High Temp HPLC
Posted: Wed Aug 14, 2013 3:50 am
Hi All:
I'm interested in elevated temperature, ballistic gradient RP-HPLC.
My column is 2.1 x 33 mm, 5 micron (PS-DVB)
Temp: 80 C
Flow rate: 1 mL/min
A = 30 mM diethylamine, water
B = 30 mM diethylamine, MeCN
My gradient is 2 to 99% B over 1 min, hold 99% B for 1 min, return to 2% B over 0.1 min, followed by 3 min re-equilibration period. The delay volume for this system is approx 0.6 mL.
The mobile phase is not pre-heated. Instead, I route pump outlet into a column oven heat exchanger, like in this picture.
http://www.spectralabsci.com/images/HP- ... rtment.jpg
The retention times for replication runs are remarkably consistent. SD < 0.5%
I'm curious to ask,
1) how will I know if the incoming mobile phase is matched to the temperature of the column oven?
2) When I run a 5 minute (2 to 99% B) gradient at ambient temperature, I find retention times (neutral, small molecule compounds) are linearly related to those measured in a high-temp (80 C) ballistic gradient (2 to 99% in 1 minute.) Is this to be expected?
Again, assuming neutral, small molecule solutes, in the absence of stationary phase degradation (I'm using PS-DVB), under what circumstances could selectivity or elution order change?
I'm interested in elevated temperature, ballistic gradient RP-HPLC.
My column is 2.1 x 33 mm, 5 micron (PS-DVB)
Temp: 80 C
Flow rate: 1 mL/min
A = 30 mM diethylamine, water
B = 30 mM diethylamine, MeCN
My gradient is 2 to 99% B over 1 min, hold 99% B for 1 min, return to 2% B over 0.1 min, followed by 3 min re-equilibration period. The delay volume for this system is approx 0.6 mL.
The mobile phase is not pre-heated. Instead, I route pump outlet into a column oven heat exchanger, like in this picture.
http://www.spectralabsci.com/images/HP- ... rtment.jpg
The retention times for replication runs are remarkably consistent. SD < 0.5%
I'm curious to ask,
1) how will I know if the incoming mobile phase is matched to the temperature of the column oven?
2) When I run a 5 minute (2 to 99% B) gradient at ambient temperature, I find retention times (neutral, small molecule compounds) are linearly related to those measured in a high-temp (80 C) ballistic gradient (2 to 99% in 1 minute.) Is this to be expected?
Again, assuming neutral, small molecule solutes, in the absence of stationary phase degradation (I'm using PS-DVB), under what circumstances could selectivity or elution order change?