Jiann-Tsyh Lin

Combined use of temperature and solvent strength in reversed-phase gradient elution. IV. Selectivity for neutral (non-ionized) samples as a function of sample type and other separation conditions

The separation of nine un-ionized samples was studied as a function of temperature (T) and gradient steepness (b). Selectivity values Δlog α∗ were obtained for 160 compounds, ranging from nonpolar hydrocarbons to very polar drugs. Selectivity varied markedly with sample type: nonpolar compounds such as aromatic hydrocarbons and fatty acid methyl esters generally showed only modest changes in band spacing as temperature or gradient steepness was varied. More polar samples exhibited larger changes in α (Δlog α∗) when temperature and/or gradient steepness wee changed, but the largest values of Δlog α∗ for these non-ionized samples are less than the average value of Δlog α∗ for the ionized samples of Part III [1]. Poly-functional silane (“polymeric”) columns exhibit slightly increased b- and/or T-selectivity for some samples.

Comparison of the endogenous gibberellins in the shoots and roots of vernalized and non-vernalized Chinese Spring wheat seedlings

Abstract Endogenous gibberellins (GAs) in Chinese Spring wheat seedlings were isolated by high performance liquid chromatography (HPLC) and identified by combined capillary gas chromatography-selected ion monitoring (GC-SIM). Gibberellins A 1 , A 3 , A 19 , A 20 , A 44 , and A 53 were identified in the shoots, A 19 and A 20 in the roots. The identification of these 13-hydroxylated GAs demonstrates the presence of the early-13-hydroxylation pathway in wheat seedlings. Based on peak area of total ion response of five characteristic ions by GC-SIM, the approximate levels of GAs in the shoots is GA 44 > GA 19 > GA 1 = GA 3 > GA 20 for the non-vernalized wheat seedlings, and GA 44 > GA 19 > GA 53 = GA 3 > GA 1 = GA 20 for the vernalized wheat seedlings. The C 20 GAs, GA 53 , GA 44 and GA 19 , are present in shoots of the vernalized (flowering) wheat seedlings at somewhat higher levels than that in the non-vernalized (rapidly growing) wheat seedlings. Approximate levels of the C 19 GAs, GA 20 , GA 1 and GA 3 were lower in the shoots of the vernalized wheat seedlings than in the non-vernalized wheat seedlings. The conversion of GA 19 to GA 20 (C 20 to C 19 GAs) may be a rate-limiting step in the vernalized wheat seedlings.

Comparison of adsorption and reversed-phase partition high-performance liquid chromatography for the separation of androgens

Abstract Sixty-nine androstane derivatives were chromatographed by adsorption and reversed-phase high-performance liquid chromatography (HPLC). The total number of hydroxyl and keto groups on the androstane molecule is the most important factor in determining their chromatographic behavior. An α,β-unsaturated keto group contributes as much to the polarity of the molecule as a hydroxyl group and more than an isolated keto group. Generally, 3-hydroxyandrostane derivatives are more polar in adsorption HPLC with n -hexane-ethanol as eluent when the hydroxyl group is equatorial than when it is axial. 17β-Hydroxyandrostane derivatives are generally more polar than their 17α-epimers in reversed-phase partition HPLC with methanol-water as eluent. Compounds inseparable by adsorption HPLC can often be separated by reversed-phase partition HPLC and vice versa .

Regulation of isopentenoid biosynthesis by plant growth retardants in Nicotiana tabacum

Abstract The effects of AMO-1618, AY-9944 and SKF-7997 on the growth of Nicotiana tabacum and on the biosynthesis of acidic and neutral isopentenoids in the seedlings were examined. All three compounds significantly retarded stem elongation but not dry wt of the plants. Incorporation of radioactivity from [2- 14 C]-mevalonate into acidic and neutral isopentenoids showed a direct relationship between the radioactivity in the two fractions and stem growth. All three compounds inhibited steps before as well as after squalene formation, but the acylic and polycyclic isopentenoids which accumulated as a result of the inhibition were not necessarily the same in each of the treatment groups. This indicates that stem growth is probably influenced not by a single product of isopentenoid biosynthesis, but rather by several products, which may even act independently in their effects on developmental processes.

Steroid Analysis by High-Performance Liquid Chromatography

Abstract Liquid-column chromatography (LCC), which was the earliest chromatographic technique to be applied to steroids, has become the most recent and most promising addition to the armamentarium of the steroid analyst in the form of high-performance liquid chromatography (HPLC). Conventional column chromatography (CC) has survived the competition with planar and gas chromatography (GC) in the steroid field, because it offers greater capacity and a greater variety of experimental conditions. Both of these advantages are important in the analysis of steroids, because in nature steroids occur as complex mixtures of disparate concentrations and polarites. By virtue of their relatively large capacity, columns can be loaded with enough crude mixture to allow the detection of trace amounts of certain components. Steroids of diverse polarities can be fractionated by a wide range of sorbents and eluents, e.g., by gradient elution.

