HPLC Products

Normal

Problem

A cycling baseline is usually caused by a pumping problem, deriving from solvent mixing problems or flow variations:

Solvent Mixing – A gradient pump with a faulty proportioning valve will give a cycling baseline. A proportioning valve is an electronic valve used in low pressure mixing pumps. It is held closed by a spring, and opened electronically. It is these valves that can be heard clicking when using a gradient pump to mix the eluent.

If a cycling baseline is observed, first test using a solvent from only one channel (ie no mixing, so the clicking of the proportioning valves should not be heard). Bear in mind that if the solvent line from the pump to the column is full of badly mixed solvent, it may take several minutes after restarting the pump before a normal flat baseline is observed. Test each valve to be sure that it is opening. If necessary, try using a different solvent on a different channel to see if the problem goes away.Try standing the solvent bottles on top of the pump to apply a little inlet pressure to the valves and ensure fre-flowing solvent supply. If the facility is available on your pump, try changing the mixing cycle time.

Check Valve sticking – this causes flow variations. If the cycling is not totally regular, suspect check valves. They can be cleaned in methanol:water in an ultrasonic bath for 15 minutes. Use about 75% water, and ultrasonicate the check valve in a beaker, in case it comes apart!

Temperature Control. A column heater with a thermostat will show cycling especially with a refractive index detector, as the temperature rises and falls. This kind of column heater should be destroyed with a hammer and replaced with a more modern unit which pulses the power, thus providing an almost continuous input of energy. Such column heaters use closed loop circuitry to monitor the temperature continuosly, making corrections to the duration of the pulses. When the heater is heating hard, the length of the pulses is increased until they join up and it heats continuously. But as it nears the target temperature, the duration of each pulse (but not their frequency) is reduced. The circuitry monitors the temperature change, calculates the rate of loss of heat, and adjusts the heating so that exactly that amount of energy is applied, keeping the temperature constant and eliminating the cycling effect on the baseline.

Air in the flow cell. A cycling baseline can sometimes be caused by an air buble in the flow cell. This may be dislodged by increasing the flow rate for a while, assuming that the column will not be damaged by the increased pressure. Otherwise, a bubble can be removed by blocking the waste pipe from the cell using a rubber septum for about 1-1.5 seconds at a flow rate of 1ml/min. Releasing suddenly sometimes causes the bubble to go.

Air in the pump. An air bubble in the pump chamber causes the flow to start and stop, as for part of the time the pump is doing nothing more than compressing the air bubble. Hence as the flow starts and stops, the back pressure goes up and down. Some detectors (especially refractive index) are sensitive to pressure changes. Even a UV detector may give a cycling baseline if the pressure is cycling. If this is the case, stop the flow, open the purge valve, and press Purge to run the pump at maximum flow and eliminate the air bubble through the waste pipe from the purge valve. Once the air is removed, stop the pump, close the purge valve, and return to pumping normally. Check there is no further solvent flow from the purge valve, and if therre is, tighten the valve to close it properly.