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.     

Steroid analysis by gas chromatography with SCOT and wide-bore WCOT columns

Glass capillary columns are ideal for analysis of urinary steroid profiles (i.e. the total steroid neutral fraction without prior separation into sub-groups). In this paper performance of both SCOT and wide-bore WCOT columns has been compared, resulting in no significant quantitative difference on urine extracts run as trimethylsilyl ether derivatives. However, because of increased efficiency the WB-WCOT column exhibited more sensitivity in measurement of small amounts of 11-ox-aetiocholanolone. Sensitivity of other steroids was greatly enhanced by use of either SCOT or WB-WCOT, leading to an almost tenfold increase over conventional packed columns (e.g. androsterone and aetiocholanolone < 40 μg per 24 h urine sample). A simple splitless injection system (based on a design of Dr. W. Greenaway, Oxford University) is presented. Experiments were also carried out using a SCOT column in conjunction with an FM-electron capture detector. Whilst efficiency was impaired due to the design of the detector, use of He (carrier) and N₂ (make up gas) showed that several steroid derivatives could be easily measured at well below the 10^?10 g level with much more rapid retention times than with a 1½m packed column. Highly successful separation of 19 amino acids (as fluoro-acyl derivatives) was achieved in 35 minutes using a SCOT (30 m) SP2100 column.     

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.     

Separation of 28 to 114°C hydrocarbons with glass capillary columns

Techniques have been developed for the rapid separation (about 20 minutes) of the 39 compounds in crude petroleums, or petroleum distillates, which boil between 28 and 114°C. A 300 meter glass column (0.25 mm i.d.) which is etched, coated with a mixture of normal hexadecane and Kel-F10157 is utilized to perform this separation at room temperature. The separations obtained with this non–polar liquid mixture and the «inert» glass surface are much more rapid than those previously obtained with stainless steel capillary columns.     

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.     

Trace analysis of halocarbons in water; Direct aqueous injection with electron capture detection

Interest in monitoring halogenated organics in drinking water and natural surface and ground water in the low ppb range continues to grow. There is a tendency to include still more volatile halocarbons, the trace determination of which is known to be rather demanding. This prompted us to re-examine the feasibility of large-volume direct aqueous injection onto capillary columns, coupled with ECD. A primary problem was to avoid simultaneous elution of water with halocarbons, since water suppresses the ground current of the ECD. The following measures contributed to the solution of this problem. Apolar, extremely inert, columns are required to elute water completely, and even before very light halocarbons. Their coatings have to be far thicker (≈? 5 mUm) than commonly employed thick films since they must permit isothermal analysis at a column temperature around 100°C in order to ensure rapid and complete elution of water. Finally, it is essential that sampling be carried out on-column for two reasons: diffusion of water vapor in the injector, resulting in delayed elution, is then eliminated, and peak distortion during splitless injection is avoided. Although we now know that persilylated columns with immobilized coatings withstand routine water injections, more longterm experience is needed to provide detailed recommendations for the handling of these columns.     

A simple terminator for high efficiency liquid chromatography columns

A comparison is made between a flat bottom column terminator and a cone terminator on. 1.27 cm O.D. and 2.54 cm O.D. columns. The cone terminator results in a doubling of the column efficiency, is simple and inexpensive, and is universally adaptable to any diameter column.     

Thermostable persilylated polar glass capillary columns by the ammonia-etching method

Static ammonia etching has been used to effect regular surface roughening of glass capillaries. Following such treatment, polar phases could be coated after persilylation with varioussilylation agents such as hexamethyl-, tetramethyldiphenyl-, and tetra- phenyldimethyldisilazane, respectively. This method produces columns of high thermostability, which is limited only by the maximum applicable temperature for the phase itself.     

