Mixed mode columns in gradient elution

[VEBA]

I'am looking after you experiences mainly in gradient elution.Using binary pumping system there is limited variability comparing quaternary systems.I have used those columns mainly in isocratic mode and it has been usually ok.
I usually use UV detection but have also tested LC -MS with formic acid/ACN gradient.Salt and PH gradient are modes
witch i would like to evaluate.
btv,I have experience with Primesep and Acclaim trinity columns

Veba

[Vlad Orlovsky]

Veba,

SIELC columns are compatible with 100% organic and 100% water so you can do any type of gradient - single, double or triple (each variation is a parameter - amount of ACN, amount of buffer and buffer pH)
In all gradients you go from weak mobile phase composition to strong mobile phase composition. In some case you need to consider some of the unique qualities of mixed-mode columns when you create gradients. Since the column you tried have polar embedded groups and ionizable groups you need to evaluate the end point of gradient. It is easy to go from mixed-mode/anion-exchange (low organic) to HILIC/ion-exchange (high organic). When you move from RP/IE to HILIC/IE you mobile phase becomes weaker and elution of compounds slows down. You also need to consider the fact that when people use only two channels they don't think about two interactions - RP and ion-exchange. We saw a lot of cases when method on mixed-mode column called for gradient of 100% of water to 100% of ACN and only channel A had ions. So effectively by going along with your gradient mobile phase was depleted of ions and elution of ionic compounds slowed down or completely stopped and compounds got stocked on the column. For all practical purposes I would recommend to start with the following gradient:
A: 5% ACN with 5 mmol ammonium formate pH 3
B: 70% ACN with 30 mmol ammonium formate pH 3
go from 100% A to 100% B in 10 minutes, hold 5 minutes, equlibration of 8 minutes (for 2.1x150 mm column). method can be shorter for shorter column, higher flow rates and stronger mobile phases.
If you retention is too short decrease amount of ACN in both channels and amount of buffer in channel B. You can also explore various pH and acids (formic and acetic for LS/MS)

we have few tools which help you with buffer selection and method development:
http://www.sielc.com/upload/file/pdf/SI ... t_2009.pdf
http://www.sielc.com/Technology_MethodDevelopment.html

If you let me know exact nature of your compounds I will advice you on column selection and the gradient to try.

P.S. example above is for RP/ion-exchange case. If you are doing RP/anion-exclusion your buffer concentration in initial point is higher than at the end point of the gradient.
P.S.S. For UV you just use UV transparent mobile phase additives

[VEBA]

Vlad,
Generally i would like to develop very generic gradient method to see if that column could be useful.I'am testing pharmaceutical compounds(basic ,acid,zwitters and neutrals).Formate buffer is ok both UV and MS systems but what
about Phosphate in low UV region?Is it big difference which cation is in use(Potassium,sodium,ammonium) to interaction
process?TFA can be alternative but i don't want to use it at least in MS .

I prefer short columns in method development and usually i use 2.1x50 format.Methods which are useful also UV and MS
would be desirable.

Veba

[danko]

That’s exactly what we want from the pharmaceutical industry. A single pill that takes care of all diseases

Best Regards

[Vlad Orlovsky]

Please send me email through our website and I will share couple of files on generic screening procedure. You can use any buffer with mixed-modes within recommended pH range. In case of phosphate you just need to watch for solubility of the salt at the end point of your gradient