Does changing the concentrations of your mobile phase affect the peak you obtain?
I was initially using methanol:phosphate buffer 80:20 % and I obtained good peaks. I'm currently doing design of experiments, so I need to vary my concentrations.
Now, I changed the concentration to 70:30 % and I'm getting a very broad peak now. How can I fix this?
Muhammad Zohaib Safdar
Adjust mobile phase composition: Changing the mobile phase composition can affect peak shape. You could try adjusting the ratio of methanol to phosphate buffer to see if this improves the peak shape. If the peak is too broad, you could try increasing the proportion of buffer to make the mobile phase more polar, which may result in sharper peaks. Change column temperature: Changing the temperature of the column can also affect peak shape. If the temperature is too low, the peak may appear broad, and increasing the temperature may help to sharpen it. Conversely, if the temperature is too high, the peak may appear too sharp, and decreasing the temperature could help to broaden it. Adjust flow rate: Changing the flow rate can also impact peak shape. If the flow rate is too high, the peak may appear broad, and decreasing the flow rate may help to sharpen it. Conversely, if the flow rate is too low, the peak may appear too sharp, and increasing the flow rate could help to broaden it. Optimize sample preparation: The way the sample is prepared can also affect peak shape. If the sample is not properly filtered or there are impurities present, this can lead to broad peaks. Ensuring that the sample is properly prepared and clean may help to improve peak shape. Change column: Sometimes changing the column can help to improve peak shape. Different columns may have different selectivities or efficiencies, and choosing a column that is better suited for the analyte of interest may result in sharper peaks.
There are several reasons why a peak may appear broad in HPLC, and changing the concentration of the mobile phase can certainly have an impact on peak shape. Here are some possible ways to fix broad peak shapes in HPLC:
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