LABORATORY EXPERIMENT pubs.acs.org/jchemeduc
Extraction and Purification of Glucoraphanin by Preparative High-Performance Liquid Chromatography (HPLC) Iris Lee and Mary C. Boyce* School of Natural Sciences, Edith Cowan University, 270 Joondalup Dve., Joondalup 6027, Perth, Western Australia, Australia
bS Supporting Information ABSTRACT: A student activity that focuses on the isolation of glucoraphanin from broccoli using preparative high-performance liquid chromatography (HPLC) is presented here. Glucoraphanin is a glucosinolate, whose byproducts are known to possess anticancer properties. It is present naturally at high levels in broccoli and other Brassica vegetables. This activity has students practicing and developing a number of skills including liquid extraction; cleanup by solidphase extraction; analytical HPLC with diode array detection to identify the peak of interest; and preparative HPLC coupled with a fraction collector to isolate the glucoraphanin fraction. It also represents an authentic application of preparative HPLC as glucoraphanin is not readily available from the main chemical suppliers. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Chromatography, Food Science, HPLC, Plant Chemistry, UV vis Spectroscopy
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hromatography and specifically high-performance liquid chromatography (HPLC) are an integral part of any analytical chemistry unit. Several excellent experiments dealing with analytical HPLC have been published in this Journal.1 5 In our analytical chemistry units there is a strong focus on HPLC as it is a technique valued and looked for in our graduates by employers. During their undergraduate studies our students complete a number of HPLC activities that provide them with a basic understanding of the technique (caffeine in cola, formaldehyde in household materials, and melamine in milk). They also complete a number of extended laboratory activities to challenge and broaden their understanding; some of these activities have a HPLC component and have been published in this Journal.6,7 In our graduate program pure glucoraphanin was required for a quantitative study utilizing capillary electrophoresis (CE).8 Glucoraphanin (Figure 1) belongs to a class of phytochemicals called glucosinolates, whose byproducts are known to possess anticarcinogenic properties.9 This group of compounds is present in appreciable quantities in cruciferous vegetables (e.g., broccoli, cabbage, cauliflower, Brussel sprouts, and kale), and consumption of these vegetables can therefore be of benefit to our health. As pure glucoraphanin is not readily available and is very expensive to purchase (U.S. $582/100 mg from the Royal Veterinary and Agricultural University, Copenhagen) its isolation from crude extracts of broccoli seeds by preparative HPLC provides an economical and alternative source. This prompted us to consider including preparative HPLC in our undergraduate program because (i) it was absent from the syllabus; (ii) this activity provided students with an authentic example of the value of preparative HPLC; (iii) it provided students with a novel activity that had not been used in previous years in our chemistry offerings; (iv) it Published 2011 by the American Chemical Society
Figure 1. Structure of glucoraphanin.
required students to practice several skills (liquid liquid extraction, solid-phase extraction, analytical HPLC), interpret data, and make decisions along the way. In designing a preparative HPLC laboratory for our undergraduate program we also noted the lack of recent preparative HPLC activities published in this Journal. Qvit et al. employ preparative HPLC when producing aspartame analogues, but it is not a focus of the activity.10 Here we present an authentic and appropriate example of preparative HPLC.
’ EXPERIMENTAL PROCEDURE The activity is divided into two 4 h laboratory sessions, and the students work in small groups (typically 3 students per group). In week 1, the students obtain a crude product from broccoli seeds by liquid extraction. As solid-phase extraction (SPE) is recommended prior to preparative HPLC of glucoraphanin, the students completed an SPE step on a portion of their crude extract. The extracts (crude and SPE extracts) are then analyzed by analytical HPLC equipped with a diode array detector (DAD). The students identify the glucoraphanin peak by using the DAD to Published: March 31, 2011 832
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scan the major peaks knowing that glucoraphanin has a maximum absorbance at 230 nm and is expected to be a major component. Students also compare the chromatograms for the extracts that had an SPE cleanup step with those that had no cleanup step and comment on the merit or otherwise of the step. In week 2, the students set up the preparative HPLC and the fraction collector to collect the glucoraphanin fraction. Students then work out at what time and for how long they should begin to collect the glucoraphanin peak. They also collect fractions before and after the glucoraphanin fraction. Having collected a set of fractions, the students then use the analytical HPLC to confirm that the fraction they have identified as containing glucoraphanin actually does contain it. If it does not, then they check the fractions on either side. In general, it takes students some time to optimize the timing and length of the fraction to be collected. As this laboratory activity requires each group to have access to two HPLC units (one fitted with a preparative column and the other with an analytical column), particularly in the second week, this extended activity is run concurrently with other extended activities that require the use of other equipment, typically gas chromatography, capillary electrophoresis, and ion chromatography. On any given week only one group is completing the HPLC activity.
Figure 2. Chromatogram recorded for broccoli seed extracts: G identifies the glucoraphanin. See text for separation details.
