1

Derivatization of Carboxylic Acids with Diazomethane and Trimethylsilyldiazomethane: Convenient Methods and Artifacts

Contents 1. Introduction 2. Experimental

2.1. Handling Precautions for Diazomethane 2.2. Preparation of Diazomethane in Larger Quantities Using Distillation 2.3. Preparation of Diazomethane and Diazomethane-C13 in Smaller Quantities Using Nitrogen “Purge

an Trap” Method and Sample Derivatization 2.4. Quantitative Measurements of Rate of Diazomethane Production with Nitrogen “Purge and Trap”

Method 2.5. Derivatization of Samples with Trimethysilyldiazomethane in Hexane Solution

3. Results and Discussion 3.1. Diazomethane Reaction Mechanism 3.2. Artifacts Formed form Diazomethane

3.2.a. Artifacts from Reaction of Diazomethane with Ketones and Aldehydes 3.2.b. Artifacts from Reaction of Diazomethane with Alcohols 3.2.c. Artifacts from Reaction of Diazomethane with Phenols 3.2.d. Artifacts form Reaction of Diazomethane with Solvents 3.2.e. Artifacts from Reaction of Diazomethane with Alkenes 3.2.f. Artifact from Reaction of Solvent with Diazomethane Precursor 3.2.g. Artifact from Reaction of Diazomethane with Amino Acids

3.3. Trimethysilyldiazomethane Reaction Mechanism 3.4. Artifacts from Trimethylsilyldiazomethane Reagent

3.4.a. Formation of Trimethylsilyl and Trimethylsilylmethyl Ester Artifacts 3.4.b. Other Artifacts Containing Trimethylsilylmethyl Groups 3.4.c. Presence of Phenol and Phenol Derivatives in Trimethylsilyldiazomethane in Hexane

Reagent 4.0 Summary 5.0 References 6.0 Tables and Figures

1.0. Introduction We frequently employ diazoalkanes such as diazomethane and trimethylsilyldiazomethane for the derivatization of carboxylic acids. The resulting methyl esters are ideal derivatives for the characterization of carboxylic acids. They are easily characterized by gas chromatography (GC) and readily identified by either interpretation or computer searching of their electron impact (EI) mass spectra. Diazomethane reacts instantaneously with carboxylic acids to yield methyl esters and forms few by-products.

R

O

OH + CH2N2 R

O

OCH3N2+

Diazomethane However, it must be prepared from a precursor and care must be taken in handling this very reactive reagent. Trimethylsilyldiazomethane reacts much slower than diazomethane with carboxylic acids and yields somewhat higher concentrations of by-products (artifacts).

2

R

O

OH+ R

O

OCH3

N2+

Trimethylsilyldiazomethane (TMSCHN2)

CH3OH+

(CH3)3SiCHN2

CH3OCH2Si(CH3)3

+

However, it is much more convenient to employ than diazomethane since it is more stable and can be purchased commercially in organic solvents such as hexane. Both diazolalkane reagents convert carboxylic acids to methyl esters in high yields. However, other functional groups will also derivatize at slower rates to yield one or multiple derivatives. We refer to these undesirable or unexpected derivatives (by-products) as artifacts. In addition, these reagents can react with solvents and/or analytes to yield artifacts. Often the formation of artifacts is accelerated by other components found in crude samples. The presence of multiple peaks or artifacts in the gas chromatographic analysis of mixtures leads to confusion about the concentration of a component or the number of components present in a sample. Thus, the identification of these components by gas chromatography-mass spectrometry (GC-MS) is critical. This report includes the types of artifacts identified in our laboratory by GC-MS and references to those found in the literature. Many times reaction conditions can be modified to avoid artifact formation. In addition, convenient methods for the preparation of diazomethane are presented and typical reaction conditions for the use of both diazoalkanes are described. 2.0. Experimental 2.1. Handling Precautions for Diazomethane

BEFORE PREPARING OR USING SOLUTIONS OF DIAZOMETHANE, THE TECHNICAL INFORMATION AND SAFETY INFORMATION1-3, 8, 9 ON DIAZOMETHANE SHOULD BE STUDIED CAREFULLY. Diazomethane is quite safe when produced in dilute solutions and in small volumes, as is the case in most analytical applications. The diazomethane is consumed very quickly since it is purged directly into the sample to be derivatized. However, one should be aware of the hazards of diazomethane and precautions for handling it safely. Diazomethane is a yellow gas at room temperature, liquefies at –23 oC, and freezes at –145 oC. It is extremely toxic and highly irritating gas which when inhaled in high concentrations can result in pulmonary edema. Long-term low-level exposure can lead to sensitization with asthma-like symptoms. Furthermore, diazomethane and some of its precursors are cited as carcinogens. Diazomethane can explode unaccountably both as a gas and as a liquid. Rough surfaces and strong light are known to detonate diazomethane. All edges of glassware used for diazomethane should be carefully firepolished and ground-glass joints cannot be employed. Care should be taken in cleaning the glassware used for diazomethane to avoid scratching the surfaces. Contact of diazomethane with alkali metals or drying agents such as calcium sulfate can result in an explosion. The recommended drying agent for diazomethane is potassium hydroxide pellets. All diazomethane reactions should be performed in an efficient fume hood and behind a sturdy safety shield. Reactions of diazomethane are best performed at room temperature or below.

