HPLC Flashcards
What is the mobile phase concentration like during isocratic elution?
The composition of the mobile phase remains the same for the entire time.
What is the mobile phase concentration like during gradient elution?
The composition of the mobile phase is adjusted according to a pre set schedule
What is the advantage of gradient elution over isocratic elution?
Gradient elution can reduce assay time and achieve better separation and peak shapes
What are the 2 most common detectors used for HPLC?
UV vis and Mass spectometry
What is the equation to measure absorbance from transmittance?
A = lg (1/T)
What is the equation to measure absorbance from light intensity?
A = lg (Io/I)
What are the two equations of beer lamberts law?
A = εbc
A = A1% bc
For the equation A = εbc, what are the units of each of the variables?
b: cm
c: mol/l
For the equation A = A1% bc, what are the units of each of the variables?
b: cm
c: g/100ml
What is the difference between normal phase and reverse phase HPLC?
Normal phase: the stationary phase is polar and the mobile phase is non polar
Reverse phase: the stationary phase is non- polar and the mobile phase is polar
What is usually used for the stationary phase for normal-phase HPLC?
- silica
- silica with polar organic moieties
What is usually used for the stationary phase for reverse-phase HPLC?
- silica with non-polar organic moieties
In reverse phase HPLC, what are the non-polar chains attached to the silica?
C8, C12, C18
What are the common solvents used in normal phase HPLC?
Hexane
Dichloromethane
Isopropanol
Methanol
What are the common solvents used in reverse phase HPLC?
Water (buffer)
Methanol
Acetonitrile
Tetrahydrofuran