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

Investigations 2 and 3: Reverse-Phase HPLC and Order of Elution of Solutes

The analysis of a complex mixture usually is carried out using some form of chromatography, which allows us to separate the mixture's components prior to their individual detection. The sample is injected into a mobile phase that moves through a column containing a stationary phase. A component of the mixture that interacts more strongly with the stationary phase takes longer to pass through the column and reach the detector, eluting at a later time than a component that interacts less strongly with the stationary phase. The resulting chromatogram consists of a series of peaks, each characterized by a retention time (tr) and a peak height (or peak area) [4].

Investigation 2. For this study we will use a reverse-phase HPLC equipped with a UV detector to monitor absorbance. What is a reverse-phase separation and how is it different from a normal-phase separation? How does the choice between a reverse-phase separation and a normal-phase separation affect the order in which analytes elute from an HPLC?

Figure 1 shows a reverse-phase HPLC chromatogram for a standard mixture of the eight main components of Danshen. The column contains a non-polar C18 stationary phase. The mobile phase is a gradient of acetonitrile and an aqueous solution of 0.04% (v/v) phosphoric acid, beginning at 15% acetonitrile and ending at 80% acetonitrile, by volume. The flow rate is 0.80 mL/min. The elution order is danshensu, rosmarinic acid, lithospermic acid, salvianolic acid A, dihydrotanshinone, cryptotanshinone, tanshinone I, and tanshinone IIA [5].

Investigation 3. Using the data in Figure 1 determine each analyte’s retention time. Based on your answers to Investigation 1 and Investigation 2, does the relative order of elution make sense? Why or why not?

[4] You can read more about chromatographic separations in general, and HPLC more specifically, in Chapter 12 of Analytical Chemistry 2.0.

[5] The data sets in this exercise are based on work described in the paper "Simultaneous extraction of hydrosoluble phenolic acids and liposoluble tanshinones from Salvia miltiorrhiza radix by an optimized microwave-assisted extraction method,"; the full reference for which is Fang, X.; Wang, J.; Zhang, S.; Zhao, Q.; Zheng, Z.; and Song, Z. Sep. Purif. Technol. 2012, 86, 149-156 (DOI).

Although some data in this exercise are drawn directly from or extrapolated from data in the original paper, other data are generated artificially or drawn from these additional sources: "Simultaneous quantification of six major phenolic acids in the roots of Salvia miltiorrhiza and four related tradition-al Chinese medicinal preparations by HPLC-DAD method,” the full reference for which is Liu, A; Li, L; Xu, M.; Lin, Y.; Guo, H.; Guo, D. J. Pharm. Biomed. Anal. 2006, 41, 48–56 (DOI); and "Simultaneous Determination of Seven Active Compounds in Radix Salviae Miltiorrhizae by Temperature-Controlled Ultrasound-Assisted Extraction and HPLC," the full reference for which is Qu, H.; Zhai, X.; Shao, Q.; Cheng, Y. Chromatographa 2007, 66, 21–27. (DOI).

The original paper also includes data for the extraction and analysis of salvianolic acid B; because its concentration in Danshen is an order of magnitude greater than Danshen’s other constituents, it complicates the presentation of data and is not included in this case study.