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IntroDuctIonDerivatization chemistry has a critical role in the analysis of organic compounds by chromatographic methods, such as when polar compounds need to acquire sufficient volatility to allow their vaporization for gas chromatography. In the emerging field of metabolomics, gas chromatography coupled with mass spectrometry (GC-MS) has a major role in the identification and quantification of all metabolites in a defined system at a defined time1, but there remains a need for improving large-scale derivatization schemes2, especially procedures that yield stable derivatives. Two basic approaches to derivatization chemistry have evolved: use of reagents that derivatize a number of differ-ent functional groups at once, and use of reagents that deriva-tize only a specific functionality. The ‘general’ reagents are useful when analyzing complex multicomponent mixtures; however, derivatizing many functional groups simultaneously often yields ambiguous information about the mixture’s individual components. In addition, the most common general derivati-zation reagents—compounds that form trimethylsilyl deriva-tives of organic acids, hydroxyls and amines—are not stable in aqueous solution, and their use can lead to undesirable artifacts, potentially causing multiple peaks for a single compound in a gas chromatographic analysis and thus confusing its interpretation3,4. Use of general reagents can also greatly increase the molecular mass and complexity of the isotopic pattern of a polyfunctional compound, an outcome that can make the interpretation of MS data in particular very challenging5.
Diazomethane has long been used in organic synthesis and was first described as a methylating agent in the late 19th century6. Today, it is one of the most
commonly used reagents for the preparation of simple methyl esters from the corresponding carboxylic acids (see Fig. 1). As a highly reactive gas, diazomethane has developed a reputation as a versatile reagent, the use of which is associated with the formation of few side products, making it ideally suited for small-scale deri-vatizations of compounds for chromatographic analysis7. Despite diazomethane’s toxicity and reactivity, which necessitate that sig-nificant precautions be taken during production and handling8, this reagent has proven to be extremely useful and is widely used in laboratory procedures and, more recently, in industrial processes (see Proctor and Warr9 and references cited therein).
As diazomethane can function as a nucleophile, electrophile or as a source of the methylene carbine, its use can lead to unwanted side reactions10–12; e.g., in our first publication on this topic, we described the slow diazomethane-catalyzed hydrolysis of amide bonds7. Diazomethane’s tendency to polymerize during stor-age13 can also be problematic, as this compound’s polymerization products can contaminate a chromatographic separation. Results from studies by several different groups9,14,15 have contributed to the compilation of the following list of desirable practices when using diazomethane as a reagent: (i) as diazomethane tends to concentrate in the vapor phase (diazomethane’s boiling point is − 23 °C) and localized concentrations as low as 5 mM are potentially explosive, a constant gas flow should be maintained during produc-tion, delivering the gaseous diazomethane either to the sample(s) to be methylated or to the solvent (usually ether) in which it will
A method for concurrent diazomethane synthesis and substrate methylation in a 96-sample formatLana S Barkawi & Jerry D Cohen
Department of Horticultural Science and Microbial and Plant Genomics Institute, University of Minnesota, Saint Paul, Minnesota, USA. Correspondence should be addressed to J.D.C. ([email protected]).
Published online 9 September 2010; doi:10.1038/nprot.2010.119
In the emerging field of metabolomics, there is an increasing need for improving sample derivatization reactions for gas chromatographic–mass spectral analysis of metabolites with large numbers of samples. this protocol details the safe direct derivatization of organic acids using diazomethane in a 96-sample format. Diazomethane is a highly reactive gas that readily forms methyl esters with carboxylic functionalities, with minimal side products or nonvolatile reaction residues. However, diazomethane’s reactivity and explosive potential make it hazardous to store and work with. In this procedure, diazomethane is generated in situ and used concurrently to methylate up to 96 samples simultaneously, thus reducing concerns about reagent stability and obviating the need for storage of solutions of the highly reactive gas. once the diazomethane generator has been assembled, processing 96 samples takes 2–3 h using this procedure.
