Preparation of glass capillary columns

Summary Glass capillary column chromatography is the most rapidly growing part of gas chromatography. There are many complex new analytical tasks and they require special capillary columns. Fortunately there is a wide range of column preparation methods available, and they make the preparation of glass capillary columns a more varied job than that of packed columns. In this paper these methods are reviewed and suggestions are given for making task-oriented columns.     

Gas chromatography of amino acids: Use of a fused-silica capillary column in the determination of plasma amino acids

A capillary chromatographic procedure using a fused silica column is described which can be used to quantitatively determine amino acids in plasma following the pre-chromatographic “clean-up” described in a recent paper [1]. In substituting this procedure for that involving a packed column, advantage has been taken of the greater resolving power to separate amino acids from background component peaks. In order to extend this advantage and provide a sound basis for quantitative analysis, the technique of cold on-column injection was employed. As a result, good precision of standard analysis was obtained with relative standard deviation (RSD) values for all amino acids of less than 4%. Application of the entire procedure to plasma samples yields RSD values of better than 10% for all amino acids with recoveries ranging from 72% to 104%. Simultaneous determination of plasma amino acid levels by gas chromatography (GC) using capillary columns and by classical ion exchange (CIE) showed reasonable agreement. Statistical evaluation showed no significant difference between twelve amino acids. Values for the remaining two, namely, phenylalanine and histidine are significantly different (p < 0.005). Comparison of the values obtained from GC capillary and packed columns reveals no significant difference between fourteen amino acids. Significant differences exist between results for phenylalanine and tyrosine (p < 0.001). It is concluded that there is good agreement between data obtained by GC capillary and CIE techniques and that differences between results for phenylalanine and histidine are method related.     

An investigation of glasses for capillary chromatography

An investigation was conducted of various glasses, other than soda lime or borosilicate, for use in glass capillary gas chromatography. The work has uncovered some unique chromatographic qualities in the use of potash soda lead and fused silica glasses as materials for making glass capillary columns. The fused silica proved to be an ideal material for capillary column construction, being inherently more inert than glass containing metal oxides. It has been shown that through the use of thin wall capillary tubing of high flexibility many of the mechanical problems associated with glass capillary columns, such as fragility and column straightening, can be avoided.     

Gas-liquid chromatography of amino acids: Columns and methodology as a basis for routine amino acid analysis using glass capillary gas chromatography

A carefully Standardized technique is described for the preparation of glass capillary columns which can be used successfully for routine quantitative amino acid analysis. Comparison is made between two different modes of sample injection. Preliminary quantitative results from “split” injection and “on-column” injection techniques are evaluated statistically and it is concluded that the “on-column” system is a prerequisite for quantitative amino acid analysis by glass capillary gas chromatography. An analysis of fish muscle protein hydrolyzate illustrates an application of this technique and results are compared with those from a packed column analysis.     

Studies of open tubular micro capillary liquid chromatography. 1. The development of open tubular micro capillary liquid chromatography

Micro capillary columns were successfully applied to liquid chromatography by employing the principles used in micro high performance liquid chromatography. Fundamental investigations on the use of capillary columns in LC were performed for the various column parameters. Good separations of five aromatic hydrocarbons and four kinds of phthalic esters were obtained on a 62 μm I.D. capillary column, coated with SE-30.     

Experiments with capillary columns having unconventional cross-sectional geometry

The potentials of unconventional capillary columns, e.g. flat, crinkled, whisker and helically coiled open-tubular columns (HOT columns) were evaluated. In comparison to circular columns, no improvement in the column performance, when used under normal capillary GC circumstances, could be observed.     

High resolution gas chromatographic determination of permanent gases with a helium ionization detector

The capability of the helium ionization detector (HID) to operate in connection with capillary columns for trace gas analyses has been evaluated. Two different capillary columns were considered: a PLOT fused silica column with molecular sleve and a thick film WCOT glass column with PS-255. The determination of trace impurities in gases can be achieved with evident advantages over classical adsorption columns, even using a split injection system. Direct on-column injections also have been investigated with promising results.     

Surface modified wall coated open tubular columns: Development and application in pesticide analysis

Filamentary crystal growth on the inner surface of a glass analytical capillary column using a modified ammonium hydrogen difiuoride procedure, produced highly efficient wall coated open tubular columns. Baseline separation of the acetate derivatives of 2,3,4,6- and 2,3,5,6-tetrachlorophenol was achieved and was instrumental in determining pentachlorophenol loadings along a watershed. The application of capillary columns, related to the analyses of organochlorine pesticide residues is also demonstrated. Parent and degradation products of twenty-five commonly monitored residues were effectively resolved on a 20 MOV-101 column.     

