Robert W. Zumwalt

Amino acid analysis : Hydrolysis, ion-exchange cleanup, derivatization, and quantitation by gas—liquid chromatography☆

Sample preparation techniques are as vital in amino acid analysis as the methods by which the amino acids are measured. Experiments were conducted to obtain a rapid, accurate, and precise procedure for protein hydrolysis and sample cleanup with subsequent gas—liquid chromatographic analysis. The use of ultrasonication to remove dissolved air while pulling a vaccumm on the sample solution prior to hydrolysis assured a good recovery for methionine and cystine. These techniques combined with a 4-h hydrolysis at 145° using 6 N HCl gave results in good agreement with the hydrolysis conditions of 18–24 h at 100°. Physiological fluids for free amino acids were prepared by precipitating the protein with saturated pieric acid followed by cation-exchange clean-up. Gas—liquid chromatographic analysis of small samples was improved by the use of a Sol-Vent device. This enables one to inject large volumes of the derivatized sample (100 μl) onto the analytical volumn, eliminate the tailing effects of the solvent and trifluoroacetic acid while still retaining the amino acids on the analytical column. The techniques for sample prepration and chromatographic analysis presented in this paper provide the chemist with valuable tools fo the analysis of amino acids in biological samples by gas—liquid chromatography.

Gas-liquid chromatography of amino acids in biological substances

Abstract The development of a gas—liquid chromatographic (GLC) method for the quantitative analysis of amino acids in complex biological substances, specifically blood plasma and urine, has been achieved. The amino acids present in these physiological fluids were quantitatively isolated by ion-exchange methods and retained on the ion-exchange resin while the substances which interfere with the GLC analysis passed through the column and were discarded. The amino acids were then eluted from the column, derivatized to their N-trifluoroacetyl (N-TFA) n -butyl esters and analyzed by GLC. Quantitative recovery of the amino acids from the cation and anion-exchange clean-up columns, and amino acids in blood and urine were successfully carried out. Also, techniques for the analysis of amino acids over a wide range of concentrations were developed. Analyses were made on aliquots of human blood plasma containing only 200 μg of total amino acids, and the results obtained at this level of concentration were both accurate and precise. Further, quantitative data were obtained with samples containing only 20 μg of total amino acids, and semiquantitative analyses were performed on samples containing 2 μg of total amino acids. The data obtained by the GLC method were in excellent agreement with results by classical ion exchange. Investigations on acylation of the amino acid n -butyl esters have shown that the optimum molar ratio of TFAA/amino acids is 50:1 with regard to reproducibility of acylation, stability of the derivative, and maintenance of a small sample volume (> 75 μl).

