Editor's note: Customers often ask about the solvent effect in the process of detecting HPLC purity of products. This article is for reference.
In chromatographic theory, the sample is introduced into the chromatographic system as a point, which does not occupy the volume and belongs to the ideal state. In practice, this is impossible to achieve. The usual practice is to dissolve the target or the matrix containing the target in the solvent, and then inject it into the chromatographic system. In HPLC, the injection volume was at0.1-100μlBetween, the vast majority of cases are1-20μlbetween(Note: the ideal injection volume depends on the column bed cross-sectional area or column bed volume), even less than a dewdrop. However, this volume is relative toHPLCThe scale of mobile phase can not be ignored. Today, guided by theory and based on actual measurement, the author expounds the influence of sample solvent on liquid chromatography separation.
1.Causes of solvent effect
. In liquid chromatography analysis, the consistency of sample solvent and separation mode should also be considered.
In liquid chromatography, the choice of sample solvent type usually has two situations: first, the mobile phase dissolves the target, which is often found in the determination of one or several target with similar polarity; Second, non mobile phase dissolves target substances, such as exploration of unknown substances in natural extracts, analysis of a variety of target substances with large polarity differences, and direct injection of some liquid substances(For example, alcohol composition analysis and water pollutant analysis)Et al.
The dissolved sample in mobile phase or initial proportion mobile phase can be regarded as the ideal state of sample processing, as long as no volume overload occurs (Note: the injection volume exceeds the column volume1%When there is a risk of volume overload) or concentration overload, generally perfect results can be obtained. When the elution intensity of sample solvent and mobile phase is quite different, the performance of liquid chromatography will encounter some trouble. When the sample solvent is inconsistent with the mobile phase, there are two cases: first, the elution intensity is significantly stronger than that of the mobile phase; Second, it is obviously weaker than the mobile phase.
In brief, the strong eluting solvent unevenly participated in the elution of the target, which led to the overall advance of the retention time of the chromatographic peak, while the insufficient diffusion of the sample in front of the column led to the distortion of the peak shape. This kind of influence will lead to peak broadening if it is light, and even peak splitting if it is serious, which will bring disaster to qualitative and quantitative analysis.
2. Influencing factors of solvent effect
A prerequisite for solvent effect to cause great disaster is:The elution intensity of the sample solvent should be significantly stronger than that of the mobile phaseOtherwise, even if the target is dissolved in the two solvents when reaching the column head, they will not be separated or even widened in the subsequent elution process due to the similar elution intensity.
In the process of sample reaching the column head, there is a process of sample diffusion to the surrounding mobile phase. For the purpose of inhibiting the expansion outside the column, the analyst hopes to inhibit this diffusion. If the target substance is dissolved in the mobile phase or a solvent with similar strength to the mobile phase, inhibiting the diffusion outside the column can improve the column efficiency and facilitate the separation. When the elution intensities of the two methods differ greatly, the solvent effect is likely to occur if the target cannot be completely diffused to the mobile phase.
The driving force of target diffusion includes three parts:Depending on the concentration difference, mass transfer from the sample solvent to the mobile phase, diffusion with the sample solvent to the mobile phase, laminar flowLaminar flow is the phenomenon that when the liquid flows in the pipeline, the central part moves fast and the edge moves slowly, making the front of the cylindrical sample protrude from the rear depression, as shown in the following figure.
The movement of molecules in the liquid is not active, and the mass transfer speed depending on the concentration difference is slow or even negligible, unless the sample can enter the chromatographic column after half a day. Nevertheless, increasing the inner diameter of the pipeline can increase the contact area, thus accelerating the mass transfer, while extending the length of the pipeline can prolong the mass transfer time. The interior of the pipeline is a smooth and regular cylindrical cavity, in which the liquid flow usually produces laminar flow, which is precisely the main driving force for the diffusion of the target to the mobile phase. Similarly, the larger the inner diameter of the pipeline, the more significant the laminar flow.The thicker and longer the pipeline in front of the post needle is, the more conducive it is for the target to diffuse to the mobile phase.Of course, even if the target completely diffuses into the mobile phase in front of the column, the elution intensity of the atmosphere in which it is located is slightly stronger than that of the mobile phase due to the participation of the sample solvent.
For a given method and system, a larger sample volume(Note: at this time, the "sample band" is long)It is not conducive to the complete diffusion of the target to the mobile phase, and the solvent effect is more obvious. The stronger the solvent elution intensity of the sample, the more serious the chromatographic peak cracking. Even if the chromatographic peak is not split due to the small volume of the sample, the retention time will move forward significantly.
2.1 Effect of injection volume
After the injection volume of the sample dissolved in mobile phase increases, the retention time remains stable and the half peak width is basically stable. .
