NMvD thanks Dr. Michael Reichelt (Max-Planck-Institute for chemical Ecology, Jena, Germany) for providing the first reference samples when she started using this method 16 years ago. Ciska Raaijmakers (NIOO-KNAW, Wageningen, the Netherlands), Sebastian Krosse (B-Ware, Nijmegen, the Netherlands), and Christian Ristok (iDiv, Leipzig) are acknowledged for improving the protocol over the course of the years. Mirka Macel and Martine Huberty (University T&#252;bingen, Germany) are acknowledged for their permission to use the Rorippa chromatogram. The authors gratefully acknowledge the support of the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, funded by the German Research Foundation (FZT 118).

1. Preparation of Solutions Needed for the Glucosinolate Extraction
<ol>
	<li>Prepare 500 mL of 70% methanol (MeOH) in water (ultrapure) in a glass bottle. For 100 samples and 5 standards, 420 mL of 70% MeOH is needed.</li>
	<li>Prepare 20 mM sodium acetate (NaOAc) (pH = 5.5) by adding 0.82 g of NaOAc or 1.36 g of NaOAc x 3 H2O in 500 mL of water; adjust the pH with hydrogen chloride (HCl). Store the NaOAc in the refrigerator. For 100 samples and 5 standards, 370 mL of 20 mM NaOAc (pH = 5.5) in water is needed.</li>
	<li>To prepare the column material, mix 10 g of cross-linked dextran gel (type G-25) with 125 mL of ultrapure water. Store the resulting mixture in a refrigerator (4 &#176;C).</li>
	<li>Preparation of the sulfatase solution
	<ol>
		<li>Dissolve 10,000 units of aryl sulfatase (type H-1 from Helix pomatia) in 30 mL of ultrapure water and add 30 mL of absolute ethanol (EtOH). Mix the solution well and transfer it to one 250-mL centrifuge tube or multiple smaller tubes, depending on availability.</li>
		<li>Centrifuge the mixture at 2,650 x g for 20 min at room temperature (RT). Transfer the supernatant(s) to a beaker; add 90 mL of EtOH and mix it. Centrifuge the mixture in one or more centrifuge tubes at 1,030 x g for 15 min at RT.</li>
		<li>Discard the supernatants. Dissolve and combine the pellets in a total of 25 mL of ultrapure water. Vortex well, dispense in 1-mL tubes, and store in a freezer at -20 &#176;C; they will keep for at least one year.</li>
	</ol>
	</li>
	<li>Preparation of the sinigrin reference solution
	<ol>
		<li>Weigh in around 9 mg of sinigrin monohydrate with 1-&#181;g accuracy (e.g., 8.769 mg). Transfer it into a 10-mL volumetric flask and dissolve the sinigrin monohydrate in 10.0 mL of ultrapure water.</li>
		<li>Calculate the molarity of the stock solution and prepare five sinigrin references between 50-750 &#181;M by diluting of the stock solution. For a detailed example, see <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55425/20160918GrosservanDamJoVEGlucosinolates_S1.docx" target="_blank">Supplementary File S1</a>.</li>
		<li>Dispense the different sinigrin references in 1.5-mL reaction tubes and freeze them at 20 &#176;C. Include a series of five sinigrin references in each extraction batch. Always use the correct molarity values for the currently used sinigrin references when calculating the concentrations.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Extraction Setup
<ol>
	<li>Prepare a column rack for the Pasteur pipette columns (for the preparation of columns, see step 3.1). Label the rack or the columns to keep track of the sample numbers (see Figure 1).</li>
	<li>Prepare a block holding the same number of labelled 2-mL microcentrifuge tubes (see step 3.3 and Figure 1)</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55425/55425fig1.jpg" /> 
Figure 1: Rack for glucosinolate extraction. Front view (left) and top view (right) of a self-made column rack and block for the glucosinolate extraction. Height: 100 mm, distance between shifted holes (to hold the Pasteur pipette columns): 30 mm (x-axis) x 15 mm (y-axis). <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55425/55425fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Preparation of the Columns and Microcentrifuge Tubes
<ol>
	<li>Prepare one column per sample and five extra columns for the five sinigrin reference samples.
	<ol>
		<li>In each glass pipette, place a piece of glass wool approximately 1 cm x 1 cm. Use a wooden or glass stick to push the glass wool down to the point where the pipette narrows. The glass wool should prevent the column material from running out, but do not use too much, as it will slow the elution of the columns. Wear gloves when working with the glass wool.</li>
	</ol>
	</li>
	<li>Place the columns on the column rack (see Figure 1). Place the column rack in a lab tray (waste tray) to catch the eluents used for the consecutive column washes.</li>
	<li>Cut the first 5 mm off the tip of a plastic 1-mL pipette tip and pipette 0.5 mL of the prepared column material (G-25 gel in water, see step 1.3) into each column. Shake the bottle containing the column material well before pipetting. Discard leaking columns (G-25 material running out through the glass wool layer) and replace with new ones.
	<ol>
		<li>After pipetting the column material into the columns, add 1 mL of ultrapure water to flush down the material.</li>
	</ol>
	</li>
	<li>Prepare one 2-mL reaction tube per sample and five tubes for the five sinigrin reference samples; label them. Use a dissecting needle to make holes in the caps for later freeze-drying and place the prepared tubes into the tube block in the exact same pattern as the columns (step 2.2, see Figure 1).</li>
</ol>
4. Extraction of Glucosinolates
<ol>
