By Anonymous on Tuesday, July 16, 2002 - 08:42 am:
Now I am using Microsorb Si column ( 250X10 mm, size 5um, pore 100 A) to seperate two position isomers 9,10 and 12, 13 diol of linoleic acid methyl ester, mobile phase is 2.5% isopropanol in hexane, 4.7 ml/min, UV detector 202 nm. Each injection is 25mg/100ul. If I increase the concentration or volume of injection, the resolution will be bad. The problem is that I want to purify about 1g raw material, that means about 50 injection at this condition. I am considering to buy a bigger column, but I don't know which one is suitable with lowest cost and I also don't know whether our equipement ( Shimazu, LC-10AT vp ) can support the bigger column or not ( such as larger flow rate or high pressure ). Any suggestion will be appreciated.
Thanks
By H W Mueller on Tuesday, July 16, 2002 - 11:41 pm:
Have you given up on your improvement of separation? It would be surprising that isopropanol is optimal for such small molecules.
You know: the greater the separation the more sample can be handled.
By A Buske on Tuesday, July 23, 2002 - 11:48 pm:
Hi Anonymus,
Your problem doesn't look to bad. To get 25 mg substance dissolved in 100 µl is really good. I have often struggeled with low solubility of analytes. What is your retention time? Do you have to clean the column between runs? Is your method isocratic? If rt is around 5 min you would need for 50 injections just 250 mins. If it is a isocratic method and there is not too much matrix, you could even inject the next run before the first one is finished. In that case I would not cosider any change.
The LC-10 delivers up to 9.99 ml/min. Backpressure should not be a problem with hexane. I have had two 25 cm columns with 3 µm material coupled together and was far away from the pump pressure limit (4 mm i.d., 1.25 ml/min).
Hans, what would you propose instead of isopropanol? It is commonly used for NP chromatography on small molecules. Addition of some acetonitrile (be carefull, there is a solunbilty gap in the system hexane: isopropanol:acetonitrile) might improve the separation.
There are a lot of other polar modifyers to consider. But your substances are isomers, they differ in shape, not in polarity, specific interactions.....
By Anonymous on Wednesday, July 24, 2002 - 07:53 am:
Thank you both.
I optimized the concentration of isopropanol in hexane. The retention time is about 15~20 min at isocratic condition. That's why i consider the change. If i try to decrease the r.t, the seperation will get worse. I also try use TFA, it does not help much for the seperation. The limitation of 10 ml/min may not support the bigger column, I guess.
By H W Mueller on Thursday, July 25, 2002 - 11:58 pm:
One could try any low viscosity solvents which are miscible with hexane, ethanol, ethyl acetate..., also replace hexane with heptane, toluene... I havn´t done this, but considered NP (analytically) for similar substances, thus, I am curious how this could be improved, or what else had been tried with less success.
Incidentally, I see red when TFA is mentioned, didn´t it attack your esters? Or did you mean THF?
By Einar Pontén - SeQuant AB on Wednesday, August 21, 2002 - 01:17 pm:
It seems that HILIC is an alternative for you. One could say that HILIC is an intermediate technique between NP and RP mode, i.e., acetonitrile or alcohols and a buffer in the mobile phase and a hydrophilic stationary phase.
The ZIC-HILIC column is available for both analytical purpose and prep scale, 1" and above.
For example, 1"x150 mm (5u, 200A) can be used at 10 mL/min and low backpressure.
http://www.sequant.com