S
O
O
N
CH3
NO
Diazald(N-methyl-N-nitroso-p-toluenesulfonamide)
+ NaOH S
O
O
O–Na+ +
Diazomethane
a
b
Sodium p-toluene sulfonate
H2C N N
H2C N NR
C
O
OH
Organic acid Diazomethane Methyl ester
N2
RC
O
OCH3
++Methanol
+ H2OH3C H3C
Figure 1 | Reaction scheme for the synthesis of diazomethane and the derivatization of organic acids. (a) Formation of diazomethane from Diazald (N-methyl-N-nitroso-p-toluenesulfonamide) in basic solution. (b) Methylation of an organic acid by diazomethane.
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be trapped and stored; (ii) to avoid decomposition, concentrated diazomethane should not be exposed to above-ambient temper-atures or to large temperature variations; (iii) headspace in the vessel in which diazomethane is generated should be limited to avoid localized concentration buildups; and finally, (iv) to avoid potentially problematic storage, diazomethane should react with its substrate as soon as it is generated.
Our protocol for the synthesis of methyl esters using diazometh-ane16 avoids many of the problems of safety and generation of arti-facts that have been noted by others. The present protocol uses high flow rates of diethyl ether–saturated nitrogen to maintain low concentrations of diazomethane in the gas phase; it avoids using potentially catalytic surfaces, such as sharp edges, ground glass and other hazardous constructions, the presence of which can increase the risk of explosions; moreover, in our protocol, diazomethane is produced in situ, so that it immediately reacts with the target compound. In addition, the protocol has been designed to operate in the standard microtiter plate 96-well format that has become common in modern laboratories, and it yields enough reagent to process 96 samples at one time. Furthermore, because of the high
gas flow used, volatile solvents and unreacted diazomethane are removed from the reaction vessel as the reaction proceeds, yielding a methylated product ready to be dissolved in an appropriate pure solvent for analysis by GC-MS.
This protocol is based on a previously published and validated method for the extraction, methylation and quantification of indole-3-acetic acid (IAA) from plant tissue16. In an accompany-ing protocol17, we have outlined the automated extraction and purification and analysis of IAA, including a detailed discus-sion of quantification by isotope dilution. The robotic option described in the report leaves IAA dissolved in methanol, ready for methylation before separation and quantification by GC-MS. Although this protocol was developed for methylation of IAA, we report here the successful methylation of other compounds with organic acid functionalities, and are confident that it can be optimized for many methanol-soluble organic acids. It is our experience that methyl IAA is not sufficiently volatile to be lost in this protocol, but those working with other, more volatile methyl esters will need to use caution and optimize the protocol to minimize loss.
MaterIalsREAGENTS
Nitrogen gas (ultrahigh purity (UHP), < 0.5 p.p.m. oxygen)Diethyl ether (HPLC grade, inhibitor free, Sigma-Aldrich, cat. no. 309966) ! cautIon Both liquid and vapor forms are extremely flammable; it forms peroxide.2-(2-Ethoxyethoxy)ethanol (di-(ethylene glycol)-ethyl ether) (Sigma-Aldrich, cat. no. 455-0) ! cautIon Both liquid and vapor forms are extremely flammable; it forms peroxide.Sodium hydroxide (ACS grade, J.T. Baker, cat. no. 3722)Diazald (N-methyl-N-nitroso-p-toluenesulfonamide, 99% purity; Sigma-Aldrich, cat. no. D28000) ! cautIon It decomposes to release diazomethane, which is toxic and explosive (see handling information in PROCEDURE).
