Help with setting up a chromatography column

[pseizure]

Hello, I am trying to extract myoglobin for a sequencing project. I have already homogenized, precipitated, and run it through dialysis. My next step is to run it through a chromatography column and collect the fractions that should be dark red. I have purchased Sephacryl 200 and an econo column from biorad with adapter and a peristaltic pump. The protocol they have for packing a column is for their XK columns which are rated for 5 atm. My columns are only up to 1 atm. I am a bit worried that maybe I need to switch to purely a gravity setup which will take much longer. Their protocol has me packing at 150 and then 270 mL/hr. I'm also running the column at 60 mL/hr. Should I be worried about pressure at these rates? Can someone advise me on the best course to take, especially with the investments into the pump in mind? I can provide some of my citations and docs if needed. I'm new here so I don't know what is preferred.

[deleted-140078]

I have reached out to some of the other experts on this board, I am not familiar with that procedure or equipment but I will try to find some answers for you.

When in doubt about hardware though always best to contact the manufacturer or if you know anybody who works in a lab setting, they may be a good resource.

[VSegarra]

Hi! We will be happy to help. Could you provide me with the catalog numbers for the items you ordered from Bio-Rad? (econo column from biorad with adapter and a peristaltic pump) I will need this to fully understand the system you are trying to set up. Also, where did you purchase the Sephacryl? GE? What is the catalog number for this material?
I look forward to helping you with this.
Veronica

[pseizure]

Thank you! The column is #737-2591 (http://www.bio-rad.com/en-us/sku/737-25 ... 2-5-100-cm)
and flow adapter: #738-0017 (http://www.bio-rad.com/en-us/sku/738-0017-flow-adaptor)

Pump is actually the NE-9000 Programmable Peristaltic Pump with the PERI-HEAD-KIT-YG4 4 roller for 1/16" tubing (http://www.katsci.com/Products/NE-9000- ... 7AodiHUAbg)

I got my 500mL of sephacryl from Sigma Aldrich (http://www.sigmaaldrich.com/catalog/pro ... &region=US) CAS Number 65546-95-4

[VSegarra]

Ok, great...I will need a little bit of time to think about this and draft a good helpful response. You will hear from me soon (no more than a couple of days). Thanks
Veronica

[VSegarra]

Hi! You are right to be thinking about these issues, matrices for size-exclusion chromatography (like Sephacryl) can be often associated with medium to high pressure systems. What you have, as you indicated will not “hold” more than 1 atm of pressure (a very modest amount). I would not use the protocol for the XK column, the specifications for those columns are different.
In general, one packs a column faster than one runs it with sample. This allows the matrix to pack well and have a relatively constant length or height. I would say the first step is to figure out how fast you can run that econo column from Biorad (maximum flow rate; mL/min) when you use the matrix of interest. I tried figuring out what that is by looking at documentation online but was not successful (do you have any documentation that provides this info?).
I think this is when you call Biorad and ask for this information. When you call have all of your information ready- including catalog numbers. Once you have that “max” flow rate given the matrix, select a flow rate close to that for packing, and one below that for running the sample. You can then assess whether you gain anything (time) by using the pump and opposed to just gravity.

Make the best of your call and info inquiry to Biorad- put together a list of questions you will ask, in that way, you will likely get all the points you want clarified.
I thought about calling for you and then relaying the info- but I think this will be a great learning experience for you!

I hope this helps you! Please keep us posted. It looks like you are troubleshooting like a pro! Way to go!

Also here is a link to a cool manual on gel filtration methods- it is from GE, it contains nifty tips.

http://kirschner.med.harvard.edu/files/ ... ration.pdf

Veronica

[donnahardy2]

Hi,

This sounds like a really great project!

You should be able to run the Sephacryl in the column using the peristaltic pump. The peristaltic pump will not overpressure the column, and if you try to run at a flow rate that is too fast, the flow adaptor will back out of the column. This is a safety feature that prevents the column from breaking.

The only problem you might experience is that some of the gel might leak from the column. The Sephacryl gel has particles that are 25 microns; the porosity of the Econo-Column frit is 30 microns. So you may notice a few resin beads leaking out when the column is newly packed, but this will stop once the gel bed is stabilized.

I recommend that you pack the column with the gel, and try running the column at 0.5 ml/min; if this works, then increase the flow rate to 1 ml/min or higher. You probably won’t be able to run the column as fast as you normally would if you had a high backpressure system, but the separation should work well.

Please go ahead and try this. However, please do let us know if
the flow rate is too slow, or if the frit gets clogged with the Sephacryl resin.

What volume of sample were you planning to apply to the column?

Donna Hardy

[pseizure]

Thanks so much for the help! I have quite a bit of sample that I could run, but I only need to run enough so I can run the collected fraction on an SDS PAGE gel. I then plan to get the protein sequenced via a mass spec lab. So if the column will let me, should I trying packing it at the suggested 150 and then 270 mL/hr rates, or just my running rate?