Q&A for Liquid Chromatography(Part-4)

This is the Part 4 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

1

Q: How can we avoid tailing?

A: First of all, we need to find out the cause of trailing, which is usually caused by the following reasons: expansion of external interface volume, packed bed pollution and interaction between analyte and bonding active site, which needs to be treated separately according to different reasons.

2

Q:How to check the cause of tailing?

A: The chromatogram can provide many clues without knowing the nature of the sample or the conditions of the chromatogram, and the remaining conditions can be used to verify the guess based on the chromatogram. First check the peak height and observe whether the column is overloaded under this chromatographic condition. In order to confirm whether it is overloaded, the sample with 1/10 concentration can be added to see whether the peak pattern is improved. If trailing remains at low concentrations, look at the chromatogram. If there are many peaks in the chromatogram, see whether each peak pattern maintains a certain trailing degree or has a consistent trend over time. If the peaks at the front of the chromatogram are trailing more than those at the back, consider the effect of out-of-column effects. If all peaks in the chromatogram have the same trailing degree, then there are two possibilities: 1) packed bed damage, 2) all samples in the chromatogram have similar chemical structure and trailing is caused by chemical effect.

3

Q: Which substances produce these chemical effects, can you explain? How to deal with it?

A:

There are several chemical effects, the most common of which is the interaction of analytes with heterogeneous active surfaces. The typical is the tailing of basic compounds in the reversed-phase column. Usually, compounds with polar or basic groups such as – COOH, – NH2, – NHR, – NR2 can have secondary adsorption with the residual silica hydroxyl and bonding phase on the packing surface, resulting in tailing.

Solutions:

1) For the analysis of basic compounds, triethylamine (TEA) can be added to the mobile phase as a tail reducing agent. TEA competes with the basic compound to bind the silica hydroxyl group to eliminate the interaction between the analyte and the residual silica hydroxyl group.

2) Tailing of acidic compounds requires lowering the pH value of the mobile phase to protonate the acid as far as possible. Competitive organic acids can be added to the mobile phase, such as 0.1% trifluoroacetic acid (TFA), which has a better result, and this additive has a low UV cut-off wavelength.

3) Increase the concentration of buffer salts in the mobile phase to inhibit ion action.

4) Add ion pair reagent to mobile phase, and generally add 0.003-0.01mol/L ion pair reagent to reverse mobile phase to improve peak type and increase compound retention.

5) Choose high purity silica gel column and completely end capped column, such as: Welch Ultisil® Polar-RP, Xtimate® C18 etc.

4

Q: In some chromatograms, We can see the chromatographic peak fronting. What causes the front?What cause the peak front?

A: First of all, we also need to find the reasons for the peak front. The peak front usually has the following reasons: external volume, packing bed pollution and solvent effect. This requires us to find the cause and solve the problem by observing the chromatogram. Of course, in the case of overload, we also verify by reducing the sample concentration. If the low concentration is still in the front, then observe the chromatogram. If there are many peaks and all peaks in the chromatogram, see whether each peak type maintains a certain front degree or has a consistent change trend with time. If the front peak in the chromatogram is more powerful than the rear peak front, The influence of out of column effect or solvent effect can be considered. If the leading edge degree of all peaks in the chromatogram is consistent, it may be caused by packing bed damage or the properties of sample substances in the chromatogram.

5

Q: How do you solve the peak front?

A:

1) For the peak front caused by solvent effect, in reversed-phase LC, if 100% organic solvent or 100% strong solvent is used, the chromatographic peak will be washed out of the chromatographic column prematurely during large volume injection, resulting in peak deformation,which can be avoided by using peak shape frontier inhibitor. In liquid chromatography, small volume injection dissolved in mobile phase is the most ideal. Or dissolve the sample with mobile phase or solvent with similar polarity to the mobile phase. If strong solvent must be used for dissolution, it is necessary to reduce the injection volume.
2) The peak front caused by the out-of-column effect, we need to reduce the dead volume of the instrument system, and then solve the front phenomenon.
3) For the peak front caused by the nature of the sample, it can be considered to increase the concentration of buffer salt in the mobile phase, increase the ionic strength in the mobile phase, reduce the front caused by electrostatic action, or add an appropriate amount of tetrahydrofuran in the mobile phase (usually within 5%). Of course, raising the column temperature is also a good choice.
4) Column vortex
The gap generated by the packing makes the flow rate of mobile phase and solute move faster than the average flow rate, resulting in peak tailing or extension. The cause of voids is improper filling or collapse of packed column bed.
5) False front
The two substances are not separated, but there is a certain separation tendency.

Peak front case analysis: C18, the mobile phase is water-methanol (55:45), the reference substance and sample peaks are all forwarded?