Improved GC/ECNI-MS sensitivity of decabromodiphenyl ether determination on 30 m columns by increasing the carrier gas flow after a modified gel permeation chromatographic cleanup protocol employing cyclohexane/ethyl acetate as eluent

BDE-209 is the predominant constituent of the commercial mixture decabromodiphenyl ether which is used as brominated flame retardant (BFR). Owing to difficulties associated with the high mass (thermal instability and low vapour pressure), short GC columns (≤15?m) have been suggested for its analysis while longer columns (30–50?m) are suggested for other polybrominated diphenyl ether (PBDE) congeners. To overcome this considerable expenditure in the analysis of PBDEs, we aimed at increasing the sensitivity of BDE-209 analysis by gas chromatography coupled with electron-capture negative ion mass spectrometry (GC/ECNI-MS) on a 30?m column. The chromatographic performance of BDE-209 on the 30?m GC column was improved by increasing the carrier gas flow from initially 1.2?mL?min^?1 to 5 or 10?mL?min^?1 after the last octabromo diphenyl ether (Br₈DE) congener was eluted. With this high carrier gas flow, the column residence time of BDE-209 was shortened by ～25%, the peak height was increased and, consequently, the limit of detection by GC/ECNI-MS in selected ion monitoring (SIM) mode was improved. When this high-flow GC/ECNI-MS-SIM method was applied to a sediment sample, we realized that gel permeation chromatography (GPC) – used for the removal of lipids and/or sulphur – provided low recovery rates for BDE-209. The large molecule BDE-209 eluted late and only 50% was recovered by our previous standard protocol for polyhalogenated compounds. Application of a modified GPC procedure with a longer collection time increased the recovery of BDE-209 in the GPC step to ～90%.     

A Novel Silicon Etching Method Using Vapor of Tetramethylammonium Hydroxide Solution

Silicon bulk etching is an important part of micro-electro-mechanical system (MEMS) technology. In this work, a novel etching method is proposed based on the vapor from tetramethylammonium hydroxide (TMAH) solution heated up to boiling point. The monocrystalline silicon wafer is positioned over the solution surface and can be anisotropically etched by the produced vapor. This etching method does not rely on the expensive vacuum equipment used in dry etching. Meanwhile, it presents several advantages like low roughness, high etching rate and high uniformity compared with the conventional wet etching methods. The etching rate and roughness can reach 2.13 μm/min and 1.02 nm, respectively. Furthermore, the diaphragm structure and Al-based pattern on the non-etched side of wafer can maintain intact without any damage during the back-cavity fabrication. Finally, an etching mechanism has been proposed to illustrate the observed experimental phenomenon. It is suggested that there is a water thin film on the etched surface during the solution evaporation. It is in this water layer that the ionization and etching reaction of TMAH proceed, facilitating the desorption of hydrogen bubble and the enhancement of molecular exchange rate. This new etching method is of great significance in the low-cost and high-quality micro-electro-mechanical system industrial fabrication.     

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.     

Disengaging solutes in shale- and petroleum-derived jet fuels by altering GC programmed temperature rates

Overlapping chromatographic peaks of components from different hydrocarbon classes can be disengaged by exploiting their shifts in relative retention behavior with changes in linear rates of programmed temperature. Many co-eluting species in complex chromatograms of shale- and petroleum-derived jet fuels can be resolved without varying stationary phase, column length, or initial column temperature. Retention indices were simultaneously determined on two bonded-phase, fused silica capillary columns of slightly different polarities at three different linear programmed temperature rates. For certain hydrocarbon types, no change in index values was observed with an alteration in programming rate. However, the indices of other hydrocarbon classes shifted uniformly with programmed temperature rates on each of the two stationary phases. When applied, this phenomenon could help resolve coeluting members of different or even the same hydrocarbon type and elucidate their probable structure. The overall precision of the retention indices, i.e., the mean standard deviation at the 95% confidence levels, was less than ±0.13 for either column at any of the three programming rates. Since the above technique is automated, it could also be a useful screening tool to search for specific hydrocarbons in a myriad of unknown components of a complex hydrocarbon mixture.     

