Part II. Separating and Analyzing Mixtures Using HPLC with UV Detection

Investigations 4–6: UV Detection and Beer's Law

The chromatogram in Figure 1 was recorded using a UV detector. Figure 2 provides representative UV spectra from 220 nm to 380 nm for four of Danshen's constituents, two of which are lipophilic and two of which are hydrophilic; you may assume these spectra are representative of Danshen's other hydrophilic and lipophilic compounds. Each spectrum is normalized so that its maximum absorbance is 1.00 [6].

Investigation 4. Based on Figure 2, are there features in these UV spectra that distinguish Danshen's hydrophilic compounds from its lipophilic compounds? What wavelength should we choose if our interest is the hydrophilic compounds only? What wavelength should we choose if our interest is the lipophilic compounds only? What is the best wavelength for detecting all of Danshen's constituents?

Although separating the analytes from each other is essential to the analysis, our ultimate goal is to determine each analyte's concentration in samples of Danshen. The height of each peak in a chromatogram is proportional to the corresponding analyte's concentration in the sample as injected.

Investigation 5. For a UV detector, what is the expected relationship between peak height and the analyte's concentration in μg/mL? For the results in Figure 1, can you assume the analyte with the smallest peak height is present at the lowest concentration [7]? Why or why not?

The standard sample for the chromatogram in Figure 1 was prepared by diluting 1.00 mL of a stock standard solution to 10.00 mL in a volumetric flask. Table 1 details the stock standard's preparation.

Table 1. Preparation of Stock Standard Solution
analyte mg diluted
to 10.00 mL
analyte mg diluted
to 10.00 ml
danshensu 06.00 dihydrotanshinone 1.51
rosmarinic acid 14.31 cryptotanshinone 2.89
lithospermic acid 13.31 tanshinone I 3.72
salvianolic acid A 04.17 tanshinone IIA 7.17

Investigation 6. Calculate the concentration, in μg/mL, for each analyte in the standard sample whose chromatogram is shown in Figure 1. Using this standard sample as a single-point external standard, calculate for each analyte the proportionality constant that relates its absorbance to its concentration in μg/mL [8]. Do your results support your answer to Investigation 5? Why or why not?

[6] The data for Figure 2 are not drawn from the original paper. The UV spectra for cryptotanshinone and for tanshinone I are adapted from "Analysis of Protocatechuic Acid, Protocatechuic Aldehyde and Tanshinones in Dan Shen Pills by HPLC," the full reference for which is Huber, U. Agilent Publication Number 5968-2882E (released 12/98; no longer available on-line, but see Aligent Publication Number 5968-2635E), and the UV spectra for danshensu and for salvianolic acid A are adapted from "Simultaneous detection of seven phenolic acids in Danshen injection using HPLC with ultraviolet detector," the full reference for which is Xu, J.; Shen, J.; Cheng, Y.; Qu, H. J. Zhejiang Univ. Sci. B. 2008, 9, 728-733 (DOI). These sources also provide UV spectra for tanshinone IIA and for rosmarinic acid, but not for dihydrotanshinone nor for lithospermic acid.

[7] You can read more about UV detection and the relationship between absorbance and concentration in Chapter 10 of Analytical Chemistry 2.0.

[8] You can read more about standardizations in general, and external standardizations more specifically, in Chapter 5 of Analytical Chemistry 2.0.