	<li>Weigh freeze-dried and finely-ground plant material (usually 50.0-100.0 mg of dry weight; the final glucosinolate concentrations in the extract should be in the range of the reference curve) to the nearest 0.1 mg in 2-mL, labeled, round-bottom reaction tubes. Add two small metal balls (3 mm in diameter) as boiling retardants to each tube. 
	NOTE: The protocol can also be applied to fresh, flash-frozen materials, that have been ground under liquid nitrogen and kept frozen until extraction. Increase the amount of material weighed for extraction and the percentage of MeOH in the extraction liquid to 85% to compensate for dilution by the water in the materials19.</li>
	<li>Pipette 1 mL of 70% MeOH into each tube and vortex briefly. Close the tubes and seal them with safety caps before placing them as quickly as possible into a hot water bath (90-92 &#176;C) for a few minutes (~5 min), until the 70% MeOH just boils. Caution: Wear safety goggles during this step!</li>
	<li>Place the sample tubes in an ultrasonic bath for 15 min. Meanwhile, take the sulfatase and the five sinigrin reference samples out of the freezer to thaw them at RT.</li>
	<li>After ultra-sonication, centrifuge the sample tubes at 2,700 x g in a benchtop centrifuge for 10 min at RT; a pellet should form in each tube. Add the supernatants to the labelled columns and pipette the five reference samples onto separate columns.
	<ol>
		<li>While pipetting the supernatants, keep the tip well over the pellet to avoid pipetting plant materials. Note that when dried samples are used, the supernatant volume will be less than 1.0 mL.</li>
	</ol>
	</li>
	<li>Add 1 mL of 70% MeOH to the remaining pellets in the sample tubes and vortex the tubes before placing them in an ultrasonic bath for 15 min. Centrifuge the tubes again, as in step 4.4, and add the supernatants to the respective columns; due to the properties of the column material, the negatively charged sulfate group of the glucosinolates will be specifically retained on the column.</li>
	<li>Wash the columns with the extracts in three sequential steps.
	<ol>
		<li>Pipette 2 x 1 mL of 70% MeOH onto each column. Wait for the column to run dry before adding the next 1 mL; this will remove more apolar compounds from the extracts (e.g., chlorophyll).</li>
		<li>Flush out the MeOH by adding 1 mL of ultrapure water to each column.</li>
		<li>Pipette 2 x 1 mL of 20 mM NaOAc buffer to each column to create the optimal conditions for the sulfatase reaction.</li>
	</ol>
	</li>
	<li>Take the rack with the columns out of the waste tray and dry the feet of the rack with a tissue. Place the rack over the block with vials and labeled tubes. Make sure that each column tip is in the corresponding, labeled, 2-mL tube (see Figure 1).</li>
	<li>Add 20 &#181;L of sulfatase solution to the columns. Ensure that the sulfatase reaches the surface of the column material. Pipette 50 &#181;L of NaOAc buffer onto each column to flush down the sulfatase. Cover the columns with aluminum foil and let stand overnight. 
	NOTE: Due to the activities of the sulfatase, the sulfate group will be removed, releasing the desulfoglucosinolates from the column so that they can elute with the water. For a detailed description, see Crocoll et al.19.</li>
	<li>The next day, elute the desulfoglucosinolates by pipetting 2 x 0.75 mL of ultrapure water onto each column. When all columns have run dry, lift the column rack and remove it from the reaction tubes.
	<ol>
		<li>Cap the tubes (make sure that there are holes in the caps) and freeze them in liquid nitrogen or in a -80 &#176;C freezer for 30 min. Freeze-dry the samples for 12-24 h (depending on the number of samples and the capacity of the freeze-drier) to remove all water.</li>
	</ol>
	</li>
	<li>After freeze-drying, re-dissolve the residue in an exact volume (usually 1.0 mL) of ultrapure water. Transfer the samples and the five sinigrin references to labeled HPLC vials. Keep the samples in a refrigerator (4 &#176;C) for up to two weeks or a freezer (-20 &#176;C) for up to one year before analyzing them with HPLC.</li>
	<li>Let the glass columns dry under the hood overnight and dispose them when dry. Recover the metal balls from the sample tubes used in step 4.5 for reuse and put the tubes in the waste disposal bin.</li>
</ol>
5. HPLC Measurements of Extracted Samples
<ol>
	<li>Separate the glucosinolates on a reversed-phase C18 column (4.6 x 150 mm, 3 &#181;m, 300 &#197;) with an acetonitrile-water gradient (see Table 1) and a flow of 0.75 mL/min and a column temperature of 40 &#176;C. Perform detection and quantification at 229 nm.</li>
	<li>Measure the sinigrin references on the same column but with an adapted gradient method to reduce solvent use (see Table 2). Start with injecting the five sinigrin references, beginning with the reference with the lowest concentration. Then inject the samples, including a methanol injection (blank) after every tenth sample to clean the column and to prevent the carryover of peaks.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time [min]</td>
			<td>Flow [mL/min]</td>
			<td>% A Water</td>
			<td>% B ACN</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.750</td>
			<td>98</td>
			<td>2</td>
		</tr>
		<tr>
			<td>35</td>
			<td>0.750</td>
			<td>65</td>
			<td>35</td>
		</tr>
		<tr>
			<td>40</td>
			<td>0.750</td>
			<td>98</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="4">Column Temperature 40 &#176;C.</td>
		</tr>
	</tbody>
</table>
Table 1: Acetonitrile-water gradient for glucosinolate separation and analysis on reversed-phase HPLC.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time [min]</td>
			<td>Flow [mL/min]</td>
			<td>% A Water</td>
