Chromatography Forum

I am trying to use bonded silica gel SPE column to get phospholipid from total lipid extract.

I would like to get glass columns instead of plastics because there are fatty acids, plasticizers contamination that makes GC interpretation difficult. 100 mg aminopropyl bonded silica packing/ 1 ml glass housing would be perfect.

I searched and could not find one, not even in other sizes for the particular packing material, neither an empty glass column at this small either.

Do you know any sources for this? An empty small glass column housing, or aminopropyl packing already filled?

Currently, I am considering to pack my column in disposable glass pastuer pipet by myself. But the only concern is I am not sure about the quality.

Thanks in advance.

Where are you located? Several companies including UCT make these products.

Don

Hi Don,


I am only able to find a 6ml glass column shell for sale on UTC's website, which is a bit too big for me.

I am in US.

I have another question on SPE columns. You mentioned that letting the column dry is not an issue in the washing step following sample loading, in this topic. viewtopic.php?f=19&t=19929

My question is a bit down in the elution steps. If there are several elution steps and I am only interested in the last portion, will it a problem if I allow the column get dry (not fully dry, just run out of eluent in the column) between fractions?

I am trying to get phospholipid fraction from total lipid (containing neutral lipids, free fatty aicds and phospholipids) using NH2 SPE column.

The typical procedure is as following:

1. Wash 100mg/ 1ml aminopropyl column using 1 ml heptane twice.
2. Apply total lipids dissolved in 0.25 ml chloroform.
3. Elute with 1 ml chloroform: isopropanol (2:1 v/v) to remove neutral lipids.
4. Elute with 1 ml 2% acetic acid in diethyl ether to remove free fatty acids.
5. Finally elute with 1 ml methanol to collect the phospholipids.

To rephrase, can I allow the column moderately dry in steps 3&4 without affecting step 5?

Actually, there is another question. Since I am only interested in the last fraction, can I skip step 3? Shouldn't the neutral lipids come out with the free fatty acids in step 4 even if I skip step 3?

Hi Don,


I am only able to find a 6ml glass column shell for sale on UTC's website, which is a bit too big for me.

I am in US.

I have another question on SPE columns. You mentioned that letting the column dry is not an issue in the washing step following sample loading, in this topic. viewtopic.php?f=19&t=19929

My question is a bit down in the elution steps. If there are several elution steps and I am only interested in the last portion, will it a problem if I allow the column get dry (not fully dry, just run out of eluent in the column) between fractions?

I am trying to get phospholipid fraction from total lipid (containing neutral lipids, free fatty aicds and phospholipids) using NH2 SPE column.

The typical procedure is as following:

1. Wash 100mg/ 1ml aminopropyl column using 1 ml heptane twice.
2. Apply total lipids dissolved in 0.25 ml chloroform.
3. Elute with 1 ml chloroform: isopropanol (2:1 v/v) to remove neutral lipids.
4. Elute with 1 ml 2% acetic acid in diethyl ether to remove free fatty acids.
5. Finally elute with 1 ml methanol to collect the phospholipids.

To rephrase, can I allow the column moderately dry in steps 3&4 without affecting step 5?

Actually, there is another question. Since I am only interested in the last fraction, can I skip step 3? Shouldn't the neutral lipids come out with the free fatty acids in step 4 even if I skip step 3?

To answer your first question; yes. You can let the sorbent dry out between elution steps. If you are going to fractionate this way, you will need to be consistent.

In step 3 your are removing neutral lipids by disrupting weak forces such as van der Waals forces, and hydrogen bonding. You are accomplishing this by using a strongly polar solvent (around 4 on the polarity index). Step 4 uses a non polar organic and might not be strong enough to remove the neutral lipids. So, if you skip step 3 and the neutral lipids remain, the lipids will elute with step 5 which uses a stronger solvent, methanol (polarity 5.1).

Don

Wow. Thank you so much. This is very helpful. I assumed the polarities of the serial solvent elutions increased in each step, without knowing how to check polarity indices.

As for the drying out between elution steps, I guess that it will be important to keep the procedure consistent. But what if I am only interested in the last eluent, is it still necessary to keep the consistency? In my case, portions 1&2 will be discarded which are equivalent to impurities in a washing step. In my mind, I am thinking these 1st two elution steps are "washing" step in many other protocols, which is lacking in this protocol.

The reason I am aksing these questions is I am trying to do something not conventional. I have about 200 samples to deal with, but our budget does not allow a vacuum manifold. So I will utilize gravity, therefore the flowrate is really slow, like 0.1 ml/min. If column drying out after sample loading is not an issue, it will be a great news as I can load a bunch of samples and still have good productivity instead of constant attention to the solvent level. In any case, this is not like forcefully drying the column out using vacuum or flushing gases, there well be still liquid holdup in the absorbent bed.

I am always afraid of drying out a column (i.e., flash chromatography) to have bubbles and channeling in bed, causing inefficient separation. I guess I just don't fully understand why column drying out is not an issue in SPE column after sample loading step.

Hi Don,

I have another question on SPE columns. You mentioned that letting the column dry is not an issue in the washing step following sample loading, in this topic. viewtopic.php?f=19&t=19929

My question is a bit down in the elution steps. If there are several elution steps and I am only interested in the last portion, will it a problem if I allow the column get dry (not fully dry, just run out of eluent in the column) between fractions?

I am trying to get phospholipid fraction from total lipid (containing neutral lipids, free fatty aicds and phospholipids) using NH2 SPE column.

The typical procedure is as following:

1. Wash 100mg/ 1ml aminopropyl column using 1 ml heptane twice.
2. Apply total lipids dissolved in 0.25 ml chloroform.
3. Elute with 1 ml chloroform: isopropanol (2:1 v/v) to remove neutral lipids.
4. Elute with 1 ml 2% acetic acid in diethyl ether to remove free fatty acids.
5. Finally elute with 1 ml methanol to collect the phospholipids.

To rephrase, can I allow the column moderately dry in steps 3&4 without affecting step 5?

Actually, there is another question. Since I am only interested in the last fraction, can I skip step 3? Shouldn't the neutral lipids come out with the free fatty acids in step 4 even if I skip step 3?

To answer your first question; yes. You can let the sorbent dry out between elution steps. If you are going to fractionate this way, you will need to be consistent.

In step 3 your are removing neutral lipids by disrupting weak forces such as van der Waals forces, and hydrogen bonding. You are accomplishing this by using a strongly polar solvent (around 4 on the polarity index). Step 4 uses a non polar organic and might not be strong enough to remove the neutral lipids. So, if you skip step 3 and the neutral lipids remain, the lipids will elute with step 5 which uses a stronger solvent, methanol (polarity 5.1).

Don

Just keep in mind that your washing steps rely on 1)polarity and 2)protonation and polarity. So, wash 2 won't start to happen until you flush out all of the solvent from wash 1. Final elution won't happen until you flush out all of the solvent from wash 2.