3

Solutions of diazomethane should not be frozen because the rough edges of crystals could cause it to explode. We employ Diazald (N-methyl-N-nitroso-p-toluenesulfonamide, Aldrich Chemical Company) as a precursor in two of our methods for preparing diazomethane. The reaction is outlined below:

CH3 S

O

O

N NO

CH3

+ KOHH2O

CH3 S

O

O

O-

+

K+

CH2N2

Diazald Diazomethane Two of our methods use 2-(2-ethoxyethoxy)ethanol as a solvent. One reaction scheme employs diethyl ether as a solvent and the other tetrahydrofuran (THF). Diazald is a severe skin irritant and all skin contact should be avoided. It should be stored in a brown bottle at room temperature. It is stable at room temperature for at least one year; however, the material should be kept refrigerated for prolonged storage. The amount of artifact formation was checked by either gas chromatography/mass spectrometry or gas chromatography with a flame ionization detector. Standards were not available for the majority of the artifacts, so their concentrations were estimated by response factors of similar compounds. 2.2 Preparation of Diazomethane in Larger Quantities Using Distillation There are many different methods and reagents used to prepare diazomethane.1-9 Our initial derivatization work employed a distillation method that produced a diethyl ether solution of diazomethane. This method is more appropriate for organic synthesis than for derivatization of samples for analytical needs such as gas chromatography (GC) and GC-MS (gas chromatography-mass spectrometry. Thus, we have switched to alternative methods in the last few years that are described in the following sections. The details of this method are not listed here but can be found in the literature.1, 2, 8, 9 The “alcohol-free” method employed Diazald, 2-(2-ethoxyethoxy)ethanol, and The Diazald Glassware Kit. This was very convenient because a very large number of samples or larger quantities of an individual sample. However, the method was much more hazardous since large quantities of diazomethane in diethyl ether are stored for an hour to several hours. 2.3. Preparation of Diazomethane and Diazomethane-C13 in Smaller Quantities Using Nitrogen “Purge and Trap” Method and Sample Derivatization The method describe here generates diazomethane in small quantities which is consumed as it is generated. Thus, the chance of an explosion or exposure is significantly decreased. The latter method was also found to be advantageous in reduced the concentration of artifacts formed. This is because a large excess of diazomethane is not present during the whole reaction. Our method is very similar to that employed by Schlenk and Gellerman,10 Walker et a.l,36 and Cohen.37 Our method could be significantly improved by incorporating several of the design features in the Cohen37 apparatus such as “Clear-Seal” standard taper joints, springs, PTFE tubing, sturdy two piece design, etc. Our apparatus is shown in Figure 1. Add enough 2-ethoxyethanol to tube B to cover approximately 1 inch of the tip of arm that extends from tube A. The arm from tube A should extend close to the bottom of tube B, but not close enough to interfere with the stirring bar. The nitrogen gas from E is used to purge the diazomethane formed in the solution in test tube B to the sample vial, M.

4

The reaction is performed in a laboratory hood behind a weighted explosion hood and gloves are worn. Start the nitrogen purge (~30-50 ml/min) and the magnetic stirrer and place a septum over H. Add 2 ml's of solution #1 to test tube B via syringe through the septum. Add 2 ml of solution #2 by syringe via the syringe through the septum and let stir. Check for Diazomethane formation by placing a blank solution of DMF in sample vial M. Diazomethane is a yellow gas and will turn the DMF solution blank yellow. Remove the DMF blank solution when diazomethane formation is indicated and start the derivatization of samples. Place samples to be derivatized at point M. Let each sample sit in the diazomethane/nitrogen stream until they turn yellow (approximately 1-5 minutes depending on the concentration of sample to be derivatized). Remove derivatized sample from point M and wash off the end of sidearm from test tube with DMF to remove residual sample. Place another sample to be derivatized in the stream. Samples for most of our work were dissolved in DMF since this solvent is very good for even very insoluble organic acids. Four or five samples can usually be derivatized with the diazomethane generated. Additional amounts of solutions #1 and #2 can be added without cleaning the glassware. Normally we add 2-4 ml’s of solution #2 without adding any additional solution #1. No additional 2-(2-ethoxyethoxy)ethanol is needed. Excess diazomethane can be easily and instantaneously destroyed by the addition of acetic acid, which generates methyl acetate as a by-product. We generally add excess diazomethane (yellow color noted) to samples and allow them to react for 30 seconds to 1 minute to insure complete reaction. Excess diazomethane is destroyed by adding small quantities of acetic acid until the yellow color of diazomethane is not observed. Also all diazomethane present in blanks or in the nitrogen purge is quenched with acetic acid. When the diazomethane generated in the purge is no longer needed, excess is quenched by bubbling the gas through several milliliters of 50% acetic acid in DMF. The purge is continued for 30 minutes to an hour to insure that all diazomethane generated is destroyed! Diazomethane labeling experiments were performed by generating 13C-labelled diazomethane. The method using no distillation was used and the N-methyl-13C labeled analogue of Diazald (Aldrich Chemical Company) was substituted for Diazald. Apparatus: (Refer to Figure 1) A, B ,C Three 6" long by 1" outside diameter test tubes w 7" x 1/4" arms with flat bottoms. They were prepared at