			<td>% B ACN</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.750</td>
			<td>98</td>
			<td>2</td>
		</tr>
		<tr>
			<td>10</td>
			<td>0.750</td>
			<td>89.3</td>
			<td>10.7</td>
		</tr>
		<tr>
			<td>11</td>
			<td>0.750</td>
			<td>98</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="4">Column Temperature 40 &#176;C.</td>
		</tr>
	</tbody>
</table>
Table 2: Shortened acetonitrile-water gradient for quantification of the five sinigrin references used for the quantification of glucosinolates.
6. Glucosinolate Identification and Quantification
<ol>
	<li>Identification
	<ol>
		<li>Compare the UV spectra and retention times of the peaks in the samples with commercially available, pure glucosinolate references. See Figure 2 for examples of UV spectra from the various glucosinolate classes.</li>
		<li>Identify unknown glucosinolates with reference standards or on LC-MS18,19, if possible.</li>
		<li>Identify unknown glucosinolates with nuclear magnetic resonance (NMR), if possible.</li>
		<li>Compare the UV spectra and retention times of the peaks with those in Figure 2 and Table 3, databases, or literature references.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55425/55425fig2.jpg" /> 
Figure 2. UV spectra of the most common glucosinolate classes. UV absorption spectra (200-350 nm) of six desulfoglucosinolates (GSL), based on solutions made of commercially available reference compounds extracted as described here, representing the most common structural classes. The common name, structural name (between brackets), and structural class are given. (A) Sinigrin (2-propenyl GSL), alkenyl; (B) glucoerucin (4-methylthiobutyl GSL), thioalkenyl; (C) glucoraphanin (4-methylsulfinlybutyl GSL), sulfinyl; (D) glucobrassicin (indol-3-ylmethyl GSL), indole; (E) gluconasturtiin (2-phenylethyl GSL), aromatic; (F) sinalbin (4-hydroxybenzyl GSL), aromatic. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55425/55425fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Quantification
	<ol>
		<li>Integrate the area under the peaks of the five sinigrin references and the samples.</li>
		<li>Calculate a calibration curve of the five measured sinigrin references based on the integrated area. Use the equation of the regression line (see Equation 1) to calculate the interpolated amount of glucosinolates (see Equation 2).</li>
		<li>To calculate the concentration of the different glucosinolates, multiply the interpolated amount (x) by the response factor (M) for detection at 229 nm from EC (1990)16, Buchner (1987)15, or Brown et al. (1993)17; the consensus response factors of the most commonly detected desulphoglucosinolates are given in Table 3. 
		NOTE: Response factors may vary between systems-this may be the reason that there are different values given by different references (see References 15-17). Nevertheless, the values are mostly quite close and in a similar range with structural classes. Following the convention, set the response factor of unidentified glucosinolates to 1, although for unknown indole glucosinolates with a UV spectrum similar to that of glucobrassicin and other indole glucosinolates (Figure 2), it would be advisable to use 0.25 as a response factor to obtain a realistic estimate of the concentration (Table 3).</li>
		<li>To calculate the final amount of glucosinolates (xt) in the sample, take the dilution factor (D) and the mass of sample (w) used for the analysis into account (see Equation 3). 
		<img alt="Equation" src="/files/ftp_upload/55425/55425eq1.jpg" /> (1) 
		<img alt="Equation" src="/files/ftp_upload/55425/55425eq2.jpg" /> (2) 
		<img alt="Equation" src="/files/ftp_upload/55425/55425eq3.jpg" /> (3) 
		y = Peak area 
		m = slope of the reference 5-point regression curve 
		c = the intercept of the regression line with the y-axis 
		x = amount of glucosinolates in the extract 
		xt = concentration of glucosinolates in the plant sample 
		D = dilution factor 
		M = response factor for the detection at 229 nm from Buchner (1987) or Brown et al. (1993) 
		w = mass of sample used for extraction</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucosinolates+and+biofumigation:+fate+of+glucosinolates+and+their+hydrolysis+products+in+soil.">Glucosinolates and biofumigation: fate of glucosinolates and their hydrolysis products in soil.</a> Phytochem Rev. 8 (1), 299-310 (2009).</li><li>Fahey, J. W., Zalcmann, A. T., Talalay, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+diversity+and+distribution+of+glucosinolates+and+isothiocyanates+among+plants.">The chemical diversity and distribution of glucosinolates and isothiocyanates among plants.</a> Phytochemistry. 56, 5-51 (2001).</li><li>de Graaf, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+4-&#945;-rhamnosyloxy+benzyl+glucosinolate+in+Noccaea+caerulescens+showing+intraspecific+variation.">Isolation and identification of 4-&#945;-rhamnosyloxy benzyl glucosinolate in Noccaea caerulescens showing intraspecific variation.</a> Phytochemistry. 110, 166-171 (2015).</li><li>van Dam, N. M., Raaijmakers, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+systemic+induced+responses+to+cabbage+root+fly+larvae+(Delia+radicum)+in+Brassica+nigra+and+B.+oleracea.">Local and systemic induced responses to cabbage root fly larvae (Delia radicum) in Brassica nigra and B. oleracea.</a> Chemoecology. 16 (1), 17-24 (2006).</li><li>Gols, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+affect+plant+chemistry+and+tritrophic+interactions.">Temporal changes affect plant chemistry and tritrophic interactions.</a> Bas Appl Ecol. 8 (5), 421-433 (2007).</li></ol>

