Speaking of bubbles, you might associate to blow out of the coke, appear above beer, or a romantic time flying, the bubbles in life, it seems, are still with so a little better, but for experiment, especially in the most won't online degassing unit configuration FPLC system of chromatography experiment, bubbles as annoying flies, the stones in the shoes, It's a lump in the throat. Failure of important samples, expensive fillers, and bubbles is a minor disaster. So the bubble is from where, and where to go, how to let them disappear from our system, the following article, to do a small discussion, throw a brick to attract jade, conspiracy countermeasures and destroy it.
As for the source of bubbles, it is nothing more than the opening of the system: the mobile phase, or the leakage point at the relative negative pressure in the flow path; If the degassing of the mobile phase is not ideal or there are problems with the connecting pipes and components of the instrument itself, the flow path will form arbitrary bubbles, causing a series of problems:
1. Component peak area and peak shape change;
2. To form a sharp or jagged noise;
3. Ghost peak appears and baseline drift fluctuation increases;
4. Abnormal pump action, pressure fluctuation, detection baseline shaking;
5. packing empty, dry column;
6. Dissolved oxygen may oxidize sample components and/or mobile and stationary phase components, distorting the detection.
The actual case
Fig.1 UV miscellaneous peaks caused by bubbles
Fig.2 System pressure fluctuation caused by bubbles
These symptoms cover most chromatographic problems, showing how damaging and annoying bubbles can be, and how they form. There are many reasons for the formation of gas in the mobile phase. The amount of dissolved air in equilibrium state is related to the nature of the solution, the type of gas, and the external conditions, the main external conditions are the following 3 kinds.
The temperature change
Generally speaking, the amount of dissolved gas in the solvent decreases with the increase of temperature, and when the solvent moves from low temperature to high temperature, the excess dissolved gas escapes in the form of bubbles. Therefore, the change of buffer temperature is easy to cause the formation of bubbles.
Low as we configure buffer will save a lot of time in tomographic ark of low temperature environment, when the experiment you need to use from the chromatography ark, the drastic changes in room temperature environment brings, change the gas solubility and, in turn, generate bubbles, impact experiment, so the use of buffer if there is the change in temperature, need to put in the experimental temperature for a period of time, Release bubbles before use; If chromatography instrument is in a constant temperature laboratory, day and night temperature difference, saved the day to finish the experiment System, the experience of night temperature change, the second day bubble is likely to appear in the System, System for wash thoroughly before experiment is very important at this time, you need to use the high velocity of the purging bubbles within the System, to avoid impact experiments.
● When the detector tank is at a higher temperature, bubbles tend to form in the tank, because as the liquid flows through the detector, the temperature increases and the pressure here decreases. If bubbles are formed in the detector cell, there will be sharp or jagged baseline noise, or even completely impossible to measure. Therefore, in the system flow path, the temperature partition between the heating component and the flow path should be maintained, and the uniformity of the temperature of the system flow path is also very important.
The pressure to reduce
As the partial pressure of the gas increases, the amount of the gas dissolved in the solvent increases. On the contrary, the solution saturated at a higher pressure will produce bubbles once the pressure decreases. This is also one of the important reasons for the formation of bubbles in the experiment, such as:
● The buffer inlet of chromatography generally has a filter design, if the filter maintenance and cleaning is not in place, the filter impurities are too much or bacteria caused by insufficient liquid flux, in the case of high flow rate, the pressure inside the pipeline is sharply reduced, at this time the bubble will shine on stage, bringing wave-like spectrum fluctuation; Therefore, before the experiment, it is necessary to check the cleanliness of the filter head of the buffer liquid inlet of the system. If there are impurities, they should be cleaned and removed immediately.
Fig.3 Buffer inlet filter head screen
● High air pressure when configuring buffer (temperature, humidity, weather and altitude will affect atmospheric pressure), the bottle is tightened and saved. If the air pressure changes during the experiment, the bubbles will be brought into the precipitation when opening the buffer for the experiment. Therefore, the degassing of the pre-configured buffer is very important. Different pipe diameter of low system flow transformation, brings the change of the flux, such as capillary tubes, in the case of high velocity, as a result of the flux increased sharply, the formation of topical negative pressure, easy to cause precipitation of bubbles or leak into, so the system pipe diameter to avoid the use of capillary tubes, if the experiment must be, should pay attention to avoid the use of high flow velocity.
● In the case of low pressure gradient, because the pressure of the unit part of the low pressure gradient is slightly lower than atmospheric pressure, and the mixing chamber is mostly installed behind the pump (high pressure side), but the actual mixing starts at the low pressure side, so the low pressure gradient is easier to produce bubbles than the high pressure gradient behind the pump. (Conventional single pump two or four channels are low pressure mixing mode)
● The pressure in the column is generally higher, the solubility of the gas increases, and it is not easy to produce bubbles in the column. Near the column outlet, however, the pressure is relatively low, the import and export of cylinder packing differential pressure will be very big, the mobile phase dissolved air bubbles due to the pressure difference, easy to precipitate in the packing, affect the column efficiency, even damaged packing, so the system will back pressure device (e.g., back pressure valve), inflicts on column after a certain amount of pressure, differential pressure, reduce the column to avoid precipitation of bubbles.