EQUIPMENTGas supply and control for methylation/evaporation
Pressure gauge (0–30 p.s.i.; Cole-Parmer, cat. no. C-68612-06)Pressure gauge (0–100 p.s.i.; Cole-Parmer, cat. no. C-68612-08)Two-way brass shutoff valves (Cole-Parmer, cat. no. C-30527-64)Flowmeter (referred to as ‘lower flow (LF) flowmeter’ in the PROCEDURE, brass valve, maximum flow rate 2,395 ml min − 1; Cole-Parmer, cat. no. C-03294-20)Flowmeter (referred to as ‘higher flow (HF) flowmeter’ in the PROCEDURE, brass valve, maximum flow rate 43,000 ml min − 1; Cole-Parmer, cat. no. C-03294-34)Male pipe adaptor elbows for 1/8-inch NPT (National Pipe Thread Taper) × 1/8-inch tubing (polypropylene; Cole-Parmer, cat. no. C-06386-05)Teflon tubing (3 mm; Cole-Parmer, cat. no. C-06407-10)
Manifold/diazomethane production apparatus for methylation/evaporation
Aluminum diazomethane generator manifold. This is a custom piece; see Figure 2 for details and a technical drawing. Although ours was machined by Dan Benson Machining, it can be made by any mechanical shop.Stainless steel frame rods (1/2-inch diameter; Cole-Parmer, cat. no C-08024-22)Thumbscrews, 1/4-inch diameter, 20 TPIScaffold foot that accommodates up to 1/2-inch rods (Cole-Parmer, cat. no. C-08024-57)
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A piece of wood, melamine or any other material that can be similarly easily drilled into (~17 cm × 30 cm × 2 cm).Teflon sheet (1/8-inch thick; Berghof America, cat. no. 15083–12125)Threaded low-pressure chromatography fittings (Upchurch, cat. no. P-130)Four flangeless ferrules (1/8 inch; Upchurch, cat. no. P-300x)Reagent bottles (250 ml; Pyrex, cat. no. 1395–250)Heavy-gauge netting (high-density polyethylene, Poly-Net protective net-ting, 2.5- to 3-inch diameter; Cole-Parmer, cat. no. C-09405-55)Teflon tape (3/4 inch × 3 mil (or 19 mm x 76.2 µm); Scientific Specialties Services, cat. no. J-7974)
Gaseous diazomethane delivery apparatus for methylation/evaporationGlass tubing (13-mm outer diameter, cut to ~12 cm in length and fire polished; Ace, cat. no. 8802-23)Teflon compression-reducing union (1/2 inch × 1/8 inch tubing outer diameter; Cole-Parmer, cat. no. SA-06373-7S K-31320-02)Teflon check valve (Cole-Parmer, cat. no. C-98553-30)Teflon T-shaped union (Cole-Parmer, cat. no. SA-06374-41)Teflon in-line union (1/8 inch, Cole-Parmer, cat. no. K-31000-71)N
2 analytical concentrator/evaporator (C/E) with a programmable heating
block (Zanntek Scientific, cat. no. ZipVap 96)Aluminum sample plate that can hold 96 GC autosampler vial inserts (250 µl) in the standard 96-well dimensions. This is a custom piece; see Figure 2 for details and a technical drawing. Although ours was machined by Dan Benson Machining, it can be made by any mechanical shop.Inserts for gas chromatography vials (250 µl; Chrom Tech, cat. no. CTI-9425). Please note that, although these are described as 250-µl inserts, they can actually hold up to 300 µl.Luna C18 column (150 mm × 4.6 mm, 5 µm particle size, 100 Å pore size; Phenomenex)Spectroflow 757 absorbance detector (ABI Analytical Kratos Division, Analytical Instruments)Silica gel G plates (10 cm × 20 cm, 250-µm thickness, Analtech)Alpha Innotech ChemImager 5500 (Cell Biosciences)Fluorchem 5500 software (v. 4.0.1, Alpha Innotech/Cell Biosciences)Waters 600E HPLC
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proceDureassembly of the high-flow diazomethane generator ● tIMInG the following steps (steps 1–23) take approximately 1–2 h to com-plete once all the materials are gath-ered. after the apparatus is built, it can be cleaned (step 33) and used repeatedly.1| Fit four stainless steel frame rods into each corner of the manifold (see Fig. 2, section B for a technical drawing and the sector of Fig. 3 labeled as ‘d’ for a drawing of its position in the overall system). Secure with thumbscrews. crItIcal step The manifold should be constructed such that once the Teflon tubing and discs and Pyrex bottles are in place, diazomethane comes in contact only with Teflon or smooth glass surfaces.