The authors have nothing to disclose.

The greatest advantages of this established and widely used method are that it is robust, rather straightforward, and relatively low-cost per sample. Most of the equipment needed for the extraction and analysis should be available in a standard laboratory or can be self-built, with the exception of the HPLC-PDA. Another advantage is that desulfoglucosinolates dissolved in water are chemically quite stable when kept cool and in air-tight (HPLC) vials, so the extracts could easily be shipped for HPLC analysis elsewhere. In contrast to LC-MS platforms, which require specialized training and extensive hands-on experience for managing the software and analyzing the data, HPLC-UV/PDAs can be easily run after a short training period. This not only reduces the costs of the procedure, but also makes this method more accessible to a broad range of scientists, including students.
Generally, when the procedures described above are followed carefully, few problems should occur. In general, the glucosinolate peaks are very well separated in the chromatogram. If this is not the case, the gradient program could be adapted by decreasing the rate of increase of acetonitrile in the eluent. Alternatively, building in a new pre-column (200-500 injections) or column (1,500 -2,000 injections) may solve the issue. Occasionally, chromatograms of single samples in a batch may show very small or no peaks. This is usually due to pipetting errors when adding the sulfatase (e.g., a column has been skipped or the sulfatase was not properly washed down into the column). Alternatively, the glucosinolate concentration in the experimental materials may have been lower than expected and too little material was used for the extraction. If the latter is the case, the injection volume may be increased to 100 &#181;L, or an exact aliquot (e.g., 800 &#181;L) of the extract could be concentrated. The latter could be achieved by freeze-drying the extract, dissolving the residue in a smaller volume (e.g., 100 &#181;L) of water, and reinjecting using the same reference curve. In the calculations for the original concentration of the extract, the numbers should be multiplied by the dilution factor. If this does not solve the issue, the materials should be extracted again using more starting material. If this is more than 100 mg, the volume of the extraction solvents and the size of the tubes should be adjusted proportionally to maintain the extraction efficiency.
An additional advantage is that this method has been well-validated. This is because it has been described as a standard method for the quantification of glucosinolates in rapeseed, for which the procedures and accuracy were confirmed in several laboratories16. In addition, the genetic background, biosynthesis, and biological functions of glucosinolates are subject to intense research efforts, in the model plant species Arabidopsis thaliana among others4,6,12. Therefore, many response factors for the exact quantification of desulfoglucosinolates in relation to sinigrin are well defined and publicly available15,17. Even though LS-MS-based protocols are more high-throughput, more sensitive, and are able to (tentatively) identify glucosinolates for which no standards are available18,19,20, the lack of universal response factors for LC-MS limits the exact quantification of glucosinolate concentrations18. Moreover, these methods usually do not include a freeze-drying step, and the amount of water in the fresh plant material is unaccounted for in the calculations, making exact quantification difficult. Lastly, because our extraction method involves a column-based purification and concentration step, it can also be applied to &#34;dirty&#34; samples with low concentrations of glucosinolates, such as soils21.
Compared to LC-MS-based methods that usually extract freshly frozen materials, use 96-well plates for extraction, and do not include a sulfatase step18,19, our method is relatively time-consuming and labor intense. With the column racks described in this paper, a single person can extract about 100-150 samples in one day. Elution (next day), freeze drying (overnight), and re-dissolving can take place within the following two days. With an automated HPLC injector, a run and equilibration time of 40-45 min per injection, and no unforeseen events, it would take 3-4 days to acquire the data for this sample set. When the HPLC software allows automatic quantification based on the sinigrin curve, a manual check of the chromatograms and peak assignments for 100 samples may only take another 1 or 2 h before the data can be used for statistical analyses.
Despite the increasing availability of glucosinolate standards, only a small fraction of the more than 130 candidates can currently be commercially bought. However, with a few references for each of the biosynthetic classes; access to literature databases specifying the compounds previously found in the plant species (e.g., Fahey et al.22); basic knowledge of chromatographic principles, such as the logic of eluotropic series (e.g., for increasing numbers of Cs on the side chain in aliphatic compounds, Figures 3 and 4); and the validation of single samples on LC-MS19 or isolated glucosinolates on NMR23, one may easily overcome this limitation. Most protocols for glucosinolate analyses use internal reference curves (i.e., a certain concentration for the extraction of sinigrin or sinalbin to the extraction solvent16,17,19). Principally, internal reference curves are more appropriate to correct for individual sample processing errors and thus theoretically yield a higher precision. Despite this advantage, we prefer to use a five-point external reference curve, as we often analyze different wild species, some of which contain high levels of sinigrin (e.g., Brassica nigra24) or sinalbin (e.g., Sinapis alba25), the two glucosinolate references for which response factors are available. Moreover, adding internal standards to each sample increases the cost of the analyses, as high-grade glucosinolate reference standards are usually quite expensive.
In conclusion, despite the time-consuming steps, this protocol provides a straightforward and accessible method to extract and quantify glucosinolates in plant samples. However, it is important to consider that the glucosinolate levels themselves are only an indication of the potential biological activity, seen as the necessity to react with myrosinase, and variation in the reaction products may arise from a single glucosinolate11. Validation assays must be performed to confirm the biological relevance.

