¹⁴C₂H₄: Its Purification for Biological Studies

Abstract

A gas chromatographic technique is described for obtaining ultra high purity 14(GH for use in biological studies. l4CHL purchased from commercial sources contained readily detectable impurities including radioactive acetylene. Following purification on two different columns, no impurities were detected by high sensitivity gas chromatographic analysis. However, shortly thereafter impurities were detected as a result of radiation decomposition. Trapping and immediately regenerating ultra high purity 14C24 from dilute, filtered Hg(CI04)2 solutions did not cause the formation of impurities, whereas additional impurities were formed when unpurified 14C2HI was used. Impurities were also formed when ultra high purity "GC-L was stored in such solutions prior to its regeneration or when it was trapped and immediately regenerated from more concentrated Hg(CI04) solutions.Surprisingly little is known about the metabolic fate of ethylene. In fact, it is not yet clear whether or not ethylene is incorporated into plant tissues, despite the fact that several studies (8-12, 14, 15) using radioactive ethylene have reported that a small but significant amount of label was incorporated by fruit and vegetative tissues. The present uncertainty, even in light of these reports, exists because of three major criticisms which have been raised (1, 2, 5) and the fact that other studies (4,6,7, 13) have failed to obtain any ethylene incorporation or metabolism. The first criticism is that adequate precautions were not taken to ensure that the label incorporated was derived from radioactive ethylene. Therefore, the reported incorporation may have been an artifact due to impurities originally present in the labeled ethylene or impurities derived from radiation decomposition (16). Second, several studies (8,14,15) was undertaken using ultra high purity "4C-labeled ethylene and aseptic conditions. This paper describes the GC' technique used throughout this study for obtaining ultra high purity "4C-labeled ethylene.Purchased samples of uniformly 14C-labeled ethylene with a reported radiochemical purity of 98% (22, 31, and 53 mCi/ mmole, Amersham/Searle) were purified twice by GC. All separations were performed on a Perkin-Elmer 900 GC connected to a Hewlett-Packard Model 7128A recorder and equipped with a fiame ionization detector and subambient accessory. Ultra high purity helium (Matheson Gas Products) served as the carrier gas while ultra high purity hydrogen (Elhygen H, generator) and oxygen (Matheson Gas Products) provided the flame. Routine ethylene analyses were performed isothermally at 110 C on a 1.83 m X 3.13 mm stainless steel activated alumina column (80-100 mesh) at a flow rate of 70 cc/ min. The sensitivity and stability of this instrument was such that 6 nl/l of ethylene could routinely be detected in a 1-cc gas sample producing a peak twice the baseline noise level with a retention time of 0.9 min.The "4C-labeled ethylene samples were purchased in 250-,4Ci lots and were shipped in small evacuated glass vials with a bre...

14C2H4: Its Incorporation and Metabolism by Pea Seedlings under Aseptic Conditions

“…The use of '4CM,4 to study its possible incorporation and metabolism by plants is not straightforward as demonstrated by the number and kind of problems encountered and alluded to above and in a previous report (5). The difficulty in working with 14CM,4 is not only attributable to its instability (5,18) but also to the relatively small amounts of radioactivity which are incorporated and converted to "4CO2 at physiological concentrations of 14CH4.…”

“…The difficulty in working with 14CM,4 is not only attributable to its instability (5,18) but also to the relatively small amounts of radioactivity which are incorporated and converted to "4CO2 at physiological concentrations of 14CH4. As the level of radioactivity decreases the likelihood increases that the label incorporated and converted to "4CO2 is an impurity rather than '4C2H4.…”

“…Uniformly labeled 4C2H4 (22 mCi/mmole) with a reported radiochemical purity of 98% was purchased from Amersham/Searle and purified first on a silver nitrate-ethylene glycol-coated Gas Chrom R column followed by further purification on a Porapak T column. The "C,H4 purified in this manner contained no impurities as evidenced by highly sensitive gas chromatography (4,5). However, shortly after purification, radiolysis products appeared (5) which were incorporated by pea seedlings.…”

“…Several of these studies (10,11,16,17) were further complicated because the "CMH4 was regenerated from mercuric perchlorate, Del. 19898. readily incorporated by plant tissues (5,14). Additional skepticism has been derived from the fact that several workers (3,6,9,15) failed to observe any ethylene incorporation or metabolism.…”

[14C]Ethylene Metabolism during Leaf Abscission in Cotton

“…Prior to placing the abscission zone or petiole segments into the incubation flasks, sufficient distilled H20 was added to the flask to cover the bottom (4 ml), and 1 ml of 1.5 N NaOH was placed in the small, side well container to trap "4CO2. The flasks were sealed, and enough purified 14C2H4 (7) injected to yield a final concentration of 20 Al/ 1 which was subsequently verified by GC analysis. The flasks were incubated on a shaker for 5.5 h in the dark at 22 C.…”