Detailed answers to the questions commonly used in the fourteen chromatograms
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1. When using HPLC for analysis, the retention time sometimes drifts, sometimes it changes rapidly. What are the reasons? How to solve it?
A: About the drift problem:
1. The temperature control is not good. The solution is to use a constant temperature device to keep the column temperature constant.
2. The mobile phase changes. The solution is to prevent evaporation, reaction, etc. of the mobile phase.
3, the column is not well balanced, the column needs to be balanced for a longer time.
About the rapid change problem
1. The flow rate changes. The solution is to reset the flow rate to keep it stable.
2. There are air bubbles in the pump, and the air bubbles can be driven out by exhausting.
3. The mobile phase is not suitable. The solution is to change the mobile phase or to mix the mobile phase in the control room.
Second, the choice of capillary column in color and quality should pay attention to what problems?
Answer: 1. The efficiency of the column should be high.
2, good thermal stability
3, strong chemical inertia
Specific choices should be noted:
1. Column polarity. The columns with different polarities were selected for analysis based on the analysis samples.
2. The inner diameter of the column. The inner diameter determines the column capacity.
3. Liquid film thickness. Analysis of the sample temperature is not the same, there are different requirements for the film thickness, the temperature of the high liquid film should be thick, the temperature of the low liquid film should be thin.
4. Column length. The longer the column, the higher the column efficiency, the better the separation effect, but there are also adsorption problems, the column is too long, and the analysis time is long, so the column length should be considered according to the sample.
5. Selection of outer coating (cylinder).
6, in addition to the instrument model, analysis objects and other factors.
3. What is the cause of tailing or double peaks in liquid chromatography?
Answer: 1. The sieve plate is blocked or the column is invalid. The solution is to back up the column, replace the sieve plate or replace the column.
2. There is interference peak. The solution is to use a longer column, change the mobile phase or replace the selected column.
4. From these aspects, can you easily determine the thermal stability of a capillary column?
Answer: 1. The capacity (or capacity factor) K is allocated. After a good column is operated at high temperature, the distribution capacity K should not drop significantly. Otherwise, the thermal stability of the column is not good.
2. The number of theoretical plates n, after the column with good thermal stability is subjected to high temperature, the number of theoretical plates should be kept basically constant.
3. The polarity of the column. The column with good thermal stability has little change in polarity before and after high temperature, and the specific performance is that there is no large change in the retention index I value.
4. Noise. When the column with good thermal stability is used at high temperatures, the noise cannot be increased.
5, the deactivation layer of the column, the capillary column before the application of the fixative liquid is generally deactivated with deactivated reagent to increase inertia. After a high-quality capillary column is heated at a high temperature, the deactivated layer should not be changed, and the adsorption property to a strongly polar sample or an acid-base sample cannot be increased.
5. Why does the glass liner in the injector affect the chromatographic behavior?
A: The glass bushing in the injector has the following main functions:
1. Provide a vaporization chamber with uniform temperature to prevent local overheating.
2. The inertness of the glass is not as good as that of stainless steel, which reduces the possibility of sample decomposition during vaporization.
3, easy to replace cleaning, in order to maintain a clean vaporization chamber surface, some trace non-volatile components will gradually accumulate in the vaporization chamber, will slowly decompose at high temperatures, so that the base flow increases, the noise increases, by cleaning the glass lining The set can eliminate this effect.
4. The glass bushing with appropriate wall thickness and inner diameter can be selected according to the need to change the volume of the vaporization chamber without replacing the entire injection heating block. From the above aspects, the reason why the glass liner affects the chromatographic behavior can be known.
6. What is the main reason for nonlinear shunting when splitting with capillary chromatography?
Answer: 1. The temperature of the injector is too low, the vaporization of the sample is not complete, and the splitting occurs.
2. The temperature of the injector is too high, some components may be thermally decomposed, and some samples may be catalytically decomposed, or the sample may be partially adsorbed on the inner surface of the injector.
3. The sample was not evenly mixed or insufficiently mixed before the split point.
4, the system's injection pad, column joints and other places leak.
Seven, the main reason for the lack of HPLC sensitivity and solutions
Answer: 1. Insufficient sample size: The solution is to increase the sample size.
2. The sample does not flow out of the column: the mobile phase or column can be changed depending on the chemical nature of the sample.
3. The sample does not match the detector: adjust the wavelength according to the chemistry of the sample or change the detector.
4. The detector is attenuated too much: adjust the attenuation.
5. The detector time constant is too large: the solution is to reduce the time parameter.
6. Detector pool window pollution: The solution is to clean the pool window.
7. There are bubbles in the detection pool: the solution is exhaust.
8. The measuring range of the recorder is improper: adjust the voltage range.
9. The flow rate of the mobile phase is not suitable: the flow rate can be adjusted.
10. The detector and recorder exceed the calibration curve: the solution is to check the recorder and detector and re-calibrate the curve.
