Computer simulation for the simultaneous optimization of any two variables and any chromatographic procedure.

Computer software that allows the simulation of any chromatographic separation as a function of simultaneous changes in any one or two variables that can affect sample separation order (selectivity) is described. For one example, an application is described for the simultaneous variation of the mobile phase pH and gradient time in reversed-phase liquid chromatography. The accuracy of such predictions is examined for a sample mixture of 17 substituted benzoic acids and anilines, and requirements for an acceptable predictive accuracy are summarized. In a second example, the separation of three peptides by capillary electrophoresis is optimized.

Sample preparation and column regeneration in biopolymer separations.

Successful use of high-performance liquid chromatography (HPLC) for biopolymer separations requires adequate sample preparation procedures for prefractionation of complex mixtures, solubilization of the compounds of interest, or removal of contaminants which would interfere with chromatography or damage the HPLC column. Rapid techniques have been described for solid-phase and liquid-liquid extraction, desalting, detergent removal, concentration, and filtration. A number of commercial products are available for off-line batch sample preparation and for on-line automated sample processing. These include ultracentrifugation cartridges and a wide variety of sorbents sold in bulk form or packed in disposable cartridges or minicolumns. In cases where HPLC column performance has degenerated to unacceptable levels, procedures have been described to achieve partial or complete recovery of performance. These include addition of material to fill voids caused by compaction or dissolution of the packing, replacement or cleaning of contaminated frits, regeneration of lost stationary phase material, and stripping of strongly retained contaminants from the stationary phase with suitable strong solvents.

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography.

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution of the common PTH amino acids. Elution conditions were developed with a conventional 150 X 4.6 mm I.D. column and transferred to a 150 X 1 mm I.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.

High-performance liquid chromatography of staphylococcal enterotoxin B.

Size-exclusion, ion-exchange, and reversed-phase chromatography were investigated for use in purification and characterization of Staphylococcal enterotoxin B (SEB), a 28,000 Mr protein associated with Staphylococcal food-borne intoxication. In all approaches chromatography of crude or purified SEB yielded one or more components which displayed toxin activity and contained the 28,000 Mr SEB protein plus other lower-molecular-weight protein species. Reversed-phase chromatography of detergent-treated SEB permitted resolution of the 28,000 Mr protein from lower-molecular-weight components.

Reverse phase liquid chromatographic determination of vitamins D2 and D3 in milk.

A single column reverse phase high pressure liquid chromatographic method is described for the determination of vitamins D2 and D3 in fluid milk. Resolution of vitamin D2 from D3 is helpful for use as an internal standard. The method involves overnight saponification at room temperature, extraction of unsaponifiables, precipitation of cholesterol, and aluminum oxide column cleanup. Sample extracts were chromatographed under isocratic conditions on a 10 micron Vydac reverse phase column using acetonitrile-methanol (90 + 10) as the mobile phase. In addition, a MicroPak MCH-5 reverse phase column with acetonitrile as the mobile phase was used with an automatic system for one product type. Thirty samples each of homogenized (3.8% fat), low fat (2.0% fat), and skim (less than or equal to 0.5% fat) milk spiked with 200, 400, and 600 IU vitamin D/qt were analyzed. Coefficient of variation (CV) and percent recovery for each product type and each spike level of vitamins D2 and D3 were calculated from 10 replicate analyses. Vitamin D2 recoveries for all product types at the 3 fortification levels varied from 85.2 to 99.7%; vitamin D3 recoveries varied from 85.9 to 98.8%. The minimum detectable quantity of vitamin D in milk was 15 IU/qt.

High-speed steric exclusion chromatography of biopolymers.

Biopolymer separations were studied on Micropak TSK type SW columns, which contain an aqueous compatible steric exclusion support. Columns of two different pore sizes, designated 2000SW and 3000SW, were compared for separation of proteins and nucleic acids covering a molecular weight range of 13,500 to 340,000 and the effect of molecular shape and denaturation upon elution volume was investigated. Use of high-speed steric exclusion chromatography as a prefractionation step prior to ion-exchange chromatography in biopolymer purification schemes is discussed.

Ion-exchange separation of nucleic acid constituents by high-performance liquid chromatography.

The high-performance liquid chromatographic separation of a large variety of nucleic acid constituents on a silica-based, weak-anion exchange column was accomplished. Using this technique it was possible to achieve some relatively difficult separations, such as the separation of 2'-, 3'-, and 5'-AMP, and the separation of a mixture of ribo- and deoxyribo-nucleosides and -nucleotides. A number of other separations are demonstrated by isocratic or gradient elution. These include the separation of a mixture of nucleoside monophosphates, the separation of a mixture of nucleoside mono-, di-, and triphosphates, the separation of a mixture of nucleosides and bases, and the separation of a mixture of nucleotide oligomers. These chromatographic separations were accomplished using relatively simple experimental procedures at ambient temperatures and involved relatively short analysis times. Excellent separations were obtained, in most cases, by adjustment of buffer concentration and pH, or by addition of an organic modifier. In some cases, it was necessary to use gradient elution to achieve optimum resolution.

