Many of the problems in liquid chromatography can be reflected in the spectra, some of which can be solved by changing the device parameters; other problems must be solved by modifying the operating procedures. The right choice for the column and mobile phase is the key to getting a good chromatogram. In the face of the often found problems with the abnormality of the HPLC spectrum, how to solve the problem truly needs to adhere to these principles: a specific problem to treat the principle; two internal principles after the peripheral; three simple and complex principles; Four single processing to comprehensive processing principles. Â
One trailing peak
1. Screen blockage: If the filter screen at both ends of the column is clogged, the sample will be blocked in the sieve plate to form a time delay, so that the peak shape will form a tail when the sample flows out after the column. Need to pass the backflush column or replace the sieve plate.
2. Column collapse: It means that the column loses the efficiency of the column due to other reasons, and can not form a retention of the substance, so that the substance does not remain on the stationary phase and flows out with the mobile phase, but there is still a little column effect, thus forming a trailing tail. . You will need to refill the column or replace the column.
3. There is pollution: the sample does not start at the same starting line, and runs from the back to reach the end point later, showing a trailing tail. Replace the column or use an organic solvent gradient for more than 1 h to rinse the column.
4. The PH value of the mobile phase is incorrectly selected: if there is a dynamic balance between the molecular type and the ionic type in some samples at a certain pH, the ionic form will gradually show a tailing to the molecular form. Adjusting the pH can inhibit molecular dissociation and improve tailing. For basic compounds, a relatively low pH value is more favorable for obtaining symmetrical peaks.
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Frontier peak
1. Sample overload: The retained samples are successively emerged before the normal peak time to form a leading edge peak. Reduce the sample content.
2. Inappropriate solvent selection: When the elution ability of the sample solvent is much stronger than that of the mobile phase, the leading edge peak appears. For example, when the nitrile is used as the sample solvent in the reverse phase chromatography, and the elution force of the mobile phase is weak. A leading edge peak will appear. The ratio of the mobile phase or the mobile phase is selected as the sample solvent.
3. Column damage: column efficiency loss, can not retain the formation of substances. Replace the column.
4. False front peak: There is a small peak appearing before the big peak, and the imaginary front peak, that is, the small peak is buried before the big peak. Adjust the mobile phase elution gradient.
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Baseline drift
1. Column temperature fluctuations: Even small temperature changes can cause fluctuations in the baseline, usually affecting the difference detector, conductivity detector, lower sensitivity UV detector or other photoelectric detector. Use a column oven to control the temperature of the column and mobile phase and use a heat exchanger before the detector.
2. Mobile phase non-uniformity: The baseline drift caused by changes in mobile phase conditions is greater than the drift caused by temperature. The HPLC grade solvent was used and the mobile phase was degassed prior to use.
3. The flow cell is contaminated or gas: flush the flow cell with methanol or other strong polar solvent. If necessary, use 1N nitric acid (do not use hydrochloric acid).
4. Improper flow ratio or flow rate change: change the ratio or flow rate. To avoid this problem, check the mobile phase composition and flow rate regularly.
5. There is a strong retained material in the sample: it is eluted as a peak of the taro, showing a gradually increasing baseline. Use a guard column and, if necessary, periodically flush the column with a strong solvent between injections or during the analysis. Â
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Four. There is a wide peak
1. The number of plates is reduced: the column is contaminated or failed, resulting in a decrease in the number of plates. Replace the same type of column. If the new column provides a symmetrical peak, flush the old column with a strong solvent.
2. The pipe is too long or the inner diameter of the pipe is too large: the pipe between the column and the detector is too long or the inner diameter of the pipe is too large. Replace short tubing with a smaller inner diameter.
3. The detector response is too large: the detector is too responsive to the reaction time or pool volume. Reduce response time or use smaller flow cells.
V. Baseline noise
1. Sharp peak: There is air in the mobile phase, detector or pump. The mobile phase is degassed and the system is flushed to remove air from the detector or pump.
2. Leakage: Check if the pipe joint is loose, if the pump leaks, if there is salt precipitation and abnormal noise. Replace the pump seal if necessary.
3. Incomplete mixing of the mobile phase: Mix well by hand or use a solvent with low viscosity.
4. Temperature effect: The column temperature is too high and the detector is not heated. Use a column oven to reduce temperature differences or add heat exchangers.
5. Accidental noise: There are other electronic devices on the same line, disconnect the LC, detector and recorder, check if the interference is from the outside, and correct it. A precision regulated power supply is used.
Six. The degree of separation is not enough
1. The mobile phase gradient elution setting is unreasonable: optimize the gradient elution procedure.
2. Mobile phase contamination or deterioration: causing a change in retention time and reconfiguring the mobile phase.
3. Protection column or analytical column blockage: Remove the guard column for analysis and replace the guard column if necessary; if the analytical column is blocked, backflush can be performed; if the problem still exists, the column may be damaged by strong retained contaminants. The regeneration procedure; if the problem persists, the inlet may be blocked, replace the sieve at the inlet or replace the column.
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