Thread Content
1 System composition and working principle of high-performance liquid chromatography instruments The system of a high-performance liquid chromatography instrument consists of a solvent reservoir, pump, injector, chromatographic column, detector, recorder, and other components. The mobile phase in the reservoir is pumped into the system by a high-pressure pump. The sample solution enters the mobile phase through the injector and is carried by this mobile phase into the chromatography column (the stationary phase). Since the various components in the sample solution have different distribution coefficients between the two phases, as they move relative to each other, repeated adsorption-desorption processes occur, resulting in significant differences in their migration speeds. Thus, the components are separated into individual components that flow out of the column one after another. As these components pass through the detector, their concentration is converted into electrical signals that are sent to a recorder, and the data is printed in the form of a spectrum. High performance liquid chromatography (HPLC) is also known as high pressure liquid chromatography, high speed liquid chromatography, high resolution liquid chromatography, and so on. It developed rapidly in the late 1960s by introducing gas chromatography theory on the basis of classical liquid chromatography. Its difference from classical liquid chromatography lies in the small and uniform size of the packing particles; these small particles result in high column efficiency, but they also cause high resistance, requiring high pressure to pump the mobile phase, which is why it is also known as high-pressure liquid chromatography. It is also called high-speed liquid chromatography due to its fast analysis speed. Features of high-performance liquid chromatography: High pressure – the pressure can reach 150–300 kg/cm2. The pressure drop per meter of the chromatography column is over 75 kg/cm2. High speed – flow rate of 0.1 to 10.0 mL/min. High efficiency – the number of plates can reach 5,000 per meter. Up to 100 components can be separated simultaneously in a single column. High sensitivity – the UV detector can achieve a sensitivity of 0.01 ng. It also consumes less sample. Compared to conventional liquid chromatography, HPLC has the following advantages: faster speed – typically, analyzing one sample takes 15–30 minutes, and for some samples it can be completed in as little as 5 minutes. High resolution – the stationary and mobile phases can be selected to achieve optimal separation. High sensitivity – the UV detector can reach 0.01 ng, while the fluorescence and electrochemical detectors can reach 0.1 pg. Chromatography columns can be reused – a single column can be used to separate different compounds. The sample volume is small, making recovery easy—the sample remains intact after passing through the chromatography column, allowing for the collection of a single component or for preparative processing. 3 Common Fault Troubleshooting for High-Performance Liquid Chromatographs Fault 1: Bubbles in the mobile phase. Turn off the pump, open the pressure relief valve, press the purge button to perform degassing; however, bubbles continue to emerge from the filter and enter the mobile phase, and no matter how many times the purge button is pressed, these bubbles cannot be eliminated. The reason is that the filter remains submerged in buffers such as ammonium acetate for an extended period of time; as a result of mold growth and reproduction inside the filter, colonies form which block the filter, preventing the buffer from flowing through it smoothly. Air enters the mobile phase through the filter under the pressure exerted by the pump. The filter to be treated can be immersed in 5% nitric acid solution and cleaned using ultrasonic waves for a few minutes; it can also be left in that solution for 12–36 hours, gently shaken several times, then washed several times with pure water. The pressure release valve should be opened, as well as the purge button, in order to carry out cleaning and degassing. If bubbles continue to emerge from the filter, it should be kept immersed in 5% nitric acid solution. If no bubbles longer appear, it means that the mold colonies inside the filter have been destroyed by the nitric acid, allowing the mobile phase to pass through the filter smoothly. Open the pressure relief valve and start the pump; adjust the flow rate to 1.0–3.0 ml/min, and rinse the filter with pure water for about 1 hour. The filter can then be cleaned thoroughly. Close the pressure relief valve and rinse with pure methanol for half an hour. Fault 2: Reasons for high column pressure (1) Salts in the buffer solution, such as ammonium acetate, accumulate within the column; (2) Contamination from the sample leads to deposition. For the first scenario, start by flushing the column with pure water at 40–50 °C at a low flow rate in the forward direction. Once the column pressure begins to drop, increase the flow rate for flushing. After the column pressure drops significantly, continue flushing with pure water at room temperature, followed by flushing the column with pure methanol for 30 minutes. For the second scenario, in the case of a C18 column contaminated due to sample deposition, flush the column in the reverse direction using pure water, then switch to methanol for flushing. Next, flush the column using a mixture of methanol and isopropanol (4 + 6); the duration of this flushing depends on the degree of contamination caused by the sample. After that, switch back to methanol for flushing, then rinse with pure water, and finally flush the column with methanol for more than 30 minutes in the forward direction. Fault 3: There is no pressure indication, and no liquid flows through. Fault 4: Pressure fluctuates greatly, and the flow rate is unstable. The reason is that there is air in the system, or foreign objects are trapped between the ball of the check valve and its seat, preventing proper sealing between them. During the processing, pay attention to the amount of the mobile phase; ensure that the stainless steel filter sinks to the bottom of the reservoir bottle to avoid air being drawn in, and the mobile phase must be thoroughly degassed [2]. If there is debris trapped between the check valve and its seat, remove the check valve and place it in a beaker containing acetone for ultrasonic cleaning.