It is estimated that plants produce over 200,000 different types of chemical compounds1. Only the minority of these compound seems to play a role in the plants&#39; primary metabolism, which fuels growth and reproduction; the majority are so-called secondary metabolites. Despite their name, secondary metabolites are often critical for plant survival and reproduction, as they serve to attract pollinators or to defend the plant against pathogens and herbivores1.
Glucosinolates are a very diverse class of secondary metabolites that have been studied for over 150 years2. To date, more than 130 structurally different glucosinolates have been identified3. Broadly, glucosinolates can be subdivided into different classes based on the amino acid from which they are synthesized. Indole glucosinolates, for example, are synthesized from the amino acid tryptophan, whereas phenylalanine provides the base skeleton for the synthesis of aromatic glucosinolates4. Within classes, there is a high level of structural diversity, which is brought about by sequential chain elongation steps in the biosynthetic pathways, such as in the class of aliphatic glucosinolates, or by side chain modifications (e.g., hydroxylation)4,5. One glucosinolate plant species may contain up to 37 different glucosinolates belonging to different structural classes6. Even though plant species have typical glucosinolate profiles, intraspecific variation for the types of glucosinolates is frequently found among individuals and populations6,7. Intact glucosinolates are stored in the vacuoles of plant cells and can be found in any aboveground or belowground organ7,8,9. Upon cell rupture (e.g., by herbivory), glucosinolates are released and are mixed with the enzyme myrosinase, setting off a mustard oil bomb10. Due to the activity of myrosinase, and depending on the structure of the glucosinolate, the reaction conditions, and the presence of modifying enzymes, different pungent, toxic, or noxious compounds are formed, such as nitriles and (iso)thiocyanates11. The reaction products have high biological activities; for example, they serve as repellents of generalist insect herbivores12. The heritability and biosynthetic pathways of glucosinolates are well studied, mainly because of their importance for herbivore and pathogen resistance, their value as flavor components in mustards and cabbages, and their negative (progoitrin) and positive (glucoraphanin) effects on human health5,13,14.
Because of the great interest in glucosinolates as biologically active compounds, extraction and detection methods based on reversed-phase high-pressure liquid chromatography (HPLC) equipped with ultraviolet (UV) or photodiode array (PDA) detectors have been commonly used since the 1980s15. Based on this method, the European Communities issued a standard protocol that was tested and validated in several labs for the analysis of glucosinolates in oilseeds (Brassica napus, colza, Canola16). Others added to this method (e.g., by determining additional response factors for glucosinolates that are not present in oilseed rape)17. Despite the increasing availability of liquid chromatography-mass spectrometry (LC-MS) platforms and high-throughput protocols for glucosinolate analysis18,19, the original HPLC-UV/PDA method is still widely used by scientists. The main reasons are that this method is straightforward, cost-effective, and relatively accessible to labs without an extensive chemical-analytical knowledge infrastructure. To serve this community, we here detail the protocol for the extraction of glucosinolates from plant materials and the analysis of their desulfo-forms with HPLC-PDA.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Methanol HiPerSolv CHROMANORM&#174; gradient grade for HPLC grade</td>
			<td>VWR</td>
			<td>20,864,320</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate (NaOAc)</td>
			<td>Sigma-Aldrich</td>
			<td>W302406-1KG-K</td>
			<td></td>
		</tr>
		<tr>
			<td>HCL</td>
			<td>VWR</td>
			<td>1,090,571,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex</td>
			<td>Sigma-Aldrich</td>
			<td>A25120-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;(&#8722;)-Sinigrin hydrate 
			from horseradish&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S1647-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Aryl Sulfatase</td>
			<td>Sigma-Aldrich</td>
			<td>S9626-10KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>20,816,298</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Pipette</td>
			<td>Carl Roth</td>
			<td>4518.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Wool</td>
			<td>Carl Roth</td>
			<td>7377.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass /wooden stick</td>
			<td>VWR</td>
			<td>HERE1080766</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL reaction tubes</td>
			<td>VWR</td>
			<td>211-2606</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting needle</td>
			<td>Carl Roth</td>
			<td>KX93.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotilabo-lab dishes</td>
			<td>Carl Roth</td>
			<td>0772.1</td>
			<td>Waste tray</td>
		</tr>
		<tr>
			<td>Freeze Dryer Freezone 12 L</td>
			<td>Labconco</td>
			<td>7960030</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitril super gradient grade</td>
			<td>VWR</td>
			<td>83,639,320</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR</td>
			<td>462-5112</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>Fisher Scientific</td>
			<td>FB 15061</td>
			<td></td>
		</tr>
		<tr>
			<td>PH Electrode</td>
			<td>Thermo Fisher Scientific</td>
			<td>STARA2115</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Heraeus Multifuge X1</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004210</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Column</td>
			<td>Thermo Fisher Scientific</td>
			<td>69697</td>
			<td>C18 column (4.6 x 10 mm, 5 &#181;m, 300 &#197;)&#160;</td>
		</tr>
		<tr>
			<td>Column Acclaim 300</td>
			<td>Thermo Fisher Scientific</td>
			<td>60266</td>
			<td>C18 column (4.6 x 150 mm, 3 &#181;m, 300 &#197;)&#160;</td>
		</tr>
		<tr>
			<td>HPLC Ultimate 3000</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>with column oven and UV or PDA detector</td>
		</tr>
		<tr>
			<td>Flask 1000 mL</td>
			<td>VWR</td>
			<td>215-1595</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucosinolate reference compounds</td>
			<td>Phytoplan</td>
			<td>various</td>
			<td>http://www.phytoplan.de/</td>
		</tr>
	</tbody>
</table>

