Sample Log P range between 1 and 5 sounds like a typical RP mode application, not HILIC. Why are you using HILIC? What functional groups are present? What pH is used? What solubility properties exist? Have your samples been analyzed before by others (web keyword search) and if so, what results were obtained? Where their methods scientifically sound or flawed? These are the types of fundamental questions posed at the start of any method development.
You may want to avoid polymeric columns (poor active surface activity and sites) and use conventional ultra-pure silica based C18 supports.
I thought c18 columns are also polymeric like ODS bonded to silica for C18 columns. Why and how do polymeric columns have poor surfave activity?. The hilic column was good for the method development as everything worked on c18 after the method was developed. I would still benefit from such literature if I decide I don't have the energy for more lab work tomorrow just to discuss how to proceed with the 150 x 4.6mm HILIC column research.
I know that HILIC is a mechanism of all trades hence the high equilibration but a c18 polymeric column takes 30 minutes to 45 to equilibrate. Is this something to do with surface wettability of polymeric Stationary phases in general. I am not confident in my material science understanding of polymeric vs monomeric columns.
Mel: You need to work on that terminology.
Polymeric columns: You are confusing true polymeric columns (e.g. polystyrene-divinylbenzene, "PSDVB") with conventional chain-style functional groups attached to silica substrates (which represent 99+% of all HPLC column types used).
Surface Area on supports: You can look up plenty of tables of available pore volume and surface area of commercially available polymeric and silica based supports. All of the polymeric supports are low and therefore show poor load-ability and resolution compared to their less expensive and more common silica supports. *If they were as good as silica supports, we would have switched over to them 50 years ago, but they represent a smaller market. Because of the much wider pH range, stability and solvent tolerance of true polymer based supports (i.e. PSDVB), they are useful in GPC work with harsh solvents and other applications areas such as where high pH (>10) mobile phase is required. Silica supports are not an option.
ODS = C18 (ODS stands for octadecylsilyl groups). Not polymeric. These are silica based supports, but you can functionalize a true polymeric support too (which has been done, and they do not work well). Silica and polymeric supports can have all kinds of chemical groups (proteins, peptides, amino acids...) linked to them.
"HILIC column": ? No such thing as a true HILIC column (even if someone sticks a marketing label on one, it is probably just a bare silica or perhaps slightly functionalized one). HILIC is a mode of chromatography, not a support type. BTW: If you thought you were using an HILIC mode to develop a method, then moved it to a C18 column, that means you did not in fact use HILIC mode on the first column at all.
"c18 polymeric column" ? think you are again referring to a silica based column.
It helps when we discuss chromatography to include all of the details (column brand, name, support, particle size, dimensions... flow, mobile phase, %composition (iso or grad), inj vol, conc, detection settings....).
Column equilibration is a function of the support type (available volume), surface type (coated vs. bound), flow rate relative to column volume, temperature and mobile phase properties. In HILIC mode (not column), it can take a long time because the column support is having trouble maintaining the thin water layer (maybe 2-3%) on the surface and ANY change in mobile phase composition (and all of the items I noted earlier) can result in instability in that layer resulting in very slow equilibration. For more conventional modes of liquid chromatography we sometimes say that a column is often equilbrated after passing 10 column volumes of mobile phase through it. However, this is just based on diffusion and a generalization. In real life, because there are so many variables (and the time needed for the detector to stabilize is another one), it will take as long as it takes.
We have one LC method that we run in our lab where the total run time is 3 minutes and the column is re-equilbrated and ready to go in 5 minutes (on a 2.1 x 30 mm, C18 column). Other methods, with larger volume columns may take 30 minutes to run, 20 minutes to wash and 30 minutes to equilibrate again (or even 3x times that). Too many variables to state "this is how long it takes". I think I said once before that "it will take as long as it takes". I have been professionally teaching chromatography for almost 30 years and that answer applies to all columns and modes of chromatography.
- Badges
- Science method