These are the ＂pass＂ of chromatographic column?

Did you ever think that the flow direction of the column could not be changed, the buffer could be washed clean by washing the column with pure water, that the new column stored in acetonitrile or methanol could be used directly, that the chromatographic column should be kept in pure acetonitrile or methanol...... Are these habitual thoughts right or wrong?
What is the effect of column contamination on the spectra? How to clean contaminated columns? Why is isopropanol indispensable? What is the best way to solve the problem of column contamination? The column efficiency is reduced, how to clean and regenerate?
Chromatographic column is the heart of chromatographic system. As can be seen from the importance, chromatographic problems are always multifarious. We should judge which faults are related to the chromatographic column and which have nothing to do with them. No experience? It doesn't matter. We'll have it next!
Column cleaning? Regeneration?
In this case, the column pressure may be increased or the column pressure may be decreased during column flushing.
In principle, stronger solvents than mobile phase should be used for cleaning chromatographic column, and each solvent should use at least 10 column volumes. The cleaning and regeneration steps are different for different types of chromatographic columns.
Washing steps of reversed phase silica gel column
1. Rinse (water / organic phase) with mobile phase without buffer salt
2. Then wash with 100% organic phase (methanol and acetonitrile)
3. 75% acetonitrile / 25% isopropanol
4. 100% isopropanol
5. Check whether the chromatographic column is recovered; if not, discard the column or consider the following operation
6. 100% dichloromethane*
7. 100% hexane*
Washing steps of reverse polymer column
When using polymer reversed phase column, we strongly recommend that the mobile phase contain at least 1% organic modifier. Long term use of 100% aqueous eluent followed by the use of organic mobile phase may affect the column efficiency; at the same time, 100% aqueous buffer can not be used to "flush" the column. Reverse flushing can be carried out. If the initial pressure is high, the flow rate can be reduced.
1. Use the current mobile phase to run the cleaning gradient, and then clean several column volumes with high proportion of organic solvent (95%). Repeat 2 to 3 times. Since the buffer may precipitate in the high organic phase, if your mobile phase contains buffer, the proportion of organic phase should not exceed 70%, or clean the buffer with the mobile phase without buffer before cleaning.
2. Try to use organic modifier with higher strength, such as TFA, to remove the pollutants with water drainage bonding.
3. Sometimes water / acetonitrile (V / V) containing 1.0% TFA can remove peptide or protein contaminants.
4. Clean properly with strong acid and alkali (including 1 M sodium hydroxide) without thermal degradation.
Recoil or cleaning steps of normal silica gel column:
For normal chromatographic columns, only organic solvents can be used. If your column is a traditional analytical 4.6 x 250 mm column, at least 50 ml of each solvent is required (equivalent to 20 column volume). Try the following solvent with increasing strength for cleaning:
1.50% methanol: 50% chloroform
2.100% ethyl acetate
Precautions after column cleaning and regeneration:
a. When switching to stronger solvents (such as dichloromethane and n-hexane) or switching back to the original mobile phase, it is necessary to pay attention to the compatibility of the solvents before and after switching back to the original mobile phase. If necessary, excessive use of isopropanol can avoid solvent immiscibility.
b. The buffer salt may precipitate and cause back pressure in the column. When this happens, hot water should be introduced slowly into the column to remove the precipitate.
c. In order to avoid precipitation, if the system does not contain nonvolatile buffer, flush with water phase mobile phase without buffer before pure organic solvent enters the system.
d. The washing solution containing propanol will produce higher operating pressure due to the increase of viscosity. During the cleaning process, the flow rate needs to be reduced to maintain a safe operating pressure.
e. When the system pressure is too high, sometimes the chromatographic column is not blocked, but some part of the system flow path (such as filter white head in the pump, injection needle, needle seat, capillary pipe in column temperature box, liquid phase protection column or online filter, etc.) is blocked. Therefore, when encountering the problem of high pressure, the blocking position should be checked first, and then the right medicine should be taken.
f. Do not attempt to replace the inlet sieve plate of the column. Because the high-efficiency process is used to fill the column, this may cause irreparable damage to the column.
g. Do not recoil the column when the particle size of packing is less than 1.8 μ M

How to improve the column efficiency of HPLC

How to improve the column efficiency of HPLC
To improve the efficiency of liquid chromatography, we can start from the following aspects. The following introduces several international popular methods for measuring and calculating column efficiency.
Methods of improving column efficiency of liquid chromatography
(1) The flow rate of the mobile phase was decreased, but the analysis time was prolonged.