Lab Glass, Kingsport, TN. D 1/4" Outside diameter nitrogen feed tube E Dry Nitrogen supply line from high purity Nitrogen tank or "house" nitrogen in laboratory F Stirring Bar G Stirring Plate, stirring mechanism removed from small stirrer and placed in Plexiglas glass box, standard

stirring plates too large to fit between test tubes H Solution supply tube with septum top I, J, K Three #4 Rubber Stoppers L Supporting plate M Sample Vial, tube immersed into solution N Diazomethane reaction solutions (Sol).:

Sol. #1: 30% KOH in water (weight/volume) Sol. #2: 20% Diazald in tetrahydrofuran (THF) (weight/volume)

2.4 Quantitative Measurements of Rate of Diazomethane Production with Nitrogen ”Purge and Trap”

Method The rate of diazomethane formation versus time is shown in Figure 2 and Table 1. This data was obtained by bubbling the nitrogen stream containing diazomethane through a sample of benzoic acid dissolved in DMF. Aliquots of the DMF solution were taken versus time and analyzed by LC with a UV detector. The amount of methyl benzoate was converted into the amount of diazomethane produced. A total yield of 48.7% was noted for diazomethane. This compared well to a 51% yield measured for the reaction by quantitative proton NMR analyses. 2.5. Derivatization of Samples with Trimethylsilyldiazomethane Solution

5

Trimethysilyldiazomethane is considered to be a stable and safe substitute for diazomethane. However, Aldrich lists the material sold as a 2.0 M solution in hexane as an irritant and neurological hazard. They suggested avoiding skin contact and inhalation and to handle and store under nitrogen. We use the material in an efficient fume hood employing laboratory gloves. The original reference outlined the following process32 for the conversion of a carboxylic acid to its methyl ester. The carboxylic acid (1 millimole) is stirred in methanol (2 ml) and benzene ( 7 ml). 1.3 millimoles of trimethylsilyldiazomethane in 1 ml of benzene are added at room temperature. The mixture is stirred for 30 minutes at room temperature and the solvent is removed as needed to concentrate the resulting methyl esters. Most current methods use 2.0 M solutions of trimethylsilyldiazomethane in hexane. This solution can be purchased from many different sources (e.g. Aldrich Chemical Company). Most of the methods34,35,38

dissolve the acid of interest in some solvent such as diethyl ether, toluene, or methanol. Methanol must be present in the reaction to obtain the methyl esters in high yield.32 Normally a reaction time of 30 minutes at room temperature is needed for complete conversion of the acids to methyl esters. However, one reference38 suggests sonicating vials covered with laboratory sealing film for 20 minutes. One reference35 suggests quenching the excess trimethylsilyldiazomethane with acetic acid. The resulting methyl esters can be taken to dryness carefully under a stream of nitrogen and dissolved in the desired solvent for GC or GC-MS analyses. Also a method was described for the N-methylation of aromatic sulfonamides that required the addition of phosphoric acid in acetone.41 We normally employ N,N-dimethylformamide (DMF) for the reactions. DMF is very good for dissolving a wide variety of aromatic carboxylic acids, which are not readily soluble in solvents such as methanol, methylene chloride, toluene, diethyl ether, etc. A typical experiment using benzoic acid is described. Benzoic acid (2 mg, 0.016 mmoles) is dissolved in 1 ml of DMF:methanol (9:1 volume:volume) and 40 microliters of a 2.0 M solution of trimethylsilyldiazomethane (0.08 mmoles) is added. The mixture is reacted in a loosely capped vial for 30 minutes at room temperature. The reaction was performed in a loosely capped vial since nitrogen gas formed during the reaction. Excess reagent was destroyed by adding approximately 5 microliters (~0.08 mmoles) of acetic acid and allowing the mixture to stand for 30 minutes at room temperature. The experiment above was also attempted with 20 microliters of the trimethylsilyldiazomethane reagent, which should have been adequate to derivatize the benzoic acid. However, after 30 minutes, there was residual benzoic acid noted by GC-MS in the sample. The excess trimethylsilyldiazomethane does not necessarily need to be destroyed with acetic acid since the reagent will chromatograph. However, other side reactions could occur with time and it is necessary to properly vent the split and septum purge of the GC to avoid personal contact with the reagent. 3.0. Results and Discussion 3.1. Diazomethane Reaction Mechanism Diazomethane is an ideal derivatization reaction. The reaction is fast, the yield is high, side reactions are minimal, the by-product is nitrogen gas, and reaction conditions are very mild. Diazomethane is a yellow gas so the progress of the reaction can easily be followed. The reaction for the conversion of carboxylic acids to methyl esters is outlined below:

6

R

O

OH+

CH2 N NR

O

O

N2

+

Diazomethane

- - ++

CH3 N N

R

O

O CH3 +

Two of the protons in the resulting methyl ester originate from the diazomethane. The other one is the “donated” acidic proton from the carboxylic acid. Diazomethane will also react with compounds containing less acidic protons including phenols, alcohols, enols, and heteroatoms. There are several excellent reviews of the chemistry of diazomethane1-3 and its use as a reagent for analytical derivatizations.4-7 Diazomethane is quite safe when handled as a dilute solution in an inert solvent. However, all users of the reagent should be aware that it is highly toxic and can be explosive if handled improperly! 3.2. Artifacts Formed from Diazomethane The majority of the observed artifacts (by-products) noted in this paper arise from known reactions of diazomethane1-3 Diazomethane will react with a wide variety of functional groups under the proper conditions. Many of these reactions require the addition of catalyst or long reaction times to occur in high yields. In addition, we have noted much higher concentrations of artifacts in crude samples containing various types of inorganic salts. We have only discussed some of the more common artifacts noted in our laboratory and in literature references. The review by Pizey1 discussing methylation, additions, homologation, and miscellaneous reactions employing diazomethane is especially useful in identifying artifacts. 3.2.a. Artifacts from Reaction of Diazomethane with Ketones and Aldehydes Ketones and aldehydes form undesirable artifacts with diazomethane by methylene insertion to yield epoxides or ketones as follows:

R

O

R' CH2N2+

+

+

O R

R'

O

R'R

R

O

R'N2

However, most of these types of reactions occur at much longer reaction times compared to the conversion of carboxylic acids to methyl esters. Several benzophenone and acetophenone model compounds dissolved in DMF showed <0.1% conversion to artifacts in a minute of exposure to a slight excess of diazomethane. However, the aldehydes were much more reactive yielding about a 6% concentration of ketone and epoxide artifacts under identical reaction conditions. For example, Compound 1 was converted to the following artifacts when exposed to a slight excess of diazomethane for 1 minute.

7

O H

O OH

+ + +CH2N2

O H

O OMe

O CH3

O OMe

O

O OMe

1 min

1 2 3 4(94%) (2%) (4%)

Compound 4 can also react with more diazomethane to give additional artifacts, but at a much slower rate than conversion of carboxylic acids to methyl esters. The structures for Artifacts 3 and 4 were confirmed by proton NMR analyses of a sample exposed to diazomethane at room temperature for 45 minutes. The electron impact mass spectra of Artifacts 3 and 4 are shown in Figure 3. Our initial derivatization reactions added a DMF solution of the aldehyde to an ether solution of diazomethane. The reverse addition of the diazomethane to the DMF solution shows much lower concentrations of artifacts. The artifacts are reduced in the latter case because it minimized the presence of excess diazomethane in the reaction mixture at the start of the derivatization reaction. The use of diazomethane-13C can be useful in determining if acetophenones or epoxides are actually present in a sample of the corresponding aldehyde. For example, our sample of Compound 5 was checked for the presence of the corresponding ketone or epoxide by this method. All the epoxide and ketone, Compounds 3 and 4, detected were found to originate from artifact formation and not from impurities present in the starting material.

O H

O OH

+ + +CH2N2

O H

O OMe

O CH3

O OMe

O

O OMe1

13 13

13

Compound 3 was noted to further react with acetic acid to give Artifacts 5 and 6. Acetic acid is used in our procedure to quench excess diazomethane forming methyl acetate as a by-product.

8

+

O

O OMe

3O OMe

O

O

CH3

OH

O OMe

OH

O

O

CH3

5 6

+

O

OH

Others have reported that fluorenones can react with diazomethane to yield the artifacts noted below:11-13

O

CH2N2

O OH

CH2N2

(5%)7

OMe

+

O

2(30%)

(1.5%)

89

The methyl ether artifact, Compound 8, is noted because the initially formed ketone artifact, Compound 7, is more stable in its enol-form. The enol is then rapidly converted to its methyl ether by the reaction with an additional mole of diazomethane. The authors offered no explanation for the mechanism of ether artifact, Compound 9. No significant concentrations of fluorenone artifacts were noted under our short reaction conditions and small excess concentrations of diazomethane. The high yields of artifacts noted in the literature12 for fluorenones were obtained with reaction times of 30 minutes to a day. Bibenzils can form a wide variety of artifacts.14 We noted under our reaction conditions that Compound 10 formed about 81% of Artifact 11 with the remaining accounted for by starting material. This result was also confirmed by proton NMR data. The electron impact mass spectra of Compounds 10 and 11 are shown in Figure 4.

9

O O

R R10

R = CO2CH3

CH2N2

O O

R R11

(81%) No significant concentrations of the other anticipated artifacts14 shown below were observed.

O O

RR

O O

R R

O

R

O

R

Ketones and some β-keto-aldehydes with α-protons that can readily enolize can form the methyl enol ether. For example, ethyl acetoacetate gave mainly the enol ether, Compound 12, with the epoxide as the minor product:

O

OCH2CH3

O

ketoenol

O

OCH2CH3

CH2N2

O

OCH2CH3

12

OH OMe

We have not compared the rate of enol-ether formation to that of the conversion of carboxylic acids to methyl esters.

3.2.b. Artifacts from Reaction of Diazomethane with Alcohols Usually alcohols are converted to their methyl ethers in high yields by employing Lewis acid catalysts such as BF3 in an appropriate solvent. Some reports16-17 have noted the conversion of alcohols containing electronegative groups to their methyl ethers by employing aliphatic hydrocarbons as solvents. Employing our standard reaction conditions in DMF, only small amounts of the methyl ether were noted.

