FAQs About HPLC Column

HPLC Columns

High-performance liquid chromatography (HPLC) method has become an important separation and analysis technology in the fields of chemistry, medicine, industry, agronomy, commodity inspection, and legal inspection.

Hawach is a well-known enterprise engaged in the production of laboratory products for more than ten years. We produce HPLC columns and other various laboratory supplies. HAWACH HPLC columns include the Xchroma series and the Echroma series. Due to the use of different polar modification techniques, the polarities of the stationary phases are significantly different and are widely used for the separation of various hydrophilic and polar compounds. Xchroma series columns use unique polar modification technology, which can provide selectivity different from conventional C18 columns, especially for the separation of compounds with very similar chemical structures, which has unique advantages.

Based on years of experience, HAWACH has summarized 30 FAQs about the HPLC column.

In view of the increasingly widespread use of HPLC in the entire industry, every laboratory analyst should be proficient in applying HPLC and should be able to analyze some common faults in order to solve problems and improve work efficiency even when running faults.

FAQs

Q1: What are the reasons for the retention time drifting or changing rapidly?

On the issue of drift:

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 and we should prevent it from evaporation, reaction, etc.;
3. The column is not well balanced, and the column needs to be balanced for a longer time.

About rapid changes

1. Regarding the change in flow rate, the operator needs to reset the flow rate to keep it stable;
2. There are air bubbles in the pump, which can be expelled by exhausting and other operations;
3. The mobile phase is not suitable. The solution is to change the mobile phase or mix the mobile phase in the control room.

Q2: What are the reasons for tailing or double peaks?

1. The sieve plate is blocked or the column is invalid: the solution is to reversely wash the column, replace the sieve plate or replace the column;
2. There are interference peaks. To solve it, the operator needs to use a longer column; change the mobile phase or replace the column with good selectivity;
3. The column may be overloaded and reduce the injection volume.

Q3: What are the main reasons and solutions for insufficient HPLC sensitivity?

1. The sample volume is insufficient: the solution is to increase the sample volume;
2. The sample did not flow out of the column. The mobile phase or column can be changed according to the chemical nature of the sample;
3. The sample does not match the detector. Adjust the wavelength or change the detector according to the chemical nature of the sample;
4. Too much attenuation of the detector. Just adjust the attenuation;
5. The time constant of the detector is too large. The solution is to reduce the time parameter;
6. Pollution of the detector pool window. The solution is to clean the pool window;
7. There are bubbles in the detection pool. The solution is to exhaust air;
8. The pressure measuring range of the recorder is inappropriate. Just adjust the voltage range;
9. Mobile phase flow is not suitable. Just adjust the flow rate;
10. The detector and recorder exceed the calibration curve. The solution is to check the recorder and detector and make the calibration curve again.

Q4: When doing HPLC analysis, the column pressure is unstable, what is the reason? How to solve it?

1. Proportional valve fails: just replace the proportional valve;
2. The pump seal is damaged: just replace the seal;
3. The bubbles in the solvent: the solution is to degas the solvent, and change the degassing method if necessary;
4. Check the leaks systematically, find the leaks, and seal them;
5. Gradient elution and pressure fluctuation are normal at this time.

Q5: The operator replaced another grade of the ODS column, but the retention time cannot be reproduced, why?

This is because the analyte may have the ability to form hydrogen bonds. Although the manufacturing technology of fillers has greatly improved in 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 of the same analyte on different ODS columns may be different. Adding a small number of competitors in the mobile phase, such as triethylamine (TEA), will saturate the bonding ability of the silanol group, thus ensuring that the relative retention time on different grade columns has good reproducibility. If the separation situation is ok, the system is stable and the system suitability requirements are met, so there is no need to reproduce the retention time.

Q6: Why is the column pressure too high during the HPLC column acceptance test?

Too high column pressure is a problem often encountered by HPLC column users. There are many reasons, and it is often not the problem of the pillar itself. You can check the cause of the problem by following the steps below.