2| Secure four scaffold feet into the four corners of a piece of wood, melamine or any other material that can be similarly easily drilled into (Fig. 2a). Place these at the appropriate position for the four frame rods to fit into.
3| Secure the four frame rods into the four scaffold feet.
4| Cut a length of 3-mm Teflon tubing that is long enough to connect the outlet port of the LF flowmeter (Fig. 3, sector c) to the manifold (Fig. 3, sector d). Using a male pipe adapter elbow, attach one end of the tubing to the outlet connection of the flowmeter.
5| On the other end of the tubing from Step 4, place a fitting and a flangeless ferrule; screw the tubing into the leftmost threaded hole at the top of the manifold (Fig. 3, sector e). The tubing should extend down from the manifold to nearly reach
b
Top view
Side view
24 TPI 24 TPI 24 TPI 20 TPI
GL45
20 TPI
c
a
127.5
85.5
11
86.5
9
127.5
9.5
9.5
12.7
9.9
Top view
Side view
d
24 T
PI
e
Figure 2 | View and schematic representation of a high-flow diazomethane generator. This system can be set up and used to methylate 96 organic acid–containing samples concurrently. (a) A photograph of the complete system. (b) The custom-designed aluminum diazomethane generator manifold. This technical drawing shows the threads per inch (TPI) measurements and the bottle thread measurements (GL45) on the manifold. It is threaded to accommodate standard reagent bottles up to 500 ml, standard low-pressure chromatography fittings and thumbscrews. The four unthreaded holes in each corner of the plate accommodate cylindrical legs to support the manifold, and they are held in place with thumbscrews. (c) A photograph of the underside of the generator manifold, showing the detail of how the inlet and outlet tubing for each bottle should be configured. The white Teflon sealing disks are also shown. (d) The custom-designed aluminum sample plate made to standard 96-well dimensions. This plate holds up to 96 GC-vial inserts containing samples to be methylated. This technical drawing shows the dimensions necessary to fit into the heating block of the 96-needle concentrator/evaporator. All the lengths shown are in millimeters. (e) A photograph of the sample plate loaded with 96 GC-vial inserts. This is a magnification of the inset shown in a.
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the bottom of the reagent bottle (~12 cm for a 250-ml bottle). This is the N2 inlet line to the first reagent bottle (Figs. 2c and 3, sector g).
6| Cut another length of tubing to connect the two reagent bottles (Fig. 3, sectors g and j). Using a fitting and a ferrule, attach one end to the second threaded hole at the top of the manifold (Fig. 3, sector f). The tubing should extend down into the reagent bottle 1 or 2 cm from the manifold (see Fig. 2d). This is the N2 line out from the first bottle (Fig. 2c).
7| Attach the other end of the tubing from Step 6 to the third threaded hole at the top of the manifold (Fig. 3, sector h) using a fitting and a ferrule. As in Step 5, connect the tubing so that it extends ~12 cm down from the manifold. This is the N2 line into the second bottle (Figs. 2c and 3, sector j).
8| Cut a length of tubing to extend between the manifold (Fig. 3, sector d) and the residue trap (Fig. 3, sector k). Connect one end of the tubing to the fourth threaded hole at the top of the manifold (Fig. 3, sector i) using a fitting and a ferrule so that, as in Step 6, it extends down 1 or 2 cm into the reagent bottle from the manifold (Fig. 2c).
9| Attach a Teflon compression-reducing union to each side of the 13-mm–outer diameter glass tubing to form the residue trap (Fig. 3, sector k).
10| Connect the other end of the tubing from Step 8 to one side of the residue trap (Fig. 3, sector k) using the reducing union. The tubing should extend more than halfway through the glass tube.
11| Cut a length of tubing to extend between the residue trap (Fig. 3, sector k) and the check valve (Fig. 3, sector l). Attach one end to the other side of the residue trap from Step 10 using the reducing union. As in Step 10, the tubing should extend more than halfway through the glass tube. The inlet and outlet tubing in the trap will therefore be out of line with each other so as to prevent the transfer of liquid or particles.
