Common problems and solutions of gradient experiment

As we all know, when doing HPLC analysis, the gradient system can greatly shorten the analysis time without affecting the separation of samples, but it is also convenient and accompanied by a variety of problems. As far as the author summarizes, there are three common problems:

(1) After mixing the two phases, a large number of bubbles are generated

As shown below:

The picture above is the bubble diagram generated by the gradient of buffered salt solution and pure methanol. For this, our company's usual solution is to use a "back pressure regulator" to make the ultraviolet detection cell have a certain pressure to prevent the formation of bubbles. Secondly, we generally do not recommend the use of pure organic phases such as pure methanol and pure acetonitrile as mobile phases.

(2) The problem of gradient baseline drift

This problem generally occurs when the gradient line shape changes, and it is basically inevitable at low wavelengths. Because methanol and acetonitrile, which are commonly used in chromatography, absorb at low wavelengths (such as 210 nm), if the baseline drifts up as the organic phase increases or decreases as the organic phase decreases, this is not only a normal phenomenon, but also Reflects the sensitivity and accuracy of the system. Therefore, this problem cannot be eliminated, and the baseline drift can only be reduced by choosing a higher quality organic solvent. For example, the industrial pollutant of acetonitrile is acetone (with strong UV absorption). Selecting a higher quality acetonitrile can effectively reduce the gradient drift. Degree.

(3) Gradient ghost peak

This problem can be divided into two. There are two kinds of gradient ghost peaks: accidental and inevitable.

Occasionally, the mobile phase is not degassed well, and dissolved oxygen can form a complex with some organic solvents with ultraviolet absorption, and strong absorption occurs. This situation is not repeatable, and can be degassed through a good mobile phase. And avoid.

The picture above shows the result of two injections of a certain sample. The non-reproducible ghost peak clearly appears at 50 minutes.

The inevitable situation is that some inexplicable peaks are formed as the gradient runs, and it has excellent repeatability, which is unavoidable, and generally occurs when the gradient changes greatly. Don't worry about this situation, just do a gradient blank control, as long as the gradient ghost peak does not affect the peak of the sample. As shown below:

The peak after 40min in the above figure is a repeatable gradient ghost peak, which does not affect the detection of the sample.

There may be some problems that cannot be summarized here. It must be discovered and summarized continuously by chemists. Solving problems is the basic work of chemists. Seeking truth from facts is the basic requirement of chemical work.

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