Eight, when doing HPLC analysis, the column pressure is unstable, what is the reason? How to solve?
A: The reasons may be:
1. There is air in the pump. The solution is to remove the air inside the pump and degas the solvent.
2. The proportional valve fails and the proportional valve can be replaced.
3. The pump seal is damaged and the gasket can be replaced.
4, the bubble in the solvent, the solution is to degas the solvent, if necessary, change the degassing method.
5, the system leak detection, find the leak point, seal it.
6. Gradient elution, where pressure fluctuations are normal.
9. How to prevent tar-like pollutants from entering the capillary column when doing PGS/MS analysis?
A: Polymers, especially those containing nitrogen, sulfur and halogens, often have tar-like substances. In order to prevent the tar-like substance from entering the capillary column and causing pollution, the performance of the column is lowered. The protective pre-column can be used, that is, a pre-column is connected between the cracker and the capillary column, and the tar-like substance is retained by controlling the temperature of the pre-column. In the pre-column, the pre-fill can be placed in the GC gasification chamber, which is easy to control the temperature and reduce the dead volume of the system. Of course, the pre-column packing needs to be replaced frequently.
X. I recently changed the ODS column of another grade. Although the separation is still possible, the retention time cannot be reproduced. Why?
A: This is because the analyte may have the ability to form hydrogen. Although the manufacturing technology of fillers has been greatly improved over the past few years, the concentration of silanol groups on the surface of ODS fillers of different manufacturers is different. It is these silanol groups that may interact with the sample. Therefore, the relative retention times of the components in the same analyte on different grades of ODS columns may be different. The addition of a small amount of competitor, such as triethylamine (TEA), to the mobile phase will saturate the bonding ability of the silanol groups, thereby ensuring good reproducibility of relative retention times on different grades of columns.
11. When I purchased the HPLC column acceptance test, the column pressure was too high. Why?
A: Excessive column pressure is the most common problem encountered by HPLC column users. There are many reasons for this, and it is often not a problem with the column itself. You can check the cause of the problem by following the steps below.
1. Remove the guard column and see if the column pressure is still high. Otherwise, it is a problem of protecting the column. If the column pressure is still high, check again.
2. Remove the column from the instrument to see if the pressure drops. Otherwise, the line is blocked and needs to be cleaned. If the pressure drops, check again.
3. Connect the inlet and outlet of the column to the instrument and flush the column with 10 times column volume of mobile phase. (Do not connect the detector at this time to prevent solid particles from entering the fluidity). At this time, if the column pressure still does not drop, check again.
4. Replace the column inlet sieve plate. If the column pressure drops, it means that your solvent or sample contains particulate impurities. It is these impurities that block the sieve plate and cause pressure rise. If the column pressure is still high, please contact the manufacturer. In general, an inline filter between the injector and the guard column avoids the problem of excessive column pressure. The Rheodyne Model 7315 filter from SGE is the best choice for this problem.
12. How long is the service life of high temperature capillary columns?
A: The life of the capillary column depends on the performance of the column itself, depending on the use, such as the temperature of use, the state of the sample, the amount of injection, etc. If the sample is clean within its temperature range, the column is not In the case of pollution, the life of the column is generally between 2-3 years.
13. If the capillary column is contaminated, the efficiency and resolution are reduced. Is there any way to recover it? Can it be solved by aging?
Answer: According to the pollution degree of the column, different methods can be used to solve the problem. If the pollution is not serious, the boiling point of the pollutant is not too high and can be solved by aging, but the aging temperature cannot exceed the maximum use temperature of the column, and generally it takes a long time ( 8-30 hours), if the pollution is serious, or the aging can not restore the performance of the column, it must be cleaned by solvent, usually by using 5 times column volume of solvent (such as n-pentane, dichloromethane, etc.) column. Of course, the more the cleaning solvent is used, the greater the damage to the performance of the column. After the cleaning, the carrier gas is aged for a certain period of time, and if the column performance is restored, it can be used continuously. It must be pointed out that only the cross-linking column can be cleaned. For non-cross-linked columns, the cleaning column will completely fail because the fixing solution is washed away. For the choice of cleaning solvent, refer to the instruction manual.
14. Can the BPX70 capillary column be used for GC/MS analysis?
A: It is completely possible. BPX70 is a polar column. It has a wide temperature range (25-260°C), a temperature programmed up to 290°C, and low loss. It is suitable for analyzing various positions and geometric isomers of fatty acid methyl esters. Structure, carbohydrates, etc. Because BPX70 is cross-linked, it is very stable for GC/MS and can be cleaned and regenerated after contamination.