12| Attach the other side of the tubing from Step 11 to the inlet side of the check valve (Fig. 3, sector l).
13| Cut a length of tubing to extend between the check valve (Fig. 3, sector l) and the T-shaped union (Fig. 3, sector m). Attach one end to the outlet side of the check valve and the other end to the T-shaped union.
14| Cut a length of tubing long enough to connect the T-shaped union (Fig. 3, sector m) with the C/E (Fig. 3, sector n). Attach one end to the T-shaped union.
15| Remove the needle valve from the manifold of the C/E (Fig. 3, sector n); even when it is fully open, the valve restricts flow to the manifold and is not necessary for this application. Attach the other end of the tubing from Step 14 to the metal tube of the C/E that has been exposed on removal of the needle valve. The tubing can be attached by slipping it over the metal tube; it should fit snugly.
N2
N2
75 °C
LF
HF
a bc
fd
e ihk
g j
l
m n
o
p q
r
I. Gas supply and control
II. Manifold/diazomethaneproduction III. Gaseous diazomethane delivery
Figure 3 | A diagram of the complete 96-sample methylation and evaporation system. For ease of assembly, the system is categorized into three major components: (I) gas supply and control; (II) manifold and diazomethane production; and (III) gaseous diazomethane delivery. The assembly of this system is described in the PROCEDURE, and all the elements are connected by Teflon tubing. Briefly, UHP nitrogen is connected to a 0–30 p.s.i. pressure gauge (a) and flows through a shutoff valve (b) to a low-flow (LF) flowmeter (c). The flowmeter is connected to the first reagent bottle (g) through the left-most threaded hole on top of the aluminum diazomethane generator manifold (e). Tubing connects the first reagent bottle to the second one ( j) by extending from the second threaded hole on top of the manifold (f) to the third (h). Both reagent bottles are fitted with protective netting, their necks are wrapped with heavy-duty Teflon tape and they are screwed into the bottom of the manifold. Tubing extends from the fourth threaded hole on the manifold (i) to the residue trap (k), which prevents any liquid or particulates from being carried on to the samples. The residue trap is then connected to a check valve (l) that prevents the high-flow, makeup nitrogen line, connected at the T-shaped union (m), from entering the diazomethane reaction in the manifold. The makeup nitrogen line, shown in the bottom row of the diagram, is meant to increase the total flow of the system so that samples can be evaporated efficiently, but bypasses the diazomethane reaction, which is too unstable for such high flow rates. The makeup line consists of UHP nitrogen connected to a 0–100 p.s.i. pressure gauge (p) flowing through a shutoff valve (q) to a high-flow (HF) flowmeter (r). The makeup nitrogen connects with the main line at the T-shaped union. Tubing extends from the union to the concentrator/evaporator (n), whose heating block contains the sample plate with organic acids to be methylated.
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16| Cut a length of tubing to reach between the outlet port of the HF flowmeter (Fig. 3, sector r) and the T-shaped union (Fig. 3, sector m); attach one end of the tubing to the outlet of the flowmeter using a male pipe adapter elbow and attach the other end to the union.
17| With Teflon tubing, connect the inlet port of the LF flowmeter (Fig. 3, sector c) to a brass shutoff valve (Fig. 3, sector b) and a 0–30 p.s.i. gauge (Fig. 3, sector a).
18| With Teflon tubing, connect the inlet port of the HF flowmeter (Fig. 3, sector r) to a brass shutoff valve (Fig. 3, sector q) and a 0–100 p.s.i. gauge (Fig. 3, sector p).
19| Connect the two gauges (0–30 and 0–100 p.s.i.) to UHP N2 tanks (Fig. 3, sectors a and p).
20| Line the threaded holes that the reagent bottles screw into with a circle of Teflon that is 4 cm in diameter with two holes to accommodate the 3-mm tubing (see Fig. 2d). These can be made from 1/8-inch-thick Teflon sheets using appropriate drill bits.
