What would you suggest on how to prep a very hydrophobic sample for reverse phase HPLC? The material in interest is a disperse dye that is insoluble in water. I was told that preparing your sample in 100% organic is a no-no but my sample is complete insoluble in aqueous phase.....What are my options? Thanks in advace for any advice.

Dissolving sample in 100% organic is OK, most of our assays do just that. The secret is keeping your injection volumes relatively small, such as 5 - 10 ul for 4.6 mm i.d. columns, 5 ul for 2.1 mm i.d. columns. Try it yourself, you'll see: larger injection volumes will make peaks look terrible. Ideally, one would dissolve sample in the mobile phase, but real life isn't always ideal.

If you cannot avoid using a 100% organic diluent, take care that the injection volume is small enough not to disturb the chromatography. If you still have problems, try a first dilution in organic, then a second one with as much as possible of water (or buffer).

Thanks for the suggestions. The problem with that dye is it forms a colloidal in aqueous media. So when I made the first dilution in 100% meoh and a second one in 60/40 meoh/water and then filter thru 0.2u, it becomes a clear solution! The actual dye is trapped in the filter...... I am injecting 10ul of sample in 100% meoh right now. Will let you guys know how it goes.

The sample should be soluble in the mobil phase if you don't want to have problems in the column

If the dye is very hydrophobic, which it almost surely is, then it will be strongly retained which will work in your favor. It is not unreasonable to dissolve the dye in anything that is miscible with water, even THF, DMF or DMSO, which is to suggest that these strong solvents will be necessary for some dyes. You will likely be able to shoot 10 uL sample from these solvents. If the dye is formulated, then one might need to worry about the fate of the dispersing agents during sample prep and separation.