(2) The amount of stationary phase was reduced, but the loading amount of sample in the column also decreased.
(3) If the particle size of the stationary phase is reduced, the permeability of the column will also decrease.
(4) The mobile phase with low viscosity is suitable for rapid mass transfer, but not for multicomponent analysis.
(5) The viscosity of the mobile phase can be reduced by properly increasing the column temperature, but the column efficiency and resolution will also decrease.
(6) The volume of the stationary phase is reduced as much as possible, but the velocity of the stationary phase is accelerated.
It can be seen from the above introduction that various factors are interrelated and restricted in the process of chromatographic analysis. Only through the tracking calculation of column efficiency value and continuous research and practice of their own analysis methods can we find the best working conditions.
Problems in tracking and calculating column efficiency
We should also remember that the column efficiency value is not enough to predict the column performance under all conditions. For most chromatographic workers, column performance refers to the ability of the column to be used for specific separation. High column efficiency alone does not guarantee this separation ability.
No matter what specific test method is used, several parameters will affect the determination of column efficiency. These parameters include: the composition and viscosity of eluent and its linear flow rate, the solute used to determine the number of trays, temperature, column length, packing method, particle size, and the selected measurement and calculation methods. The measurement and calculation methods play an important role in determining the column efficiency.
Several methods of measuring and calculating column efficiency
Because the chromatographic peak is the distribution of the sample band which is obtained by assuming that the sample concentration is normally distributed in the mobile phase and the stationary phase, the peak type is often regarded as the normal curve to calculate the number of theoretical plates. Therefore, the formula for calculating column efficiency (in the unit of theoretical plate number n) is conventionally defined as:
Where TR is the retention time of chromatographic peak; σ 2 is the deviation of chromatographic peak measured in time; a is the constant related to the peak height (measured from the baseline of peak width), and ω B is the peak width, which represents the distance between the two intersection points of the peak top and the inflection point on both sides of the peak and the base line of the peak bottom.
conclusion
If a chromatographic peak is really normal, then each calculation method will get the same result. However, even if some ideal instruments and solutes tend to get symmetrical peaks, the non normal peaks will appear due to the slots or voids in the column. Therefore, different calculation methods will get a large difference in the value of n. Usually, the peak pattern deviating from the normal model is expressed as "forward" or "trailing". For these peak patterns, the higher the height of the peak, the greater the calculated theoretical tray value (the lower the accuracy). In many cases, chromatographic workers need to be able to reflect the column efficiency value of the whole peak type (including tailing). At the same time, in order to ensure quantitative repeatability, chromatographic peak symmetry is also needed. At this time, the most sensitive calculation method to the asymmetry of chromatographic peaks is the most suitable. If the purpose is only to monitor the column efficiency from the first use to the end of its service life, then any of the above methods can be used, and the simplest method should be selected.
How to improve the column efficiency of gas chromatography
In practical work, through the selection of carrier gas flow rate, injection technology, gasifier temperature, chromatographic column, column temperature and detector temperature, we have effectively improved the column efficiency, made the chromatographic peak shape normal, no peak expansion, tailing, peak missing detection and other adverse phenomena, high resolution, thus improving the accuracy of the analysis results.
The column efficiency is to achieve satisfactory analysis results with short columns in a short time. In order to improve the efficiency of chromatographic column and reduce the phenomenon of expansion, tailing and missing detection of chromatographic peak, we discussed the selection of column operation conditions from the following six aspects in practical work.
Selection of carrier gas velocity
The most commonly used carrier gases for gas chromatography are hydrogen, nitrogen, argon and helium.
According to the rate theory, slow carrier gas flow rate is conducive to mass transfer and separation of components, but the analysis time will be longer; if the carrier gas flow rate is fast, it is conducive to accelerate the analysis speed, reduce the diffusion of components, but the degree of separation is reduced. Sometimes in order to shorten the analysis time, increase the flow rate, but the separation effect is not good at this time. It can be seen that the speed of carrier gas flow will reduce the column efficiency. After a long time of experiment, it is found that the carrier gas flow rate is 20-100ml / min for general chromatograph. At present, we use thermal conductivity detector to analyze liquefied gas, hydrogen is used as carrier gas, and its flow control is 30 ml / min. Hydrogen flame ionization detector is used to analyze pentane foaming agent, and nitrogen, combustion gas, hydrogen and oxygen are used as carrier gas. The volume ratio of these three gases is nitrogen: hydrogen: oxygen is 1:1:10.
Selection of injection technology
In gas chromatography analysis, syringe or six way valve injection is generally used. When considering the injection technology, we mainly focus on syringe injection.