21| Wrap the neck of each reagent bottle with heavy-duty Teflon tape (Fig. 3, sectors g and j). This will help prevent leaks and will prevent the glass threads from chipping against the aluminum manifold.! cautIon Sharp glass edges, such as those formed by chips in the threads of the reagent bottle, can cause diazomethane to explode. Lining threads and bottlenecks with heavy-duty Teflon tape will help prevent formation of such chips.
22| Encase each reagent bottle (Fig. 3, sectors g and j) in heavy-gauge netting as a safety precaution to protect against flying glass in case the diazomethane causes an explosion in the bottle.
23| Place the high-flow diazomethane generator apparatus in an explosion-proof ventilated hood. Optionally, an in-line union can be placed between any of the major components of the system, such as between parts I, II and III in Figure 3. Because in-line unions are easily taken apart, this allows the system to be modular and easily assembled and disassembled.! cautIon Methylation will be performed using diazomethane, which is a toxic and explosive gas. Diazomethane should only be used in a well-ventilated hood using standard safe laboratory practices such as wearing goggles and gloves. crItIcal step The manifold is designed such that, once the Teflon tubing and discs and Pyrex bottles are in place, diazomethane comes in contact only with Teflon or smooth glass surfaces. It is therefore important to follow the assembly steps carefully.
High-flow, simultaneous diazomethane generation and sample derivatization and concentration ● tIMInG the following steps (steps 24–31) should take 2–3 h to complete24| Prepare sample plate containing up to 96 samples of organic acids dissolved in up to 300 µl of methanol. Methanol is the necessary solvent for this procedure because it has been shown to efficiently catalyze the methylation reaction18. crItIcal step Moisture will substantially interfere with the methylation reaction. Best results will be obtained with samples that are as free of water as possible.
25| Place the sample plate in the heating block of the C/E.
26| Set the heating block to 75 °C. Please note that, although room temperature (22 °C) works well for methylating IAA19, 75 °C is the temperature that we empirically determined works well for the concurrent methylation and concentration of IAA-containing methanol-based extracts from plant tissues. Other substrates may require different temperatures. This can be determined by attempting to methylate and evaporate samples at a lower temperature and tracking whether the samples evaporate within a reasonable time and give measurable data by GC-MS.
27| Unscrew the first reaction bottle (′g′ in Fig. 3) from the manifold assembly and add ~100 ml of diethyl ether to it. Ensure that the bottle’s neck is thoroughly wrapped in heavy-duty Teflon tape and fit the bottle back into the first position of the generator manifold.
28| Unscrew the second reaction bottle (′j′ in Fig. 3) from the manifold assembly and add 35 ml of 2-(2-ethoxyethoxy) ethanol, 35 ml of diethyl ether and 35 ml of 7 N NaOH to it. Add 4.5 g of Diazald and fit the bottle back into the second position of the generator manifold. Ensure that the bottle’s neck is thoroughly protected by Teflon tape.
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crItIcal step Add the reagents to the second reaction bottle within 1 or 2 min of beginning the nitrogen flow. Allowing some or all of the components to settle for a longer amount of time might prevent the vigorous production of diazomethane. For example, we have noted that when 2-(2-ethoxyethoxy)ethanol and NaOH are allowed to react together for more than a few minutes, the mixture turns a dark brown and interferes with good methylation once the other reagents are added.
29| After placing the sample plate in the heating block, begin the nitrogen flow through the system at ~3,300 ml min − 1 of total flow (~900 ml min − 1 for the low-flow line and ~2,400 ml min − 1 for the high-flow line). The high-flow line is a makeup nitrogen line that serves to help evaporate the samples rapidly. It bypasses the diazomethane generator so as not to introduce high pressure to the diazomethane reaction. Please note that this nitrogen flow setting was empirically determined for the methylation of IAA-containing extracts from plant tissues. Other substrates or solvents may require faster or slower settings to evaporate samples within a reasonable time frame. Although our extraction protocol leaves our samples in 300 µl of metha-nol before this methylation step, others might be able to use a smaller volume of methanol with a more volatile solvent such as ether, dicholoromethane or pentane, noting that it is important to have methanol present for the methylation reaction to occur18.