1. Remove the protection column to see if the HPLC column pressure is still high, otherwise, it is a problem for the protection column.
2. Remove the HPLC chromatographic column from the instrument to check whether the pressure drops. If not, the pipeline is blocked and needs to be cleaned. If yes, check again;
3. Connect the inlet and outlet of the column to the instrument in reverse and rinse the column with 10 times the volume of the mobile phase (do not connect the detector at this time to prevent solid particles from entering the flow cell). At this time, if the column pressure still does not drop, check again, which is only for the used column.
4. Replace the sieve plate at the HPLC column entrance. If the column pressure drops, it means the solvent or sample contains particulate impurities. It is these impurities that block the sieve plate and cause the pressure to rise. If the column pressure is still high, please contact the manufacturer. Under normal circumstances, an online filter can be connected between the injector and the HPLC guard column to avoid the problem of excessive column pressure.

Q7: Will the mobile phase in the column drain?

Many people doing chromatographic separation experiments have encountered such a situation that the mobile phase in the solvent bottle is sucked dry by accident and the mobile phase in the solvent bottle is inadvertently replenished, and the HPLC system stops working. Will this damage the column? Has the pump drained all mobile phases from the HPLC column? Can the HPLC column be used? In fact, if the pump dries the mobile phase, it will not cause damage to the column. Even if the pump is filled with air, the pump will not exhaust the air into the HPLC column. Because the pump can only deliver liquid, not air.

In contrast, another situation that is more likely to happen is to forget to close the sealing caps on both ends of the column or the cap is too loose and the HPLC column dries. Similarly, it is not easy for the entire column to dry up. It is possible that only a few millimeters at both ends of the column have dried out. It takes a long time for the column to dry out because all the solvent has evaporated.

You can try to flush the column with a completely degassed solvent with low surface tension (such as methanol degassed with helium) to remove the air. Lower surface tension helps to wet the surface of the filler; the degassed solvent should be able to dissolve and remove the air trapped in the filler. The HPLC column takes approximately one hour or more (at a flow rate of 1 ml /min) to be thoroughly infiltrated and returned to normal.

Q8: What are the causes and solutions of baseline drift?

Cause analysis

1. Column temperature fluctuates. Even a small temperature change will cause the baseline to fluctuate. It usually affects differential detectors, conductivity detectors, lower-sensitivity UV detectors, or other photoelectric detectors.
2. The mobile phase is not uniform.
3. The flow cell has air or is contaminated.
4. The detector outlet is blocked. The high pressure causes the flow cell window to rupture, producing a noise baseline.
5. Improper matching of mobile phase or change of flow rate.
6. Slow column equilibrium, especially when the mobile phase changes.
7. Mobile phase pollution, deterioration, or made up of low-quality solvents.
8. Substances with strong retention (high K’ value) in the sample. The sample is eluted, thus showing a gradually rising baseline.
9. The use of recycled solvents is not recommended. The detector is not adjusted.
10. The detector is not set at the maximum absorption wavelength.

Solution

1. Control the temperature of the column and mobile phase, and use a heat exchanger before the detector.
2. Use HPLC-grade solvent, high-purity salt, and additives. The mobile phase is degassed before use, and online degassing or helium degassing is used during use.
3. Flush the flow cell with methanol or other strong polar solvents. If necessary, 1N nitric acid can be used. (Do not use hydrochloric acid).
4. Remove the obstruction or replace the tube. Replace the flow cell window, according to the User Manual.
5. Change the ratio or flow rate. To avoid this problem, periodically check the mobile phase composition and flow rate.
6. Rinse with a medium-strength solvent. When changing the mobile phase, rinse with 10-20 times the volume of the new mobile relative HPLC column before analysis. When using ion-pair reagents and buffer salts, attention should be paid to the equilibration HPLC column.
7. Check the composition of the mobile phase. Use high-quality chemical reagents and HPLC-grade solvents to change the analysis conditions. Use guard HPLC columns, if necessary, periodically flush the HPLC column with a strong solvent between injections or during analysis.
8. Reset the baseline. Use a new mobile phase.
9. Adjust the wavelength to the maximum absorption wavelength. Reselect the detection wavelength.

