Chromatography Forum

Hi all

I'm using a Supel™-Select SPE HLB 20 ml tubes to separate hydrophylic and hydrophobic peptides from a peptide mixture sample of 0.1 g diluted in 1 mL of water. In the process of separation we use a vacuum pump to speed up the all process, but it seems not to be working.

I can not find anybody working with these tubes...so Please if you are working with them and even when the question is really simple. Do you use the pump?? maybe we are speeding the process too much? What are the things that can be failing during this separation process?

I would really appreciate all answers


Thanks a lot


PS the protocol we follow (apart from the pump) is the one recomemded
1.100% methanol 2.water 3.sample 4.water 5.10%methanol 6.50 % methanol to collect the second fraction

What you are trying to accomplish is a separation of 2 classes of compounds by liquid chromatography. This is different than SPE in many ways. In this method you are applying theoretical plates, flow rate and polarity to accomplish your separations.

Fast is usually not a good approach to this type of chromatography. Gravity is the preferred method if possible. Otherwise, I recommend 5mL/min as a starting point.

I'm curious how hydrophobic peptides can be soluble in water? 2-propanol might be a better choice.

What do you mean "it doesn't work"? Do your peptides elute at all? Do they co-elute? What?

Resins sometimes have so-called "active sites" that bind sample irreversibly. Consider conditioning the cart with a mg or so of an inexpensive sacrificial peptide/g resin.

5.10%methanol 6.50 % methanol to collect the second fraction

To clarify, you're trying to elute one peptide using 5.1% MeOH eluent, the second with 6.5% MeOH from an SPE column? Forgetaboutit! Use SPE to clean up a sample then let HPLC do the separating. That is not to say SPE cannot separate these two, but you're going to need to exploit differences other than hydrophobicity in order to do it. However, I can't begin to suggest an alternative SPE scheme without information related to peptide sequences or even just AA content.. OK to use "H" for "hydrophobic AA" "A", "B" for acidic, basic residues, respectively, but without this critical information it is impossible to help further