column chromatography - how to make the buffer input rate constant? (Mar/29/2007 )
Hi All! I have this question, how to make the buffer input rate constant in a column chromatography? After loading the elution buffer, the height of the elution buffer above the matrix decreases with time and therefore the pressure exerted by this buffer also decreases. When we load more elution buffer, the pressure increases back. Is there any simple way to make sure this height remains constant without the use of some specialised apparatus or the addition of the buffer constantly throughout the procedure which may take hours?
-yiklim-
QUOTE (yiklim @ Mar 30 2007, 06:07 AM)
Hi All! I have this question, how to make the buffer input rate constant in a column chromatography? After loading the elution buffer, the height of the elution buffer above the matrix decreases with time and therefore the pressure exerted by this buffer also decreases. When we load more elution buffer, the pressure increases back. Is there any simple way to make sure this height remains constant without the use of some specialised apparatus or the addition of the buffer constantly throughout the procedure which may take hours?
you can use a peristaltic pump which constantly fills your column with fresh elution buffer; if you have no pump, filling can be done by gravity flow where you gear the flow with a valve or a clamp at the tube; filling flow and column flow must be synchronized
-The Bearer-
QUOTE (yiklim @ Mar 30 2007, 12:07 AM)
Hi All! I have this question, how to make the buffer input rate constant in a column chromatography? After loading the elution buffer, the height of the elution buffer above the matrix decreases with time and therefore the pressure exerted by this buffer also decreases. When we load more elution buffer, the pressure increases back. Is there any simple way to make sure this height remains constant without the use of some specialised apparatus or the addition of the buffer constantly throughout the procedure which may take hours?
the simple way to maintain constant pressure and flow would be tu use a mariotte tube on your buffer bottle.
you can find a description of how to do this at: mariotte_bottle1.pdf (you may also find info on this in the gel filtration booklet from ge healthcare).
the pressure head will be from the bottom of the mariotte (air) tube to where the drop forms post-column and will remain there until the buffer level falls below the bottom of the tube.
-mdfenko-
Thanks a lot!!
Next Question, i'm using silica and sepharose to make two columns respectively. Usually how long does it take for the matrix to settle before the loading of samples? Also, which of these two matrices is generally better?
-yiklim-
QUOTE (yiklim @ Apr 3 2007, 08:26 PM)
Thanks a lot!!
Next Question, i'm using silica and sepharose to make two columns respectively. Usually how long does it take for the matrix to settle before the loading of samples? Also, which of these two matrices is generally better?
Next Question, i'm using silica and sepharose to make two columns respectively. Usually how long does it take for the matrix to settle before the loading of samples? Also, which of these two matrices is generally better?
Equilibrate the column with your loading buffer near 3-4 ( better 10 if you have a time) volume column at the flow rate the same or nearby which you choose for your separation. Adjust adapter directly to resin ( and 2-3 ml into resin) if you see that resin height decreased. Do it until the height of your resin in column stop to decrease under your flow rate. Adjust adapter finally. Check pressure and do use extreme flow rates ( see instructions for the column ) it is especially concerned Sepharose also for CL.
About choice of matrix - it depends on your aims and tasks. Sepharose better for protein and may be DNA GF/SEC ( better CL cross linked, see also GE Healthcare catalog info) Non-modified Silica as stationary phase is normal phase chromatography variant. So it is usefull for polar compounds separation( better low molecular). For proteins - high possibility of non-specific adsorbtion. What material do you want to purify?
-circlepoint-