Derivatization of Carboxylic Acids With Diazomethane

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 “Purg
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   1 Derivatization of Carboxylic Acids with Diazomethane and Trimethylsilyldiazomethane: ConvenientMethods and ArtifactsContents 1. Introduction2. Experimental2.1.   Handling Precautions for Diazomethane2.2.   Preparation of Diazomethane in Larger Quantities Using Distillation2.3.   Preparation of Diazomethane and Diazomethane-C13 in Smaller Quantities Using Nitrogen “Purgean Trap” Method and Sample Derivatization2.4.   Quantitative Measurements of Rate of Diazomethane Production with Nitrogen “Purge and Trap”Method 2.5.   Derivatization of Samples with Trimethysilyldiazomethane in Hexane Solution3. Results and Discussion3.1.   Diazomethane Reaction Mechanism3.2.   Artifacts Formed form Diazomethane3.2.a.   Artifacts from Reaction of Diazomethane with Ketones and Aldehydes3.2.b.   Artifacts from Reaction of Diazomethane with Alcohols3.2.c.   Artifacts from Reaction of Diazomethane with Phenols3.2.d.   Artifacts form Reaction of Diazomethane with Solvents3.2.e.   Artifacts from Reaction of Diazomethane with Alkenes3.2.f.   Artifact from Reaction of Solvent with Diazomethane Precursor 3.2.g.   Artifact from Reaction of Diazomethane with Amino Acids3.3.   Trimethysilyldiazomethane Reaction Mechanism3.4.   Artifacts from Trimethylsilyldiazomethane Reagent3.4.a.   Formation of Trimethylsilyl and Trimethylsilylmethyl Ester Artifacts3.4.b.   Other Artifacts Containing Trimethylsilylmethyl Groups3.4.c.   Presence of Phenol and Phenol Derivatives in Trimethylsilyldiazomethane in HexaneReagent4.0   Summary5.0   References6.0   Tables and Figures 1.0. Introduction We frequently employ diazoalkanes such as diazomethane and trimethylsilyldiazomethane for thederivatization of carboxylic acids. The resulting methyl esters are ideal derivatives for the characterizationof carboxylic acids. They are easily characterized by gas chromatography (GC) and readily identified byeither 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 OOH+CH 2  N 2 R OOCH 3  N 2 + Diazomethane  However, it must be prepared from a precursor and care must be taken in handling this very reactivereagent.Trimethylsilyldiazomethane reacts much slower than diazomethane with carboxylic acids and yieldssomewhat higher concentrations of by-products (artifacts).   2  ROOH+ROOCH 3 N 2 + Trimethylsilyldiazomethane(TMSCHN 2 ) CH 3 OH+(CH 3 ) 3 SiCHN 2 CH 3 OCH 2 Si(CH 3 ) 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 theseundesirable or unexpected derivatives (by-products) as artifacts. In addition, these reagents can react withsolvents 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 toconfusion 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 thosefound in the literature. Many times reaction conditions can be modified to avoid artifact formation. Inaddition, convenient methods for the preparation of diazomethane are presented and typical reactionconditions 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 TECHNICALINFORMATION AND SAFETY INFORMATION 1-3, 8, 9 ON DIAZOMETHANE SHOULD BE STUDIEDCAREFULLY.Diazomethane is quite safe when produced in dilute solutions and in small volumes, as is the case inmost analytical applications. The diazomethane is consumed very quickly since it is purged directly intothe 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 o C, and freezes at –145 o C. It isextremely 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 explodeunaccountably both as a gas and as a liquid. Rough surfaces and strong light are known to detonatediazomethane.All edges of glassware used for diazomethane should be carefully firepolished and ground-glass jointscannot 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 calciumsulfate can result in an explosion. The recommended drying agent for diazomethane is potassiumhydroxide pellets. All diazomethane reactions should be performed in an efficient fume hood and behind asturdy safety shield. Reactions of diazomethane are best performed at room temperature or below.   3Solutions of diazomethane should not be frozen because the rough edges of crystals could cause it toexplode.   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: CH 3 SOO NNOCH 3 +KOHH 2 OCH 3 SOOO-+K +CH 2  N 2 DiazaldDiazomethane  Two of our methods use 2-(2-ethoxyethoxy)ethanol as a solvent. One reaction scheme employs diethylether 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 materialshould be kept refrigerated for prolonged storage.The amount of artifact formation was checked by either gas chromatography/mass spectrometry or gaschromatography with a flame ionization detector. Standards were not available for the majority of theartifacts, 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 initialderivatization 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 analyticalneeds such as gas chromatography (GC) and GC-MS (gas chromatography-mass spectrometry. Thus, wehave 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 veryconvenient 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 beadvantageous 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 bySchlenk and Gellerman, 10 Walker  et a.l , 36 and Cohen. 37 Our method could be significantly improved byincorporating several of the design features in the Cohen 37 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 tipof arm that extends from tube A. The arm from tube A should extend close to the bottom of tube B, but not closeenough to interfere with the stirring bar. The nitrogen gas from E is used to purge the diazomethane formed in thesolution 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 thenitrogen purge (~30-50 ml/min) and the magnetic stirrer and place a septum over H. Add 2 ml's of solution #1 to testtube B via syringe through the septum. Add 2 ml of solution #2 by syringe via the syringe through the septum and letstir. Check for Diazomethane formation by placing a blank solution of DMF in sample vial M. Diazomethane is ayellow 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 turnyellow (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. Placeanother sample to be derivatized in the stream. Samples for most of our work were dissolved in DMF since this solventis 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 anyadditional 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 generatesmethyl acetate as a by-product. We generally add excess diazomethane (yellow color noted) to samples and allowthem to react for 30 seconds to 1 minute to insure complete reaction. Excess diazomethane is destroyed by addingsmall 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 nolonger 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 13 C-labelled diazomethane. Themethod using no distillation was used and the N-methyl- 13 C labeled analogue of Diazald (Aldrich ChemicalCompany) 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 atLab Glass, Kingsport, TN.D 1/4 Outside diameter nitrogen feed tubeE Dry Nitrogen supply line from high purity Nitrogen tank or house nitrogen in laboratoryF 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 tubesH Solution supply tube with septum topI, J, K Three #4 Rubber StoppersL Supporting plateM 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 wasobtained 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 UVdetector. The amount of methyl benzoate was converted into the amount of diazomethane produced. Atotal yield of 48.7% was noted for diazomethane. This compared well to a 51% yield measured for thereaction by quantitative proton NMR analyses. 2.5. Derivatization of Samples with Trimethylsilyldiazomethane Solution
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