I have a problem on sepration of hydroxyzine and its related compound according to USP method there are overlap between these compounds

I don't have experience with this method, but I took a look at the USP method. The method uses 2 silica columns with a flow rate of 0.4. I would check the flow rate accuracy of your system. Maybe it is pumping too fast. Are you using 5 or 10 micron silica?

Any information you can provide about what you have tried would be helpful to us in order to give you good advice.

1. Silica is not silica.
2. Silica can be wet or dry.

Anon:

Would you please explain what you mean by silica is not silica. How would this affect the separation above. Aren't all columns packed in a slurry?

dear mel
we use .4 flow according to usp method and MN col NUCLOSIL Si 100-10 and usp solvent we changed the composition of usp solvent but it couldnot change anything the retention time according to usp method is about 12 min! but we can't see any seperation

What did you change the compostion too. Are you being careful to not get any water into the column?
Melissa

Which column is recommended by USP?

It recommends an L3 packing which is bare silica. You can use any manufacturer of an L3 packing.

Dear mel
the composition of usp solvent is acetonitril and .12 N sulfuric acid (90:10) it has water!
we use NUCLEOSIL Si 100 col. I think this is L3 packing but I amnot sure
af

There is nothing wrong with using water on a silica column. What does the USP say you are supposed to get? And what are you not getting? It is not clear to me...
If possible please describe the retention times that you got, what the USp numbers are supposed to be, what you changed, and what effect it had. Maybe then we understand what is going on and can help.

Dear Mr. Neue:

I apologize if I gave out wrong information concerning water and silica. I had always been told that you had to be very careful with how much water was in the mobile phase.

Melissa

dear Neue
there isn't any specific RT for Hyd. and its related on USP but our test RT is 12 min with .4 ml/min flow USP wants a resolution about 2 but we cann't seperate the Hyd. and its relation
af

Hi af,

You say you can't separate the active and its rel subst. So you mean you have tried some experiments. I guess thats what uwe means, what exactly have you tried already and what effects does this have on your separation?

af,
do you have to test this sample to USP specifications? the di-HCl salt method uses reverse phase eluents on a porous silica column, which you have got, but (from memory) another monograph for aqueous solutions of hydroxyzine di-HCl runs with water/methanol on a C18 column. ie. if you have the salt and don't have to test to USP spec, the other method could be suitable.

forgot to include in previous message;

are bare silica supports robust under acidic aqueous conditions?

Do not be concerned about the stability of the column under these circumstances. Silica is no different than glass, and acids (except for HF) are commonly stored in glass bottles. I'd prefer a silica column in water over a bonded phase any day...

Dr. Neue:

I have read often that glass is borosilicate. That is, it's a silicate. Silica is silicone dioxide. Isn't there any differnece between them? Please give me more info.

OK, to everybody concerned about the solubility of silica in water: maybe the sand on the beach does not exist, because it has dissolved already in the ocean...
Silica is "rock" stable at any pH from at least 8 down to concentrated sulfuric acid. Nothing to worry about...

thank you Uwe.

is running a silica column with this aqueous acid/acetonitrile mobile phase still defined as Reversed-Phase chromatography?

when somebody mentioned earlier to avoid getting water on a silica column, i posed my question so that this situation would be clarified. IMO the original poster was associating the use of a silica column with a normal-phase technique whereby water would not be desired, especially with cellulose/amylose phases used in a lot of chiral columns.

along with cyano and amino, another example of a common stationary phase which can be used in either NP or RP mode.

The most often used name for this kind of chromatography is hydrophilic interaction chromatography (HILIC). The word was created by Andy Alpert.

It is not that difficult to get water out of silica columns. Of course, if you are working with a stationary phase that is only adorbed, the washing of the column with water could be deadly. Not recommend for these types of columns!

There seems to be some confusion in this thread about the use of water with silica columns/ and the presence of water in normal phase eluents with silica columns.

Clear from what Dr Neue says is that water will not damage an ordinary bare silica column ( strange adsorbed stationary phases are another question!).
However, another point in this thread is that in classical normal phase chromatography, using silica columns in conjunction with solvents like hexane, dichloromethane etc, the presence of water can really mess up a separation. This is because in this mode water is a really strong solvent. In some cases this will mean complete retention of analytes in dry mobile phases but rapid elution in solvents saturated with water. To get round this and to make things more reproducible, some workers recommend drying the solvents and then adding small quantities of alcohol (eg methanol, isopropanol) to control the solvent stregth.

David

Hi David,

Yes, this discussion focussed on the use of silica columns in an aqueous environment. There is nothing wrong with this. However, in standard normal phase chromatography, water is a deactivating agent for silica. In order to get reproducible retention, the water content of the mobile phase needs to be controlled.
There have been various proposals in the literature, how to do this best, from mobile phases half-saturated with water to the addition of other polar solvents.

so if you get water, or another highly polar protic solvent, into a normal-phase eluent system is the lower retention of polar analytes due to them liking the mobile phase more, or not being adsorbed onto the silica surface because that is deactivated by water (H-bonding??)

Uwe - as far as I remeber from inorganic chemistry textbooks, SiO2 crystals are grown in hot water (maybe under increased pressure). Then there was a time where many people used "saturation precolumns" in RP chromatography. I am not saying that this is relevant for HPLC.
johnboy - In some older LC textbooks you will find the term liquid-liquid chromatography: that desctribes inorganic support, like silica gel,that is covered by a layer of water ( or pores are filled with water) and elution by unpolar solvents. its basically a liquid-liquid partition mechanism. I think this principle is still used in sample preparation (SPE or better liquid phase extraction on solid support).

hehe, i really love chromatography and i'm trying not to get confused. Uwe Neue said in his October 31 message that "water is a deactivating agent for silica" so i was still thinking the main mechanism of retention was adsorption, now you tell me its liquid-liquid partitioning :-)

One can do all kinds of things with silica. One can use it as an adsorbent for doing adsorption chromatography. An example is the separation of aromatic hydrocarbons with rather dry hexane as the mobile phase. Or one can fill the pores with a solvent that is immiscible with the organic mobile phase. You can fill the pores of silica with water with or without other stuff in it, and use a water immiscible mobile phase like methylene chloride with stuff in it. With the former approach, you need to worry about the water content of the mobile phase and the silica, with the latter approach you use water. And finally, you can use silica with aqueous mobile phases. The primary mode is HILIC, as described above, but you can also use silica as a weak cation exchanger.
I am aware of the use of saturation columns. I have never done it, I never believed in it. If it did anything, I think it protects the analytical column from debris from pumps and solvent reservoirs.
To get back to the original question: has the problem been resolved?