Reproducible Chromatograms Research Articles

Fast and reliable method to estimate global DNA methylation in plants and fungi with high-pressure liquid chromatography (HPLC)-ultraviolet detection and even more sensitive one with HPLC-mass spectrometry.

DNA (Deoxyribonucleic acid) methylation is one of the epigenetic modifications of DNA, acting as a bridge between genotype and phenotype. Thus, disruption of DNA methylation pattern has tremendous consequences for organism development. Current methods to determine DNA methylation suffer from methodological drawbacks like high requirement of DNA and poor reproducibility of chromatograms. Here we provide a fast and reliable method using high-pressure liquid chromatography (HPLC)-ultraviolet (UV) detector and even more sensitive one with HPLC- mass spectrometry (MS) and we test this method with various plant and fungal DNA isolates. We optimized the preparation of the DNA degradation step to decrease background noise, we improved separation conditions to provide reliable and reproducible chromatograms and conditions to measure nucleotides in HPLC-MS. We showed that global DNA methylation level can be accurately and reproducibly measured with as little as 0.2µM for HPLC-UV and 0.02µM for HPLC-MS of methylated cytosine.

Ion-Exclusion High-Performance Liquid Chromatography of Aliphatic Organic Acids Using a Surfactant-Modified C18 Column.

Ion exclusion chromatography (IELC) of short chain aliphatic carboxylic acids is normally done using a cation exchange column under standard HPLC conditions but not in the ultra-HPLC (UHPLC) mode. A novel IELC method for the separation of this class of carboxylic acids by either HPLC or UHPLC utilizing a C18 column dynamically modified with sodium dodecyl sulfate has been developed. The sample capacity is estimated to be near 10 mM for a 20 µL injection or 0.2 µmol using a 150 × 4.6 mm column. The optimum mobile phase determined for three standard mixtures of organic acids is 1.84 mM sulfuric acid at pH 2.43 and a flow rate of 0.6 mL/min. Under optimized conditions, a HPLC separation of four aliphatic carboxylic acids such as tartaric, malonic, lactic and acetic can be achieved in under 4 min and in <2 min in the UHPLC mode at 2.1 mL/min. A variety of fruit juice and soft drink samples are analyzed. Stability of the column as measured by the retention order of maleic and fumaric acid is estimated to be ∼4,000 column volumes using HPLC and 600 by UHPLC. Reproducible chromatograms are achieved over at least a 2-month period. This study shows that the utility of a C18 column can be easily extended when needed to IELC under either standard or UHPLC conditions.

Physicochemical Analysis of the Aqueous Extracts of Six Nigerian Medicinal Plants

Purpose: Extracts of Picralima nitida seeds, Detarium microcarpum stem bark, Aframomum melagueta seeds, Terminalia catappa leaves, Acacia nilotica pods, and Morinda lucida stem bark, are under consideration for development into suitable dosage forms for treating diabetes mellitus, sickle cell anemia and malaria. This study aimed at evaluating the extracts for features that would influence decisions on them in the course of the project. Methods: Physicochemical determinations, including proximate analysis, were done by sensory examination, and gravimetric and electrochemical techniques. Thin layer chromatography was carried out with normal silica plates using various solvent systems. Metallic content analyses were carried out by atomic absorption spectroscopy. Results: The extracts were dry but hygroscopic, with a loss on drying range of 0.26 – 12.00 %w/w. The pH of the 5 - 10 % solutions ranged 5 - 7. No harsh sensory effects, such as lacrimation, were detected in any of the extracts. Total ash ranged from 3.79 – 20.68 %w/w, while acid insoluble ash values were below detection. The extracts yielded reproducible chromatograms on normal silica plates developed with various solvent systems. Copper, present at 0.16 - 0.58 mg/100g, was the lowest occurring microelement while calcium content was highest, at 41 - 216 mg/100g. The level of lead, a heavy metal, was 0.05 - 0.22 mg/100g. Conclusion: The results confirm that the extracts require no special handling, possess characteristics that would allow their possible development into solid dosage forms, and that their lead contents complied with official limits. Keywords: Aqueous extract, Picralima nitida, Detarium microcarpum, Aframomum melagueta, Terminalia catappa, Acacia nilotica, Morinda lucida.

