Chromatography Forum

Hello everyone,

I am experiencing issues with my HPLC analysis; Increasing pressure and loss of resolution, but replacing the guard-column resolved this. I expect issues with my sample preparation.

So a quick question:
* What would be good steps that can be undertaken to remove polysaccharides and proteins from water-based plant extracts?

I look forward to seeing your replies and suggestions.
Thanks in advance!

*Edited post to remove unnecessary details.

I don't have a lot of experience working with polysaccharides but if you are primarily interested in the amino acids themselves you might subject your extract to protein denaturation conditions such as 6M Urea, a very low or high pH aqueous solution or you can even attempt high salt to precipitate the proteins in hopes that it does not precipitate the amino acids (look up salting out for proteins). If you can precipitate the proteins in the extract you would just perform another centrifugation step and discard the pellet.

Do they make syringe filters that have a small enough pore size to catch proteins but let small molecules pass? Similar to the idea behind dialysis where the pore size allows molecules up to a certain size to pass through but larger molecules (such as proteins) cannot.

Just some ideas as I have never performed this kind of analysis...but hopefully this can provide insight or spark an idea from someone else.

Zoraku wrote:
I don't have a lot of experience working with polysaccharides but if you are primarily interested in the amino acids themselves you might subject your extract to protein denaturation conditions such as 6M Urea, a very low or high pH aqueous solution or you can even attempt high salt to precipitate the proteins in hopes that it does not precipitate the amino acids (look up salting out for proteins). If you can precipitate the proteins in the extract you would just perform another centrifugation step and discard the pellet.

Do they make syringe filters that have a small enough pore size to catch proteins but let small molecules pass? Similar to the idea behind dialysis where the pore size allows molecules up to a certain size to pass through but larger molecules (such as proteins) cannot.

Just some ideas as I have never performed this kind of analysis...but hopefully this can provide insight or spark an idea from someone else.

Thank you for your reply Zoraku.
Your suggestions give me leads to look for in literature, and I had not thought of looking into these size-exclusion filters myself, so thank you for the insights.

As you already remarked, these procedures might also influence the recovery of the AAs, but that is something that has to be tested out..

2 commonly used procedures come to mind to precipitate these macromolecules in aqueous extracts, followed by filtration or centrifugation:

- Add acetonitrile or MeOH, at least in a 1:1 ratio organic:water
- Use Carrez I & II, which are aqueous metal salt solutions

As Zoraku pointed out, there are more options.

Rndirk wrote:
2 commonly used procedures come to mind to precipitate these macromolecules in aqueous extracts, followed by filtration or centrifugation:

- Add acetonitrile or MeOH, at least in a 1:1 ratio organic:water
- Use Carrez I & II, which are aqueous metal salt solutions

As Zoraku pointed out, there are more options.

Hello Rndirk, thank you for the reply.
Just to clarify; Can acetonitrile or methanol be used for the simultaneous precipitation of both macromolecules?

Furthermore, I have so read elsewhere that acetone can be used for proteins precipitation. Do you know if this will also be effective to precipitate the polysaccharides?

I think it depends what polysaccharides we are talking about. The larger (for example starch) the easier they will precipitate. But starch has a low solubility in water anyway..

There's a lot of literature on protein precipitation, as it is widely used as simple & quick sample preparation in clinical analysis (blood, urine).

Fremouwski wrote:

Rndirk wrote:
2 commonly used procedures come to mind to precipitate these macromolecules in aqueous extracts, followed by filtration or centrifugation:

- Add acetonitrile or MeOH, at least in a 1:1 ratio organic:water
- Use Carrez I & II, which are aqueous metal salt solutions

As Zoraku pointed out, there are more options.

Hello Rndirk, thank you for the reply.
Just to clarify; Can acetonitrile or methanol be used for the simultaneous precipitation of both macromolecules?

Furthermore, I have so read elsewhere that acetone can be used for proteins precipitation. Do you know if this will also be effective to precipitate the polysaccharides?

You can use acetone at a rough 3:1 ratio of acetone:sample solution but I have no idea how it will impact polysaccharides.

If you already know what sort of recovery you get with your normal extraction process, running a few of these precipitation reactions and comparing them to a normal extraction should help you gauge how much the recovery is affected by these other reactions.

I would also suggest trying to perform some of the precipitation reactions in a cold room, or with cold solvents (~4-8ºC) if you don't see success at room temperature.

Zoraku wrote:

Fremouwski wrote:

Rndirk wrote:
2 commonly used procedures come to mind to precipitate these macromolecules in aqueous extracts, followed by filtration or centrifugation:

- Add acetonitrile or MeOH, at least in a 1:1 ratio organic:water
- Use Carrez I & II, which are aqueous metal salt solutions

As Zoraku pointed out, there are more options.

Hello Rndirk, thank you for the reply.
Just to clarify; Can acetonitrile or methanol be used for the simultaneous precipitation of both macromolecules?

Furthermore, I have so read elsewhere that acetone can be used for proteins precipitation. Do you know if this will also be effective to precipitate the polysaccharides?

You can use acetone at a rough 3:1 ratio of acetone:sample solution but I have no idea how it will impact polysaccharides.

If you already know what sort of recovery you get with your normal extraction process, running a few of these precipitation reactions and comparing them to a normal extraction should help you gauge how much the recovery is affected by these other reactions.

I would also suggest trying to perform some of the precipitation reactions in a cold room, or with cold solvents (~4-8ºC) if you don't see success at room temperature.

Thank you, valuable suggestions!

Keep in mind that, if you're doing reversed-phase HPLC, there's a big chance peak shapes will suffer if you go from injecting aqueous extracts to organic extracts.

That's what I like about the carrez solutions; it doesn't add any solvent. But I can't tell if it works for amino acids. We use it for several HPLC sample preps on food extracts: antioxidants, sweeteners, ..

Hi all,

Just a precautionary note about using acetone with HPLC. It will perish pump seals, and will likely damage the stationary phase phase of your column over time. Best to use another solvent, or solvent exchange to a more suitable one if possible.

Has anyone noticed if Carrez Reagents have a damaging effect on HPLC Columns?
I am using an SupelcoSil LC-NH2 column for sugars analysis with a warmed ACN:H2O (30:70) extraction solution, centrifugation then 0.45µm nylon filtration. We occasionally use the Carrez reagents when samples appear cloudy, but are not sure if it has derogatory effect on our column. We use a guard column, which requires replacement approx every 3 months.

Many Thanks.