# Part II. Separating and Analyzing Mixtures Using HPLC


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?

In a reverse-phase HPLC separation, the stationary phase is non-polar and the mobile phase is polar. For a normal-phase separation, the stationary phase is polar and the mobile phase is non-polar. Separations in HPLC depend on a difference in the solubility of the analytes in the mobile phase and in the stationary phase. In a reverse-phase separation, for example, analytes of lower polarity are more soluble in the non-polar stationary phase, spending more time in the stationary phase and eluting at a later time than more polar analytes. In a normal-phase separation, the order of elution is reversed, with less polar analytes spending more time in the mobile phase and eluting before more polar analytes.

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 order make sense? Why or why not?

The retention times for the analytes are:

hydrophilic compounds

tr (min)

lipophilic compounds

tr (min)

danshensu

4.81

dihydrotanshinone

50.50

rosmarinic acid

27.93

cryptotanshinone

55.21

lithospermic acid

29.44

tanshinone I

56.47

salvianolic acid A

35.80

tanshinone IIA

62.60

As seen in Investigation 2, for a reverse-phase HPLC separation, we expect more polar compounds to elute earlier than less polar compounds, a trend we see here as all four hydrophilic compounds elute before the four lipophilic compounds. The trend in retention times within each group is harder to discern, particularly given the changing composition of the mobile phase; however, danshensu is significantly more soluble in water than the other hydrophilic compounds and elutes much earlier.

Note: The data used to create Figure 1 are not drawn directly from the original paper. Instead, the retention times and the relationships between peak height and analyte concentrations, in μg/mL, were determined using the HPLC data in Figure 8b and the corresponding extraction yields, in mg/g, from the first row of Table 3, obtained using a 1.00-g sample of Danshen and 35.0 mL of solvent. The resulting values for k in the equation A = kC were used to generate the data for this chromatogram and for all subsequent chromatograms. Details on the standard used to generate Figure 1 are included in Investigation 7. Although the original paper reports peak areas instead of peak heights, the latter is used in this exercise as it is easier for students to measure.

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?

The UV spectra for the lipophilic compounds cryptotanshinone and tanshinone I show a single strong absorption band between 240 nm and 270 nm. The hydrophilic compounds danshensu and salvianolic acid A, on the other hand, have strong adsorption bands at wavelengths below 240 nm and at wavelengths above 270 nm. Clearly choosing a single wavelength for this analysis requires a compromise. Any wavelength in the immediate vicinity of 280 nm is an appropriate choice as the absorbance value for salvianolic acid A is strong, and the absorbance values for tanshinone I, cryptotanshinone, and danshensu are similar in magnitude. At wavelengths greater than 285 nm the absorbance of tanshinone I, cryptotanshinone, and danshensu decrease in value, and the absorbance of danshensu decreases toward zero as the wavelength approaches 250 nm. All four compounds absorb strongly at wavelengths below 230 nm, but interference from the many other constituents of Danshen extracts may present problems. The data in the figures that follow were obtained using a wavelength of 280 nm.

Note: 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 and available at https://www.chem.agilent.com/Library...s/59682882.pdf), 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:10.1631/jzus.B0820095). These sources also provide UV spectra for tanshinone IIA and for rosmarinic acid, but not for dihydrotanshinone nor for lithospermic acid.

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? Why or why not?

For a UV detector, we expect the absorbance to follow Beer’s law, A = kC, where A is the analyte’s absorbance, C is the analyte’s concentration, and k is a proportionality constant that accounts for the analyte’s wavelength-dependent absorptivity and the detector’s pathlength. Because each analyte has a different value for k, we cannot assume that the analyte with the smallest peak height is also the analyte present at the lowest concentration.

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 the proportionality constant for each analyte that relates its absorbance to its concentration in μg/mL. Do your results support your answer to Investigation 5? Why or why not?

The table below shows the absorbance values (in mAU) for each analyte from Figure 1, the analyte’s concentration in the standard sample, and its value for k.

analyte

absorbance (mAU)

C (μg/mL)

k (mAU•mL/μg)

danshensu

96.3

60.0

1.605

rosmarinic acid

125.6

143.1

0.878

lithospermic acid

71.4

133.1

0.536

salvianolic acid A

66.1

41.7

1.585

dihydrotanshinone

442.9

15.1

2.841

cryptotanshinone

54.4

28.9

1.882

tanshinone I

59.5

37.2

1.599

tanshinone IIA

105.2

71.7

1.467

Using danshensu as an example, concentrations are derived from the data for the stock standard, accounting for its dilution and converting from mg to μg

$C= \mathrm{\dfrac{6.00\: mg}{10.00\: mL}\times\dfrac{1.00\: mL}{10.00\: mL}\times\dfrac{1000\: μg}{mg}=60.0\: μg/mL}\nonumber$

and k is calculated as

$k=\dfrac{A}{C}=\mathrm{\dfrac{96.3\: mAU}{60.0\: μg/mL}=1.605\: mAU•mL/μg}\nonumber$

Although dihydrotanshinone is present at the lowest concentration and has the smallest peak height, it has the largest value for k and is the strongest absorbing analyte. If, for example, dihydrotanshinone is present at a concentration of 25.0 μg/mL (a concentration smaller than the other seven compounds), its absorbance of 71.0 mAU will be greater than that for lithospermic acid, salvianolic acid A, cryptotanshinone, and tanshinone I. This is consistent with our expectations from Investigation 5.

Note: See the comments for Investigation 3 for details on the data used in this investigation.

Part II. Separating and Analyzing Mixtures Using HPLC is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by David Harvey.