The use of glass capillary columns for the analysis of polar natural products

Two published procedures for glass capillary column production are applied to produce capillaries for chromatography of polar natural compounds. Soda lime glass capillaries, after leaching with aqueous HCL, are either treated with colloidal silicic acid or with barium carbonate and coated with either Silar 7CP or SE 52. A test mixture as well as polar carbohydrate and peptide mixtures are chromatographed on the different columns and their chromatographic properties are evaluated. Combined capillary gas chromatography-electron impact and chemical ionization mass spectrometry are used for identification of the peptide sequence.     

Evaluation of packed capillary liquid chromatography columns and comparison with conventional-size columns

Apart from extracolumn effects peak dispersion in liquid chromatographic columns is caused by the column inlet, the packed bed, and the column outlet. A strategy applicable for independent evaluation of the individual sources of column band broadening was developed on the basis of the linear extrapolation method (LEM). This method was applied to compare the performance of packed capillary LC columns from various commercial suppliers with conventional-size columns. The columns differed widely in their performance with respect to peak shapes and widths for standard substances. The capillary columns were found well packed, but in some cases overall performance would benefit from improving the design of the area between the packed bed and the connecting capillaries, containing frits as well as dead volumes.     

Separation of fluorinated amino acids and oligopeptides from their non-fluorinated counterparts using high-performance liquid chromatography

Chromatographic conditions for the separation of fluorinated amino acids and oligopeptides from their non-fluorinated counterparts were explored. The separation of six pairs of analytes, including both aromatic and aliphatic fluorocarbons, was investigated at various temperatures using both hydrocarbon and fluorocarbon columns and eluents. Our results show that when hydrocarbon eluents are used, fluorocarbon column provides better separation of fluorinated amino acids or oligopeptides from their non-fluorinated counterparts; when fluorocarbon eluents are used, hydrocarbon column provides better separation of fluorinated amino acids or oligopeptides from their non-fluorinated counterparts. These chromatographic behaviors reflect the fluorophilicity possessed by fluorinated amino acids and oligopeptides.     

Review. Use of laser detectors in capillary liquid chromatography

The main relationship of high-performance liquid chromatography (HPLC) are considered. It is shown that the optimum conditions of ultrasensitive trace analysis should be achieved by using packed capillary columns manufactured from flexible quartz capillaries with dc approximately less than 0.2 mm. The main features of these columns (v opt = 0.6 v opt of that for conventional HPLC columns with double the hydraulic permeability) make it possible to obtain two or three times higher plate numbers for the same analysis time and column pressure characteristic of conventional HPLC, as a result of using a submicrometre sorbent. The main features of laser detection in capillary liquid chromatography (laser-induced fluorescence and cross-beam thermal lens absorption detectors) are considered. The requirements that should be met by a modern capillary liquid chromatograph based on using flexible quartz capillary columns with a submicrometre sorbent and laser detectors are formulated. Examples of using these systems for femtomole and attomole analyses of biological samples (amino acids and prostaglandins) are given.     

Capillary gas chromatographic analysis of amino acids in blood and protein hydrolysates as tert.-butyldimethylsilyl derivatives

A method is given for a one-step derivatization and gas chromatography of amino acids in blood and protein hydrolysates. Blood samples are partially purified by solvent extraction. Protein hydrolysates are neutralized with a triethylamine solution. Then tert.-butyldimethylsilyl derivatives of the amino acids are prepared in a one-step procedure and separated on a 30-m fused-silica SE-30 capillary column. Except for tryptophan and cystine, amino acids are eluted within 30 min. Amino acids are derivatized more rapidly than their corresponding trimethylsilyl derivatives and do not degrade on the long fused-silica columns.     

Coupling miniaturized liquid chromatography to capillary gas chromatography (micro-LC-CGC): Possibilities of reversed phase LC

Retention gaps with different polarity treatments were evaluated for reversed phase solvents. Aminopropyl- and cyanopropyl-deactivated retention gaps showed the best results for methanol-water mixtures. A reversed phase packed fused silica capillary LC column is connected on-line with a capillary gas chromatography column. The combination was used for the analysis of diazepam in urine. Volume overloading on packed fused silica columns without loss of too much efficiency was demonstrated for propranolol.     

