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Troubleshooting of air bubbles in isocratic system of high performance liquid chromatography
Troubleshooting of air bubbles in isocratic system of high performance liquid chromatography
Symptom:
1. Causes of failure: When the solvent is mixed, bubbles will be generated due to the change of the thermodynamic volume of the two liquids; when mixing, it is easy to generate bubbles when exothermic or endothermic. For example, mixing methanol and water is exothermic, and mixing acetonitrile and water is endothermic. . Generally, after mixing with a measuring cylinder, the volume increases and decreases obviously, and many small bubbles are generated, hanging on the wall of the bottle, or shaking it, you can see many small bubbles in the liquid.
Troubleshooting: Solvent filtration, ultrasonic degassing (commonly used), especially after mixing acetonitrile and water, the ultrasonic degassing time is longer, and can also be degassed by other methods, such as installing an online degasser, nitrogen-filled degassing, etc. . The treated liquid should keep the indoor temperature constant during use.
2. The cause of the failure: the solvent filter head is contaminated, which results in uneven suction of the pump during the liquid suction process and forced liquid suction, resulting in tiny vacuum bubbles. At this time, many small bubbles will be observed on the wall of the liquid inlet tube, some will stay on the wall of the tube, or the flow rate will be smaller than the set value, and the pressure will be unstable.
Troubleshooting: Observe the phenomenon to confirm, and then eliminate. Soak in 5% -10% dilute nitric acid overnight (afraid of ultrasonic filter head) or ultrasonic treatment for several hours (ultrasonic filter head), and then soak in pure water overnight or ultrasonic treatment, pay attention to change the water for several times, wash To remove the acid residues, finally use organic phase methanol soaking treatment or ultrasonic. If the above treatment fails, a new solvent filter head needs to be replaced.
3. Cause of failure: After the mobile phase is placed, there is no exhaust, and the pump starts to run, which is easy to generate bubbles.
Troubleshooting: After placing the mobile phase, open the bypass valve and slowly suck in the liquid with a syringe. After observing that there is no air bubble in the inlet tube, suck 10ml again to drive the air in the flow path clean. Tighten the bypass valve before running the pump. Or open the bypass valve to drain at a high flow rate, observe that there is no air bubble in the inlet pipe, and then exhaust the air for 2 minutes to drive the air in the flow path clean, tighten the bypass valve, and then run the pump.
4. Cause of failure: contamination inside the check valve or pump head. (The pump pressure fluctuation is generally considered to be above 100psi.) The pump head is the place where the pressure changes most drastically and is also the most prone to bubbles. The pump head absorbs liquid by negative pressure, and the negative pressure will inevitably make the mobile phase Small bubbles grow up, when the pump cavity becomes positive pressure, the grown bubbles may not all become smaller and flow into the subsequent liquid path, then the bubble will accumulate in the pump cavity, thus affecting the suction accuracy.
Troubleshooting: Carefully remove the pump head, use methanol to ultrasonically clean the check valve and the internal seal ring and the entire pump head, and wipe the plunger rod with cotton and methanol. Replace check valve, seal ring, plunger rod, etc. if necessary.
5. Reasons for failure: Bring bubbles into the sample, and bubbles into the needle during aspiration
Troubleshooting: Before injecting the sample, pay attention to expelling the air in the syringe, put the syringe needle vertically upward, use the filter paper on the finger pad to press the needle, and push it up. The air bubbles can easily go up and be discharged.
6. Cause of failure: Chromatographic column air bubbles
Troubleshooting: Flush the reversed-phase chromatographic column with pure methanol at low flow rate (0.2-0.3ml / min) for a long time, and then gradually increase the flow rate to a maximum of 1ml / min until the column pressure stabilizes. Or replace the column.
7. Cause of failure: The flow cell is a place where bubbles can easily accumulate, which has a greater impact on the baseline. After the mobile phase flows into the chromatographic column, the pressure becomes smaller and smaller, tiny bubbles will gradually grow, and the cross-sectional area of ​​the flow cell is relatively large. Then, the bubbles are likely to grow in the pool. In the absence of external pressure, it is difficult to reduce the diameter of the pipeline to flow out of the flow pool, which exists in the pool, resulting in a messy baseline.
Troubleshooting: add a back pressure regulator (the pressure is generally around 150psi) at the outlet of the waste liquid to provide a back pressure for the flow cell; followed by blocking the discharge end of the waste liquid pipe with your fingers, seeing the pump column pressure rise (about 2- 3s), release, and at the same time watch the change of absorbance value on the detector display. If there are air bubbles in the pool, the waste liquid tube is blocked by the finger, and the absorbance value will change drastically. When the hand is released, the absorbance value will increase again, which proves that there are bubbles in the pool but not discharged. When the absorbance value is basically unchanged, it proves that the exhaust bubble is successful, and then the baseline will be stable (this process needs to be repeated 10-20 times).
The above is the troubleshooting of the isocratic system bubble. I hope you can give me advice and discussion. Give corrections and communicate in a timely manner if there is anything wrong.