Question

Experimental Organic Chemistry 1

HPLC Questions

- What's the difference between single point calibrations and five point calibrations?

- How could you improve your resolution on a chromatograph?

- What's the difference between isocratic runs and gradient runs?

Answer 1

1.) Single-Point calibrations aren't that useful for quantitative analysis and there is more room for error in result readings as compared to a 5-Point calibration.

2.) there are two parameters to adjust in order to improve the resolution. One of them is the temperature ramp. this parameter may vary the resolution but in very small amount. The parameter who adjust the resolution more drastically is the column flow. It depends not only of the flow value but also depends on the carrier gas. The best carrier gas is hydrogen, and the best compromise is Helium. Also, if you are using a FID detector, one important parameter to be taken into account is the makeup gas flow. This parameter is a flow value from the same carrier you are using to analyze your samples declared for the detector

3.)

• Isocratic means that the concentration of the strong solvent remains constant whereas Gradient is done by increasing the concentration of the strongest solvent
• Isocratic mode is most commonly used for assay, dissolution, and routine analysis where you want to separate 2 or 3 compounds in a single run whereas Gradient elution is very useful for impurity profiling, degradation studies, as well as analysis of complex herbal extracts where analytes are having mix polarities.