Gas-liquid chromatography of protein amino acid trimethylsilyl derivatives

Abstract The purpose of the investigation was to make a thorough study of the chemistry of derivatization of the twenty protein amino acids as their N-trimethylsilyl trimethylsilyl (TMS) esters. Major emphasis was directed toward chromatographic separation of the derivatives, precision and accuracy of the method, silylation as a function of reaction temperature and time, molar excess of reactants, stability of the TMS derivatives, quantitative analysis of a synthetic amino acid mixture, and application to biological samples. The gas-liquid chromatographic separation of the N-trimethylsilyl TMS esters of the twenty protein amino acids was achieved after evaluation of a number of combinations of siloxane liquid phases. The final chromatographic conditions used for the total separation on a single column for all twenty of the amino acids consisted of a mixed liquid phase of 3.0 w/w% OV-7 and 1.5 w/w% OV-22 coated on high-performance 100 120 mesh Chromosorb G in a 1.75 m × 4 mm I.D. U-shaped borosilicate glass column. Phenanthrene was a suitable internal standard as it was completely resolved from the TMS amino acids. The instrumental settings were 75°, initial hold 7 min, program rate 2°/min, and carrier flow (N 2 ) of 42 ml/min for fourteen of the amino acids, and 100° initial column temperature for the other six. Prior to chromatography, it is essential to analyze performance blanks under the same chromatographic and instrumental conditions to establish the purity of all chemical reagents. The reaction conditions were investigated for the quantitative silylation of the twenty amino acids. Fourteen of the amino acids were reproducibly converted to the respective TMS derivatives in 15 min at 135° in a closed vial using a 30 molar excess of bis(trimethylsilyl)trifluoroacetamide (BSTFA)/total amino acids. A comparison of various silylation temperatures showed that silylation at 135° produced the most atmosphere in a closed tube. After evaporating the sample to dryness at room temperature with a rotary evaporator, the sample was transferred to a 50-ml volumetric flask and brought to volume with 0.1 N HCl. Five milliliter aliquots of this stock ribonuclease solution were then transferred to 16 mm × 75 mm glass reaction tubes, dried, and derivatized as described in the section experimental . The chromatograms obtained on silylation at 135° for 15 min and 4 h, respectively, are presented in Figs. 11 and 12. The data obtained from three independent GLC analyses are given in Table X, and the results are in good agreement with those from classical ion-exchange analysis. Preliminary investigations on the GLC analysis of cation and anion-exchange cleaned human urine by the TMS technique have shown that some problems still exist. The urine samples contained a large amount of glycine, and difficulty in obtaining a single peak for glycine was noted. Both the di-trimethylsilyl (GLY 2 ) derivative and the tri-trimethylsilyl (GLY 3 , ca. 10%) derivative were obtained when the samples were derivatized at 135° for both 10 and 15 min. The GLY 3 peak interfered with the resolution of TMS leucine and TMs proline due to the large quantity of glycine in the sample. Further investigations are needed to obviate this problem.

The complete gas—liquid chromatographic separation of the twenty protien amino acids☆

The complete gas—liquid chromatographic separation of the twenty protein amino acids is presented. In our previous publications, we reported on in effective chromatographic separation of seventeen N-trifluoroacetyl n-butyl esters of the amino acids on a packing composed of stabilized grade EGA and acid-washed Chromosorb W (heated at 140° for 12 h). A new column packing has been found to replace the OV-17; it is composed of a mixed phase of OV-17 and OV-210. This is a superior packing and shows quantitative elution, and highly efficient and complete separation of histidine, internal standard tranexamic acid, lysine, arginine, tryptophan, n-butyl stearate (I.S.), and cystine. No longer is it necessary to make a separate “subtraction-calculation” for histidine. With these two packings, EGA and mixed siloxane phases, one can now simultaneously analyze and separate the 20 protein amino acids in 30 min or less with automatic electronic integration of all peaks. A second internal standard (tranexamic acid) is introduced which contains both -NH2 and -COOH functional groups. It can be used to follow the performance of ion-exchange cleanup, thereby increasing the reliability of analysis of complex biological materials. The use of two columns for separation of the twenty amino acids has important advantages over a single column system with respect to resolution, cross confirmation, and identification of the eluted compounds.

Chromatography of nucleosides

Abstract The chromatographic parameters affectingthe reversed-phase high-performance liquid chromatographic (HPLC) separation of major and modified nucleosides with a μBondapak C 18 column have been studied. This investigation has resulted in the HPLC separation of eighteen nucleosides in a single analysis. The parameters studied include: the mobile phase flow-rate, pH, methanol concentration, column temperature and injection volume. Each parameter was investigated individually to observe the effect on the chromatographic behavior of the nucleosides. The relationships which we have established for the elution of the nucleosides as a function of the respective parameters investigated can be used to predict their separation. From these experiments, the chromatographic conditions for the separation of urinary nucleosides were optimized using both isocratic and step gradient conditions. The step gradient system is more suitable for determining the nucleoside composition of tRNA hydrolysates, and the complete separation of the major ribo- and deoxyribonucleosides can be accomplished. Also, we have studied the storage stability of urinary nucleosides, and have looked for nucleotides and oligonucleotides in normal and cancer patient uring and found none. In addition, we report a rapid isocratic system for the separation of m 2 2 G and t 6 A. A most significant aspect of this research is the determination of the effects of various chromatographic parameters on the reversed-phase HPLC separation of the nucleosides. These findings provide great flexibility in the analysis of nucleosides in that these data form a guide for finding optimal conditions for nucleosides separations. This chromatography is of importance in the accurate determination of tRNA composition, especially to scientists investigating tRNA biosynthesis, function and sequence, and also for investigations on the purity of RNA and DNA isolations, and research on DNA and its modification.