Q9: What are the reasons for regular baseline noise?

Causes

1. There is air in the mobile phase, detector, or pump (sharp peak).
2. Leakage.
3. The mobile phase is not completely mixed.
4. Temperature influence (column temperature is too high, the detector is not heated).
5. There are other electronic devices on the same line (accidental noise).
6. Pump vibration.

Solution

1. Deair mobile phase. Flush the system to remove air from the detector or pump.
2. Check whether the pipe joint is loose, whether the pump is leaking, and whether there is salt precipitation and abnormal noise. If necessary, replace the pump seal.
3. Shake by hand to mix evenly or use low-viscosity solvent.
4. Reduce the difference or add a heat exchanger.
5. Disconnect the LC, detector, and recorder, check whether the interference comes from outside, and correct it. Adopt precision-grade regulated power supply.
6. A pulse damper is added to the system.

Q10: What are the reasons for irregular baseline noise?

Causes

1. Leakage.
2. The mobile phase is contaminated, deteriorated, or made up of low-quality solvents.
3. The solvents in the mobile phase are not miscible.
4. There are problems with the electronic components of the detector/recorder
5. There are bubbles in the system.
6. There are bubbles in the detector.
7. Pollution of the flow cell (Even a few pollutants will generate noise.)
8. Insufficient energy of the detector lamp.
9. Loss or blockage of HPLC chromatographic column packing.
10. Uneven mixing of mobile phase or abnormal working of the mixer.

Solution

1. Check whether the joint is loose, whether the pump is leaking, and whether there is salt precipitation and abnormal noise. If necessary, replace the seal. Check whether the flow cell leaks.
2. Check the composition of the mobile phase.
3. Choose mobile phases that are miscible.
4. Disconnect power to the detector and recorder, check, and correct.
5. Clean the system with a strong polar solution.
6. Clean the detector and install a background pressure regulator behind the detector.
7. Clean the flow cell with 1N nitric acid (no phosphoric acid).
8. Replace the lamp.
9. Replace the HPLC column.
10. Repair or replace the mixer. When the mobile phase does not follow the gradient, it is recommended not to use the mixing device of the pump.

Q11: What are the reasons for retention time drift?

There are two different situations for retention time that does not reproduce: retention time drift and retention time fluctuation. The former means that the retention time only changes in one direction, while the latter means that there is no fixed fluctuation in retention time. Separating these two situations is often helpful in finding the cause of the problem.

For example, retention time drift is often caused by HPLC column aging, which is unlikely to cause irregular fluctuations in retention time. In fact, most of the reasons for the retention time drift are column aging of different mechanisms, such as loss of stationary phase (for example, through hydrolysis), HPLC column contamination (caused by the sample or mobile phase), etc.

Several common reasons for retention time drift are as follows:

1. HPLC Chromatographic column equilibrium

   If we observe retention time drift, we should first consider whether the HPLC column has been completely equilibrated with the mobile phase. Usually, 10-20 column volumes of mobile phase are required for equilibration, but if a small number of additives (such as ion-pair reagents) are added to the mobile phase, it takes a considerable amount of time to equilibrate the HPLC column.
   Mobile phase contamination may also be one of the reasons. A small number of contaminants dissolved in the mobile phase may slowly accumulate on the HPLC column, causing drift in retention time.

2. The stability of the stationary phase

   The stability of the stationary phase is limited, even if it is used within the recommended pH range, the stationary phase will slowly hydrolyze. For example, the silica gel matrix has good hydrolytic stability at pH 4.
   The rate of hydrolysis depends on the type of mobile phase and the ligand. Bifunctional ligands and trifunctional ligands are more stable than monofunctional ligands; long-chain bonds are more stable than short-chain bonds; alkyl bonds are more stable than cyano bonds. Cleaning the column frequently will also accelerate the hydrolysis of the HPLC column stationary phase. Other silica-based bonded phases can also undergo hydrolysis in aqueous solutions, such as amino bonding.
   

