Hi,

Choose a column of appropriate size for the application. The volume of the

column should be 20 to 50 fold that of the sample to be applied. Usually tall thin

columns with lengths 20-40 times their diameter are used.

Try to maintain a constant temperature without any drafts, so that turbulence or

thermal gradients do not effect the flow.

When packing the column, fill the column about one third full with elution buffer.

Open the valve at the bottom of the column so the liquid starts to drip out. Then,

using a pipette, add a dilute slurry of the column matrix material to the top of the

column while stirring gently. Keep adding a slurry to the column while gently

stirring intermittently. The goal is to produce a uniform bed of desired height using

a minimal amount of Sephadex G-75. After the desired height has been reached,

equilibrate the column with several column volume of elution buffer.

The sample should be applied in a narrow band at the top of the column. At some

point stop adding buffer to the column and allow the buffer to flow until it reaches

the top of the bed. Stop the flow by closing the valve, and gently add the sample

against the wall of the column without disturbing the bed. Once added, open the

valve and let sample flow into the bed. Now, close the valve and gently add a

small amount of elution buffer using the same technique you used for sample

loading. Repeat this procedure with buffer and finally add a larger quantity of

buffer without disturbing the bed.

After equilibration, use 0.5 ml of the sample. You are now ready to elute the fractions  You can measure 2 ml of distilled water into a test tube and

use it as a reference for fraction collection..

Usually the column length should be longer for Gel filtration chromatography. For ex. for laboratory scale the length of the column should be at least or more than 70 cm depending on your amount of sample. It is better to load very small volumes, for ex. 1-1.5 ml. Because,  all proteins should be at the possibly same position (1 cm zone) when the elution starts. So, in your column dimensions I don't see any problem apart from diameter.

Allow the equilibration buffer to pass into the gel matrix just below matrix level (not to allow the column matrix to dry), then load the sample. After loading the sample allow it to enter into the matrix level completely (again taking precaution not to dry the matrix), after that you can add the elution buffer and start collecting the fractions ...... it is better to maintain a continuous flow once you pack the matrix into the column and continue it until the elution is done, without stopping in between. 

Hi there,

The usual size for preparative CES is 1.6cm*60cm (bed volume 120mL), Each linear cm of column will contain 2mL. If you compare with your dimensions you will note your column is longer and narrower and the bed volume is smaller (90mL). 1cm of column will contain  0.75mL. It means that for running similar experiments with your column you will have to inject roughly 3 times less volume. The question is can you adapt your protocol to such condition?

I think this length is  not suitable for your experiment  too long 60-70 cm with diameter 1.5cm is good to use the most  important is  Mw of your enzyme should be within the range of  exclusion of  the sephadex limit

I agreed with all comments in that your column longer and thinner than usual. I dont agree with Bahman Momen in stopping of valve after sample application. Elution must not disturbed by closing and opening of valve.

u can go ahead with the column.take 1 to 2ml of sample and continue till the sample is eluted completed.

There are several considerations that you need to think about before making your decision. You should know the approximate size of your enzyme and possible contaminants. Also this step often dilutes your protein a lot. Ion exchange is often easier and more powerful. Gel filtration is normally used as a final step. Good luck

To answer your question precisely: it all depends upon what protein you are attempting to purify. You decide the column dimensions and sephadex quantity, base on the protein that you are trying to purify.

I'm currently working with a column 1.5cm diameter and 120cm tall using gel filtration (sephadex G-150). If you can avoid working with such a long column I would recommend it simply because it is awkward to pour and use (especially inside a large refrigerator as I am doing). You want to pour the column at the same temperature it will be run.

I decided to use this column as the first of 4 steps in purifying my protein of interest (105 kD). I needed a column of this length to accommodate and adequately separate the relatively large volume of my crude cell extract (15-20 mL). I poured the column to a height that allowed me to use as much of the column length as possible while leaving enough head space at the top to load my entire sample at once.

Before pouring the gel, add buffer through the bottom of the column to dislodge any trapped air bubbles and close the stopcock with a small amount of buffer still at the bottom of the column. I found when pouring a column of this size, it was easiest to use a large (60cc) syringe with rubber tubing at the end long enough to reach down into the column to allow for slow, even pouring of the gel. Be sure to avoid any bubbles or letting the column dry out and crack because repouring is extremely tedious with this size column.

For my purposes, I'm not terribly worried about extremely good separation and resolution. I am using this column simply to exclude a fair amount of the crude cell extract in order to have a relatively pure fraction before loading it on an immunoaffinity column. We just don't want to shorten the life of the antibody resin by having so much junk in our sample. 

Depending on what you're doing, a column of this size can be appropriate, but fairly awkward to work with.

The column height 120 sm is excellent height for gel-filtration. However diameter 1 sm will produce signifivant  "'wall-effect". I would suggest diameter 2.5 sm

It again depends on the level of purification of the desired protein that you have achieved so far. You can initially use a 1.5 x 80 cm column with about 1.5 ml of the sample and then decide based on the separation you get.

Of course, the longer the column is the better. However, diameter also counts. As Niyaz points out rightly, 1 cm in diameter might cause significant "wall-effect" by the end of separation, if the length is 120 cm. You would need thicker column; as "basic" idea try to keep the 1.6 cm x 60 cm ratio for the ID x L.

I would also suggest de-airation of buffers and sample before loading on the column.

Otherwise all important points have been mentioned above by colleagues.

If you are separating proteins of close molecular size and your sample volume is small (ca 200 uL), such a long column is desirable. However, from the stated diameter, you may have to be worry about flow rate vs tolerable pressure of your column material.

Other respondents have already given you a lot of good advice about HOW to run your Sephadex column. You also asked if this is an appropriate method. It is certainly a well-tried and gentle method, but the real answer is that like each one of us, you have to find out if it does a good job for you and your protein. One has to approach these questions empirically. I also agree very much  with Leif Bulow - on average, ion exchange (and particularly anion exchange) may be a better bet. The fact that the protein does not stick to the gel filtration beads imposes a limit on the degree of resolution you can get, whereas with a gradient elution on an ion-echange column you can achieve a high degree of resolution and typically much higher purification factors than on a Sephadex column. .....But Sephadex could still do a good job for you!

You allready have a good advice. Gel filtration separate molecules by molecular weight, the column lengh is very  important (120 cm is ok), but also buffer flow, the lower the better (overnight chromatography in a cool room is ok, 5-8 mL/h), the fraction size is also important (the lower the better), your column has 94 ml volume, it means that with 95 fractions of 1 ml you will finish the chromatograpy. After chromatograpy you have to check purification by PAGE electrophoresis, and also measurement of specific activity with give factr purification obtained. if any peak is not pure enough you can tray by ion exchaenge, hidrophobic or affinity chromatography. HNS

you need 1.6*80 is suitable for you with sephadex 75

I'd disagree with Hector Nolasco. As van Deemter equation tells us, the lower flow rate is not always better. Or rather, there is a limit to that.

The length of column is decided by the resolution you want to achieve since the resolution of two separated proteins in gel filtration increases as the square root of column length also the time taken to elute the proteins will increases as the column length increase .The column length  usually not exceeded 1 m  60 to 100 cm range is good.However the diameter of the column is important which is governed by the sample size which usually  between ;5-1% of the bed volume so depend upon the size of sample you want to load on your column it usually acceptable column diameter to column length between 1:20 up to 1:100  so try to use column 1 cm diameter and 100cm length

wishing you my best