By Anonymous on Monday, June 28, 2004 - 10:05 am:

I have a Knauer K-1001 pump with quaternary low pressure mixing. If I prepare a mobile phase (50:50 ACN:water) and pump through one channel, my retention times are shorter than when I have the pump mix the same mobile phase. What might cause this? I know the pump is not mixing properly, but I don't know how to fix it. Now I am concerned that when I run a solvent gradient that I am not really running the gradient I think I am.

By Anonymous on Monday, June 28, 2004 - 12:49 pm:

normaly it is the inlet checkvalve causing it. exhange or wash with 5-10% HNO3 (water before and after) and without colum.

By Anonymous on Tuesday, June 29, 2004 - 06:15 am:

Make sure that the solvents are well degassed also. This is a problem with low pressure gradient systems. You can verify that the GPV's are or are not working correctly by running a GPV test.

By tom on Tuesday, June 29, 2004 - 09:50 am:

Before doing a tear-down of the system, it might be a good idea to actually *check* the proportioning accuracy of your system:
- remove the column and go directly from the injector to the detector with a connecting tube (add enough pressure restriction to enable the pump to operate properly).
- put water or methanol in the A reservoir
- put water or methanol with 0.1% acetone in the B reservoir (should give you about 1 AU of absorbance at 265 nm)
- run a "stairstep" gradient profile (5 or 10% steps). Make the hold at each step long enough to be sure that it flattens out.
- examine the signal to see that each step is equal.

If each step shows an equal absorbance change (within the specifications of the pump), then the proportioning system is working OK. That suggests that the "manually prepared" mobile phase may have been incorrect.

If steps vary, then your pump is indeed malfunctioning and needs service.