buffers effect on size exclusion chromatography.. - amm bicarb, phospate with or without salt/ethanol. (Jun/26/2008 )
hii.. can some1 explain to me the differences and reasons for use of the following in Size exclusion chromatogarphy?
Mobile phase:
1. Ammonium bicarbonate buffer, pH 7 at a flow rate of 0.5 ml/min, detection at 215 nm.
2. Phosphate buffer,Ph 7
3. Phosphate buffer with 110 mM sodium chloride, Ph 7
4. Phosphate buffer with 110 mM sodium chloride, Ph 7 with 10% ethanol
all 4 with Same column, at a flow rate of 0.5 ml/min, detection at 215 nm. for all.
and what is the best prefered diluent in all these cases?
please let me know why for all!
thanks!
-alice!-
QUOTE (alice! @ Jun 26 2008, 01:34 AM)
hii.. can some1 explain to me the differences and reasons for use of the following in Size exclusion chromatogarphy?
Mobile phase:
1. Ammonium bicarbonate buffer, pH 7 at a flow rate of 0.5 ml/min, detection at 215 nm.
2. Phosphate buffer,Ph 7
3. Phosphate buffer with 110 mM sodium chloride, Ph 7
4. Phosphate buffer with 110 mM sodium chloride, Ph 7 with 10% ethanol
all 4 with Same column, at a flow rate of 0.5 ml/min, detection at 215 nm. for all.
and what is the best prefered diluent in all these cases?
please let me know why for all!
thanks!
Mobile phase:
1. Ammonium bicarbonate buffer, pH 7 at a flow rate of 0.5 ml/min, detection at 215 nm.
2. Phosphate buffer,Ph 7
3. Phosphate buffer with 110 mM sodium chloride, Ph 7
4. Phosphate buffer with 110 mM sodium chloride, Ph 7 with 10% ethanol
all 4 with Same column, at a flow rate of 0.5 ml/min, detection at 215 nm. for all.
and what is the best prefered diluent in all these cases?
please let me know why for all!
thanks!
buffer 1 has the advantage for downstream processing, because you can get rid of the salt during lyopholization step. However, I dont like it as it may evolve bubbles during the run, particlularly at high temp, like rt. Once that happens, you need to repack your column.
2 and 3 are nearly the same, except 3 is cheaper to make and yet it has sufficient buffering capacity.
4. never used before, may have something to do with improving the solubility or keep bacs from growing? can your protein survive this is the question?
-genehunter-1-
QUOTE (genehunter-1 @ Jun 26 2008, 08:46 PM)
QUOTE (alice! @ Jun 26 2008, 01:34 AM)
hii.. can some1 explain to me the differences and reasons for use of the following in Size exclusion chromatogarphy?
Mobile phase:
1. Ammonium bicarbonate buffer, pH 7 at a flow rate of 0.5 ml/min, detection at 215 nm.
2. Phosphate buffer,Ph 7
3. Phosphate buffer with 110 mM sodium chloride, Ph 7
4. Phosphate buffer with 110 mM sodium chloride, Ph 7 with 10% ethanol
all 4 with Same column, at a flow rate of 0.5 ml/min, detection at 215 nm. for all.
and what is the best prefered diluent in all these cases?
please let me know why for all!
thanks!
Mobile phase:
1. Ammonium bicarbonate buffer, pH 7 at a flow rate of 0.5 ml/min, detection at 215 nm.
2. Phosphate buffer,Ph 7
3. Phosphate buffer with 110 mM sodium chloride, Ph 7
4. Phosphate buffer with 110 mM sodium chloride, Ph 7 with 10% ethanol
all 4 with Same column, at a flow rate of 0.5 ml/min, detection at 215 nm. for all.
and what is the best prefered diluent in all these cases?
please let me know why for all!
thanks!
buffer 1 has the advantage for downstream processing, because you can get rid of the salt during lyopholization step. However, I dont like it as it may evolve bubbles during the run, particlularly at high temp, like rt. Once that happens, you need to repack your column.
2 and 3 are nearly the same, except 3 is cheaper to make and yet it has sufficient buffering capacity.
4. never used before, may have something to do with improving the solubility or keep bacs from growing? can your protein survive this is the question?
yaa.. but mine is an analytical SEC, not preparative.
2, 3 wouldnt salt decrease non-specific interatcion?
ethanol decreases tailing.. but how and why?
-alice!-