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That is very interesting, tried several times, but thanks god, developed good assay:
Column: C18, 250X4.6 mm, 5 um.
Mobile Phase: 0.1 % H3PO4, use H2O as solvent.
Flow rate: 1.0 ml/min, 200 nm.
Today, I was asked :" Why not use 0.1 % H3PO4 as mobile phase or Why use buffer as mobile phase?
OOOPsss.. That is really good questions. Now I still try my best to answer this question in one words, however I failed all the time.
(1) Covalent attachment of the stationary phase yields a thermally and hydrolytically stable bonded phase.
(2) Hydrophobic Surface, Particle Size Distribution, Porosity, Pore Size and Surface Area.
(3) Less Polar ( more Hydrophobic) analytes are more attracted to the hydrophobic bonded phase. So more hydrophobic spends more time associated with the bonded phase... then are eluted last... That is the I always Love Methanol: it is a active solvent.
(4) Hydrophobic Theory...Partition Theory..Adsorption Theory [Chromatography of “cavities” in solvent created by hydrophobic portion of analyte molecule Surface Tension, Interaction of polar functions with solvent, Stationary phase is passive]
Cool, See that "Surface Tension" ... that is the reason why we use the Sodium Phosphate/Potasium Phosphate...
At the same time, always remember that "Water is “weak” solvent, Increased organic ---> decreased retention"
Today, met a technical issue how to analyze the purity of Creatine series.
Yes, General Assay: 0.1 M ~ 0.02 mM Buffer, pH=2.9- 4.5, 205 / 210 nm.
Why the Quantitation by Normalization for the purity testing did not work?
:>) Smile..
So Happy.... April-21-2008: Today finished the 1st GC assay @ IN.
VIP Tips: (1) The temperature of FID should higher than the final temperature of column, otherwise the baseline would have a little / slight increase.
(2) General Ramp Temp Setting: 50 C (2 min) to 150 C at 10 C/min hold 20 min, then to 250 C at 20 C/min, hold 5 min.
Adjusting the Initial Temperature and Hold Time
To improve the resolution of earlier eluting peaks, decrease the initial temperature or increase the initial hold time. Decreasing the initial temperature usually results in the largest resolution improvement, but analysis times are substantially increased. In addition, cool down times between runs can be significantly increased especially when cooling below 50°C.
However, It is often impossible to cool a GC oven below 35°C in most laboratory environments without using cryogenic oven cooling. The resolution of the later eluting peaks are minimally affected by lowering the initial temperature especially for longer length columns. If excessive resolution is obtained with the original linear temperature program, increase the initial temperature to reduce the resolution and analysis time. The resolution of later eluting peaks may also be
reduced upon increasing the initial temperature.
That is very interesting, tried several times, but thanks god, developed good assay:
