There is no problem. What you have observed is only partially related to viscosity, but is in fact due to a chemical reaction between water and the organic solvent, ACN or MeOH. When you mix these together an endothermic or exothermic (respectively for each organic solvent) occurs which is evidence of the chemical reaction taking place. This is something that you should observe when first learning about HPLC, as solutions of MeOH and Water (and ACN and Water) are commonly used. Plot the pressure profile of each mixture from low water to high water concentrations and you will be in for a surprise! They are not linear! Water/MeOH will show peak backpressure ~ 50% and Water/ACN will peak ~ 70%. This is a normal result, but extremely important for novices to learn about early so they can understand what "normal" back pressures look like when running RP chromatography, esp gradients. This specific phenomena is something that I teach in the entry level HPLC classes and is something that all chromatographers need to understand in order to perform troubleshooting as well as just understanding the basic concepts of liquid chromatography.

*This is a great classroom exercise to try yourself. Take the time to set up a range of compositions [suggest 10, 20, 30, 40, 50, 60, 70. 80, 90 % Water vs either Methanol or ACN]. Let each one stabilize, then observe the back pressure readings. Record and plot them to see the effect.

ACN has a much lower viscosity than MeOH, thus lower backpressure. As long as the separation works I won't worry about this issue.

As I know the solvents have different viscosities. So I'm agree with above answer.

There is no problem. What you have observed is only partially related to viscosity, but is in fact due to a chemical reaction between water and the organic solvent, ACN or MeOH. When you mix these together an endothermic or exothermic (respectively for each organic solvent) occurs which is evidence of the chemical reaction taking place. This is something that you should observe when first learning about HPLC, as solutions of MeOH and Water (and ACN and Water) are commonly used. Plot the pressure profile of each mixture from low water to high water concentrations and you will be in for a surprise! They are not linear! Water/MeOH will show peak backpressure ~ 50% and Water/ACN will peak ~ 70%. This is a normal result, but extremely important for novices to learn about early so they can understand what "normal" back pressures look like when running RP chromatography, esp gradients. This specific phenomena is something that I teach in the entry level HPLC classes and is something that all chromatographers need to understand in order to perform troubleshooting as well as just understanding the basic concepts of liquid chromatography.

*This is a great classroom exercise to try yourself. Take the time to set up a range of compositions [suggest 10, 20, 30, 40, 50, 60, 70. 80, 90 % Water vs either Methanol or ACN]. Let each one stabilize, then observe the back pressure readings. Record and plot them to see the effect.

200 bars is not that high. Don't worry about it. Of course you can reduce a bit the flow rate.

No need to reduce the flow rate when the pressure observed (and reasons for it) is normal.

Additionally for the above anawer, I suggest to use ingredient Modo to select the appropriate composition for the mobi phase, this, will be easier for you to limit the time and solvent .

It is normal, don't worry. You must avoid reaching the maximum pressure of the pump.

Thank you all very much! You were very helpful!

Agree with William

"This is a great classroom exercise to try yourself. Take the time to set up a range of compositions [suggest 10, 20, 30, 40, 50, 60, 70. 80, 90 % Waters vs either Methanol or ACN. Let each one stabilize, then observe the back pressure readings. Record and plot them to see the effect. "

Zhiqian Liu I very much agree with you on the pressure. With that being said, how much premixing and degassing are you doing? Furthermore, with 4.6 x 250mm ; 5-micron packed with C18 it doesn't matter what you run through it; the pressure is still going to be significantly higher than if you are a shorter column with a different packing.

I do want to point out, something (this will come as a shock to most) make sure all your lines are primed with solns and are pre-primed with solvent prior to starting. In my lab there was an issue where a student decided to change MPs after a run from ACN to MeOH... long story short it creates a vapor lock in the pistons and check valves are shot.

So I'm agree with above answer.