a straightforward method for glucosinolate extraction and analysis with high-pressure liquid chromatography (hplc)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

This method enables the detection and separation of commonly found desulfoglucosinolates in all structural classes (Figure 3). The detrimental 2-hydroxyglucosinolate progoitrin comes relatively early in the chromatogram and is clearly separated from the beneficial glucosinolate glucoraphanin, the only methylsulfinylglucosinolate in this sample. Sinigrin, gluconapin, and glucobrassicanapin form an eluotropic series of alkenyl glucosinolates with increasing side-chain lengths (C3, C4, and C5, respectively). A similar logical series is visible for the two methylthioalkenyl glucosinolates, glucoiberverin (C3) and glucoerucin (C4). The peaks of the three indole glucosinolates, glucobrassicin and its derivatives 4-hydroxy and 1-methoxyglucobrassicin (neoglucobrassicin), are also clearly separated. It should be noted that the peaks of neoglucobrassicin and glucoerucin, as well as gluconasturtiin, the only aromatic glucosinolate in this sample, are particularly high in this Brassica extract due to the addition of root extracts to the mix9.
<img alt="Figure 3" src="/files/ftp_upload/55425/55425fig3.jpg" /> 
Figure 3: Chromatogram of a glucosinolate extract. Detail (1-32 min) of an HPLC chromatogram resulting from the analysis of combined root and shoot samples from Brassica nigra, B. rapa, and B. oleracea. The peak labels indicate the retention time and the abbreviations for identified desulfoglucosinolates (GSL). PRO = progoitrin (2-OH-3-butenyl GSL); RAPH = glucoraphanin (4-methylsulfinlybutyl GSL); SIN = sinigrin (2-propenyl GSL), GNA = gluconapin (3-butenyl GSL); 4OH = 4-hydroxyglucobrassicin; IBV = glucoiberverin (3-methylthiopropyl GSL); GBN = glucobrassicanapin (4-pentenyl GSL); ERU = glucoerucin (4-methylthiobutyl GSL); GBC = glucobrassicin (indol-3-ylmethyl GSL); NAS = gluconasturtiin (2-phenylethyl GSL); NEO = neoglucobrassicin (1-MeOH-glucobrassicin). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55425/55425fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Longer chain methylsulfinyl glucosinolates, which are commonly found in the model plant Arabidopsis, also show an eluotropic series, as seen in a root extract of Rorippa austriaca. Glucohesperalin (C6), glucosiberin (C7), glucohirsutin (C8), and glucoarabin (C9) appear at regular intervals on the chromatogram (Figure 4). Together with the UV spectra of the peaks, such eluotropic logical series may be used to classify, and provisionally identify, unknown glucosinolates.
<img alt="Figure 4" src="/files/ftp_upload/55425/55425fig4.jpg" /> 
Figure 4: Chromatogram (frame 9-30 min) of the desulfoglucosinolates in a Rorippa austriaca root extract. The peak labels indicate the retention time and the abbreviations for identified desulfoglucosinolates (GSL). HES = glucohesperin (6-methylsulfinylhexyl GSL); SBE = glucosiberin (7-methylsulfinylheptyl GSL); GBC = glucobrassicin (indol-3-ylmethyl GSL); HIR = glucohirsutin (8-methylsulfinlyoctyl GSL); ARA = glucoarabin (9-methylsulfinylnonylglucosinolate); NEO = neoglucobrassicin (1-MeOH-glucobrassicin). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55425/55425fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Common name</td>
			<td>Side chain structure</td>
			<td>Rt (min)*</td>
			<td>229 nm</td>
			<td>reference#</td>
		</tr>
		<tr>
			<td>aliphatic glucosinolates</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucocapparin</td>
			<td>methyl</td>
			<td>3.5</td>
			<td>1</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Sinigrin</td>
			<td>2-propenyl</td>
			<td>5.5</td>
			<td>1</td>
			<td>Brown, EC</td>
		</tr>
		<tr>
			<td>Gluconapin</td>
			<td>3-butenyl</td>
			<td>9.5</td>
			<td>1.11</td>
			<td>EC</td>
		</tr>
		<tr>
			<td>Glucobrassicanapin</td>
			<td>4-pentenyl</td>
			<td>13.5</td>
			<td>1.15</td>
			<td>EC</td>
		</tr>
		<tr>
			<td>Glucoiberverin</td>
			<td>3-methylthiopropyl</td>
			<td>10.9</td>
			<td>0.8</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Glucoerucin</td>
