Chromatography Forum

for high flow rate up 10ml/min, what is the most efficient way to control temperature.

You need - at least - to tell us what are the column dimensions.

Best Regards

I use a 20L plastic bucket filled with water, and a standard water-circulating laboratory waterbath thermo-circulator ( such as Techne, Grant, Haake models ) controlling the bath temperature.

I have the column suspended vertically, with entry ( via a guard column ) at the bottom, and 1/16 widebore SS line going down through the water to preheat the mobile phase before entering the guard.

It's advisable to have larger-diameter prep columns in the vertical orientation.

Bruce Hamilton

sorry, that is id 21mm*length 25cm

You need - at least - to tell us what are the column dimensions.

Best Regards

what kind of diameter column you have?
since you are doing prep separation, could you do me a favor tell me how large mixer volume you have to ensure enough mobile phase mixing.
or you just use isocratic or step gradient?

thanks a lot.

I use a 20L plastic bucket filled with water, and a standard water-circulating laboratory waterbath thermo-circulator ( such as Techne, Grant, Haake models ) controlling the bath temperature.

I have the column suspended vertically, with entry ( via a guard column ) at the bottom, and 1/16 widebore SS line going down through the water to preheat the mobile phase before entering the guard.

It's advisable to have larger-diameter prep columns in the vertical orientation.

Bruce Hamilton

why vertical position is good? any reason? Thanks.

I use a 20L plastic bucket filled with water, and a standard water-circulating laboratory waterbath thermo-circulator ( such as Techne, Grant, Haake models ) controlling the bath temperature.

I have the column suspended vertically, with entry ( via a guard column ) at the bottom, and 1/16 widebore SS line going down through the water to preheat the mobile phase before entering the guard.

It's advisable to have larger-diameter prep columns in the vertical orientation.

Bruce Hamilton

Hi Jiang295,

The flow rate you’re using is actually quite slow - in the utilized column diameter context. So, any temperature controlling device would do the job. In case of large diameter columns (and actually in any case, but especially when thick columns are utilized) I would recommend some kind of preheating. But it all depends on the delta temperature between the mobile phase and the separation/column temperatures. The bigger difference the more need for preheating.

Best Regards

the reason why Ｉuse so slow flow rate is because of pressure that I cannot handle.
and I really do not know what kind of mixer volume I need to use for continuous gradient elution. right now, I am only using 2.6ml mixer. do you know what kind of mixer we need for high flow rate, like 20ml-40ml/min.

Thanks a lot.

Hi Jiang295,

The flow rate you’re using is actually quite slow - in the utilized column diameter context. So, any temperature controlling device would do the job. In case of large diameter columns (and actually in any case, but especially when thick columns are utilized) I would recommend some kind of preheating. But it all depends on the delta temperature between the mobile phase and the separation/column temperatures. The bigger difference the more need for preheating.

Best Regards

what kind of diameter column you have?
since you are doing prep separation, could you do me a favor tell me how large mixer volume you have to ensure enough mobile phase mixing.
or you just use isocratic or step gradient?

Typically, 21, 30, 50 mm diameter x 250mm long, with guards.
Mixer volume is <2 ml for up to 100 ml/min, but mixer design will depend on system, and planned solvents.

I mainly use isocratic for clean samples, and gradients for dirty samples, as I have two Agilent 1100 preparative pumps for the mobile phase components, each capable of 100 ml/min, and would normally use 25-40 ml/min for 21 and 30mm diameter columns. I use step mode to flush retained material from columns at end of runs or batches.

Reason for vertical columns is to help ensure horizontal solvent front, especially with step gradients and larger particle size columns.

Jiang295,

The mixer you use is adequate. The pressure? I wonder what is the reason for the pessure problems you are experiancing - especially in the context of the applied flow rate. How high is the pressure anyway? Also, what particle size is the column material?

Best Regards

Hi Danko,

I am using 5um particle size, that is the reason why I experienced pressure problem using 20ml/min for 21mm*30cm column. pressure went beyond 300 bar at 50% methanol, which is up limit of LC-8A pump.

Jiang295,

The mixer you use is adequate. The pressure? I wonder what is the reason for the pessure problems you are experiancing - especially in the context of the applied flow rate. How high is the pressure anyway? Also, what particle size is the column material?

Best Regards

Hi Bruce,

thanks a lot for your detailed answer!
another question for you:)
if you use gradient method, do you use step gradient or continuous gradient? I am experiencing difficulty using continuous gradient, it is difficult to control retention time very precisely using continuous gradient.

what kind of diameter column you have?
since you are doing prep separation, could you do me a favor tell me how large mixer volume you have to ensure enough mobile phase mixing.
or you just use isocratic or step gradient?

Typically, 21, 30, 50 mm diameter x 250mm long, with guards.
Mixer volume is <2 ml for up to 100 ml/min, but mixer design will depend on system, and planned solvents.

I mainly use isocratic for clean samples, and gradients for dirty samples, as I have two Agilent 1100 preparative pumps for the mobile phase components, each capable of 100 ml/min, and would normally use 25-40 ml/min for 21 and 30mm diameter columns. I use step mode to flush retained material from columns at end of runs or batches.

Reason for vertical columns is to help ensure horizontal solvent front, especially with step gradients and larger particle size columns.

if you use gradient method, do you use step gradient or continuous gradient? I am experiencing difficulty using continuous gradient, it is difficult to control retention time very precisely using continuous gradient.

In prep HPLC, good temperature control of injector loop and column usually produces consistent retention. It's also advisable to have the injection solvent composition similar to initial mobile phase.

If peaks are dancing around, and you are confident that both the gradient composition and column temperature control are consistent, you should investigate whether the sample solvent and mobile phase are suitably buffered.

Because loadings are higher, mismatches between sample solvent and mobile phase can have adverse effects on retention. Also, always ensure that your sample remains soluble throughout the analysis.

The column back pressure can indicate if sample material is being deposited, but pressure changes won't be so obvious with your lower flowrates.

C18 reverse phase preparative HPLC is often working near the solubility limit of compounds in mobile phase, in which case small changes in temperature can have profound effects on retention ( and yield ).

thanks a lot.
learn a lot from you.

if you use gradient method, do you use step gradient or continuous gradient? I am experiencing difficulty using continuous gradient, it is difficult to control retention time very precisely using continuous gradient.

In prep HPLC, good temperature control of injector loop and column usually produces consistent retention. It's also advisable to have the injection solvent composition similar to initial mobile phase.

If peaks are dancing around, and you are confident that both the gradient composition and column temperature control are consistent, you should investigate whether the sample solvent and mobile phase are suitably buffered.

Because loadings are higher, mismatches between sample solvent and mobile phase can have adverse effects on retention. Also, always ensure that your sample remains soluble throughout the analysis.

The column back pressure can indicate if sample material is being deposited, but pressure changes won't be so obvious with your lower flowrates.

C18 reverse phase preparative HPLC is often working near the solubility limit of compounds in mobile phase, in which case small changes in temperature can have profound effects on retention ( and yield ).