This is the Part 5 of our following series “Q&A about Liquid Chromatography” that was published before. Which will cover some more Q&A, help you to know more about liquid Chromatography

Q1. If the selectivity of liquid chromatography is in increased, what to do?

Mobile phase
(1) The type and ratio of organic solvents in the mobile phase will affect the selectivity of chromatographic separation. For reversed-phase chromatography mode, commonly used organic solvents are methanol, acetonitrile and tetrahydrofuran. The selectivity and resolution can be changed by changing the type of organic phase in the mobile phase or adjusting its ratio.

(2) The pH value of the mobile phase affects the resolution. For neutral compounds, changes in the pH of the mobile phase will not have much impact on their selectivity; for acidic compounds and basic compounds (dissociable compounds), the impact is greater.

(3) The ionic strength of the mobile phase can be adjusted by adding buffer salts to improve the retention between the target compound and the stationary phase.

Stationary phase
The type of bonded phase, the pore size of filler, the type of end capping and the particle size and size of chromatographic column will affect the results of chromatographic separation. The most important factor is the selection of bonding phase. When you can’t get good separation results with C18 stationary phase, you can consider C8 stationary phase with strong polarity, or phenylhexyl column with great difference in selectivity, or use terminated chromatographic column to replace unsealed chromatographic column.

Hardware system
Adjusting some parameters of the hardware system can also improve the chromatographic separation results to a certain extent. For example, higher sampling rate; A smaller inner diameter pipeline and a smaller flow cell are used to reduce the diffusion volume and delay volume and reduce the broadening of chromatographic peaks; Or improve the resolution by increasing the column temperature of the chromatographic column.

Q2: What if the peak area of the sample becomes smaller?

1. The response of all components changed to approximately the same extent, which may be the reason
(1) Injection problem: check the sample bottle and injection needle to ensure that the sample concentration and injection volume do not change.
(2) Detector problem: check all parameter settings of the detector. If the baseline noise is large, it is usually not the detector but the system is polluted.
(3) Whether the component structure is stable and may degrade during injection.
(4) Whether the chromatographic conditions have changed, such as mobile phase ratio, pH value, etc.
(5) When the chromatographic column changes, the selectivity changes, and the effect value will also change.

2. Only part of the component response decreased
(1) Check whether they have similar volatile or functional groups. For different types of samples, the inspection angle can be different: samples with polar functional groups (such as – Oh, – NH, – SH) are easy to be adsorbed, and the polluted system can be simply maintained.
(2) Whether the properties of these components are stable and whether degradation occurs in the detection process.

Q3: When the same sample is injected repeatedly,Why does the peak area fluctuate?

There are many reasons for this result:

If the sample is stable, first eliminate the fault of the instrument, bubble is the most important kind, the generation of bubble will lead to the irregular change of volume between the samples.

If the concentrations vary greatly between samples and the peak area of the same peak remains constant after 2-3 injections, it may be sample residue.

The decrease in peak area may also be caused by temperature, but the effect is very small, less than 2%. If you take the sample out of the refrigerator and put it in the sampler, it will slowly heat up and get bigger. In this way, the injection volume before and after is different.

The random change of flow velocity will also lead to the change of peak area. Another potential cause of flow rate changes is leakage at the high pressure part of the system, which should be easy to spot.

Complex samples with a acromion (locally separated from the target peak) can make it difficult for the software to determine where the baseline is. Manual integration or automatic integration with mandatory baselines will improve the reproducibility of the integration. The sample itself also causes the change of peak area. In reversephase chromatography, some proteins are not completely elute in the initial gradient, and residues appear in the following blank gradient. These ghost peaks are only present in specific protein and elution procedures. If so, add a blank after analyzing the sample. In addition, proteins would irreversibly adhere to some columns, and the peak area of the sample would gradually increase with the increase of sample injection.

Q4: Pressure fluctuation, how to make the pressure normal?

A: Pressure fluctuation is a common problem in daily work. Generally speaking, the relationship between pressure fluctuation and chromatographic column is not large, but mainly the problem of instrument system. The following two points deserve attention:

  1. There are bubbles in the pump;
  2. Leakage of check valve or gasket.
    The solutions are as follows:
  3. Ultrasonic degassing after solvent filtration; Use on-line degasser; Open purge valve to discharge bubbles, or fill helium in solvent;
  4. Clean and replace the check valve; Replace the pump gasket.

Q5: How to store the liquid chromatographic column correctly?

1. Short-term storage of chromatographic columns. Reversed phase column: Use buffer or mobile phase containing buffer salts. After the experiment is completed, use 20-30 column volumes of methanol or a 10:90 mixture of acetonitrile and water for washing, and use a 90:10 mixture of methanol or acetonitrile and water. Perform flushing. If overnight storage is required, the flow rate can be maintained at 0.1 to 0.2 mL/min.

Normal phase column: Wash the sample with the mobile phase, then rinse the column with a high ratio of n-hexane: isopropanol, and store it.