A Review of Synthetic Polymer Characterization by Pyrolysis–GC–MS

Pyrolysis–GC with mass spectrometry detection (Py–GC–MSD) study of the thermal degradation products of synthetic polymers is reviewed. Due to the high heating speed, accurate temperature reproducibility and a wide temperature range, Py–GC–MSD has been applied successfully for polymer characterization. Introduction of samples using the pyrolysis carrier gas through the split injection port, followed by sub-ambient cryofocusing of the pyrolysis products, has shown to give reproducible chromatograms (pyrograms). One of the advantages of this method is that all compositions of the polymers and additives can be investigated without any pretreatment, providing important compositional and structural information in a simple way. The method is a convenient method for compositional analysis of complex polymer materials. The aim of this review is to describe the kinds of applications for which Py–GC–MSD has been found to be suitable; to present guidelines for method optimization; to survey innovations that have recently been developed or are currently being researched; to point to problems in our understanding of the pyrograms; and to suggest areas in which research efforts might be most effective in realizing the full potential of this technique.

Application of Fluorogenic Derivatization-Liquid Chromatography-Tandem Mass Spectrometric Proteome Method to Skeletal Muscle Proteins in Fast Thoroughbred Horses

To extend the applicability of the fluorogenic derivatization-high performance liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) method, which consists of fluorogenic derivatization (FD), separation by liquid chromatography (LC), and identification by LC-tandem mass spectrometric (MS/MS) proteomic analysis, we applied it to Thoroughbred horse muscle. With the optimization of the protein extraction and separation procedure, reproducible chromatograms were obtained and the changes in protein expressions during exercise were able to be analyzed. To quantify the changed protein expressions, the training-to-detraining (+/-) ratios for proteins were calculated, and the correlation of the ratio with the percentage of maximum oxygen consumptions (VO2max; the indicator of the running speed) was investigated. Sixteen proteins involved in energy supply, especially in anaerobic energy production, increased with an increase in VO2max, suggesting that this method was able to suggest the biochemical events in the faster-running horse and would be useful for evaluating the training effect in Thoroughbred horses.

Gas chromatographic–mass spectrometric characterisation of plant gums in samples from painted works of art

This paper presents an analytical GC–MS procedure to study the chemical composition of plant gums, determining aldoses and uronic acids in one step. The procedure is based on the silylation of aldoses and uronic acids, released from plant gums by microwave assisted hydrolysis, and previously converted into the corresponding diethyl-dithioacetals and diethyl-dithioacetal lactones. Using this method only one peak for each compound is obtained, thus providing simple and highly reproducible chromatograms. The analytical procedure was optimised using reference samples of raw plant gums (arabic, karaya, ghatti, guar, locust bean and tragacanth, cherry, plum and peach gums), commercial watercolours and paint layers prepared according to ancient recipes at the Opificio delle Pietre Dure of Florence (Italy). To identify gum media in samples of unknown composition, a decisional schema for the gum identification and the principal component analysis of the relative sugar percentage contents were employed. The procedure was used to study samples collected from wall paintings from Macedonian tombs (4th–3rd centuries bc) and from the Mycenaean “Palace of Nestor” (13th century bc) in Pylos, Greece. The presence of carbohydrates was ascertained and plant gum binders (fruit and a mixture of tragacanth and fruit tree gums) were identified in some of the samples.

Analysis of phenylurea herbicides from plants by GC/MS

Seven phenylurea herbicides (chlorbromuron, fluometuron, diuron, linuron, metobromuron, monolinuron, monuron) were isolated from weed plant materials by extraction with acidic aqueous solutions—the aqueous extract preconcentrated and cleaned-up by using a continuous SPE module. Elution with ethyl acetate resulted in >95% recovery of the herbicides from the LiChrolut-EN sorbent column. Separation of the seven herbicides was easily achieved working under fixed gas-chromatographic conditions and, although other peaks were also detected (corresponding to isocyanate degradation products), they showed different retention times and lower intensity than the parent compounds; in addition, as reproducible chromatograms in terms of herbicide-to-isocyanate product peak areas ratio were obtained, no derivatization was required. The analytical figures of merit of the GC/MS method in the selective ion monitoring mode are as follows: limits of detection from 0.5 to 5.0 ng ml −1; precision (R.S.D.) ≈7.0%; and preconcentration factor 100 (using 10 ml of sample extract). The proposed GC/MS method offers better resolution and selectivity than the conventional LC-UV method.