Gibberellin Content of Immature Apple Seeds from Paclobutrazol-Treated Trees over Three Seasons

Seeds from heavily fruiting (“on-year”), mature untreated, and paclobutrazol-treated apple trees (Malus domestica Borkh. cv. Spartan) were sampled in mid-June 1987, mid-July 1987, and mid-July 1990. After seeds were freeze-dried, gibberellins (GAs) were extracted, purified, and fractionated via C18 reversed-phase high-performance liquid chromatography (HPLC). Nine GAs (GA1, GA3, GA4, GA7, GA8, GA9, GA19, GA20, and GA53) were quantified by the use of deuterated GA internal standards. Paclobutrazol trunk drench treatments reduced vegetative shoot elongation in the seasons that seeds were sampled by 55% or more. Between June 17, 1987 and July 15, 1987, the dry weight of seeds from both untreated and treated trees increased about 2.5 times and there were reductions, on a per seed basis, of GA4 in seeds from both untreated and treated trees, of GA7 in seeds from treated trees, and of GA9 in seeds from untreated trees. However, GA9 increased in seeds from treated trees. Changes in levels of some of the early-13-hydroxylation pathway GAs (GA15 GA3, GA8, GA19, GA20, and GA53) also occurred during the month. For mid-July harvested seeds, the pattern, with some exceptions, was that 2 years after paclobutrazol treatment (1987), levels of early-13-hydroxylation pathway GAs in seeds from treated trees were lower compared to controls but after 5 years (1990) their levels tended to increase. For the non-13-hydroxylated GAs (GA4, GA7, and GA9), 2 years after paclobutrazol treatment, GA4 levels were equal in seeds from untreated and treated trees, GA7 decreased in seeds from treated trees compared with controls, but GA9 levels increased. Levels of these three GAs were higher in seeds from treated trees 5 years after treatment (1990) but levels of GA4, GA7, and GA9 dramatically increased in seeds from treated trees 4 years after treatment (1989), as we previously reported.

High-Performance liquid chromatography of steroidal sapogenins

Abstract Thirty steroidal sapogenins were chromatographed by normal-phase and reversed-phase high-performance liquid chromatography (HPLC). These two HPLC systems complement each other and as result most sapogenins can be separated. The total number of hydroxyl and keto groups plays the most important role in the polarity of spirostane derivatives in both normal-phase and reversed-phase HPLC. Generally, 3-hydroxysapogenins are more polar in the normal-phase HPLC with hexane—ethanol as eluent when the hydroxyl group is equatorial than when it is axial. 25-Epimers of sapogenins cannot be separated well by either system.

Conversion of 4-androstene-3,17-dione to testosterone by Pisum sativum

Abstract 4-Androstene-3,17-dione-[4-14C] was applied to the leaves of growing pea plants, Pisum sativum. Within a week, 28% of the administered steroid was specifically reduced to testosterone. Part of the testosterone was present in esterified form, and 5α-androstane-3β,17β-diol was also identified as a metabolite, but neither epitestosterone nor estrogens were detected.

SEPARATION OF INTACT PHOSPHATIDYLCHOLINE MOLECULAR SPECIES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Identification of the molecular species of phosphatidylcholines (PCs) is important for studies of lipid metabolism and the structure of cell membranes. The high performance liquid chromatography (HPLC) separations of the intact molecular species of PCs used earlier were mostly on reversed-phase C18 (octadecyl silica) columns, usually with isocratic elution. The eluent used contained a silanol suppressing agent. Volatile silanol suppressing agents are preferred for the purification of PCs because they can be removed from the HPLC fractions easily by nitrogen stream. They must be used when liquid chromatography-mass spectrometry (LC-MS) and evaporative light scattering detector (ELSD) are used because non-volatile silanol suppressing agents cause high background. For the identification of radioactive metabolites, using both the UV detector at 205 nm and radioactivity flow detector sequentially, we recommend the separation on a C8 (octyl silica) column, eluted with a gradient of methanol-water containing conc. NH4OH as silanol suppressing agent.

Reversed-phase C18 and normal-phase silica high-performance liquid chromatography of gibberellins and their methyl esters

Abstract Twenty-three gibberellins and their methyl esters were chromatographed by gradient reversed-phase C18 partition and isocratic normal-phase silica adsorption high-perforamnce liquid chromatography (HPLC). These four complementary HPLC systems allowed for the necessary separation to resolve these gibberellins. The four HPLC systems can be used to identify radioactive metabolites in metabolism studies by co-chromatography of radioactive labelled gibberellins with authentic standards using UV and radioactive flow detectors. The highest separatio efficiency for these gibberellins was achieved with the reversed-phase C18 HPLC of their methyl esters.