2. Store the chromatographic column such as the chromatographic column for a long time. Must be stored in a suitable solvent. The reverse phase column can be stored in pure methanol or acetonitrile, the normal phase column can be stored in pure n-hexane after strict dehydration, and the ion exchange column can be stored in water (containing the preservative sodium azide or thimerosal). And plug the plug that was included when purchasing a new chromatographic column. For storage of special types of chromatographic columns, please refer to the column instructions and use the solvents recommended by the manufacturer.

Q6: The retention time of the liquid chromatographic peak is prolonged, how to solve it?

1. Flow rate reduction
Check and reset the flow rate; Check whether there are bubbles in the pump; Check whether the pump gasket is leaking, and whether the check valve and other systems are leaking.

2. Change of mobile phase composition
Check whether there are liquid leakage points in the system; Remove system bubbles; Replenish the solvent bottle; Ensure that the gradient system scale is correct; The premixed mobile phase was eluted in equal gradient; Mobile phase composition. In reversed phase chromatography, the retention factor K is exponentially related to the proportion of organic phase in the mobile phase. Generally, the organic phase changes by 1%, and the retention time changes by 5% – 15%, generally 10%. A pH change of 0.1 will lead to a retention time change of 10%, so the pH should be measured accurately and the pH meter should be calibrated. In reverse phase chromatography, with the increase of pH, the retention time of acidic compounds will be shortened and that of alkaline compounds will be prolonged; The concentration of ion pair reagent will affect the retention time of ionic compound components. The retention time of analytes with opposite charge to the ion pair reagent will be shortened and those with the same charge will be prolonged. The retention time of normal phase chromatography is very sensitive to the polar components in the mobile phase. In any case, water is a trouble. Non polar solvents such as n-hexane and dichloromethane with semi saturated water are used to avoid this problem.

3. Loss of bonding phase
For common silica gel chromatographic columns, keep the mobile phase between pH2 ~ 8; For very high pH (> 10) or very low pH (< 2), use high stability stationary phase, polymer or high stability stationary phase column.

4. Activation sites of silica gel fillers
Using mobile phase modifier; Competitive base is added to the mobile phase; Filler with higher coverage for stationary phase.

5. Temperature reduction
Control column temperature. Generally, the retention time changes by 1% – 2% for every 1 ℃ change in temperature. Generally, the impact is not so great. Obviously, this problem can be avoided by using column temperature box.

Q7: How to solve the problem of shortening the retention time of liquid chromatographic peaks?

1. Active sites of chromatographic column packing
Use mobile phase modifiers to compete with alkalis (basic compounds such as triethylamine) or increase buffer strength, and use high coverage column packing.

2. Change of mobile phase composition
Check whether there are liquid leakage points in the system; Remove system bubbles; Replenish the solvent bottle; Ensure that the gradient system scale is correct; The premixed mobile phase was eluted in equal gradient.

3. Sample overload
Reduce the sample size or use a larger inner diameter or longer column.

4. Loss of bonded phase or silica gel base
Use a mobile phase of mild pH; Use special silica gel columns, polymer columns or other high / low pH columns resistant to high pH or low pH.

5. Chromatographic column aging
Use protective columns, or high stability bonded phase polymers, hybrid or high stability columns.

Q8: How to check if the baseline fluctuation affects the sample test?

1. The liquid phase system is not well balanced, and the mobile phase in the column is changing all the time, resulting in baseline fluctuation;

2. For instruments without degasser, when the mobile phase is not degassed or degassed completely, spikes will appear at the baseline;

3. When the chromatographic column is polluted, it will also cause baseline fluctuation;

4. The flow cell of the detector is polluted, resulting in unsteady absorption. At this time, the flow cell needs to be cleaned;

5. When the energy of the detector lamp is insufficient, the absorption will become unstable, and the baseline is generally unstable, especially at low wavelength;

6. When using low wavelength detection, sometimes the mobile phase will use two-phase or more equivalency. At this time, the small mixing proportion error of the mixer will be amplified, which is likely to make the baseline fluctuate;

7. The cut-off wavelength of the organic phase in the mobile phase should preferably be less than the detection wavelength by more than 20 nm;

8. The laboratory environment is unstable (such as temperature rising and falling, air flow changing, etc.);

9. Problems with the instrument can also cause baseline fluctuations, in which case pressure instability usually occurs. For example, the mobile phase filter head is blocked, the filter element of the inlet active valve is polluted, the one-way valve is blocked by pollutants, the pump head has bubbles, and the system flow path leaks, such as the leakage caused by the cracking of the pipeline and the incomplete connection of the peek connector to the chromatographic column.

Q9: How much is the service life of chromatographic column?

A: The service life of the chromatographic column is about 1000 needles, while the service life of the protective column is about 280 needles. It is mainly affected by chromatographic conditions and storage conditions. Manifestations: increase of column pressure, decrease of column efficiency, tailing of peak shape, widening of peak shape, change of retention time, loss of stationary phase, etc.

