Poor Peak Shape, and How to Improve It
When the injection volume of test substance within the range of the security, changing injection volume will affect the peak height and peak area accordingly. But the influence of retention time, peak width, and resolution is slight. When injection volume or the quality is excessive, it will cause the overload phenomenon. With the peak width increases, poor peak shape and lower resolution is inevitable.Overload
When the injection volume of test substance within the range of the security, changing injection volume will affect the peak height and peak area accordingly. But the influence of retention time, peak width, and resolution is slight. When injection volume or the quality is excessive, it will cause the overload phenomenon. With the peak width increases, poor peak shape and lower resolution is inevitable.
For columns whose length is 50 to 250mm and inner diameter is 4 to 5mm, injection quality of a single sample injection should be less than 50 μg and the injection volume should be less than 25 μL. Overload can be divided into two kinds: volume overload and quality overload, the following will introduce the two kinds of overload’s impact on separation.
(1) Volume overload: The figure below shows the chromatogram of 1mg/mL forsythiin with 10, 40, 60, 80 and 100 μL, respectively. It can be seen that 10μL has the best peak shape while 40 μL has a leading area and it will become more obvious with the increase of the injection volume.
(2) Quality overload: The following figure is a determination of amino acids and their derivatives, showing peak shape in condition of normal and supermassive quality.
Overload can affect chromatographic peak shape, only if the volume and quality of the sample are in the appropriate range, can we get good peak shape. Now, we will continue to discuss what factors can lead to leading area or tailing area.
Leading area: (Tailing factor<0.95)
1. Influence of solvent effect
(1) Dissolve the sample with mobile phase: (curcumin standard substance)
(2) First dissolve the sample with strong solvent and then diluted with a mobile phase :(huperzine standard)
2. Improper buffering effect: (inject with matrine)
3. Improper instrument system
When the Ultisil® XB-C18 column (4.6×50mm, 1.8μm) is used on a conventional liquid chromatograph, its peak shape will be affected by the large dead volume of the system. As shown in the figure of roflumilast below, the peaks of the chromatograph can be seen to delay.
Tailing area: (Tailing factor<0.95)
1. Reduce pH and inhibit the ionization of the silica hydroxyl group.
2. Increase buffer salt and increase buffer effect
3. Since the buffer capacity of each buffer salt is different, it can be adjusted by changing the buffer salt
4. Influence of shielding silica (such as adding triethylamine)
In our qualitative and quantitative detection and analysis, peak shape has a great impact on our experimental results, so to ensure a good peak shape is the key to the success of our experiment, and the detection and solving of abnormal peak shape is of course an important skill for our experimental analysis.
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