3. HPLC Chromatographic column pollution

   Another common cause of retention time drift is column contamination. HPLC chromatographic column is a very effective adsorption filter, which can filter and adsorb any substance carried by the mobile phase. Sources of contamination can be the mobile phase itself, the mobile phase container, connecting tubes, pumps, injectors and instrument seals, and samples. The source of pollution can usually be determined through experiments.
   If there is a strong component remaining on the HPLC column in the sample, it may be a potential source of retention time drift. These roots are usually the sample matrix. A simple way to avoid column contamination is to prevent it from happening. A strong solvent under the given chromatographic conditions is usually used, but not all contaminants can be dissolved in the mobile phase. Using guard columns is a very effective method. Backflushing the column is only a last resort.

4. Composition of the mobile phase

   The slow change in mobile phase composition is also a common cause of retention time drift. For example, the volatilization of volatile components in the mobile phase is equal to the circulating flow.

5. Hydrophobic collapse

   When a reversed-phase packing HPLC column with a small pore size and good end-group sealing uses nearly 100% water as the mobile phase, sometimes a sudden loss of separation occurs and the retention of the analyte is significantly reduced or not retained at all. This is called hydrophobic collapse.
   This phenomenon is caused by the mobile phase not infiltrating the surface of the stationary phase. The salvage method is to infiltrate the stationary phase with a mobile phase containing a large number of organic components and then balance it with a mobile phase with high water content.
   This phenomenon can also occur with long-term column storage. The use of reversed-phase HPLC columns with embedded polar groups (such as Waters SymmetryShield RP HPLC columns) or non-end-sealed HPLC columns (such as Waters Resolve HPLC columns) can also avoid collapse.

Q12: Why are there shoulders or forks?

1. The sample volume is too large: use the mobile phase to prepare the sample, the total sample volume is less than 15% of the first peak;
2. Sample solvent is too strong: use a weak sample solvent;
3. The HPLC column collapses or forms a short-circuit channel: replace the HPLC chromatographic column, using weaker corrosive conditions;
4. The sintered stainless steel in the HPLC column fails: replace the sintered stainless steel, add an online filter, and filter the sample;
5. The injector is damaged: replace the injector rotor.

Q13: Why is there a ghost peak?

1. Residual peaks of the injection valve: clean the valve with a strong solvent after each use to improve the cleaning of the valve and the sample;
2. Unknown in the sample: processing the sample;
3. The column is not equilibrated: re-equilibrate the HPLC column, using the mobile phase as the sample solvent (especially ion pair chromatography);
4. Trifluoroacetic acid (TFA) oxidation: freshly prepared every day, with antioxidants;
5. Water pollution (reverse phase): check the water quality by changing the equilibrium time, and using HPLC-grade water.

Q14: Why is there peak tailing?

1. Column overload: reduce the sample volume, increase the column diameter, and use a higher capacity stationary phase;
2. Peak interference: clean the sample and adjust the mobile phase;
3. The role of silicon hydroxyl group-add triethylamine: passivate the HPLC column with alkali, increase the concentration of buffer or salt, lower the PH value of the mobile phase, and purify the sample;
4. The sintered stainless steel in the HPLC column fails: replace the sintered stainless steel, add an online filter, and filter the sample;
5. The column collapses or forms a short-circuit channel: replace the HPLC chromatographic column, using weaker corrosive conditions;
6. The dead volume or the volume outside the column is too large: the connection point is reduced to very low. Make appropriate adjustments to all the connection points, and use the connection pipe with a thin inner diameter as much as possible;
7. Decreased column efficiency: replace columns with lower corrosion conditions and use protective columns.

Q15: In addition to the flow rate, what other factors can cause pressure changes?