1. There are two basic chromatographic conditions:
(1) In the same experiment, the chromatographic column used before has a long service life. In this case, it should be checked whether the chromatographic conditions are consistent. The composition of the sample and the strongly absorbed pollutants in the sample will destroy the column efficiency of the chromatographic column. Or whether the sealing ring of the pipeline is intact? The falling seal ring will block the top layer of column filter and packing, thus affecting the distribution of samples. If it can be confirmed that the chromatographic conditions have not changed, it can be speculated that it is the reason for the loosening of the column bed. During the laboratory and transportation, the violent vibration of the chromatographic column will lead to the loosening of the column bed;
(2) All chromatographic columns used in this experiment are damaged after the same number of times, and the most likely reason is that the components in the sample are adsorbed on the top of the chromatographic column. It may be insoluble precipitates in the mobile phase or substances with strong absorption;
(3) Handle the samples using appropriate sample preparation techniques. Solid phase extraction (SPE) was performed with a solid phase extraction column similar to the analytical column;
(4) Use protective posts. The protective column is installed in front of the chromatographic column as a sacrificial column and will be replaced in case of problems. The packing and filling technology of the protective column must be the same as that of the analytical column;
(5) Recoil column. The current filling technology can make the column bear recoil and better remove the pollutants at the column head end.

2. The use method is incorrect, and it is usually rare to use the column according to the manufacturer’s instructions
(1) The pH of the mobile phase is out of the range of use, resulting in column bond and phase hydrolysis or matrix dissolution. It also occurs when the sample is dissolved with strong acid or alkali;
(2) High temperature will also cause damage to the chromatographic column. For example, high temperature will accelerate the dissolution of the bonded phase;
(3) Special column precautions, such as amino column can react with aldehydes and ketones. The amino column will be strongly alkaline in aqueous solution without buffer salt and decompose part of silica gel;
(4) Columns exposed to the wrong solvent will also collapse. Because these columns are based on a very loose structure. They are partially bonded by the adhesion between particles. If placed in a mobile phase that can break this adhesion, the column bed is likely to collapse. This problem sometimes occurs when cyano column is in neutral solution and phenyl column is in THF. This is another reason not to wash the column.

Q10: Cause analysis of peak bifurcation

1. Column contamination
No problem at the beginning, but after a period of use, this problem may be caused by pollution. At this time, it is necessary to strengthen the flushing of the chromatographic column, that is, use a stronger solvent than the method mobile phase to flush the chromatographic column.

2. The protection column fails
If the sample matrix is ​​dirty, after a period of use, problems such as peak bifurcation or tailing may be caused by the failure of the guard column. Generally speaking, if the number of plates, pressure or resolution changes by more than 10%, the guard column needs to be replaced. The guard column is a consumable, it is recommended to replace it directly without regeneration.

3. The connector is incorrectly connected
If there is a problem with the peak shape of each peak during the use of the chromatographic column, first check whether there is a connection problem. The pipeline may be too long or too short, which may cause leakage or peak bifurcation/tailing. If the pipeline is too long and the sealing ring is in the wrong position, leakage will occur. If the distance that the pipeline is pushed out is not enough, a dead space will be created, forming a mixing cavity, resulting in extra-column volume, and deteriorating peak shape.

4. Solvent effect
In reversed-phase LC, such as using 100% organic solvents or 100% strong solvents, when large-volume injections, the chromatographic peak will elute from the column prematurely, resulting in peak distortion. The injection solvent is diluted with water, which is more compatible with the mobile phase, and there will be no peak distortion even if the injection volume is large. Peaks that elute earlier or peaks near the solvent front are more prone to tailing. In liquid chromatography, it is ideal to use a small volume injection dissolved in the mobile phase.

5. Sample overload
When the injection volume exceeds the capacity of the column, the peak of the sample exhibits bifurcation or tailing, and the solution is to reduce the injection volume.

6. Collapse of the chromatographic column
The peak bifurcation caused by column collapse is difficult to repair. If the mobile phase pH>7 or long-term use near the critical point of the chromatographic column pH tolerance, it is easier to cause the silica gel packing to dissolve and collapse. If the column collapses, you will see that the shape of each peak in the chromatogram will change (peak tailing, broadening, or bifurcation), and it can run in the opposite direction in a short time. It is recommended to replace the column directly. Pay attention to lowering the temperature below 40°C during daily use, especially when using high pH mobile phases.

7. The sieve plate in front of the column is partially blocked for long-term analysis of dirty samples
If there is no guard column or on-line pre-column filter, particulate matter and strong adsorbents are likely to block the sieve plate at the column inlet, and the pressure rises at the same time. The solution is generally column backflushing.

8. The performance of the chromatographic column is degraded
When using a chromatographic column, it is used to analyze various target substances, use various mobile phases and separate different sample matrices. After a long period of use, the chromatographic column will be affected by these substances and undergo some subtle changes, which will cause peak separation. Forking, tailing, or change in retention time.

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