10

CH2N2

O OH

OH

O OMe

OMe

O

OH

OMe

+

(99.9%) (0.1%)

1 minroom temperature

3.2.c. Artifacts from Reaction of Diazomethane with Phenols Phenols can readily be derivatized18 when a Lewis acid catalyst is employed with diazomethane. Without the catalyst, the rate of conversion of a phenolic group to its methyl ether can vary drastically. We tested the derivatization of several phenols with very short reaction times and the yields are noted in parenthesis below:

O

OH

CH2N2

O

OH

+

O

OMe

(97%) (3%)

OH OMe OMe

O

OH

Br

CH2N2

O

OH

Br

+

O

OMe

Br

(0%) (100%)13

OH OMe OMe

Br

OH O

OH CH2N2

Br

OH O

OMe+

Br

OMe O

OMe

(100%) (0%)14

11

Our results were as expected. The phenolic group in Compound 13 reacts with diazomethane because the presence of the ortho-bromine group increases its acidity. Compound 14, which contains a bromine atom in conjugation with the phenol, showed no reaction. This is expected because studies have shown29-31 that the rate of methyl ether formation is very slow when the phenolic hydrogen can form an intramolecular hydrogen bond with an adjacent carbonyl group. Two different approaches can be used to avoid multiple components due to incomplete conversion of phenols to their corresponding methyl ethers. The first is to derivatize the phenol in diethyl ether containing BF3, which converts the phenol to its methyl ether in high yields. The second is to cool the reaction mixture to below 0 oC and minimize reaction times to minimize ether formation.19-20 Caution should be exercised in freezing diazomethane solutions since they could be shock sensitive and explode. 3.2.d. Artifacts from Reaction of Diazomethane with Solvents Diazomethane reactions can be performed in a variety of solvents. Typical solvents include methylene chloride, dioxane, diethyl ether, toluene, xylene, benzene, hydrocarbons, and diethyl ether/methanol. However, many organic acids are insoluble in these solvents, and thus, their rate of conversion to methyl esters is very low. We have tried two polar solvents, N, N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO) which increases the solubility of many acids. DMF worked very well with only small concentrations of artifacts noted. However, DMSO formed relatively high concentrations of artifacts. A few literature references reporting using DMSO as a solvent, but no artifact formation was mentioned.21-23 DMSO forms methylthiomethyl ester artifacts. For example, the reaction products of benzoic acid dissolved in DMSO and reacted with diazomethane are shown below:

O OH

CH2N2

O OMe O OCH2SCH3

+

15

DMSO

S

O

+

The methylthiomethyl group in Compound 15 could have come totally from the DMSO or could have incorporated the methylene group from the diazomethane. The origin of the artifact was probed by substituting DMSO-d6 for DMSO in the reaction. The formation of Compound 15a demonstrated that the methylene and methyl groups in the methylthiomethyl group are obtained entirely from the DMSO and none from the diazomethane. The mass spectrum of the methylthiomethyl ester of benzoic acid is shown in Figure 5.

O OH

CH2N2

O OMe O CD2SCD3

+

15a

DMSO-d6

D3CS

O

CD3+

Several other by-products from the reaction were identified as dimethyl disulfide (MW 94) and 1-methylthio-1-methylmethane (MW 92). We didn’t scan the mass spectrometer low enough to detect methanol.

12

Our results are consistent with the formation of the methylthiomethyl ester artifacts by a mechanism24 similar to a Pummerer-type reaction.

S

O

+ CH2N2

OS S

OMe

S

OMe

R

O

OH+

R

O

OCH2SCH3

+ CH3OH

There were also some minor artifacts noted when using DMF as the solvent for diazomethane reactions. The DMF artifacts appeared to be formaldehyde type oligomers. Their electron impact spectra typically showed ions at m/z 45, 61, 75, 89, 91, 105, 119, 121, 135, 149, etc. They were tentatively identified as the following structures:

H

O

O O n

Me

n = 7,12

O O n Me Me

n = 7,12 The molecular weights and formulae were determined27 by accurate mass ammonia chemical ionization data and consistent with the proposed structures. The ammonia chemical ionization data showed primarily M + NH4 ions for all the components. In addition, the quasi-molecular ions showed subsequent neutral losses of several molecules of formaldehyde. The concentrations of these impurities varied significantly from sample to sample even though the temperatures, reaction times, quality of solvents employed were controlled. The exact mechanism for the formation of these artifacts is unknown, but it is suspected to be related to the DMF since many of them are thought to be formate esters. Isotopic labeling would be very useful in understanding the mechanism for their formation and possible devising methods for controlling their formation. We have never employed THF (tetrahydrofuran) as a solvent for derivatization reactions. However, it is employed as a solvent in the preparation of the diazomethane. Some of this THF is purged with nitrogen into our samples during the derivatization procedure. We have noted that the artifact is formed at about the 500-1000 ppm-level relative to acid being derivatized. For example, the derivatization of Sorbic Acid yielded the following components plus smaller concentrations of other artifacts.

OH

OCH2N2

O+

THF

O

OMe

O

OOMe

+

The structure of the impurity was confirmed by substituting THF-d8 for THF to give the following compound.