一种多通道匀浆装填毛细管色谱柱的新装置及应用

针对目前毛细管色谱柱装柱效率低、不同批次装填的毛细管色谱柱之间性能差异大的问题,我们发展了一种多通道匀浆装填毛细管色谱柱的新装置。该装置以液相色谱泵提供压力、采用磁力搅拌保持匀浆液均匀分散,一次可装填多达6根毛细管色谱柱。以牛血清白蛋白(BSA)的胰蛋白酶酶切肽段混合物为样本,选择峰容量、蛋白覆盖率、3个特定离子的保留时间以及毛细管色谱柱柱压为指标,在毛细管液相色谱-质谱联用系统上对装填的反相毛细管色谱柱的性能进行了评价。分别考察了一次装填的6根毛细管色谱柱、两次装填的12根毛细管色谱柱以及一次装填1根与一次装填6根毛细管色谱柱的性能及稳定性。实验结果表明:同一批次装填的6根毛细管色谱柱的性能相近;不同批次装填的12根毛细管色谱柱的峰容量和覆盖率没有明显的区别,但保留时间和毛细管色谱柱柱压的稳定性较差;一次装填1根和一次装填6根毛细管色谱柱柱性能的稳定性与两次分别装填6根毛细管色谱柱的稳定性相近,即采用本装置可显著提高毛细管色谱柱的装填效率且每次装填毛细管色谱柱的数量不会对柱性能产生影响。     

Determination of 4-aminobutyric acid, aspartate, glutamate and glutamine and their 13C stable-isotopic enrichment in brain tissue by gas chromatography-mass spectrometry

A selected-ion monitoring method was developed for measuring 4-aminobutyric acid, aspartate, glutamate, and glutamine in brain tissue. Natural isotopes of these amino acids and their stable-isotopic enrichment following intravenous infusion of a precursor, [13C]glucose, were quantitated. Frozen mouse brain tissue was homogenized in cold 80% ethanol, and the supernatant, equivalent to 1 mg of wet weight brain tissue, was extracted using solid-phase bonded silica ion-exchange columns. Aspartate and glutamate (dicarboxylic acids) were isolated from strong anion-exchange columns, whereas 4-aminobutyric acid and glutamine (neutral amino acids) were isolated from strong-cation exchange columns. n-Butyl ester pentafluoropropionyl amide derivatives of these amino acids were analyzed by gas chromatography-mass spectrometry using a methane positive chemical ionization mode after gas chromatographic separation on a wide-bore, fused-silica capillary column. The method is applicable to determination of brain concentrations of these amino acids as well as their fluxes following administration of a stable-isotopic tracer.     

Seperation of protein amino acids as their N(O)-Acyl alkyl ester derivatives on glass capillary columns

A comparison of the elution properties of the major protein amino acids as their N(O)-acyl alkyl ester derivatives (O-n-propyl, -n-butyl, -isopentyl; N(O)-trifluoroacetyl, -heptafluorobutyryl) on open-tubular glass capillary columns coated with SE-30, OV-17, OV-210 and EGA is described. A single-column separation to the baseline of the protein amino acids as their N(O)-heptafluorobutyryl n-butyl ester derivatives in less than 35 min was obtained on the SE-30 column. OV-210 columns have properties complementary to those of SE-30 columns and can be used as an aid to compound identification from retention time data. Separations of the amino acids from beer and dialysate from uremic patients are used to illustrate the practical posibilities of the method.     

Twenty years of glass capillary columns. An empirical model for their preparation and properties

Both the invention of the capillary drawing machine and the preparation and successful use of the first glass capillary column were achieved by Desty and his group in 1959. This paper attempts to describe the glass capillary column in a general way, including the spreading of liquid phases of different polarity and solid surfaces with different chemical and geometrical structures, and surface activity (as a source of essential column characteristjcs such as film stability, adsorptive and catalytic activity, acidity, and thermostability). The model is entirely based on experimental evidence. It is shown that today's apolar columns may closely approach ideal performance, such as is conceivable at the present time, while a severe gap still exists between the actual and the ideal characteristics of polar columns. The problem lies in the necessity of using considerably active support surfaces for polar coatings, yielding columns with reduced upper temperature limits, and of restricted suitability concerning the nature of sample. It is hoped that progress in the field of surface roughening may produce an inert support which is wettable by polar phases.     

Persilylation of glass capillary columns. Part 4: Discussion of parameters

Persilylated glass capillary columns are the most universal GC columns available. They are relatively easy to prepare, but there is nevertheless a great need for helpful practical instructions. In the recent literature, methods and conditions for the preparation of persilylated columns have been recommended, but these might confuse the individual column maker, since they seem to originate from limited observations rather than from comprehensive investigation. They will hardly help to make column preparation safer. This paper attempts a summarized view of the present experience with persilylation and provides basic information for column makers.