A Study of the Thermal Degradation Products of Styrene-Butadiene Type Rubber by Pyrolysis/GC/MS

Abstract Pyrolysis coupled with GC/MS was used to study thermal degradation products of styrene—butadiene rubber (SBR). Introduction of samples, using the pyrolysis carrier gas through the split injection port, followed by sub-ambient focusing of pyrolysis products gave reproducible chromatograms. The styrene content of styrene—butadiene copolymer was determined by plotting the GC areas of styrene and butadiene dimer (4-vinlycyclohexene) vs the percent bound styrene measured by refractive index and infrared spectroscopy. The accuracy and ease of use of the technique in determining the styrene content of styrene—butadiene copolymer is also compared to that of the refractive index and infrared spectroscopy methods. Finally, the effects of carbon black and other fillers on the thermal degradation products of the styrene—butadiene copolymer are also discussed.

Improvement of a method for chain-length distribution analysis of wheat amylopectin

For the chain-length distribution analysis of wheat amylopectin using high-performance anion-exchange chromatography (HPAEC) under alkaline conditions, the reduction of amylopectin isoamylolyzate with sodium borohydride facilitated obtaining reproducible chromatograms without both alkaline degradation and epimerization of maltooligosaccharides in aqueous NaOH solution. In a comparison of the chromatograms of isoamylolyzates of wheat starch and its fractionated amylopectin, reproducible results were also obtained. The improved method presented here, based on the use of starch isoamylolyzates reduced with sodium borohydride, is available for performing automated analysis of the chain-length distribution of wheat amylopectin by HPAEC equipped with an autosampler.

Chromatographic tank designed to obtain highly reproducible high-performance thin-layer chromatograms of gangliosides and neutral glycosphingolipids

Although high-performance thin-layer chromatography (HPTLC) is one of the most useful methods in glycosphingolipid studies, there are some difficulties with the reproducibility of the chromatographic patterns. When mixtures of solvents with different vapour pressures are used as the mobile phase, saturation of the tank is a critical point for obtaining reproducible chromatograms. A chromatographic tank designed to give optimal saturation conditions, resulting in highly reproducible chromatograms, is described.

Size exclusion chromatography of nylon-6

A new size exclusion chromatography (SEC) system for nylon-6 has been developed which uses the universal calibration peak retention of narrow dispersity poly(methyl methacrylate) (PMMA) in 2,2,2-trifluoroethanol (TFEtOH). The SEC system, operated at room temperature with silanized silica columns, obviates the solvent incompatibility of TFEtOH with styrene-gel packings and gives simple and reproducible chromatograms of polyamide-6. The method provides molecular weight accuracy within ±10% without correction for dispersion. To support the universal calibration for nylon-6, it was necessary to investigate the macromolecular hydrodynamics of PMMA in TFEtOH; results are represented well by the Stockmayer-Fixman viscosity-molecular weight theory. Poly(methylmethacrylate) in TFEtOH exhibits a Mark-Houwink exponent of 0.88 and a polymer-solvent interaction parameter, χ 12 equal to 0.38.

High performance size exclusion chromatography of polyamic acid

AbstractMolecular weight studies of polyamic acids are complicated by polyelectrolyte effects. Although early work on the molecular weight determination of polyamic acids employed salts to suppress this polyelectrolyte effect, recent publications reveal that such salts can cause precipitation of polyamic acids from solution, however, a mobile phase consisting of 0.03M LiBr/0.03M H3PO4/1% vol THF in dimethyl formamide was reported to successfully suppress the noted polyelectrolyte effect without causing the precipitation of polyamic acid and give satisfactory size exclusion chromatograms using columns packed with “macro‐porous” glasses. However, the development time was long (ca. 4 h) and considering the lability of the polyamic acids, the results must be viewed with skepticism. We find that use of a similar “mixed” mobile phase with Zorbax® PSM‐Bimodal columns using size exclusion chromatography in the high performance mode (HPSEC) provides well resolved and reproducible chromatograms and molecular weight data with development times of &lt; 15 min. Aside from the avoidance of long development times, which can lead to questionable results, this procedure permits the facile routine analysis of polyamic acid on a daily basis. The procedure has great utility as an analytical tool to support fundamental studies of polyamic acid chemistry and two examples of this application are given.