13

O

OCD2CD2CD2CD2-O-CH3

The mass spectra of the deuterated and undeuterated artifacts are shown in Figure 6. A literature reference25 notes a similar type reaction to yield a methoxybutyl enol ether by the reaction of diazomethane with a substituted 1,3-indandione in the presence of THF. 3.2.e. Artifacts from Reaction of Diazomethane with Alkenes A wide variety of alkenes can add diazomethane to yield pyrazolines, cyclopropane, or C-methyl artifacts.26 For example, Sorbic Acid dissolved in DMF and reacted with diazomethane for 1 minute gave the following artifacts at a concentration between 50-100 ppm:39

OH

O

NH N

O

OMe

N NH

O

OMe

Pyrazoline Artifact

Sorbic Acid

CH2N2

O

OMePrimary Product

+

+

+ O

OMe

O

OMe

The electron impact mass spectrum of the pyrazoline artifact of Sorbic Acid is shown in Figure 7. It is very useful to run derivatization reactions with excess diazomethane for 45-60 minutes instead of the normal 1-minute reaction time. The changing profile makes it easy to detect artifacts and to identify them. In the Sorbic Acid derivatization reaction, an internal standard, decanoic acid, was added to the Sorbic Acid sample at the 100 ppm-level. The decanoic acid forms methyl decanoate quickly and does not form any artifacts in this time frame, and thus its concentration is constant. It was then easy to see which components were decreasing or increasing versus time. 3.2.f. Artifact from Reaction of Solvent with Diazomethane Precursor Several trace-level components were noted as artifacts in the derivatization of samples. One artifact was identified by its mass spectrum and accurate mass ammonia chemical ionization data27 as:

14

S

O

CH3

OCH2CH2OCH2CH2OCH2CH3

O

Apparently it is formed by the reaction of one of one of the solvents, 2-(2-ethoxyethoxy)ethanol, and the Diazald by-product in our diazomethane preparation. It must somehow be entrained in the ether distillation step in the production of diazomethane. Include table of contents. 3.2.g. Artifact from Reaction of Diazomethane with Amino Acids Amino acids were shown to form artifacts with diazomethane. Both N-methyl and N,N-dimethyl artifacts were observed.40 3.3. Trimethylsilyldiazomethane Reaction Mechanism Trimethylsilyldiazomethane is a convenient substitute for diazomethane since it can be purchased commercially and is much safer to use than diazomethane. Trimethylsilyldiazomethane is a greenish-yellow liquid, which is stable in a hydrocarbon solution.33 and in pure form. However, the conversion of carboxylic acids to methyl esters with this reagent requires around 30 minutes (an hour if excess reagent quenched). In addition, we have noted more chemical noise originating from the hydrocarbon reagent solution, reaction by-products, and artifacts formed from undesirable side reactions. Nevertheless, we have successfully substituted it many times in derivatization reactions previously employing diazomethane. The reaction of trimethylsilyldiazomethane with carboxylic acids is proposed32,33 to occur by a significantly different reaction mechanism than that of diazomethane with carboxylic acids. The reaction must have methanol present to get good yields of the desired methyl ester.

R

O

OH

+

N2

+

Trimethysilyldiazomethane

++

CH3 N NR

O

O CH3+

CHSi

CH3

CH3

CH3

N N-

R

O

O+

CH2

Si

CH3

CH3

CH3

NN-

CH3OH CH3OH

++

R

O

O-

SiOCH3 CH3

CH3

CH3

++

SiOCH3 CH3

CH3

CH3 One of the protons in the resulting methyl ester originates from the diazomethane, one from methanol, and the remaining one is the “donated” acidic proton from the carboxylic acid. 3.4. Artifacts from Trimethysilyldiazomethane Reagent

15

Many of the types of artifacts noted with diazomethane should also be formed with trimethylsilyldiazomethane. We have not used trimethylsilyldiazomethane as frequently as we have diazomethane, so the artifacts noted to date will be somewhat limited. 3.4.a. Formation of Trimethylsilyl and Trimethylsilylmethyl Ester Artifacts The methanol in the reaction suppresses the following mechanism:33

R

O

OH

++

+CH3 N NR

O

OSi

CHSi

CH3

CH3

CH3

N N-

R

O

O+

CH2

Si

CH3

CH3

CH3

NN-

CH3OH

+

R

O

O Si CH3

CH3

CH3

-N2

R

O

OH

R

O

O-

-N2

R

O

OCH3

+

However, the acid to derivatize can “donate” two protons to form the methyl ester, the trimethylsilyl, and the trimethylsilylmethyl ester of the acid being derivatized. Figures 8 and 9 show the electron impact mass spectra of three trimethylsilymethyl ester artifacts formed by this mechanism. 3.4.b. Other Artifacts Containing Trimethylsilylmethyl Groups Many of the artifacts noted with diazomethane can be also be noted with trimethylsilyldiazomethane. In contrast, many of them can also include the trimethylsilyl group. For example, trimethylsilyldiazomethane was noted to undergo a 1,3-dipolar addition with acrylonitrile:33

++ CHSi

CH3

CH3

CH3

N N-

N NN

N

Si

H

The resulting material was extremely unstable to hydrolysis by atmospheric moisture. Other Similar reactions were noted with other substrates, but the products could not be isolated and many polymerized.33 Trimethylsilyldiazomethane was noted to add to cyclohexene in the presence of copper (I) chloride to yield two isomers (syn and anti):33

16

++ CHSi

CH3

CH3

CH3

N N- Si

H

Trimethylsilyldiazomethane was also noted to decompose33 in the copper (I) chloride reaction to yield two isomeric cyclopropanes whose structure was confirmed by proton-NMR data.