For the sample dissolved by strong elution solvent: when the injection volume is small, the target substance diffused to the mobile phase accounts for the vast majority, except that the retention time is slightly advanced, and the peak shape and peak width are basically the same as when the mobile phase is dissolved. When the volume increases to a certain extent, the chromatographic peaks diverge obviously when the number of the target substance diffusing to the mobile phase atmosphere is equivalent to that remaining in the solvent atmosphere.
When the sample volume is further increased, most of the target substances are dissolved in the solvent when entering the chromatographic column, and the front peak is the main peak(As shown below)At this time, it may give the analyst an illusion:The solvent effect was solved by increasing the injection volume or through a thinner pipeline. .In any case, please keep in mind that if some samples are not dissolved in the mobile phase, the analyst does not even know where the real retention time is!
2.2 Influence of pre column pipeline
The larger the inner diameter of the pre column pipeline and the longer the pipeline are, the more conducive it is for the target to diffuse to the mobile phase,And usingThicker lines are more effective than longer lines(Note: assuming the same dead volume)Although the thick pipeline aggravates the out of column broadening under isocratic conditions, which is harmful to column efficiency, compared with the harm of peak bifurcation, the former is acceptable.
As shown in the figure below, the black curve uses0.12mm. ID * 500 mmThe pink curve uses the0.25mm ID. * 500 mmThe sample solvent was acetonitrile, and the injection volume was2μlIn the system with thin inner diameter pipeline, the peak started to bifurcate, while the thick inner diameter pipeline could maintain an acceptable peak shape, although the latter had peak broadening.
Therefore, the solvent effect is more obvious in some systems. This is not because the instrument is not good, but because the system controls the pipeline too well, the peak shape deformation caused by the solvent effect is more obvious!
2.3 Other influencing factors
Increasing the elution volume, such as extending the column length, or decreasing the elution intensity of the mobile phase to increase the retention factor of the target, can inhibit the solvent effect. Moreover, after the chromatographic column is extended, the decrease in column efficiency caused by the pre column pipeline can be compensated. This method is equivalent to reducing the injection volume without changing the column specification.
2.4 Special instructions
In the gradient elution mode, the solvent effect is not obvious. If it does exist, it only affects the chromatographic peak with earlier peak.This is because under gradient elution, the initial proportion mobile phase cannot elute the target, and the strong elution solvent cannot make it move rapidly. They will be enriched in the column head, and the compounds will be eluted gradually when the elution intensity of the mobile phase increases to a certain extent.
For some compounds with weak retention, the closer its elution intensity is to the initial proportional mobile phase, the more susceptible it is to solvent effect. Therefore, in order to take care of the target with early peak, even if gradient elution is used, do not use strong elution solvent to dissolve(If the solvent with low elution strength can completely dissolve all the target substances)。(There is no picture here. Readers are advised to try it, or most of them have encountered it for a long time.)
3. Harm of solvent effect
(1) Lead to column efficiency reduction and hazard separation;
(2) Hazard peak shape, quantitative hazard, and even qualitative hazard; For example, inLCMSIn this case, when scanning precursor ions, we don't even know which peak is the target;
(3) When the solvents of the standard sample and the sample are inconsistent, the retention time is not consistent, and the same hazard is qualitative; For example, in the analysis of alcohol composition, the standard sample is dissolved in pure water, and the sample is filtered wine. The latter solvent is ethanol water, and its retention time must be earlier than that of the standard sample dissolved in pure water; Another example is the analysis of pollutants in water. The target substance is dissolved in organic phase to obtain a standard sample, and the sample is filtered natural water;
(4) The impurities extracted from the matrix by the strongly eluting solvent will precipitate when encountering the mobile phase, blocking the needle base, pre column pipeline or chromatographic column.
4. solutions
How to identify solvent effects?
In reverse phase chromatography, the polarity of solvent is weaker than that of mobile phase; In normal phase chromatography, the polarity of solvent is stronger than that of mobile phase. There is a risk of solvent effects in both cases. There are two characteristics of peak splitting in solvent effect. The splitting peak is in front of the normal chromatographic peak, and the earlier the peak, the more obvious the splitting of compounds, and the compounds with late peak will not even be affected. (If the sieve plate is blocked)The resulting peak splits and bifurcates appear to the right of the normal peak, and this effect will affect all compounds.
