Chromatography Forum

Dear all,

Merry Christmas to all of you. I am a new member and I have a question for everyone.

I came across a method ( USP monograph -method) for estimation of Methylprednisolone in Methylprednisolone Sodium Succinate for Injection which uses following mobile phase.

Mobile phase:A filtered mixture of butyl Chloride , water- saturated butyl chloride, THF , methanol and glacial acetic acid ( 95:95:14:7:6).

Column : 300 x 4 mm,5µm -, Porous silica particles

I was wondering why water saturated butyl chloride and ordinary butyl chloride is used.

I looked for answers and asked many people . One of the answers I got is even in Normal phase chromatography there is a minimum amount of water is required to get proper resolution. Is it true? But Why??? Then why we spend so much time in taking change over from reverse phase to normal phase .( Purging the system with Methanol , IPA etc.)

One more interesting fact I tried to omit the water saturated part of mobile phase preparation. I was getting the almost same Retention time. Some one suggested to me that the acetic acid contains enough water in it to make the butyl chloride saturated. Is it true??



Thanks in advance for your answers.

In general (if I remember that in right way) water saturation will help you to keep method robustly independent from possible changes in water content, that can have different sources (for example - hasn't glacial acetic acid to be water-free per definition? Are you going to perform KF analysis on your solvents each time you run this HPLC method?). IMHO, You have to turn your HPLC system from RP to NP mode, keep your solvents dry - assure that there are no other sources of water, except controlled water saturation.

Other question to forum (sorry, anirbanroychowdhury, it's your topic) - in that case (water saturation), should one consider to turn this column to "dedicated", i.e. is it reversable?

The proposal for the use of normal-phase solvents that are half-saturated with water go back to the studies of Heinz Engelhardt and Helmut Elgass. The idea is to keep retention times reasonably constant, when fresh mobile phase is prepared.

The addition and removal of water from the surface of silica is completely reversible. It can even be done with dry hexane, but it will take a week to stabilize retention in dry hexane. In addition, if you switch from one bottle of dry hexane to another bottle of dry hexane, your retention may change again. To avoid this problem is the basic idea behind the Engelhardt/Elgass procedure.

Hello

Most people believe that water is a strict no-no in normal phase. This is not true.

Water is sometimes used in a normal phase separation, at low levels. This is because water tends to decrease retention times, when used in normal phase. Therefore used to enhance resolution, in trace amounts.

Also, many normal phase HPLC methods are a carryover from TLC methods in which water-saturated organic phases are commonly used - a typical example being Butanol-acetic acid-water ( 4:1:5, upper phase).

Glacial Acetic acid is used as a co-solvent in such mobile phases. Although it may be water-free to start with, HOAc is very hygroscopic and may still contain traces of water, if improperly stored.

As for Uwe's comment - I didn't know about the Engelhardt/Elgass procedure, so I guess I've learnt something myself. Thanks Uwe!

Rgds,