A nanogram and picogram method for amino acid analysis by gas-liquid chromatography.

A gas—liquid chromatographic method has been developed for the analysis of samples containing nanogram to picogram amounts of amino acids. The procedural details of the method are presented with the reaction conditions for conversion of 1 to 1000 ng of amino acids to their N-trifluoroacetyl n-butyl esters. The developed method was found to be essentially quantitative. Recoveries were greater than 80% when 5 ng of each amino acid were taken through the total method by comparison with diluted macro standard solutions. Also, an injection port solvent vent-chromatographic system was invented which allows injection of the total derivatized samples (up to 100 μl) on a standard packed analytical column. This device prevents the solvent and excess acylating reagent from traversing the column and entering the detector, while allowing quantitative transport of the amino acid derivatives through the column. Samples containing seventeen amino acids at the 50 and 5 ng levels were taken through the total derivatization and chromatographic procedure, and then analyzed by GLC incorporating the solvent vent device. To achieve higher sensitivities, the detection of the N-trifluoroacetyl and N-heptafluorobutyryl n-butyl esters of selected amino acids by 03Ni electron capture was studied. The minimum detectible amounts of various amino acid derivatives were determined by demonstrating that 1 to 50 pg can be clearly observed by this method. Studies were made on the N-TFA n-butyl ester derivatives of eight amino acids, and on the N-HFB n-butyl esters of methionine and cysteine. Analyses of the water extracts of the Returned Lunar Samples (Apollo flights 11 and 12) are presented with none of the common amino acids being detected above the background detection limit of ca. 4 to 5 ng/gram. However, several unidentified chromatographic peaks were observed, particularly in the Apollo 12 sample, which expected content of the compounds of interest is extremely small. The broad spectrum of application for this technique has already included the search for amino acids in the Returned Lunar Samples, and would range from studies at the cellular level, investigations on the molecular basis of disease, to the analysis for trace amounts of environmental pollutants.

Quantitative gas-liquid chromatography of histidine

Abstract The quantitative gas—liquid chromatographic analysis of histidine was easily accomplished using two different methods and the mono and diacyl N-TFA n-butyl esters of histidine as the derivatives. To obtain quantitation, histidine must be present entirely as either the monoacyl or as the diacyl derivatives. There are certain advantages in using the diacyl derivative, but this derivative was not separated from the N-TFA n-butyl ester of aspartic acid on any of the siloxane columns evaluated (OV-1 to OV-25). A quantitative, reproducible method has been developed and is presented by which histidine can be analyzed as its diacyl N-TFA n-butyl ester. In this method the diacyl derivative formed during acylation was converted to the monoacyl derivative by evaporation of the excess acetylating reagent, TFAA. Thus, histidine is present as the monoacyl derivative when the sample is injected ∝on column’. Then, the monoacyl derivative was converted to the diacyl derivative by direct ∝on column’ injection of 4 μl of TFAA. When the TFAA was injected immediately after the methionine peak, the diacyl derivative synthesized on the column was precisely eluted between tyrosine and glutamic acid. No interference was observed with any of the other amino acid derivatives. The separation of the diacyl derivative of histidine from the aspartic acid deriviative was made possible because the retention temperature of the monoacyl derivative is different from that of the diacyl histidine derivative or the derivative for aspartic acid. The quantitative elution of all twenty of the protein amino acids as their N-TFA n-butyl esters has been achieved on a dual column system of 0.325 w/w % of stabilized EGA on heat-treated Chromosorb G and 1.5 w/w % OV-17 on high performance Chromosorb G as the stationary phases. Alternatively, for sixteen amino acids, a 1.5 m glass column with packing of 0.65 w/w % of stabilized EGA on 80 100 mesh acid-washed Chromosorb W dried at 140° for 12 h can be used. In another approach, sixteen of the protein amino acids can be determined as their N-TFA n-butyl esters on a 1.5 m × 4 mm I.D. glass column consisting of 0.325 w/w % of stabilized EGA on heat-treated Chromosorb G (or 0.65 w/w % of stabilized EGA on 80 100 mesh acid-washed Chromosorb W dried at 140° for 12 h) while arginine, histidine, tryptophan, and cystine are chromatographed as their trimethylsilyl (TMS) derivatives on a 1.75 m × 4 mm I.D. glass column containing a mixed phase of 3.0 w/w % OV-7 and 1.5 w/w % OV-22. The TMS derivative offers a unique advantage in that the derivatization reaction is completed at 135° for 4 h in a closed tube with no transfers or removal of reagents. Silylation of amino acids to their TMS derivatives holds considerable promise not only for the quantitative analysis of arginin, histidine, tryptophan, and known mixtures and ribonuclease as the TMS derivative is reported. An average recovery of 102.5% was obtained. The reaction conditions and chromatography of the TMS derivatives of the twenty protein amino acids and some non protein amino acids are the subject of a separate manuscript. A simplified determination of histidine can be made by a calculation of the AREAHIS on the OV-22 column from the AREAHIS+ASP on the same column.