+CHSi

CH3

CH3

CH3

N N-

Si

SiSi3

-N2

We have noted an artifact in our reactions (only one isomer) that could be one of these compounds. Its EI spectrum is shown in Figure 9. Two components were noted from the dimerization of trimethylsilyldiazomethane:

SiSi Si

Si

3.4.c. Presence of Phenol and Phenol Derivatives in Trimethylsilyldiazomethane in Hexane Reagent The trimethylsilyldiazomethane (in hexane) reagent purchased from Aldrich includes phenol. Aldrich would not reveal their method of synthesis, but indicated that the phenol was a by-product from the synthesis. The phenol was noted in all samples and varying amounts of the trimethylsilyl phenyl ether and methyl phenyl ether. Aldrich could be using a synthesis employing a phenyl phosphate ester. Another vendor’s reagent might not contain phenol as a by-product if it was synthesized by another approach.39

OH O

Si

O

CH3

4.0. Summary Normally derivatization reactions of carboxylic acids with diazomethane and TMSCHN2 yield primarily the desired methyl ester. However, many different artifacts can be formed at lower levels for some compounds. In addition, multiple peaks can be noted due to incomplete methylation of compounds. Identifying the artifact or partially derivatized component often leads to a means of avoiding the artifact.

17

Several ways of avoiding artifacts were noted in our studies and literature references. Ones that we have found generally useful are summarized below: -The first step is to characterize all components in the mixture by electron impact GC/MS. In some cases, additional analyses were required by chemical ionization GC-MS, accurate mass, and isotope labeling to identify unknowns. In addition, reaction times were often increased from 1 minute to 45-90 minutes to increase the concentration of the artifacts to facilitate identifications. Reaction conditions can only be efficiently optimized if components are identified and reactions leading to their formation understood. -Reaction times and temperatures should be optimized for the components/functional groups of interest. For many compounds, derivatization reactions are complete upon mixing with diazomethane. Diazomethane reactions should be performed at room temperature or below. For safety, solvents should not be used at or below their freezing points (see precautions on handling diazomethane!). -Select another solvent for the derivatization. Artifacts are often noted by reactions of the solvent with analytes and/or diazoalkanes. -Derivatization methods developed for pure standards often yield different products than those noted for crude samples containing additional solvents, inorganic acids, inorganic salts, etc. Therefore if is best to develop derivatization procedures for samples with matrices identical or similar to the targeted process samples. -The order or addition of the diazomethane to sample is important. Lower levels of artifacts for aldehydes were observed when diazomethane is added to the sample in solution as opposed to adding the sample in solution to diazomethane dissolved in an organic solvent. -Drive incomplete reactions to completion by the use of an appropriate catalyst/solvent mixture. -There are a multitude4-7, 28 of other derivatization reactions for conversion of acids to methyl esters including methanol employing acid catalysts, N,N-dimethylformamide dimethyl acetal, tetraaklyammonium reagents. Thus, consider a totally different class of derivatization reagent.

18

5.0. References 1. J. S. Pizey, Synthetic Reagents, Volume II, John Wiley & Sons, pp. 65-142 (1974). 2. H. B. Hopps, Aldrichim. Acta., 3(4) 9, Aldrich Chemical Company (1970). 3. T. H. Black, Aldrichim. Acta., 16 (1), 3, Aldrich Chemical Company (1983). 4. Daniel R. Knapp, Handbook of Analytical Derivatization Reactions, John Wiley & Sons, 1979. 5. Graham S. King, Karl Blau, Handbook of Derivatives for Chromatography, Heyden & Son, Ltd., 1977. 6. Karl Blau, John M. Halket, Handbook of Derivatives for Chromatography, Second Edition, John Wiley

& Sons, 1993. 7. J. Drozd, Chemical Derivatization in Gas Chromatography, Journal of Chromatography Library,

Elsevier, 1981. 8. Aldrich Technical Information Bulletin No AL-113. 9. Aldrich Technical Information Bulletin No AL-103. 10. H. Schlenk, J. L. Gellerman, Anal. Chem., 45, 1412 (1960). 11. Reference No. 1, p. 76. 12. R. F. Schultz et al., J. American Chem. Society, 62, p 2902 (1940). 13. B. Eistert and M. El-Chawawi, Monatsh., 98, p 941 (1967). 14. Reference No. 1, pp. 97-98. 15. Reference No. 1, pp. 75, 83. 16. Reference No. 1, pp. 70-73. 17. H. Meerwein, T. Bersin, W. Burneleit, Ber. 62,999 (1929). 18. Reference No. 1, pp. 71-78. 19. B. Plazonnet, W. J. A. VandenHeuvel, J. Chromatogra., 142, p 587 (1977). 20. J. R. Watson, P. Crescuolo, F. Matsui, J. Pharm. Sci., 60, p 455 (1971). 21. V. Zitko, C. T. Bishop, Can. J. Chem., 44, p. 1275 (1966) 22. B. A. Dmitriev, L. V. Backinowshy, Yu A. Knirel, V. L. Lvov, N. K. Kochetkov, Izv., Akad. Nauk

SSSR, Ser. Khim., p. 2235 (1974). 23. Reference 6, p. 117. 24. S. Oae et al, Tetrahedron, 19, pp. 29-30 (1963). 25. K. Buggle et al., Chem. Ind. (London), Vol. 8, p. 343 (1974). 26. Reference No. 1, pp. 86-102. 27. W. F. Haddon et al., “A General Method for Accurate Mass Measurement of Ammonia CI Spectra

Using PFK Internal Reference,” Proceedings of the 36th Annual Conference on Mass Spectrometry and Allied Topics, 1988, pp 1396-1397.