			<td>4-methylthiobutyl</td>
			<td>14.0</td>
			<td>0.9</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Glucoiberin</td>
			<td>3-methylsulfinylpropyl</td>
			<td>3.7</td>
			<td>1.2</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Glucoraphanin</td>
			<td>4-methylsulfinylbutyl</td>
			<td>4.9</td>
			<td>0.9</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Glucoalyssin</td>
			<td>5-methylsulfinylpentyl</td>
			<td>7.6</td>
			<td>0.9</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Glucohesperin</td>
			<td>6-methylsulfinylhexyl</td>
			<td>10.5</td>
			<td>1</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Glucosiberin</td>
			<td>7-methylsulfinylheptyl</td>
			<td>13.5</td>
			<td>1</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Glucohirsutin</td>
			<td>8-methylsulfinyloctyl</td>
			<td>16.8</td>
			<td>1.1</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Glucoarabin</td>
			<td>9-methylsulfinylnonyl</td>
			<td>20.5</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucocheirolin</td>
			<td>3-methylsulfonylpropyl</td>
			<td>4.2</td>
			<td>0.9</td>
			<td>Brown</td>
		</tr>
		<tr>
			<td>Progoitrin</td>
			<td>2(R)-OH-3-butenyl</td>
			<td>4.5</td>
			<td>1.09</td>
			<td>Buchner, EC</td>
		</tr>
		<tr>
			<td>Gluconapoleiferin</td>
			<td>2-OH-5-pentenyl</td>
			<td>8.3</td>
			<td>1</td>
			<td>EC</td>
		</tr>
		<tr>
			<td>indole glucosinolates</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-hydroxyglucobrassicin</td>
			<td>4-hydroxyindol-3-ylmethyl</td>
			<td>11.2</td>
			<td>0.28</td>
			<td>Buchner, EC</td>
		</tr>
		<tr>
			<td>Glucobrassicin</td>
			<td>indol-3-ylmethyl</td>
			<td>15.3</td>
			<td>0.29</td>
			<td>Buchner, EC</td>
		</tr>
		<tr>
			<td>4-Methoxyglucobrassicin</td>
			<td>4-methoxyindol-3-ylmethyl</td>
			<td>18.2</td>
			<td>0.25</td>
			<td>Buchner, EC</td>
		</tr>
		<tr>
			<td>Neoglucobrassicin</td>
			<td>1-methoxyindol-3-ylmethyl</td>
			<td>22.5</td>
			<td>0.2</td>
			<td>Buchner, EC</td>
		</tr>
		<tr>
			<td>aromatic glucosinolates</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sinalbin</td>
			<td>4-hydroxybenzyl</td>
			<td>8.1</td>
			<td>0.5</td>
			<td>Buchner</td>
		</tr>
		<tr>
			<td>Glucosibarin</td>
			<td>2(R)-OH-2-phenylethyl</td>
			<td>12.1</td>
			<td>0.95</td>
			<td>see next</td>
		</tr>
		<tr>
			<td>Glucobarbarin</td>
			<td>2(S)-OH-2-phenylethyl</td>
			<td>12.7</td>
			<td>0.95</td>
			<td>Buchner</td>
		</tr>
		<tr>
			<td>Glucotropaeolin</td>
			<td>benzyl</td>
			<td>13.8</td>
			<td>0.95</td>
			<td>Buchner, EC</td>
		</tr>
		<tr>
			<td>Gluconasturtiin</td>
			<td>2-phenylethyl</td>
			<td>18.0</td>
			<td>0.95</td>
			<td>Buchner, EC</td>
		</tr>
		<tr>
			<td>unknown - aliphatic/aromatic like UV spectrum</td>
			<td></td>
			<td></td>
			<td>1</td>
			<td>EC</td>
		</tr>
		<tr>
			<td>unknown - indole like spectrum</td>
			<td></td>
			<td></td>
			<td>0.25</td>
			<td>EC</td>
		</tr>
		<tr>
			<td colspan="5">* approximate retention time (Rt) rounded to nearest 0.1 min (&#177; 0.3 min depending on the column, eluent quality). Retention times are determined on ThermoFisher/Dionex Ultimate HPLC platforms equipped with an C18 column (150 x 4.6 mm, 3 micrometer particle size) plus C18 precolumn (10 x 4.6 mm, 5 micrometer particle size) with a gradient program as in Table 1.</td>
		</tr>
		<tr>
			<td colspan="5"># References for response factors: Buchner, R. in Glucosinolates in rapeseed (ed J.P. Wathelet) 50-58 (Martinus Nijhoff Publishers, 1987); Brown, P. D., Tokuhisa, J. G., Reichelt, M. &#38; Gershenzon, J. Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry. 62 (3), 471-481, doi:10.1016/S0031-9422(02)00549-6, (2003); EC. Oil seeds - determination of glucosinolates High Performance Liquid Chromatography. Official Journal of the European Communities. L 170/28. Annex VIII 03.07.27-34 (1990).</td>
		</tr>
	</tbody>
</table>
Table 3: Response factors of the most commonly found desulfo-glucosinolates in plant extracts and their approximate retention times on C18 columns. Eluents, gradient, column temperature, and flow rate as in Table 1.