Here are some suggestions given by the author:
(1) Without affecting the solubility of the target substance, try to use the mobile phase, the initial proportion mobile phase or the liquid with the lowest elution intensity as the sample solvent;
(2) Without affecting the dissolution of the target, dilute the sample with a solvent with weak elution strength, such as adding water to the organic solvent under reverse phase chromatography;
(3) In order to meet the extraction or solubility of a variety of target substances, it is necessary to reduce the injection volume without affecting the sensitivity when using strong elution solvents, such as natural product analysis;
(4) If you don't want to reduce the injection volume or change the solvent, you can set the elution intensity of the initial mobile phase higher to make the elution intensity of the initial mobile phase close to that of the solvent. However, this method may endanger the separation of compounds;
(5) Without affecting the separation, the pipeline with a larger inner diameter should be used in front of the column;(Note: if it is isocratic elution, this method will reduce the column efficiency; If it is gradient elution, the column efficiency will not be affected)。
(6) Use the pre mixing function of the sampler.After aspirating, the needle does not return to the needle base, hovers in the air, and the metering pump aspirates-row-absorb-Row several times to make the target completely diffuse to the mobile phase.(Note:If it is isocratic elution, this method will reduce the column efficiency; If it is gradient elution, the column efficiency will not be affected)。
5. Influence of solvents with low elution strength
In the mainstream literature, it is generally believed that solvents with low elution intensity usually do not affect the chromatographic performance of compounds, and the same is true. However, in some extreme cases, small effects can also be found.
When the target in low-intensity solvent(green)When entering the chromatographic column, this part of the target will basically not move forward, because low-intensity solvent cannot elute it, and the latter is enriched in the column head(The area width is compressed, similar to the initial proportional mobile phase in gradient elution)。The low-intensity solvent will continue to move forward, catch up with the front part of the target, and slightly reduce the moving speed of the front part of the target in a small period of time.The maximum time difference between the front part of the target center and the middle part of the target center is less than or equal to the sample volume/Flow rate.
Subsequently, the target dissolved in the mobile phase later also enters the chromatographic column, which is similar to the target enriched in the column head(The elliptical band on the left of the figure below),(Note:This part is displayed in a wide oval shape to show that this part of the target accounts for the majority, which does not mean that its area width is wider, on the contrary, its width is narrower than the normal chromatographic peak)。It should be noted that the retention time of the band merged in the middle and later parts is slightly later than the normal retention time. The larger the injection volume, the more obvious the delay in retention time.
Compared with the case of strong elution solvent dissolution, peak splitting basically does not occur at low elution intensity. Especially important is that, as shown in the above figure, the peak composed of the middle and rear parts is always the main peak and can maintain a good peak shape. Even if the peak splits in the front and middle and rear parts occur, we can still find the main peak.
On the premise that the low-intensity solvent cannot elute the target, it can be boldly predicted that when the sample is actively injected, all the target will diffuse to the mobile phase before entering the chromatographic column, and the peak shape is completely normal(This is similar to when using strong elution solvents)When the injection volume is positive, the proportion of target substances diffusing to the mobile phase is relatively low before entering the chromatographic column, the proportion of target substances in the front part is smaller, the main peak is obvious, and there will be a small splitting peak in front. Only when the injection volume is not too large or small, there will be an obvious splitting peak. In general, low elution solvent is less harmful to chromatographic peaks than strong elution solvent.
Dissolved in mobile phase and pure water were compared(Low intensity)Ofcaffeine, the same conditions as the previous analysis.
It can be seen from the above figure that the injection volume is0.1μl、0.5μl 、1.0μl. The injection volume was1.0μl、5.0μl、10μlIn the analysis of, the chromatographic peak splits into one small peak and one large peak, and the front small peak is close to the real retention time(take0.1μlThe injection of was regarded as the real retention time). This is consistent with the previous mechanism of low-intensity solvent effect.
The analyst injects samples for the purpose of improving sensitivity10μl, which is important for2.1mm. ID, 50 mm,The column is obviously overloaded. In order to obtain a good peak shape, the elution condition was changed to gradient elution, and the initial mobile phase was pure water(Consistent with the sample solvent, i.e., pure water). the problem is solved perfectly(As shown below)。
6, summary
(1) The solvent effect caused by strong elution solvent is mainly that the retention time is advanced, and when the peak splits, the split peak is on the left. .
(2) The solvent effect can be eliminated by using mobile phase to dissolve the sample or reducing the injection volume; Diluting the sample with low-intensity solvent or gradient elution is also a good way to inhibit the solvent effect; Using a thick inner diameter pipeline before column or pre mixing before sample injection can diffuse the target into the mobile phase, which can rescue the peak shape. If it is gradient elution, this will not even affect the column efficiency. .
Note: there are many pictures in the original text that more clearly illustrate various situations and principles. For further understanding, please read the original text,
A long article allows you to fully understand the solvent effect in liquid chromatography.pdf