There are four factors that will bring a change of pressure. Change the composition and temperature of the mobile phase; change the column length, the HPLC column inner diameter, and the particle size of the column; the column’s sudden blocking pressure increases (normally, the column pressure increases gradually with other conditions unchanged).

Q16: What are strong solvents and weak solvents?

The peak capacity factor and retention time can be changed by changing the composition of the mobile phase or the strength of the solvent. Under certain conditions, the solvent that reduces the retention time or shortens the analysis time is a strong solvent, and the solvent that increases the retention time or extends the analysis time is a weak solvent.

Q17: How can the peak position be rearranged?

When analyzing multi-component samples, only the strength (composition percentage) of the mobile phase is changed without changing its composition. Generally, only the retention time of all components is changed, and the rearrangement of peak positions does not occur.

The rearrangement of the peak position may occur under the following conditions: the mobile phase is replaced with a strong solvent; the pH value is changed; the column packing is changed; the column temperature is changed; the composition of the mobile phase is changed (such as the addition of ion pair reagent triethylamine, etc.).

Q18: In addition to online degassing, what are the commonly used laboratory degassing methods?

Heating and reflux degassing effects are ideal, but they cannot be maintained; helium degassing has a good degassing effect and can remove more than 90% of the air, but helium is too expensive, so it is not used much; vacuum degassing effect is second only to helium degassing, but it is easy to cause volatile loss of sample solution during degassing; ultrasonic degassing can only remove about 30% of the air, but it is generally used in the laboratory. At present, we still strive to use online degassing, which is convenient and effective.

Q19: Occurrence and treatment of chromatographic double peaks

In HPLC analysis, when the chromatographic column is normal, the sample sensitivity is sufficient, the analysis method is appropriate, and the peak shape should be symmetrical and sharp under the condition that the peak time is short (excluding gradient).

However, when the sample is not well understood, the method is improper, the sample processing method and the injection method are unreasonable, various unexpected problems will occur, and it is difficult to make a reasonable interpretation of the chromatographic peak, especially for novices. The chromatographic double peak refers to a substance that clearly appears, but a double peak appears in the chromatogram, indicating that it contains two substances. There are four reasons for this situation.

1. Chromatography column

If you analyze the sample and find that there are double peaks in each chromatographic peak (the faster the peak, the possibility of double peaks will be reduced), especially when using a single pure substance, you can be sure that the column has a problem: the column head is damaged or the column head stationary phase dirty or lost.

If the injection volume is small, the original chromatographic column is normal, and the shape of the chromatographic peak is mostly a large peak with a small peak, not necessarily tailing, which should generally be the column head is blocked. Connect the column in reverse and rinse or pickle with a mobile phase or other solvents to wash away the residue that is stuck in the column head, and then reverse. In general, it will do.

And sometimes the recoil is normal. If the peak is tailed, the difference between the strength of the two peaks is not large, the column head stationary phase is dirty or more likely to be lost. At this time, the injection head can be unscrewed, the microporous filter can be sonicated, and the column header can be scraped off a part of the packing. Fill it with new packing and tighten it, but this kind of work requires a certain technique, and it cannot do frequently. Otherwise, it will be scrapped due to low efficiency.

2. Solvent polarity and injection volume

The general HPLC books and literature will not mention this content, and this is indeed a very important reason for the occurrence of double peaks. At present, HPLC analysis is mostly reversed-phase chromatography, and the mobile phase is mostly methanol, acetonitrile, water, and various additives which are added to improve separation performance. The sample is generally dissolved in a solvent that is compatible with the mobile phase. The recommended dissolution method is to dissolve with the mobile phase, but in many cases it is inconsistent.

When using reagents with strong polar strength of solvents, such as pure methanol, pure acetonitrile, and pure ethanol, and the analysis system is mainly water, the sample injection volume is large, such as the 20ul quantitative tube. Under this condition, it is completely certain pure materials produce double peaks, the second peak is smaller than the first peak (not the same every time), and the tail. The retention time will be advanced (relative to the small injection volume), reducing the injection volume by more than half, and the peak will become normal.