Large-scale automated isolation of Escherichia coli mutants with thermosensitive DNA replication.

We have screened about 1.4 million colonies of Escherichia coli K-12 for their ability to grow on nutrient agar at 30 degrees and 41 degrees. Among the 2266 temperature-sensitive mutants found, 110 were defective in DNA synthesis but not in protein synthesis at 41 degrees. Three of these dna mutations mapped at two previously undescribed loci on the E. coli genetic map and may represent new genes involved in DNA replication in E. coli. The mutant isolation was aided by novel automatic machinery that inoculated agar-filled petri dishes with mutagenized E. coli cells laid down in square arrays of evenly spaced rows and columns on the agar. Time-lapse photographs taken before and after a temperature shift were used to find colonies of temperature-sensitive mutants. These mutations were mapped by interrupted conjugation and viral cotransduction methods, and the mutants were divided into three classes according to the kinetics of DNA synthesis at the restricted temperature. Some of the mutants exhibited mutator activity at partially restrictive temperatures. It is argued that some genes involved in DNA synthesis remain to be discovered.

Characteristics of cold-sensitive mutants of Escherichia coli K-12 defective in deoxyribonucleic acid replication.

Four cold-sensitive mutants of Escherichia coli have been isolated which show a reduced ability to synthesize deoxyribonucleic acid at low temperature. The mutants also have a reduced ability to incorporate nucleoside triphosphates into deoxyribonucleic acid at low temperature in cell preparations made permeable with toluene. All four mutations are located at or near the dnaA locus on the E. coli genetic map. They are recessive to the wild-type allele and two of them can be integratively suppressed by F episomes.

Isolation and properties of a ribonuclease-deficient mutant of Salmonella typhimurium.

A mutant of Salmonella typhimurium has been isolated that has less than 5% of the ribonuclease activity of the parent strain. Mutant screening and enzyme assays were done in the presence of ethylenediaminetetraacetic acid, a substance that activates ribonuclease I and inhibits other known microbial nucleases. Genetic mapping indicates that the mutation is located between the purE and gal genes on the Salmonella chromosome. A ribonuclease-deficient mutant that carries a deletion in the pyrF gene is unable to utilize ribonucleic acid as a pyrimidine source, whereas the pyrF parent with normal ribonuclease activity will grow. This suggests that the enzyme may perform a scavenge function in the utilization of exogenous ribonucleic acid. Loss of this enzyme seems to have no detrimental effects on the growth of Salmonella.

Effect of putative deoxyribonucleic acid inhibitors on macromolecular synthesis in Saccharomyces cerevisiae.

The effects of inhibitors of bacterial deoxyribonucleic acid (DNA) synthesis upon logarithmically growing cultures of Saccharomyces cerevisiae were investigated. Cell division, ribonucleic acid (RNA) synthesis, and DNA synthesis were measured after addition of nalidixic acid, fluorodeoxyuridine, or phenethyl alcohol to cultures of yeast growing in defined and complex media. Both nalidixic acid and fluorodeoxyuridine had only temporary effects on nucleic acid synthesis in cultures growing in defined medium, and little or no observable effect on cultures growing in complex medium. Neither compound inhibited colony formation on complex solid medium, although growth was slow on defined solid medium. Phenethyl alcohol caused complete inhibition of DNA synthesis, RNA synthesis, and cell division in cultures growing in defined medium. In cultures growing in complex medium, RNA synthesis and cell division were inhibited to a lesser extent. A slight increase in DNA was observed in the presence of the inhibitor.

Macromolecular synthesis in Saccharomyces cerevisiae in different growth media.

Synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein was determined in Saccharomyces cerevisiae during amino acid and pyrimidine starvation and during shift-up and shift-down conditions. During amino acid starvation, cell mass, cell number, and RNA continued to increase for varying periods. During amino acid and pyrimidine starvation, cell mass and RNA showed little increase, whereas total DNA increased 11 to 17%. After a shift from broth medium to a minimal defined medium, increase in RNA and protein remained at the preshift rate before assuming a lower rate. DNA increase remained at an intermediate rate during shift-down, and then dropped to a low rate. During shift-up from minimal to broth medium, increase in cell number, protein, and DNA showed varying lag periods before increasing to the new rate characteristic of broth medium; each of these quantities exhibited a step sometime in the first 2 hr after transfer to rich medium, suggesting a partial synchronous division. Immediately after shift-up, RNA synthesis assumed a high rate, and then dropped to a rate characteristic of growth in the rich medium after about 1 hr.