Determination of infrared spectra on microgram quantities of some plant growth-regulating compounds.

Infrared spectroscopy is a valuable tool for the characterization of organic compounds, ibut only recently has it been possible to obtain detailed ispectra on quantities of a few micrograms of material. In the past ifew years papers have been lpublished describing several methods for doing this (2, 3, 4, 5,6, 8,9). Some techniques have been, considerably more successful than others. One of the best is th,e use of small KBr pellets (3). This method has been described in detaill by Mason (5). He employed 0.5 and 1.5 mm KRr pellets, the former enabling him to obtain informative spectra on less than 1 microgram of material. Recently we have been interested in applying infrared techniques to small quantities of plant hormones (7). Herein is descrilbed a method which employs 1.5 mm KB!r fpellets, permitting good spectra on 5 to 10 ,ug of material. The technique is essentialily that of Mason (5), but we have modified it so that the preparation of the sample is much simplified, requiring a minimum of time and special equipment. All of our wotk has been done with a PerkinElmer Model 337 sipectrophotometer. The instrument was routinely employed with a Perkin-Elmer 4x refracting beam condenser with KBr optics in the sample beam, and with an attenuator in the reference beam. to equilize beam energy. The 1.5 mm KBr pelilets were prepared using the Per~kin-Elmer Ultra Micro Die. The metlhod has been employed for obtaining spectra on a number of organic compounds, among them being IAA,, several gibberellins and l(+) abscisin II. The steps for preparing the 1.5 mm KBr pellets are as follows, using IAA as an example of the compound to be analiyzed: A) Clean all glassware and metal equipment that might come in contact with the sample. We often use nothing more than a water rinse, followed by an acetone rinse, then drying. However, for ultra-clean equipment the method of Behringer (1) may be used. Glassware is cooked for an hour in 800 2 N HNO,, rinsed thoroughly with distilled water, then cooked another hour in 800 2 N HCl, rinsed 15 or more tinmes with high 'purity distililed wateir, and finally baked for 1 hour at 1000. Metal parts may be soaked in water to remove any KBr, rinsed with ethyl alcohol to remove water, then soaked in boiling benzene for 5 to 10 minutes to degrease them. Finally, they may be placed in a vacuum chanzrber to remove volatiles. Forceps are used to handle cleansed equipment. B) 1AA is transferred in a Ifew jAl of solvent to a commercialily available borosilicate culture tube, 10 X 75 mm. The carrier solivent is evaporated, and the IAA residue dissolved in 10 jl of ethyl alcohol. C) Three mg of IR grade KBr dissolved in 90 ILI of glass diistilled 'water are added to the IAA solution. Controls containing only aqueous KBr are also prepared. D) The samples are lyophilized by placing them in the liquid state in a vacuum desiccator. No cold tralp is employed between pump and desiccator, since only a few ul of water are involrved. Lyophilization is complete in 30 to 45 minutes. It is imiportant that the sample be a finely divided, 'fluffy mass at the end of the lyophilization period, rather than a crystalline residue which sticks tightly to the sides of the cuilture tube; otherwise a transparent pellet may not be obtained. E) Working under a heat lamp to minimize water absorption, the lyophilized KBr-IAA sample is loosened ifrom the bottom of the lyophilization tube with a small metal spatula and transiferred to the mi'cro die where the 1.5 mm pellet is'made. F) The IJR spectrum of the pellet is obtained in a conventional manner, except that a beam condenser i's generally employed.