The amount of dissolved gas is related to the type of gas, and also to the type of solvent. Generally, the gas with low polarity is easy to dissolve in the solvent with low polarity, and the gas with high polarity is easy to dissolve in the solvent with high polarity. Usually when two different solvents are mixed, the amount of air dissolved in the mixture will also change. A large number of experiments show that the amount of dissolved air in the mixed solvent is smaller than that of each pure solvent.
Low configuration buffer, mostly will be a mixture of different liquid, liquid volume change, thermodynamics and heat absorption of heat, will produce bubbles, such as configuration 20% ethanol solution, acid solution, the salt solution and acetonitrile solution, there was an obvious bubbles will produce, at this point you need to let stand or degassing operation of solution, only can be used in the experiment.
Low in chromatography experiments, there are a lot of solution mixed operation, such as the replacement cushion, elution, and so on, so some mixed solution need to avoid as far as possible, such as ethanol solution and salt solution, need to use pure water is separated, otherwise easy to cause bubbles precipitation, so when ethanol solution to save the system need to experiment, such as PBS buffer is salt solution, We need to rinse the system with pure water and then fill the PBS.
Fig.4 Bubbles precipitated in buffer
If the experiment design, the inevitable need two different polar solvent mixture, to avoid air bubbles, the need to find a way to, from mixed two-phase solvent mixture of general chromatography system is in a dynamic mixing pool with stirring device, mixed, easy separation bubble, can consider to change the dynamic mixer to static mixer, at this time Let the solvent mixing mode from violent to mild, will have a better effect of avoiding bubbles.
Fig.5 Dynamic mixer and static mixer
Of funnelled into, in addition, in addition to the solution system will also introduce the bubbles, non pressure of low pressure flow, especially in pipe fitting, valve and rotor separation structure, easy to produce funnelled baseline fluctuation caused by system if found to have bubbles, and import and found no visible bubbles in solution, you need to find funnelled within the system, An effective method is to replace the opaque Peek tube with transparent ETFE tube and observe which section of the bubble appears, so as to determine the leak point and deal with it in time.
Fig.6 Leaking air bubbles in the pipeline
There are two commonly used solvent degassing methods: ultrasonic degassing and negative pressure degassing; Ultrasonic degassing is the use of ultrasonic pot or ultrasonic probe, when high power ultrasonic coupled to the liquid, the liquid is compressed and expanded in high pressure and low pressure cycles respectively, in the low pressure cycle, the formation of tiny vacuum bubbles (so-called cavitation bubbles), these bubbles will grow in multiple pressure cycles. During those periods of bubble growth, dissolved gases from the liquid enter the vacuum bubble, which becomes a growing bubble. Ultrasonic degassing is characterized by convenient operation, high efficiency and fast degassing. Generally, 10-15 minutes of ultrasonic can remove most of the bubbles in the solution, but the disadvantage is that it is easy to heat up and need to cool down. Note that when using ultrasonic pot to degassing, the solvent cap should be loosened or removed; Vacuum degassing is the use of mechanical separation, negative pressure in the way to make the solution bubble general analytical chromatography or need high precision testing instrument, online degassing unit in the flow configuration, are to be carried out by using the principle of negative pressure degassing, laboratory commonly used is the vacuum suction filter device, which can filter the solution, and degassing and high practicability.
Fig.7 Ultrasonic degassing
Fig.8 Vacuum extraction and filtration device
If chromatography system with the bubble in flow, first is must carry on the flow velocity of the system for wash, can purge out most of the bubble, some tiny air bubbles, however, is still not easy to rush out, visible in the transparent tube tapping let them out of line of fine bubbles can be used outside force inside and out, but within the system, especially the pump cavity, one-way valve, Flow pool of detector tiny air bubbles, invisible, also can't knock force, so I can't determine whether rinse thoroughly, stranded in the plunger pump check valve or flow pool of detector of tiny air bubbles will still bring chromatography graph swings, at this time we can do by using anhydrous methanol or ethanol system for wash, best to add a certain amount of pressure, Because methanol or ethanol reagent reduces the gas-liquid interfacial tension, methanol (ethanol) molecules can not form a solid film, so the bubble burst or fall off from the inner wall of the flow path, thus bringing out various fine bubbles in the flow path, to achieve the purpose of system degassing; System, on the other hand, the internal flow of clean degree is also a important factor affecting the bubbles were stranded, FPLC systems often have complex biomass samples, if you don't fully daily washing, flow internal easy to form the dirt, increases the stubborn adsorption of tiny air bubbles, impact experiments, so comprehensive cleaning, regular system to eliminate air bubbles is also crucial.
To sum up, for quick preparation chromatography chromatography, gets a bubble is a dozen of instability, but if we know the reason, ready to fully, in the right way, the bubbles can be completely away from the system, make the experiment more smoothly, make map more smooth, let precious samples perfect separation, expensive packing material.
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