ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
27
N-Methylimidazole-Catalyzed Acetylation of Hydroxy Compounds Prior to Gas Chromatographic Separation and Determination Roman Wachowiak' and Kenneth A. Connors' School of Pharmacy. University of Wisconsin, Madison, Wisconsin 53706
Alcohols, phenols, glycols, and sugars were quantitatively acetylated in about 5 min at room temperature with acetic anhydride in the presence of N-methylimidazole. Gas chromatographic conditions are described for the separation and determination of the acetylated products.
Hydroxl compounds, especially polyols and high molecular weight alcohols, are often converted to their acetate esters before gas chromatographic separation: for example hletcalfe ( 1 ) considers acetates t o be t h e best derivatives for t h e gas chromatography of long-chain fatty alcohols. T h e usual procedure is to treat the sample with acetic anhydride iAc20) and pyridine (pyrj. Reaction times tend to be inconveniently long; t h e following are typical conditions. Sorbitol and mannitol are acetylated hy refluxing for 1 h in 1:l A c , O / p y (2, 3) or by heating in 5 1 .4c20/pyr a t 100 "C for 30 min ( 4 ) . Resorcinol monoacetate (5) is acetylated in 30 min on a steam b a t h (4:15 Ac,O/pyr); ethyl pantothenate a n d pantothenol (6) are converted t o their acetates by 1:l Ac20/p>-ra t room temperature for 1 h. Some phenothiazines containing hydrox) groups were treated with 1:l Ac20/pyr a t room temperature for 3 h ( 7 ) . Alditols were acetylated with 1:l Ac20/pyr by refluxing overnight (8). PbTidine serves as a nucleophilic catalyst of the acyl transfer reaction ( 9 ) as well as a general base to accept the proton produced. Since it is not a powerful catalyst (as indicated b> t h e cited reaction times), a more effective catalyst would be desirable. Recently 4-dimethylaminopyridine (DMAPj has been used for this purpose ( I O , 11). This substance is a very effective catalyst but, since it is a solid, it must be incorporated into a solvent, and pyridine serves this function. In a n active laboratory, pyridine may be regarded as a particularly unpleasant substance, and its removal from acetylation reagents would be desirable. This laboratory recently introduced .V-methylimidazole as a n acetylation catalyst for hydroxy determination with a titrimetric finish (12). 'V-Methylimidazole (NMIM) is a liquid, a n d therefore can serve as catalyst, base, and solvent. Its catalytic efficiency is intermediate t o t h a t of pyridine and D M A P , t h e relative rates for the catalyzed acetylation of isopropyl alcohol being 1:360:17000 (pyridine:NMIM:DMAP) (13). T h e present paper describes t h e application of Xmethylimidazole as a catalyst for acetylations of hydroxy compounds before gas chromatographic separation and determination. EXPERIMENTAL Chromatography. A Tracor 550 gas chromatograph with flame ionization detector was used; the 4-mm i.d. glass columns were 4 ft long. h-itrogen was the carrier gas. Gas flow rates were: nitrogen, 6 M 5 mL/min; hydrogen, 40 mL/min; air, 300 mL, min. Present address, Department of Pharmaceutical Chemistry. Institute of Chemistry and Analytics, Medical Academg . Poznan, Poland. 0003-2700/79/035 1-0027$01 0010
The packing support for most colurnns was Chromosorb W / AWIDMCS,80-100 mesh; liquid phases were OV-17 (5% wjw). Carbowax 2OiLI (5% w/w), or SE-30 (10% w./w). Liquid phases were applied to the support in chloroform solution, the solvent was allowed to evaporate without stirring at room temperature, the packing was air-dried at room temperature for 12 h, and the packed column was conditioned at 100 "C for 2 h. then 180 " C for \5 h. and at 30 "C less than the stationary phase temperature limit until the base line was stable. For sucrose octaacetate separations, the support was 8G100mesh glass beads, coated with 0.25% SE-30. Operating temperatures differed for the several kinds of samples: details are given in Tables I-V. Acylation Conditions. Only a small excess ilG-207~) of acetic anhydride is needed to ensure complete reaction, and the general proportions, on an equivalent basis, of reactants were about 1:1.2:2 (hydroxy sample:Ac,O:NMIM j . Thus, for monohydroxy alcohols. reactants were mixed in volume ratio 1:1.5:2 (alcohol:Ac20: NMIM). For glycols, 0.05 mL of glycol, 0.20 mL of Ac20. and 0.3 mL of NMIM were mixed (for glycerol these volumes were 0.05 mL. 0.3 mL, 0.4 mL). If the sample contains water. sufficient anhydride must be used t o react with the sample compound plus the water. For example, a 2 0 7 ~aqueous solution of glucose was acylated with the following proportions: 0.05-0.3 mL glucose solution, 2 mL Ac20,0.02 mL glycerol triacetate or 0.02 g caffeine as internal standard, and NMIh'I to bring the total volume to 5.0 mL. It was found convenient, for most samples, to dissolve the hydroxy compound in NMIM and then to add the acetic anhydride. Acetylation was complete in 5 min (or less) at room temperature (10 min for sucrose). (The reaction is exothermic and the temperature of the reaction mixture rises from this cause.) An appropriate sample (2-1 pL) of the reaction mixture is then injected directly into the column. For quantitative studies, internal standards were added to the reaction mixture; usually an acetate ester of another alcohol served as a suitable standard. Peak areas were calculated as the product of peak height and peak width at half the maximum height. Linear calibration plots were observed over the approximate sample range of 10-150 pg of sample injected. Materials. Acetic anhydride (Fisher) and &V-methylimidazole (1-methylimidazole. Aldrich) were used directly. Hydroxy compounds and their acetate esters were from commercial sources and were used directly. Synthesis of D-Glucose Pentaacetate. D-Glucose, 0.5 g, was dissolved in 5 mL of NMIM and 1.5 mL of acetic anhydride was added. After 15 min, 5 mL of water was added. In 5-10 min, glucose pentaacetate crystallized, yield over 9 0 7 ~ . It was recrystallized from ethanol as needles, m p 112-113 " C (lit. (14) mp 112-113 "C).
