C 18 column

Basic questions from students; resources for projects and reports.

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*why c18 column is preferred for reverse phase HPLC and why is it called C 18?

*does it always contains silica gel packing?

*how is it packed inside the stainless steel tube column...slurry method and introduced at high pressure?

*which detector is preferred ...UV ...flouresence...differential refractrometer...conductivity measurement and why?

*how to analyse a drug that exist in isomeric forms...how come we determine which isomer is eluting now...and under which stress condition the drug changes from one isomer to other...

*is it fine to analyse a drug in a mobile phase composition in which the drug is not soluble...the solubilty affects partition coefficient and which in turn is the basis for liquid chromatography?

*why c18 column is preferred for reverse phase HPLC and why is it called C 18?
C-18 provides good retention in reverse phase, most manufactures have considerable experience with C-18 and can achieve good lot to lot reproducibility, it is hydrophobic enough to provide some shielding of the siloxane bond and can yield extremely long column lifetimes.

*does it always contains silica gel packing?
No. There are different substrates available. Zirconica, polymers, silica-orgainic hybrids, etc. Even with silica there are gel and sol forms.

*how is it packed inside the stainless steel tube column...slurry method and introduced at high pressure?
I don't work for a col. manufacturer but I'm sure the method varies by manufacturer. Constant flow, constant pressure, non-linear flow program, etc.

*which detector is preferred ...UV ...flouresence...differential refractrometer...conductivity measurement and why?
It depends! Where it is applicable, UV has a lot going for it. Fairly sensitive, good linearity over a wide range, etc. Not all compounds absorb in the UV however. Luckily, most drugs have some absorbance in the UV.

*how to analyse a drug that exist in isomeric forms...how come we determine which isomer is eluting now...and under which stress condition the drug changes from one isomer to other...
This is a large area. There are structural and configurational isomers that may or may not be easy to detect with reverse phase. There are various chiral columns that are designed to help separate configuration isomers. There are conformational isomers that can lead to difficulties in a separation. Drugs can also be polymorphic, that is exist in more than one crystalline form. These forms can convert on stability and have different physical properties. Of course, the crystalline form cannot be detected by hplc. Chiral column may give you a reason to believe which isomer should be eluting first. If you have to have confirmation you may need nmr and/or MS data.

*is it fine to analyse a drug in a mobile phase composition in which the drug is not soluble...the solubilty affects partition coefficient and which in turn is the basis for liquid chromatography?
You need some solubility, but it definately does not have to be highly soluble. With most small molecule pharmaceuticals you should have very high recoveries in reverse phase column/no column studies.
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