Simultaneous Extraction and Derivatization of Carbohydrates from Green Plant Tissues for Analysis by Gas–Liquid Chromatography

Abstract

Simultaneous extraction and derivatization of carbohydrates was performed by mixing dry ground plant tissue with derivatization reagents in pyridine; trimethylsilyl derivatives were analyzed by gas–liquid chromatography. This “direct analysis” was compared to analysis of samples prepared by exhaustive ethanol extraction of the same ground plant tissues. Comparisons included leaf blades from apple, grape, corn, and tomato and leaf blade, petiole, stem, and pod tissues from soybean plants. Direct analysis gave superior quantification of sucrose, glucose, and fructose because of sucrose hydrolysis during ethanol extraction. Sucrose hydrolysis was highly variable among plant species and use of hot ethanol at the first extraction step reduced sucrose hydrolysis but did not always abolish it. Sucrose hydrolysis was probably due to the activity of hydrolytic enzymes in 75% ethanol at room temperature. Direct analysis was inferior for the quantification of cyclitols in the fibrous tissues of soybean but provided acceptable results for cyclitol analysis in leaf blade tissue. When the time for extraction/reaction was extended from 40 to 60 min, some improvement in recovery of cyclitols was observed, but recovery remained 10 to 20% below that obtained with exhaustive ethanol extraction. Mannitol was vacuum infiltrated into the five types of leaf tissue and recovery averaged 100% by the direct method relative to ETOH extraction for apple, grape, corn, and soybean leaves but was only 76% for tomato leaves. Direct analysis provides very large time savings and is clearly the method of choice when the analysis of large numbers of samples of plant tissues for carbohydrate composition is required.