Synthesis of Sucrose Octaacetate. Sucrose, 0.5 g, was dissolved with heating in 7 mL of NMIM. After cooling to room temperature, 1.2 mL of Ac20 !vas added. After 10 min, 15 mL of ice water was added and the mixture was allowed to stand overnight. The precipitate was removed by filtration, and was + 59.3 "C recrystallized from 95% ethanol, mp 8:5-87"C, [aIzoD (chloroform, C = 107~), yield 6 2 7 ~(lit. ( 1 5 ) mp 69 "C, [nIzoD+ 59.6 "C). RESULTS AND DISCUSSION
Alcohols. Although simple alcohols can be chromatographed without first converting them to derivatives, they were C 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, J A N U A R Y 1979
Table I. Product Yields and Retention Times for Primary Alcohol Acetylation Products ~
compound ethyl acetate n-propyl acetate n-butyl acetate n-pentylacetate
t R , min yield, % Carbo______ wax std. 20Ma OV-17b meanC dev.
1.9
3.0 5.2 8.2
2.0 7.0 11.0
15.0
100.9 3.1 101.1 4.5 101.1 4.0 (internal standard ) 100.4 4.5 99.6 2.8
n-hexyl acetate 11.2 18.0 n-heptyl acetate 1 3 . 8 22.0 n-octyl acetate 15.8 24.0 ... ... n-nonyl acetate 17.8 27.0 97.7 3.3 acetic anhydride 10.2 10.0 acetic acid 14.8 4.0 N-methylimidazole 19.5 20.5 70-210 ^ C at 5 'C/min; inlet 240 "C, outlet 1 7 0 'C, detector 2 1 0 -C, 40-200 'C at 3 "Cimin; inlet 200 'C, outlet 160 -C, detector 210 "C. Mean of 5 determinations.
peak
area ratio
05,
io
40
,bo rio
io
60
tlrnm /rnin
I40
IbO
'
'
Figure 1. Extent of acetylation (expressed as peak area ratios) of some hydroxy compounds, as a function of time, at room temperature. Curve 1, isopropyl alcohol and NMIM; 2, n-propyl alcohol and pyridine; 3, ethylene glycol and pyridine; 4, isopropyl alcohol and pyridine I 2
3
Table 11. Acetylation and Chromatography of Isomeric Alcohols alcohol n-propyl isopropyl n-butyl isobutyl sec-butyl n-pentyl isopentyl
tR,
mina
9.5b 7.0b
5
6.5 4.5 4.0 11.0
78
9.0
reagents acetic acid acetic anhydride N-methylimidazole
4
2.0
5.0 12.5
6
* On
OV-17; 40-200 ^ C a t 5 ^ / m i n ;inlet 230 + C ,outlet 160 ^C, detector 210 "C. For the propyl compounds, temperature programming was begun after the isopropyl acetate was eluted, giving t R 2.0 (acetic acid), 8 . 0 (Ac;O), 20.0 min (NMIM).
chosen as the initial sample compounds in this study because their reactions are uncomplicated, and the chromatographic conditions could be easily optimized. Use of the smallest possible excess of acetic anhydride minimizes discoloration of the reaction mixture, though this discoloration does not appear to affect the acetylation process or the chromatography. Table I lists primary alcohols subjected to NMIM-catalyzed acetylation, with chromatographic conditions, retention times. and reaction yields (which were evaluated by comparing peak areas of the products with those of authentic acetates, referred t o n-pentyl acetate as an internal standard). Some isomeric alcohols were treated in the same way. Retention times are given in Table 11. Usually an acetate ester of another alcohol was added to a mixture, prior to acetylation, t o serve as internal standard; thus see-butyl acetate served in this role for the propyl isomers, n-pentyl acetate for t h e butyl isomers. and n-hexyl acetate for the pentyl isomers. Note that the peaks for isobutyl and see-butyl acetates are not resolved on this column. Calibration plots of peak area ratio against amount of sample compound were linear, hence are not reproduced. A comparison of rates of acetylation was made using NMIM and pyridine as catalysts. Figure 1 shows the extent of acetylation as a function of time for the pyridine-catalyzed reaction of n-propyl alcohol, isopropyl alcohol, and ethylene glycol; the figure also gives data for the NMIM-catalyzed
I
0
8
l
5
