chromatographic peak

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