Watch this Scientific Journal Video about A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC at JoVE.com

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

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Nanyang Technological University

Research

JoVE Journal

Chemistry

25.0K Views.  German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig. Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography. 

Video: A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. Specifically, the sapphire cell enables visual data collection from multiple loadings to solve a set of material balances to precisely determine phase composition. Ternary phase diagrams can then be established to determine the proportion of each component in each phase at a given condition. In principle, any ternary system can be studied although ternary systems (gas-liquid-liquid) are the specific examples discussed herein. For instance, the ternary THF-Water-CO2 system was studied at 25 and 40&#160;&#176;C and is described herein. Of key importance, this technique does not require sampling. Circumventing the possible disturbance of the system equilibrium upon sampling, inherent measurement errors, and technical difficulties of physically sampling under pressure is a significant benefit of this technique. Perhaps as important, the sapphire cell also enables the direct visual observation of the phase behavior. In fact, as the CO2 pressure is increased, the homogeneous THF-Water solution phase splits at about 2 MPa. With this technique, it was possible to easily and clearly observe the cloud point and determine the composition of the newly formed phases as a function of pressure.
The data acquired with the sapphire cell technique can be used for many applications. In our case, we measured swelling and composition for tunable solvents, like gas-expanded liquids, gas-expanded ionic liquids and Organic Aqueous Tunable Systems (OATS)1-4. For the latest system, OATS, the high-pressure sapphire cell enabled the study of (1) phase behavior as a function of pressure and temperature, (2) composition of each phase (gas-liquid-liquid) as a function of pressure and temperature and (3) catalyst partitioning in the two liquid phases as a function of pressure and composition. Finally, the sapphire cell is an especially effective tool to gather accurate and reproducible measurements in a timely fashion.

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus is a unique tool to study, without sampling, phase behavior under a wide range of pressures. Using a cathetometer, very precise volume measurements can be recorded to measure liquid expansion and phase composition. Thus, this synthetic method enables the study of (1) phase equilibria of multi-component mixtures and (2) the partition behavior of catalyst or model compounds as a function of pressure.

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. ...

Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.

This article describes a rapid supercritical extraction method for fabricating silica aerogels. By utilizing a confined mold and hydraulic hot press, monolithic aerogels can be made in eight hours or less.

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The ...

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

High Pressure Single Crystal Diffraction at PX^2

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the GSECARS 13-BM-C beamline at the Advanced Photon Source. The DAC program at 13-BM-C is part of the Partnership for Extreme Xtallography (PX^2) project. BX-90 type DACs with conical-type diamond anvils and backing plates are recommended for these experiments. The sample chamber should be loaded with noble gas to maintain a hydrostatic pressure environment. The sample is aligned to the rotation center of the diffraction goniometer. The MARCCD area detector is calibrated with a powder diffraction pattern from LaB6. The sample diffraction peaks are analyzed with the ATREX software program, and are then indexed with the RSV software program. RSV is used to refine the UB matrix of the single crystal, and with this information and the peak prediction function, more diffraction peaks can be located. Representative single crystal diffraction data from an omphacite (Ca0.51Na0.48)(Mg0.44Al0.44Fe2+0.14Fe3+0.02)Si2O6 sample were collected. Analysis of the data gave a monoclinic lattice with P2/n space group at 0.35 GPa, and the lattice parameters were found to be: a = 9.496 &#177;0.006 &#197;, b = 8.761 &#177;0.004 &#197;, c = 5.248 &#177;0.001 &#197;, &#946; = 105.06 &#177;0.03&#186;, &#945; = &#947; = 90&#186;.

In this report, we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell at the GSECARS 13-BM-C beamline at the Advanced Photon Source. ATREX and RSV programs are used to analyze the data.

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction is to modulate the lipophilicity of the compound. In our group, we are interested in the study of the impact of fluorination on lipophilicity of aliphatic fluorohydrins and fluorinated carbohydrates. These are not UV-active, resulting in a challenging lipophilicity determination. Here, we present a straightforward method for the measurement of lipophilicity of fluorinated compounds by 19F NMR spectroscopy. This method requires no UV-activity. Accurate solute mass, solvent and aliquot volume are also not required to be measured. Using this method, we measured the lipophilicities of a large number of fluorinated alkanols and carbohydrates.

A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy

A novel and straightforward variation of the shake-flask method was developed for accurate lipophilicity measurement of fluorinated compounds by 19F NMR spectroscopy.

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction ...

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...