On-column injection onto capillary columns. Part 2: Study of sampling conditions; practical recommendations

During one year continuous use of on-column injection, the typical advantages described in our first report have fully been confirmed. In addition the analysis of large sample volumes has proved promising. Only minor modifications have been applied to the on-column injector device. Broad evidence has been gathered showing that full separation efficiency of the capillary columns after on-column injection is attained only when cold trapping or the solvent effect, as band shortening mechanisms, are working- While, under the conditions of on-column injection, cold trapping is less efficient than with other injection techniques, the opposite holds true for the solvent effect. Compared with splitless injection, the danger of excessive solvent condensation on the column is increased. A working rule is presented for establishing the optimal chromatographic conditions for handling large sample volumes while ensuring full separation efficiency yet avoiding harm to the column.     

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.     

On-line hydrogenation of fatty acid methyl esters in capillary gas chromatography

An injection splitter in front of a glass capillary column was used for the hydrogenation of fatty acid methyl esters (FAME) mixtures. Hydrogenation followed by gas chromatographic analysis on capillary columns permitted detection and identification in complicated natural mixtures of branched fatty acids, showing minor structural differences, in quantities down to 10^?8g. The technique described, apart from its suitability for FAME analysis, shows promise for structure determination studies of other classes of compounds.     

Technique for etching glass capillaries simultaneously with the drawing of the tubes

Summary A method is described for the etching of the inner surface of glass capillaries by methyl 2-chloro-1,1,2-trifluoroethyl ether vapour carried out simultaneously with the drawing of the capillary tube. The influence of the vapour concentration on the level of etching of the capillary surface and on column efficiency was studied with glasses having different chemical composition.     

Flexible soft glass capillary columns vs. flexible fused silica capillary columns for gas chromatography-a critique

The gas chromatographic use of flexible thin walled soft glass capillary columns coated with non-polar stationary phases is compared to similar columns made of fused silica glass. With non-polar soft glass columns, the use of surface roughening viagaseous HCI followed by a Carbowax 20 M pretreatment gave adsorptive phenomena, and thermal instability. With very polar soft glass columns where a variety of cyanopropyl silicone phases were coated directly onto the NaCI crystal matrix, adsorptive effects were again prominent and frequent break-down in film stability with time, was also observed. These undesirable effects were due to the presence of metal oxides in the soft glass. Attempts to remove these materials from the thin walled soft glass surface by means of acid leaching produced significant brittleness. This deleterious result was further increased by attempts at high temperature silylation or polysiloxane deactivation. In sharp contrast, the fused silica surface was essentially free of metal oxides and the surface silanol groups are easily neutralized by silylation or polysiloxane deactivation techniques. No brittleness was observed following these procedures. An increasing series of high molecular weight, viscous, polymeric vinyl containing non-polar and highly polar stationary phases have been produced which readily wet the surface of the fused silica and are easily crosslinked in the presence of free radical generators. These columns are essentially free of all the problems noted with flexible thin walled soft glass. When all of the parameters involved in the fabrication of a glass capillary column are assessed, it appears at this time, that the flexible fused silica glass column with cross linked phases approaches the “ideal” capillary column.     

Studies on Optimal Gas Supply For a Maskless Etching System with Micro- Discharge Plasma Operated at Atmospheric Pressure

An optimal gas supply method for the micro discharge plasma generated along a quartz glass electrode, which was useful for the maskless fabrication of electrode grooves for surface electrodes on solar cells, was examined. We here constructed an electrode system with gas inlet and outlet holes. The gas supply directly to the plasma region contributed to reduce byproducts on the surface being etched, and then it was confirmed that the uniform etching was achieved in the case where the micro-discharge plasma locally produced at the etching area.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.     

A novel method of preparing of glass capillary columns: Low-temperature plasma etching and polymerization

A novel method is described for the preparation of stable glass capillary columns (glass open tubular columns), including the etching and formation of a polymer film on the inner glass capillary surfaces. The approach used here is based on low-temperature plasma etching and polymerization. Under the influence of a field of radio frequency discharge, low pressure gases of fluoric compounds, introduced into the glass capillary tube, generate excited fluorine radicals which etch the inner surface. The plasma of organosilicone monomer in the glass capillary yields a uniform polymerized film on the inner surface. The resultant material functions as a good stationary phase for glass capillary gas chromatography (GC²). The inner surfaces treated with such a plasma, can be studied by means of a scanning electron microscope (SEM). The flexibility of this method permits the use of various stationary phases and surface modification.