You have observed one of the most fundamental aspects of HPLC operation. It relates to both injector operation AND Column Void Volume.
Two common reasons usually account for the early change in amplitude observed near the start of an analysis (positive and negative, "blip").
(1) In all cases: At or around the Tzero time a pressure pulse should be observed in the HPLC chromatogram. This "peak" occurs from the sudden change in pressure when the injector valve switches from the "inline" to "inject" position (pressure drops then rises). This is normal and very useful as we sometime use the presence of the alternating "peak" to help estimate the Tzero time and/or confirm the injector switched.
(2) A related peak may be seen at or near the Tzero time due to the different chemical and/or physical properties of the injection solution vs the mobile phase (a positive, negative or if overlaid with the pressure peak, positive/negative "blip"). Ideally, your injection solution should always BE the mobile phase. With no change in the solution composition, "ideally" no change would be expected or shown by most types of detectors (e.g. UV/VIS) from the introduction of this new solution to the flow path. Only the pressure "blip" would be observed. However, even tiny changes in temperature, small differences in solvent composition, refractive index changes etc may be detected by the system and a second "blip" or "peak" seen at this same moment.
- "Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero)"; https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
Bill explained well. Here are some additional comments: Please note that the flow velocity within a tubing exhibits an uneven distribution profile across the radial direction, with the highest velocity occurring at the center. Within the UV/Vis detector's flow cell, a concentration gradient of the sample solvent forms in the radial direction as the solvent band passes through due to that uneven flow velocity in radial direction. When there is a difference in refractive index between the sample solvent and the mobile phase, this gradient causes the liquid inside the cell to behave like a lens. This can lead to variations in the amount of light passing through the flow cell, resulting in the appearance of two adjacent peaks. These peaks may occur in a positive-negative or negative-positive sequence, corresponding to the leading and trailing edges of the solvent band, respectively.
In chromatography, the positive peak is usually caused by compounds that are more to the stationary phase, while the negative peak is caused by compounds that are less attracted. It is all about the different interactions between the compounds and the stationary phase. Lesser affinity run fast from column and higher affinity retain more time with stationary phase cause positive peak.
Mitali Dalwadi I do not think you understood the question posed. The up/down "peak" is from the injector.
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