The FPLC columns usually have specified some recommended flow rate, but for (U)HPLC it's common to have only maximum pressure, if at all.
So how do you choose the flow rate? Do you always go for the maximum with the recommended pressure? Or do you have some rule of thumb for whatever?
We used on our 2.1x50mm 1.8u columns 0.4ml/min with the pressure between 40-50MPa. Now we purchased 2.1u core-shell column that has much lower pressure, so I increased the flow rate to 0.5 ml/min, mainly because I was afraid the peaks would elute in the injection, but that moves to the front as well, right?
And I must say, although it's illogical and everything, I'd feel somehow guilty and bad for having too short analysis. Something like that I'm cheating or doing something wrong.
OK, so how do you choose the flow rate? Is it just some arbitrary number you "randomly" choose and if it works well enough, you use that?
How do you decide then, whether to use lower concentration of B or lower flow rate to achieve better separation?
Hello!
Basically, in the HPLC analysis, the recommended flow rate is not so important because you're working with very small particle sizes and higher flows generated no particular deterioration of the resolution (bottom number).
Core Shell columns have a specific low back pressure. 2.1u particle size of Core Shell column generated a similar return pressure as a fully porous (normal) column with 2.1u!
Only the resolution (bottom number) of Core Shell column is better than at a normal column. One should always choose a higher flow rate to the system limit (HPLC, connections etc) is achieved because you want very quickly runs. Two disadvantages of a Core Shell Column 1. slightly lower selectivity 2. the column must not be overloaded, ie. you should very little injecting (perhaps only 0,5uL) otherwise you overload the column.
It is also important to know. Which mobile phase you have. Do as the mobile phase acetonitrile / water?
Many greetings
Burghard Cordes
Sorry!
Core Shell columns have not a specific low back pressure.
Many greetings
Burghard Cordes
Hi Tomas, don't feel guilty for short Analysis times as These depend on your specific aims and challenges. We're running a Standard Gradient on a 2.1x50 1.8 um column with flow rates of 1 mL/min (60°C water/ACN) and Gradient times of 1.5 min. Throughput is the challenge here and not so much Separation. Peaks become sharper with higher flow rates (sensitivity!) so, according to Farooq, as long as you can afford smaller Peak differences ist all fine.
I must say I'm surprised. I thought you will be all Go for the maximum. Why? Because we can! but you're advocation rather the lower flow rates :)
Hi Tomas
There are a number of advantages to using core shell technology columns, mostly by achieving excellent resolution without the extreme backpressure of smaller particle size stationary phase material. The disadvantage in many cases are the dead volume of HPLC systems as opposed to UPLC systems where flow paths and detector systems have been optimized for lower flow rates by reducing the dead volumes although they work well at high flow rates as well due to the pressure they can sustain.
By using higher flow rates with a core shell material you eliminate some of the effects of the larger dead volume of the LC system.
The larger the dead volumes (some HPLC sytems have more than 1.8 ml), the longer it takes for a gradient formed at the pump heads but before the pressure dampers and mixers, to reach the column where the separation actually takes place. If the flow rate is increased this time for the gradient to reach the column is reduced. The problem is that if the flow rate is too high, then the equilibration of the analyte and stationary phase is not effective and your resolution starts degrading. Also at the very high flow rates there are thermal effects due to the friction of the mobile phase that causes thermal heating that cannot be effectively removed and temperature convection effects cause degradation of the efficiency of the column.
The advantage of the core shell columns is the rapid equilibration rate due to the limited distance that the analyte needs to move into the particle before returning to the moving mobile phase. The larger particle allows lower back-pressures so you get two benefits. The down side of this material is that the capacity of the column is less than for fully porous particles so you need to watch for column overloading and that you do not loose sensitivity just to be able to run the separation faster. Chromatography is always a compromise!
Regards
Duncan
Hi,
Flow depends on trial & error and system suitability parameters and go through following link how flow rate is set to maintain the linear velocity & you can vary the same based on your trials on your product, it is all in house.
http://hplctips.blogspot.in/2013/04/using-smaller-diameter-hplc-columns.html
It seems that you use UV detection, then the flow rate does not matter too much as long as the dead volume of your UV cell, and connection system is compatible with a ca 0.2 mL/min flow rate. If your detector is a MS, and depending on the MS ionization mode, additional factors should be considered.
When I was writing manuscript for limonene UPLC paper, the wise Waters representative, Denise Kent, recommended that the flow rate "sweet spot" for resolution on the H-class system with the core-shell column 1.7 micron particle, 100 Angstrom pore, 100 mm length and 2.1 mm diameter was going to be around 0.7 ml/min. I followed her advice. As mentioned above, throughput is paramount, so you do not want to stress your equipment early on, because filters will get clogged eventually, raising backpressure and blowing off a connection or causing some other overpressure failure. Therefore I am not a fan of operating at max pressure. Crank up the column heater as much as analyte stability will allow. If anyone is interested and does not have access to J. AOAC International, I can send the pdf.
HPLC flow rates are NOT determined by system pressure (unless it is a limiting factor or results in heating). HPLC column ID and particle size determine flow rate in HPLC. Flow rate shall be optimized for each method (too high or low equals loss of resolution). For "STD" sized HPLC columns, columns with particle sizes of 3 to 5u (and larger), we select a flow rate that is optimized per van Deemter plot (see table below for general guidance). Start with these flow rates for general use.
- Column I.D. (mm) Flow Rate (ul/min)
4.6 mm 1,000 ul/min
2.1 mm 208 ul/min
1.0 mm 47 ul/min
0.3 mm 4 ul/min
0.15 mm 1 ul/min
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