Analysis of Ultraviolet-Absorbing Compounds in Human Urine by High Performance Liquid Chromatography

Abstract Ultraviolet (UV)-absorbing compounds in human urine were analyzed by means of high-performance liquid chromatography using the column of macroreticular anion exchange resin maintained at 60°C. The sample was eluted with a linear gradient of water to 0.25 M ammonium perchlorate-acetonitrile (85:15) at 0–50 min and with 0.25 M ammonium perchlorate-acetonitrile (85:15) at 50–70 min and monitored with absorption at 254 nm. Reproducible chromatograms were obtained and 68 well resoluted peaks were numbered. The storage of a urine sample at −-20°C for 7 weeks did not significantly affect the chromatogram. The correlation coefficients of every pair of the numbered peaks were calculated to examine the daily variations and the individual difference of the UV-absorbing urine components.

Analysis of Human Urine by High Performance Liquid Chromatography with Ultraviolet Detection Using Octadecylsilica Column

Ultraviolet absorbing compounds in human urine were analyzed by means of high-performance liquid chromatography using the column of octadecylsilica(TSK-gel ODS-120A, Toyo Soda). The sample was eluted with a linear gradient of 0.1 M potassium dihydrogen phosphate(pH, 2.12)to 0.1 M potassium dihydrogen phosphate-acetonitrile(1:1)at O-60 min and O.1 M potassium dihydrogen phosphate-acetonitrile(1:1)at 60-80 min and monitored with the absorption at 254 nm. Reproducible chromatograms were obtained and 55 well-resoluted peaks were numbered. A number of urine samples collected from healthy females with and without the administration of diuretics were analyzed, The correlation coefficients between every pair of peaks among the numbered peaks were calculated to examine the daily variations, the individual difference and the effects of diuretics on the UV-absorbing components. From the cross correlation matrix thus obtained, a few peaks which correlated closely with the peak of creatinine were identified. The results showed the feasibility of the HPLC analysis of urine concerning the diagnostic purposes.

Application of temperature-programmed desorption to surfaces with physically adsorbed species

Temperature-programmed desorption has been applied to physisorbed aliphatic alcohols and acids on a rutile pigment surface. Reproducible desorption chromatograms for a series of alcohol adsorbates indicate that the same sites are involved in adsorbing the series and that the adsorbate orientation from the surface deviates progressively from normal as the chain length increases. The data also infer a reduction in the activation energy of desorption with increasing chain length. Acid adsorbates also generate reproducible chromatograms, those for formic and acetic acid showing characteristic peaks near 300 °C. The reason for the very high stability of these adsorbates has not yet been resolved.

Some fundamental aspects of centrifugally accelerated paper chromatography

A study has been made of some of the fundamental factors involved in obtaining good separations and reproducible chromatograms, with the technique of contrifugally accelerated paper chromatography. A study was also made of variables which might influence RF values. Mixtures of bromphenol blue, methyl orange and methyl red were fractionated as well as solutions of lencine, methionine and glycine.For the separation of the dyes, Whatman No. 1 filter paper was used with a veronal buffer solution of ionic strength 0.05 and having a pH of 8.7. For this system, it was found that the RF values of the dyes were essentially unaffected by changes in rotational speed of the rotor from 300 to 925 r.p.m. It was found that the rate of addition of solvent was important in developing satisfactory chromatograms. A flow rate of 1.15 ml/min was found to be satisfactory at all rotational speeds above 250 r.p.m. The shape of the developed chromatogram was elliptical rather than circular, for all four types of paper which were studied. For Whatman No.1, the major axis of the ellipse was parallel to the machine direction of the paper. The RF values for the dye components were essentially the same for the following papers: Whatman No. 1, Eaton-Dikeman No. 613, and Cremer-Tiselius-Munktells.The amino-acid mixture was fractionated using Whatman-3MM paper and a solvent system of butanol-glacial acetic acid and water, in the volume ratio 40:10:10, respectively. The disk was rotated at 975 r.p.m. With a solvent flow rate of 1.2 to 2.1 ml/min, sharply defined zones were obtained. The time of development was 15 min for a flow rate of 1.2 ml/min and 7 min for a flow rate of 2.1 ml/min. The RF values obtained were 0.85 for lencine, 0.56 for methionine and 0.20 for glycine.

A Technique for Examining Large Numbers of Bacterial Culture Filtrates by Partition Chromatography

SUMMARY: A description is given of apparatus and methods which have been used to produce large numbers of satisfactory and reproducible chromatograms of bacterial culture filtrates. Some of the factors which influence the preparation and development of chromatograms are discussed.