28. Alan E. Pierce, Silylation of Organic Compounds, Pierce Chemical Company, 1968. 29. Reference No. 1, p. 46. 30. A. Schonberg and A Mustafa, A. J. Chem. Soc., p. 746 (1946). 31. A. G. Perkin, R. C. Storey, J. Chem. Soc., p. 233 (1928). 32. “New methods and reagents in organic synthesis. 14. A simple efficient preparation of methyl esters

with trimethylsilyldiazomethane (TMSCHN2) and its application to gas chromatographic analysis of fatty acids,” Hashimoto, Norio; Aoyama, Toyohiro; Shioiri, Takayuki. Fac. Pharm. Sci., Nagoya City Univ., Nagoya, Japan. Chem. Pharm. Bull. (1981), 29(5), 1475-8

33. “Trimethylsilyl-substituted Diazoalkanes I. Trimethysilyldiazomethane,” Dietmar Seyferth, Horst Menzel, Alaan W. Dow, and Thomas C. Flood, Journal of Organometallic Chemistry, 44, p 279-290 (1972).

34. Sehat, N. et al. Lipids, 33, 217-221 (1998). 35. “Methodology Test for Total CLA and CLA Isomers, (Internet Document),” William W. Christie

Scottish Crop Research Institute, Invergowrie, Dundee (DD2 5DA), Scotland, Jean Louis Sebedio and Pierre Juaneda Unité de Nutrition Lipidique, 17 rue Sully, B.V. 1540, 21034 Dijon Cedex, France

36. M. A. Walker, D. R. Roberts,, and E. B. Dumbroff, J. Chromatogrl, 241, (!982) 390. 37. J. D. Cohen, " Convenient Apparatus for the Generation of Small Amounts of Diazomethane " J.

Chromatog. v.303 p193-196 ( 1984). 38. D. A. Rimmer, P. D. Johnson, R. H. Brown, “Determination of phenoxy acid herbicides in vegetation, utilizing high-resolution gel permeation chromatographic clean-up and methylation with trimethylsilyldiazomethane prior to gas chromatographic analysis with mass-selective detection,” Journal of Chromomatography A, 755 (1966) 245-250.

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39. “New methods and reagents in organic synthesis. 28. A convenient and efficient preparation of trimethylsilyldiazomethane (TMSCHN2) using diphenyl phosphorazidate (DPPA),” Mori, Shigehiro; Sakai, Izumi; Aoyama, Toyohiko; Shioiri, Takayuki. Fac. Pharm. Sci., Nagoya City Univ., Nagoya, Japan. Chem. Pharm. Bull. (1982), 30(9), 3380-2. 40. N-Methylation and N,N-dimethylation of amino acids. An artifact production in the analysis of organic acids using diazomethane as derivatizing agent. Liebich, Hartmut M.; Foerst, Claudia. Med. Universitaetsklin., Tuebingen, D-7400, Fed. Rep. Ger. J. Chromatogr. (1985), 338(1), 33-40. 41. Determination of Residues of Cloransulam-methyl in Soybeans and Soybean Forage, Hay, and Processed Commodities by Capillary Gas Chromatography with Mass Spectrometric Detection. Shackelford, Darcy D.; Duebelbeis, Dennis O.; Snell, Brian E. Global Environmental Chemistry Laboratories-Indianapolis Lab, DowElanco, Indianapolis, IN, USA. J. Agric. Food Chem. (1996), 44(11), 3570-3575.

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Table 1: Data for Conversion of Benzoic Acid to Methyl Benzoate Using Diazomethane

Time Diazomethane moles methyl benzoate (ug/ml)

0.0 0.0 0.0

1.0 3.26E-05 4.44

1.5 1.33E-04 18.11

2.0 2.34E-04 31.86

2.5 3.14E-04 42.76

3.0 3.92E-04 53.39

4.0 5.05E-04 68.8

4.5 5.58E-04 75.93

5.0 6.09E-04 82.93

5.5 6.61E-04 90.03

6.0 6.79E-04 92.5

7.0 7.30E-04 99.44

8.0 7.71E-04 104.99

9.0 8.06E-04 109.8

10.0 8.15E-04 111.02

11.0 8.35E-04 113.64

12.0 8.60E-04 117.06

13.0 8.87E-04 120.75

14.0 8.88E-04 120.95

15.0 8.82E-04 120.08

20.0 8.73E-04 118.82

25.0 9.03E-04 122.91

30.0 8.95E-04 121.91

35.0 9.08E-04 123.64

40.0 9.05E-04 123.16

45.0 9.11E-04 124.07

50.0 9.14E-04 124.43

60.0 9.07E-04 123.46

21

22

23

24

25

26

27