The chromatogram in Box 25-3 shows the supercritical fluid chromatography separation of seven steroids monitored by three detectors.

(a) In the middle chromatogram, ultraviolet detection provides near universal response for the steroids, whereas in the lower chromatogram the ultraviolet detector provides a selective response for a few of the steroids. How can ultraviolet detection act as either a selective or universal detector?

(b) Why is a sloping baseline observed at 210 nm, but the baseline is flat at 254 nm?

(c) Use the baseline disturbance early in the 254 nm chromatogram to measure t_m. How does the measured value compare with that predicted using Equation 25-5 given that the column is 25 × 0.46 cm and the flow rate is 2.0 mL/min.

In the middle chromatogram, ultraviolet detection provides near universal response for the steroids, whereas in the lower chromatogram the ultraviolet detector provides a selective response for a few of the steroids. An ultraviolet detector using a flow cell such is the most common HPLC detector because many solutes absorb ultraviolet light. Simple systems employing the intense 254-nm emission of a mercury vapor lamp were the backbone of early HPLC systems, but are little used today. More versatile, variable-wavelength detectors have broadband deuterium, xenon, or tungsten lamps and a monochromator, are used now a days. At wavelengths above 210 nm, detection is selective for compounds with an absorbing chromophore. Many compounds absorb wavelengths below 210nm and so ultraviolet detection below 210 nm is nearly universal whereas above that wavelength it acts as a selective detector.

The increasing gradient of CH₃OH absorbs weakly at 210 nm. So, sloping baseline is observed at 210 nm, but the baseline is flat at 254 nm.

For calculating t_m the formula need to be used is shown below

$$\begin{gathered} t_m=\frac{L{d_c}^2}{2F} \\ L=Columnlength \\ {d}_c=Columndiameter \\ F=Flowrate \end{gathered}$$

Therefore, from the given data the following can be calculated

$\begin{array}{rcl}L& =& 25cm\\ d_c& =& 0.46cm\\ F& =& 2mL/min\\ t_m& =& \frac{25\times {0.46}^2}{2\times 2}min\\ & =& 1.29min\end{array}$

The measured value of t_m is 1.29 min, and the predicted value of t_m is 1.32 min.