Injection volume: if the injection volume is large in the injection process, the separation degree is small, the retention value change is difficult to be qualitative, and the peak height and peak area are not linear with the injection volume, and can not be quantified. The injection volume is related to gasification temperature, column capacity and linear response range of the instrument. The injection volume should be controlled in the instantaneous gasification to meet the specified separation requirements and the allowable range of linear response. The instantaneous injection volume of packed column flushing method: the liquid sample or solid sample solution is generally 0.01 ~ 10 μ L, and the gas sample is generally 0.11 ~ 10 ml. in the quantitative analysis, attention should be paid to the accurate reading of the injection volume.
Elimination of air in syringe: take liquid sample with micro syringe, as long as the liquid is repeatedly pumped into the syringe and quickly discharged back to the sample bottle, the air can be eliminated. Another better method is to replace the syringe 3-5 times with about 2 times of the planned injection volume. After each sample is taken, lift the syringe vertically with the needle tip upward. The air left in the syringe should run to the top of the syringe. Push the syringe plug, and all the air will be discharged.
Ensure the accuracy of injection volume: use the replaced syringe to take about 2 times of the planned injection volume of the sample, pick up the syringe vertically with the needle tip upward, and let the needle pass through a layer of gauze. In this way, the gauze can absorb the liquid discharged from the needle tip. Push the syringe plug until the desired value is read. Dry the tip of the needle with gauze. At this point, the accurate liquid volume has been measured, and it is necessary to pump air into the syringe. If the plunger is pushed carelessly, the air can protect the liquid from being discharged.
Injection technique: hold syringe in both hands. Use one hand (usually the left hand) to hold the needle into the gasket to inject a large volume sample (i.e. gas sample) or when the pre column pressure is extremely high, it is necessary to prevent the pressure from the gas chromatograph injector to pop out the injector piston (i.e., press the top of the piston with the thumb of the right hand). Let the needle tip pass through the gasket as deep as possible into the injection port, press down the syringe piston, hold for 1 second, and then extract the needle tip as quickly and stably as possible (continue to press the syringe piston while pulling out).
Injection time: the injection time has a great influence on the column efficiency. If the injection time is too long, the chromatographic region will be widened and the column efficiency will be reduced. Therefore, for flushing chromatography, the shorter the injection time, the better, generally less than 1 second.
Selection of gasification chamber temperature
The temperature of the gasifier depends on the chemical and thermal stability of the sample, the boiling range, the type of injection port, etc. A suitable temperature in the gasifier can keep the sample completely gasified instantly without causing sample decomposition. When the temperature is too low, the gasification speed is relatively slow, resulting in irregular peak shape, flat head peak or tongue extending peak; if the temperature is too high, the number of peaks will change, leading to the generation of forward extended peaks, and even sample decomposition. In order to select the appropriate temperature of the gasifier, we found that the temperature of the gasifier is 50-100 ℃ higher than the column temperature or 50-70 ℃ higher than the highest boiling point of the sample components. If the temperature is too high or too low, the column efficiency will be affected.Netizens' advice
Generally, the column efficiency is not good, first eliminate other problems, if there is no problem, the column is not good.
If the column is relatively old, you can reverse the small flow rate to wash, I have done the effect is very good! (only for old columns, a little risky).
The effect is very good by digging out the filler with color change on the column head and filling it with new filler of the same kind.
If the dead volume in the column increases, the new column should be replaced.
Chromatographic column is a consumable. Theoretically speaking, the chromatographic column with low column efficiency can not be saved. But there is a trick. When you find that the chromatographic column is not saved, you can use it. It is the backwash column. However, you should pay attention to that the flow rate should not be set too high, so that it can be persisted for a while. If that doesn't work, you can buy it again.
If the column is really old, try reverse flushing. It may still be used for a period of time, but not too long. After that, you can only replace the filler or the new column.
We were not good at the column effect some time ago, others are quite normal. Later, the column was reactivated, and the column efficiency increased from 2000 to 4000. But it didn't work after a month.
summary
The temperature of the first column can be increased, and the second column with small inner diameter can be selected. The third column is smaller and better, and the fourth is ultra pure silica gel. The fifth deuterium lamp should choose high energy and the sixth one should use hollow light transmitting lamp. Seventh, the pH of the eighth mobile phase can be improved by changing the organic phase ratio. The ninth is to select the bonding phase, and the tenth is to change the organic additives. The detection time is shortened by eleven matching and the response time is changed.
For more related technologies, please consult MS technologies technicians