This is caused by the polar difference between the solvent of the sample and the mobile phase, and the mobile phase is too late to dilute it to equilibrium. One of the reasons mentioned above is that the injection volume causes double peaks. The other reason is that the injection volume is not necessarily large, but the absolute volume is large. The double peaks on the chromatogram are close together, basically at the same height, without tailing (If the peak appears quickly, it may be a column problem). It is sufficient to dilute the sample and re-inject it. This is due to the excessive injection volume and the overload of the column.

3. Characteristics of the sample

Some samples have tautomerism due to the chemical structure, and this tautomer cannot be separated but exists in a dynamic equilibrium. In the chromatographic analysis, under a specific condition, a substance will have double peaks, which are close together, basically high, with no tailing. The conditions change slightly, especially pH, and the double peak phenomenon will disappear, such as Erythromycin, etc. Some samples can not see double peaks on the ultraviolet chromatogram, but under LC-MS, using a mass spectrometer, the total ion current of the mass spectrum is more obvious, for example, the pesticide acetamiprid ).

4. Parameters

The recorded parameters are generally internal and do not need to be modified, but the parameters of GC and HPLC are not completely consistent. For example, the general recording time interval on the C-R3A data recorder is 2ms for GC and 5ms for the HPLC column. One peak will become two or more peaks.

Q20:  What are the basic indicators for evaluating a column?

The basic indicators for evaluating a chromatographic column are the number of plates, peak asymmetry factor, column pressure drop, applicable range and concentration of bonded phase, and peak capacity.

Q21: Why is there peak broadening?

1. The sample volume is too large: use the mobile phase to match the sample, the total sample volume is less than 15% of the first peak;
2. Cause peak expansion in the injection valve: discharge bubbles before and after injection to reduce diffusion;
3. The sampling rate of the data system is too slow: the set rate should be greater than 10 points per peak;
4. The time constant of the detector is too large: set the time constant to 10% of the half-width of the first peak of interest;
5. The viscosity of the mobile phase is too high: increase the HPLC column temperature and use a low-viscosity mobile phase;
6. The volume of the detection pool is too large: use a small volume pool to remove the heat exchanger;
7. Retention time is too long: increase the content of strong solvent during isocratic elution, gradient elution can also be used;
8. The external volume of the HPLC column is too large: reduce the connecting pipe diameter and connecting pipe length to very small;
9. Sample overload: enter small concentration and small volume samples.

Q22: Why do peaks sometimes appear during the experiment?

The mobile phase used has absorption at the detection wavelength, and if the solution has no absorption at this wavelength or absorbs lower than the mobile phase, caves will appear in the mobile phase and peaks will appear after passing through the HPLC column.

Q23: Why are there “fat” peaks and “flat” peaks? How to avoid it?

Injecting a large volume of sample with a strength greater than that of the mobile phase usually damages the quality of the chromatogram and results in “fat” and flat peaks. The following rules should be followed to choose a solvent to dissolve the sample:

1. Dissolve the sample with the mobile phase and inject it.
2. Use a large volume of weak solvent to dissolve the sample. For example, reverse-phase chromatography uses water to dissolve the sample for injection. The main disadvantage is that after each injection, a large negative peak appears at the beginning of the chromatogram, and sometimes the sample peak is also affected.
3. Dissolve the sample with a strong solvent when needed.

Q24: Why does the front extension peak occur and how to solve it?

The HPLC column temperature is easy to cause the front extension peak. Some samples show the front extension peak at room temperature, and the phenomenon of the front extension peak disappears after increasing the temperature. In ion-pair chromatography, another reason for the extension of the peak is the use of the non-mobile phase as the sample solvent.