Gas-liquid chromatography of protein amino acids - Separation factors.

Abstract This research reports on chromatographic and instrumental studies which will permit one to accurately, rapidly, and routinely analyze the twenty natural protein amino acids in biological substances. A number of factors influencing the performance of the chromatographic system were evaluated, and the separation characteristics of various polyesters of neopentyl glycol were evaluated. Also, an evaluation was made of support materials which had undergone various heat treatments. It was shown that the optimum chromatographic performance for neopentyl glycol polyesters was observed at a carbon chain length of ten (neopentyl glycol sebacate). The separation ability of ethylene glycol adipate as a liquid phase was found to be superior to neopentyl glycol sebacate, consistent, and reproducible with respect to time and temperature. Arginine, histidine, and cystine were not reproducibly eluted from this column. This is a result of interaction between the substrate phase and the amino acid derivative. However, seventeen of the amino acid derivatives were well separated and quantitatively eluted in 33 min from columns containing 0.325 w/w % ethylene glycol adipate coated on 80/100 mesh acid-washed heat-treated Chromosorb G. In general, the retention temperatures for the amino acids were lower and a significant improvement in resolution was noted when columns were prepared with Chromosorb G which had been treated at 450° to 600° for 15 h. For the analysis of arginine, histidine and cystine, columns containing 1.5 w/w % OV-17 coated on high performance 80/100 mesh Chromosorb G were used, Di-acyl histidine was converted to the mono-acyl derivative by injection of n -butanol immediately after injection of the sample. The di-acyl derivative of histidine does not interfere with any of the amino acids on the EGA column. A dual column chromatographic system of ethylene glycol adipate and OV-17 as the liquid phases is described which quantitatively separates that protein amino acids in 55 min. The quantitative gas-liquid chromatographic analysis of the amino acids in ribonuclease is reported.

Applications of a gas—liquid chromatographic method for amino acid analysis : A system for analysis of nanogram amounts☆

Abstract The quantitative gas—liquid chromatographic determination of the protein amino acid content of biological substances has been clearly demonstrated with analyses of corn grain, soybean oil meal, blood plasma, and human urine. The precision and accuracy of the gas—liquid chromatographic technique was found to be equal to that of classical ion-exchange chromatography, and superior in some instances. Further, an instrumental-chromatographic system has been invented which allows the injection of 100 μl or more on a standard analytical column. This device eliminates the TFA peak during analysis of amino acids on the EGA column; results in a more stable baseline due to the decreased amount of solvent and reagents traversing the column; greatly simplifies analysis for nanogram amounts of amino acids; and should find a wide range of applications in many gas—liquid chromatographic and gas chromatography—mass spectrometry investigations. A superior mixed phase chromatographic column is reported for the separation of His, Lys, Arg, Trp and Cys.

Determination of polyamines in human urine by an automated ion-exchange method

Abstract A sensitive and reliable automated cation-exchange method for the determination of polyamines in human urine has been developed and applied to samples from both normal subjects and patients bearing various types of cancer. A relatively large number of cancer patients urine samples are currently being analyzed, and these data will be the subject of a later paper.