1) Whether the sample is overloaded. Reduce the injection concentration to see if the peak shape is improved. It is generally considered that the peak height is about 100mAU, which will not affect the peak shape due to overload.
2) Check whether the sample is dissolved in mobile phase. The elution ability of the solvent (such as pure methanol) dissolving the sample is stronger than that of the mobile phase, and the peak will be delayed. The specific mechanism is that the normal peak shape should be obtained when the sample moves forward evenly on the chromatographic column, and the concentration distribution is normal at any time during the whole process of passing through the chromatographic packing bed. The time for the sample solution to reach the chromatographic column after injection is very short, and it should not be fully diluted by the mobile phase. The local presence of sample solvent with stronger elution ability will accelerate the elution speed of some samples, resulting in peak delay.
3) Increase the concentration of buffer salt in the mobile phase. Increasing the concentration of buffer salt can increase the ionic strength in the mobile phase and reduce the forward delay caused by electrostatic action (which may exist between sample molecules or between sample molecules and packing surface).
4) Add an appropriate amount of tetrahydrofuran into the mobile phase. Adding a small amount of tetrahydrofuran to the mobile phase can sometimes improve the peak shape and increase the resolution. Many chromatographic workers know and use it, but its mechanism seems to be rarely mentioned. Usually the amount added is less than 5%, and a larger amount can be added when necessary.

6

Q: How should the liquid chromatographic column be activated?

A: For liquid chromatographic columns, each column is tested before shipment and stored in the test eluent for transportation. Therefore, in the first use, it is recommended that 80% methanol be washed with the flow rate of 1 / 2 of the test sample for 4 hours, and then the chromatographic column can be thoroughly balanced with the mobile phase. If mobile phase additives (such as buffer or ion pair reagent) are used, it is recommended to use the mobile phase with the original proportion but without these additives for intermediate transition, and then replace it with the mobile phase of the analytical sample after 10 to 20 column volumes. For columns with short chemical chain (e.g. C8, phenyl, CN) bonded phases, care should be taken to ensure that they are thoroughly balanced before using the column. This ensures repeatability and helps prevent drift in retention time.
Normal phase solvent and reverse phase solvent are immiscible, which can not be ignored. For the newly purchased column, please first open the analysis and test manual to understand the storage solvent of the column. If the storage solvent is immiscible with the mobile phase you will use, please transition with isopropanol first. During the transition process, pay attention to the increase of column pressure due to the high viscosity of isopropanol, and appropriately reduce the flow rate. If the mobile phase contains buffer salts, first use the mobile phase with the same proportion without buffer salts to avoid the precipitation of buffer salts.

7

Q: When I use amino column to analyze sugars, why is the retention time of the target unstable and moving forward gradually?

A: This is caused by the characteristics of the amino column. When analyzing sugars, the typical mobile phase of the amino column is 60% ~ 90% acetonitrile water mixture. During use, the high concentration of amino groups in the packing gap is alkaline, resulting in the slow hydrolysis of silica gel and bonding phase. With the passage of time, more and more bonding phases fall off, which will lead to the change of the retention time of the target, At the same time, this is also the reason why the life of amino column becomes shorter under reversed-phase conditions.

8

Q: High column pressure?

A: The pressure rise of chromatographic column is a common problem in the practical application of liquid phase workers, and “blockage” is considered first. The main reasons for the increased pressure are summarized as follows:

1. The sieve plate at the inlet of chromatographic column is blocked;
2. The sample or mobile phase buffer salt precipitates in the chromatographic column;
3. Chromatographic column contamination;
4. The viscosity of mobile phase is too high;
5. The in-line filter or the guard column is blocked;
6. Pipeline blockage;
7. Polymer column: swelling caused by solvent change.

Solutions:

1. Backflush the chromatographic column with a flow rate of 1/4 of the standard flow rate without connecting the detector to remove the clogging of the sieve plate. (Except for 1.8μm particle chromatographic column)
2. Try to use the mobile phase as the sample solvent to reduce the possibility of sample precipitation. Reduce the salt concentration in the mobile phase as much as possible. After using the mobile phase with salt, the column should be washed with 10 to 20 column volumes of ultrapure water and organic phase in the same proportion of the salt in the mobile phase, and then stored in a suitable solvent.
3. The chromatographic column is contaminated, and the chromatographic column needs to be cleaned and regenerated.
4. Try to choose a solvent with a low viscosity as the mobile phase, or increase the column temperature.
5. Check the filter head of the in-line filter and the column core of the guard column, and replace if necessary.
6. Disassemble the pipeline for verification and replace if necessary.
7. For polymer-based columns, information on solvent compatibility is required.

9

Q: Low column pressure?

A: When the pressure of chromatographic column decreases, the “leakage” shall be considered first. The main reasons for low pressure rise are summarized as follows:

1. The solvent inlet filter element is blocked;
2. Leakage of connecting pipeline or other spare parts (pump head gasket);
3. Change of solvent or flow rate;
4. Pump inlet valve fails;
5. Pump outlet valve fails;
6. Chromatographic column failure and loss of stationary phase.
   Solution:
7. Check all pipelines, gaskets and other spare parts;
8. Replace the chromatographic column;
9. Check whether the chromatographic conditions have changed;
10. Check that the pump flow is accurate.

10

Q: What is dead volume and delayed volume of liquid phase?

A:

1) Dead volume

It refers to the volume from the effective injection point to the effective detection point that excludes the portion of the column containing the stationary phase. It consists of 4 parts: the volume of the pipe from the injector to the chromatographic column, the space between the stationary phase particles in the column (occupied by the mobile phase, Vm), the volume of the column outlet pipe, and the volume of the detector flow cell. Among them, only Vm participates in the chromatographic equilibrium process, and the other three parts only play a role in peak expansion. To prevent peak expansion, the volume of these three parts should be minimized.

2) Delayed volume

The delay volume refers to the volume between the solvent mixing point (usually in the mixing chamber or proportional valve of a liquid chromatograph) and the head of the LC column.

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