Ultra Performance Liquid Chromatography coupled with UV/Visible Spectrophotometry

Ultra Performance Liquid Chromatography is a technique similar to that of HPLC when it comes to separating different components of a sample, as well as identifying, quantifying and separating the components of a mixture. The major difference in UPLC is the particle size in the column which is less than 2 µm providing better separation than in HPLC where the size of these is limited to 5 µm. These smaller particles require higher pump pressures (100 MPa vs 40 M Pa) which makes this method very efficient with rapid analysis and higher resolution.

On leaving the UPLC, the separated compounds enter the UV/Visible detector which is commonly used in Liquid Chromatography. We have a double beam photometer in the platform as a type of detection.

In practice, the polychromatic light of a deuterium lamp (D2) is focused on the entry slit of a monochromator. This selectively transmits a narrow band of chosen wavelengths via an array to the output slot. In general, the wavelength is set to the maximum absorbance of the analyte. A wrong choice of wavelength can lead to a reduction in the size of the peaks and therefore distort the results.

This monochromatic light is separated into two beams before reaching the flow cell. One of the beams passes through a reference tank, the mobile phase, located off the axis of the Flow Cell. While the other beam passes through the sample (in the Flow Cell). A mirror system makes it possible to send these two beams to the same sensor which therefore alternately receives the reference beam I0 and the beam transmitted by the sample I.

When no analyte passes through the detector, the light passes through the flow cell and generates a maximum signal at the light sensor, which corresponds to the baseline of the chromatogram I0. On the other hand, when an analyte passes, the amount of light I reaching the sensor is reduced and causes a modification of the detector signal. We know that the absorbance A = log I0/I and that the signal is processed by the data system causing the appearance of a positive peak in the chromatogram.

We also know that the absorbance is proportional to the concentration of the chemical according to the Beer-Lambert law: A = e * L * C where (C) is the concentration of the analyte, (A) its absorbance, (e) its molar absorbance coefficient and (L) the length of the path of the light through the Flow Cell.