Improve the resolution of the chromatographic peak

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 CIMacTm 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 µL
Analysis time : 8 min
Temperature : 22 °C.
DAD: wavelenght 280 mAU and bandwing 4 nm without reference

Thanks :)

  • Hello, could you share your chromatogram?

    Also, have you tried increasing flow rate or decrease injection volume?

  • 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.

  • Hi Wolney,
    1-No is a new column
    2- the binding buffer is 50 mM Tris-HCl + 0.15 M NaCl (A), pH 7, and the eluent is 20 mM Citric acid, 100mM NaCl pH 2.5 (B), with a gradient of 100A for 2.5 minutes, then 100B maintained for 2.5 minutes, and back to 100A for the remaining 2.5 minutes
    3-Yes, now I'm trying with less volume like also 2 ul, the shape it's better, but still broad peak
    4- No, not to much.

    Another question: which buffer I should use for prepare the blank solution ? both 1:1 (A,B) or only the elution buffer?

  • At this point, we should stablish some baseline.

    1- We need to make sure the system is working fine, maybe a PM should be in order to verify overall status of instrument, specially the MCGV, considering you have a quaternary pump since it is possible the system is not being mixed at the right proportion. When was the last time your system was serviced? What did they do?

    2- We need to know what are the modules in your system? Could you please share the part number of all your modules (pump, sampler, detector, tcc, others)?

    3- Has this method ever worked? Could you share a good chromatogram?

    4- You said the retention time hasn`t shifted too much. How much exactly has it shifted?

    5- I still think it might be related to the column. Can you confirm the connections to the column are tight enough? How new is this column?

    Unfortunately I am not comfortable giving advice in regards to the buffer question. You would highly benefit from a consult/training from Agilent personnel to help with your situation specifically.

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