30| Allow the derivatization reaction and the concentration of samples to proceed for ~25 min or until the samples are com-pletely dry (this may be especially important for GC column phases that are sensitive to alcohols). Although our experience has shown that methyl IAA will not be significantly lost in this step, those working with compounds in which the methyl- ester form is much more volatile than the free acid will need to use caution and optimize this step to prevent loss.! cautIon Methylation is performed using diazomethane, which is a toxic and explosive gas. Use standard safe laboratory practices, such as wearing goggles and gloves, throughout the reaction. Please be especially vigilant that there are no sharp edges—e.g., ground glass joints, scratched or broken glass, or crystals that form when solvents solidify in the reaction vessel, as these surfaces can catalyze diazomethane’s explosion.
31| Resuspend the samples in a solvent appropriate for chromatographic analysis; place each insert in a GC vial, and then cap and analyze the methylated samples. An 8- or 12-channel pipettor is useful in this step for resuspending 8 or 12 samples at once; the pipette tips must be rinsed with alcohol and dried before use. This will help remove phthalates from the surface of the tips. Phthalates can cause interference with chromatographic analysis.
32| Waste disposal: To consume any remaining diazomethane in the reaction bottle, keep the bottle in the ventilated hood and add small amounts of acetic acid until the yellow color from diazomethane is no longer visible15. This will produce methyl acetate, which, along with p-toluene sulfonic acid sodium salt, diethyl ether, 2-(2-ethoxyethoxy)ethanol and excess NaOH, will be what remains in the reaction bottle. After waiting several hours to ensure that the reaction is complete, dispose of this mixture in a way that complies with your institution’s waste disposal policies.
33| Maintenance: After each use, the reagent bottles should be washed well with soap and water. The gas-delivery needles of the C/E should be soaked in water, then methanol, and then fully dried. When residue can be seen building up on the residue trap, it should be disassembled, washed with soap and water, and dried well. Residue can also build up in the Teflon tubing downstream of the generator. Once a month, this stretch of Teflon tubing should be rinsed with water, then with methanol, and then dried well. Every year or so, the tubing should be replaced.
34| Estimating yield: The most reliable way to estimate yield is to use a stably labeled internal standard and isotope dilution. This method is fully discussed in the accompanying report17.
● tIMInGSteps 1–23, Assembly of the diazomethane generator: this is a one-time project that should take 1–2 h once all the materials have been gatheredSteps 24–31, Simultaneous diazomethane generation and sample derivatization and evaporation: 2–3 hSteps 32–34: Variable. Waste management and maintenance typically can be accomplished in less than 1 h per week in a laboratory using the system daily. Yield estimation is usually part of the downstream analytical procedures and does not contribute to the time required for methylation per se.
? trouBlesHootInGTroubleshooting advice can be found in table 1.
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antIcIpateD resultsIn a trial experiment, ten methanol-soluble organic acids were methylated by the procedure outlined above, without specific optimization for each except for IAA16, and analyzed either by HPLC or thin-layer chromatography (TLC), depending on their suitability for each. The following compounds were analyzed by HPLC, with their methylation efficiencies given in parenthe-ses: anthranilic acid (93%), caffeic acid (100%), trans-cinnamic acid (85%) (Fig. 4), IAA (98%) and naphthalene acetic acid (99%). The compounds analyzed by TLC were as follows: citric acid (100%), gibberellic acid (74%), jasmonic acid (83%), malic acid (100%) and salicylic acid (100%).
Each compound was dissolved in methanol at a concentration of 3 mg ml − 1; 300 µl of each solution was placed into a vial insert and the inserts were placed into an aluminum sample plate. Evaporation and methylation were performed as described in the procedure for 20 min. Thereafter, each compound was resuspended in 1 ml of 50% methanol (vol/vol) and analyzed for the efficiency of methylation.