’ LABORATORY REQUIREMENTS Extraction and SPE Cleanup Procedure
We use water for the extraction to minimize handling of organic solvents and to enable us to perform SPE to remove organic contaminants. However, extractions with pure methanol and varying ratios of methanol water have been reported.11 13 We use broccoli seeds rather than florets as glucoraphanin is present in higher concentrations (typically 1.3 g/100 g fresh weight).8 For SPE cleanup, 300 mg C18 cartridges are used, but any packing bed size is suitable providing it is not overloaded. The SPE cartridges are activated with approximately 3 mL of methanol followed by approximately 3 mL of deionized water. After applying 3 mL of the crude glucosinolate extract, residual glucosinolate is eluted with approximately 3 mL of deionized water. Minimum HPLC Instrumentation Requirements
For analytical HPLC an instrument fitted with an isocratic pump, a manual injector setup (20 μL loop), and a photodiode array detector is required. For preparative HPLC analyses a unit fitted with an isocratic pump, a manual injector set up (100 μL loop), and fixed-wavelength UV vis detector (set at 230 nm) is required. A fraction collector is preferable, but manual collection of fractions into test tubes is a viable option. For the analytical analyses, a 5 μm C18, 250 mm 4.6 mm column is suitable with an isocratic mobile phase of 5 mM tetramethylammonium bromide (TMAB) in 2% v/v methanol/ water and a flow rate of 1 mL/min. Other mobile phases have been tried,14 but we selected TMAB because its ion-pairing interactions with glucoraphanin provided good resolution from other peaks in the chromatograms. Furthermore, minimal organic solvents are required to prepare the mobile phase. For preparative HPLC analyses, a 5 μm C18, 250 mm 10 mm column with a preparative guard, 5 μm C18, 33 mm 7 mm is suitable with an isocratic mobile phase of 1% v/v acetonitrile/ water (both with 0.1% v/v formic acid) and a flow rate of 5 mL/ min. The glucoraphanin fractions are collected by a fraction
Figure 3. UV vis spectrum of glucoraphanin.
collector at a collection rate of 0.5 min/tube or as dictated by the student.
’ HAZARDS Hazards are minimal in this activity; however, safety precautions should be exercised when handling the HPLC mobile phases that contain small amounts of acetonitrile and methanol to minimize skin contact and inhalation. Acetonitrile may cause skin irritation and damage to the respiratory system, kidneys, and nervous system. Methanol is a flammable liquid and may cause central nervous system depression. Tetramethylammonium bromide may cause irritation to skin and eyes and is slightly hazardous if ingested. Students wear safety glasses at all times and gloves when handling solvents. ’ RESULTS AND DISCUSSION The chromatogram of the crude extract separated on the analytical column is shown in Figure 2. The peak at 7 min is tentatively identified as glucoraphanin based on its UV vis spectrum (Figure 3) and comparison with chromatograms from the literature. At this point the advantage of liquid chromatography (LC) with mass spectroscopy (MS) detection in confirming the peak of interest is a discussion point. The usual method of spiking the extract with pure glucoraphanin is not made available to the students given the expense of the standard and that the lack of a readily available standard is a reason for this activity. A comparison of the chromatograms obtained for the SPE treated extracts and the crude extracts clearly demonstrates to the 833
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Figure 4. The analytical HPLC chromatogram of the (A) fraction collected before the main glucoraphanin fraction, which contains small quantities of glurcoraphanin; (B) fraction containing glucoraphanin, and (C) fraction collected after the main glucoraphanin fraction: G identifies the glucoraphanin. See text for separation details.
students that there are no significant differences and the cleanup step is not necessary. However, this result may depend on the variety of broccoli seed used and should be checked before the SPE step is omitted. The analytical HPLC chromatograms of the glucoraphanin fraction and those fractions collected on either side are displayed in Figure 4 and represent a typical student result. Glucoraphanin was isolated in the first two fractions; however, glucoraphanin was present in highest concentrations in the second fraction and was contaminated with another analyte in the first fraction. A student’s first attempt at isolating glucoraphanin can often have the glucoraphanin peak eluting with another peak or with the glucoraphanin peak being collected across two fractions or no glucoraphanin collected. This activity, as outlined here, requires two, 4 h lab periods and two HPLC units, one fitted with a fraction collector. As indicated previously, over several weeks we rotate the student groups through a number of different laboratory activities to avoid needing more than two HPLC units in any given laboratory period. However, we have also run this laboratory activity in different formats in response to time and instrument constraints. When the laboratory time was 3 h per week, we excluded the SPE step (as we have found this step, contrary to the research, to be unnecessary) in week 1. When only one HPLC unit was available, we used capillary electrophoresis, and specifically micellar electrokinetic chromatography, as an alternative to analytical HPLC for checking the fractions.8 Experimental details and a typical electropherogram are provided in the Supporting Information. If accessing a second separation unit is an ongoing issue, then a UV vis spectrophotometer could be used to identify the fraction which contains the highest absorbance and hence the most glucoraphanin. A fraction collector is only necessary if the aim is to repeatedly collect the glucoraphanin fraction in an attempt to pool the fractions and generate a sizable amount of glucoraphanin. In most instances, manual collection of the fractions into test tubes is sufficient to demonstrate the approach. If dedicating two laboratory periods to this activity is problematic, students could, having extracted the glucoraphanin, inject the crude extract (no SPE step) directly onto the preparative column and use DAD to identify the glucoraphanin peak. Having identified the peak of interest, the students then attempt to collect the glucoraphanin fraction. The collected fractions can be tested using UV vis spectrophotometry. This simplified experiment also has the advantage of being less-instrument intensive.
’ CONCLUSION This activity allowed us to include preparative HPLC in our curriculum in a meaningful way. It provided students with an authentic and relevant activity that gave them new skills but also allowed them to practice and combine other skills, in particular, peak identification using UV vis, solid-phase extraction, and analytical HPLC. ’ ASSOCIATED CONTENT
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Supporting Information Student handout; notes for the instructor; criteria used to assess laboratory reports; information for capillary electrophoresis as an alternative to analytical HPLC; a typical electropherogram. This material is available via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
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