Therefore, in ion-pair chromatography, only the mobile phase is required to dissolve the sample, and the injection volume should not be too large, otherwise, it will cause pre-posted peaks or other problems. In RP-HPLC, the strength of the sample solution is greater than that of the mobile phase, which causes a prolonged peak. Increasing the strength of the mobile phase, reducing the strength of the sample solution, and increasing the ionic strength in the ion-pair chromatography can overcome the effect of prolonged peaks. In addition, using a mobile phase to dissolve samples is a simple and practical method.

Q25: How to simply judge whether the proportional valve is leaking?

Set the pump to use a separate channel, open the purge valve at a flow rate of 5ml/min, lift the solvent filter head in other solvent bottles until it leaves the liquid level, and observe whether the solvent in these channels is flowing, and should not flow under normal conditions

Q26: What are the reasons for the peak broadening?

1. The HPLC column itself degrades during use, gradually reducing HPLC column efficiency;
2. Peak width effect outside the HPLC column. A good dedicated HPLC column used in another liquid chromatography system causes a decrease in the number of plates, indicating that the new system has a great effect on peak width outside the HPLC column;
3. The chemical effect is mostly caused by the interaction between the mobile phase and the stationary phase. Changing the mobile phase can improve the broad peak.

Q27: What are the common reasons for the slow HPLC column equilibrium?

The common reason for slow HPLC column equilibration is that the components strongly adsorb to the column in the old or new mobile phase, or the concentration in the new mobile phase is small or even zero.

A: mobile phase contains amine modifier;
B: mobile phase contains ion pair reagent;
C: silica gel HPLC column;
D: tetrahydrofuran in mobile phase.

A special HPLC column can be considered for special methods. When not in use, the HPLC column should be folded down, filled with an appropriate solvent or mobile phase, sealed, and stored, and no other analysis is required.

Q28: What are the requirements for internal standards?

1. The structure or physical and chemical properties of the internal standard should be similar or similar to the analyzed component;
2. The retention value of the internal standard should be slightly larger or smaller than the retention of the analyte, and the difference should not be too large;
3. The peak of the internal standard should have a good resolution with the peaks of all analytes (R is greater than 1.5), and the internal standard must not be an interference;
4. There is no internal standard with a similar structure and similar internal standards can be retained;
5. The response of the instrument to the analyte is basically the same as the internal standard, and the size of the peak area cannot be very different.

Q29: What is the “infinite diameter effect” of HPLC?

Due to the use of a high-efficiency particulate stationary phase and high-pressure mobile phase in HPLC analysis, after the sample is injected into the chromatographic HPLC column with a plunger, the molecular diffusion of the sample molecule in the HPLC column is very small due to the large resistance of the column until it flows out from the HPLC chromatographic column. It is not in contact with the inner wall of the HPLC column, so the peak shape expansion caused by the chromatogram is very small, which can maintain high column efficiency.

Q30: How to evaluate a detector?

A. Noise: Generally, noise refers to the high-frequency noise and irregular fluctuations of the baseline caused by the electrical components of the instrument, temperature fluctuations, linear pulses of voltage, and other non-solute effects;
B. Baseline drift: Drift is a slow upward or downward movement of the baseline, which can be observed for a long time (0.5~1h). It can mask noise and small peaks. Drift is related to the entire liquid chromatography system, not only caused by the detector;
C. Sensitivity (smaller detection concentration or smaller detection amount): In a particular separation work, whether the detector has sufficient sensitivity is very important. When comparing detectors, the performance index of sensitivity is often used. Sensitivity refers to the concentration or quality of the solute that enters the detector within a unit of time when the signal-to-noise ratio (signal-to-noise ratio) is equal to 2;
D. Linear range: When performing quantitative analysis, the detector is expected to have a wide linear range, so that the main components and trace components can be detected simultaneously in one analysis;
E. Cell volume of the detector: it should be less than 1/10 of the elution volume of the dead-time chromatographic peak that eluted earlier, otherwise severe off-column band expansion will occur.

The end. You also can choose C30 HPLC Columns, Phenyl HPLC Columns, SCX HPLC Columns, PAH HPLC Column, C1 HPLC Column, and other HPLC Columns using.