Because the analysis of methylated compounds depends on the particular properties of the target compounds, the fol-lowing is not meant to be a part of the protocol of this paper, but rather a report of how we analyzed these trial samples. However, others may use these methods as a starting point to optimize the steps for their needs. In all cases, an aliquot of the unmethylated compound solution was analyzed and compared with the methylated one. Five of the compounds were analyzed on a Waters 600E HPLC using a 150 mm × 4.6 mm Luna C18 column of 5-µm particle size and 100 Å pore size (Phenomenex). Detection was carried out using a Spectroflow 757 absorbance detector (ABI Analytical Kratos Division) with the wavelength set appropriately for each compound (anthranilic acid: 330 nm; caffeic acid: 323 nm; trans-cinnamic acid: 265 nm; IAA and naphthalene acetic acid: 282 nm). Solvent A (1% acetic acid (vol/vol), 10% methanol/water (vol/vol)) and solvent B (100% methanol (vol/vol)) were used at a 1 ml min − 1 flow rate with the following gradient: 0–30 min, initially 83% solvent A, linear gradient to 100% solvent B; 30–35 min hold at 100% solvent B. Data were analyzed using BaseLine Chromatography Data System software. The remain-ing five compounds were analyzed by TLC on Silica gel G plates (10 cm × 20 cm, 250-µm thickness, Analtech), with a mobile phase of ethanol:ammonium hydroxide:water (20:5:3 vol/vol). The plates were allowed to develop for 1 h, after which they were sprayed with a chromic acid–sulfuric acid reagent (0.17 M potassium dichromate in 40% sulfuric acid (vol/vol)) and charred at 150 °C for 1 h (ref. 20). The plates were then analyzed on a light box (Alpha Innotech Chem-Imager 5500, Cell Biosciences) using white or ultraviolet light, and spot densities were measured using Fluorchem 5500 software (v. 4.0.1, Alpha Innotech/Cell Biosciences).
taBle 1 | Troubleshooting table.
problem possible reason solution
Low yield of the methyl ester(s)
Poor solubility of the organic acid in methanol
Attempt to repeat the derivatization reaction with a mixture of methanol and a solvent suitable for the target compound (water is not an option), keeping in mind that pure methanol has been shown to be the appropriate solvent to catalyze methylation by diazometh-ane17. If a solvent mixture does not work, or for compounds not soluble in methanol or a methanol-solvent mixture, diazomethane may not be the appropriate methylation reagent
Moisture interferes with the methylation reaction
Repeat the reaction with fresh reagents and a sample that has been well dried (e.g., by placing over magnesium sulfate crystals before phase separation by filtration)
Derivatized product is left behind on the walls of the glass inserts
If the compound is confirmed to be highly soluble in the reagent used for resuspension, a more thorough resuspension, concentrating on rinsing the walls of the sample-containing insert, should solve this problem
Low gas flow or the flow going in the wrong direction
Tubing plugged by reaction residue
Rinse the tubing with water and methanol. Periodically replace the Teflon tubing with a new one
200
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Figure 4 | A sample HPLC chromatogram showing the separation of trans-cinnamic acid and trans-cinnamic acid methyl ester after a 20-min methylation using the HPLC system described in the text.
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1626 | VOL.5 NO.10 | 2010 | nature protocols
acknowleDGMents We thank J. Normanly, A.Culler, W.-P. Chen and Y. Mohammed for help in trials of the diazomethane generator, and M. Emerick for help with metal design and fabrication. This work was supported by grants from the US National Science Foundation (DBI 0077769, DBI 0606666, MCB 0725149 and IOS 0923960) and the US Department of Agriculture National Research Initiative (2005-35318-16197).
autHor contrIButIons J.D.C. and L.S.B. conceived the protocol. The protocol is based on a previous diazomethane generator design by J.D.C. that he redesigned for higher flows and volumes. L.S.B. conducted the experiments, tested the protocol and wrote the paper with assistance from J.D.C.
coMpetInG Interests stateMent The authors declare no competing financial interests.
Published online at http://www.natureprotocols.com/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/.
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