Lecture #6
HPLC have mobile phase , column , pump and detector .
HPLC (High Pressure Liquid Cromatography) → maen that I have pump .
HPLC (High Performance Liquid Chromatgraphy) → mean that give good
resolution of the peak .
HPLC mobile phase needs : HPLC good solvent which contain aqueous part
and organic modifier (Why?)
Because here we use reverse phase (not normal, polar→water ) .. but if
mobile phase all of it is polar and the column is non-polar there will be no
competition ,so we use organic modifier (which means :water soluble
organic solvent , semi-polar) like: ethanol , methanol , acetonitrate and tetra
hydrochloric .
Acetonitrate and methanol are most commonly used after (microfiltration) ..
Methanol manufacturing :
We use sugar source like grabs and bacteria to make fermentation and
convert the glucose to ethanol (96% ethanol) why?
Because alcohol always have a 4% of water during distillation process .
To have 100% ehanol they make isosbestic distillation by adding benzene to
ethanol with make eutectic mix then benzene and ethanol evaporate
together leaving water , then separation ethanol from benzene by simple
distillation to have absolute ethanol , but it is expensive (high purity )
Eutectic mix : when we mix 2 substances together , it will cause boiling point
elevation or melting point depression.
HPLC code :
Solvent should be free from insoluble impurities to avoid scattering of UV ,
AND free of soluble impurities to avoid chromophore impurities which may
lead to interference with UV absorbtion .
Cut-off value :
↓λ ↑energy
Higher energy → absorbtion of α bond
Lower energy → absorbtion of π bond
Methanol should be transparent on π region (low energy) to prevent making
At (blue) higher absorbtion ,, it means that the methanol absorbtion is higher than
Blank sample → low absorbance ,, if it was high absorbance analyte will not be clear.
if mobile phase (blank) →high absorbance at low energy and will make excitation of
analyte .. analyte won't be clear
methanol absorbtion at low λ → due to impurities
cut-off value → λ → better when it lower
so we need solvent with high purity (free of soluble and insoluble impurities ) and we
can relay on cut-off value .