Capillary columns retention time reproducibility

The discussion of IL-based stationary phases up to this point has centered around ILs that are either coated as a thin film on a capillary wall or on a solid support. Although ILs exhibit a variety of properties that allow them to be unique stationary phases, their most significant drawback lies with their drop in viscosity with increasing temperature. This results in an increased propensity for flowing of the IL within the capillary, which often produces pooling of the stationary phase and nonuniform film thickness throughout the column. These factors often contribute to diminished analyte retention time reproducibility as well as detrimental effects on separation efficiency. 

It is particularly difficult when the gradient nano LC flow rate is below 1 /tL/min at which any small plumbing leakage could significantly affect retention time and gradient reproducibility. A 3 cm capillary column packed with 1.5 to 3 /tm particles can be run at a high flow rate to reach... 

The retention index of a compound obtained on a given stationary phase under given experimental conditions constitutes worthwhile information. However, if several indices of the same compound obtained on different stationary phases are available, better identification of this compound can be made. Because of the excellent reproducibility of retention times on modern chromatographs, this method is reliable for known control compounds. While obtaining retention indices does not constitute absolute identification of a compound, this method can be quite useful to identify unknowns if the proper retention indices tables are available on the most common stationary phases (Squalane, Apiezon, SE30, Carbowax 20M). However, the use of retention indices is now of lesser interest because of capillary columns that involve new stationary phases. This limits the information that can be obtained from the retention indices. Hyphenated techniques are currently more popular. They represent excellent methods for compound identification but depend on instruments that are more complex and more expensive. 

Although both reproducible preparation and operation of CEC columns are extremely important issues that will further stimulate the development and the acceptance of this technique, only a few groups have reported data on column-to-col-umn, run-to-run, and day-to-day reproducibility of monolithic capillary columns. Palm and Novotny showed reproducibility data for migration times tr, efficiencies, and retention factors for a number of analytes on acrylamide-based monoliths [35], The relative standard deviations (RSD) were smaller for run-to-run compared to day-to-day measurements. For example, the average run-to-run RSD for 6 analytes was... 

Tests of the reproducibility of retention times, retention factors, separation selec-tivities, and column efficiencies for our methacrylate monolithic capillary columns are summarized in Table 6.2. This table shows averaged data obtained for 9 different analytes injected 14 times repeatedly every other day over a period of 6 days, as well as for 7 different capillary columns prepared from the same polymerization mixture. As expected, both injection-to-injection and day-to-day reproducibilities measured for the same column are very good. Slightly larger RSD values were observed for col-umn-to-column reproducibility. While the selectivity effectively did not change, larger differences were found for the efficiencies of the columns. 

The most critical properties of a capillary column are resolution, support inertness, retention reproducibility, thermal stability, and column bleed. To provide fast, reliable, and accurate analysis, it is important that the stationary phase, internal diameter (ID) of the column, film thickness, and length of the column be chosen with a view to the particular application. CWC-related chemicals differ greatly from each other in their chemical and physical properties and thus the selection of the stationary phase is in most cases a compromise between resolution and analysis time. The most suitable stationary phases for the separation of chemicals related to the CWC are listed in Table 1, along with their structures and polarities (24). 

In the test, a sample aliquot diluted with a viscosity-reducing solvent is introduced into the gas chromatographic system, which uses a nonpolar open tubular capillary gas chromatographic column for eluting the hydrocarbon components of the sample in the order of increasing boiling point. The column oven temperature is raised at a reproducible linear rate to effect separation of the hydrocarbons. Quantitation is achieved with a flame ionization detector. The sample retention times are compared with those of known hydrocarbon mixtures, and the cumulative corrected area of the sample determined to the 371°C (700°F) retention time is used to calculate the percentage of oil volatilized at 371°C (700°F). 

Zhang and Shamsi reported the combination of a chiral column tapered at the outlet end and coupled to ESI-MS for simultaneous analysis of ( )-warfarin and ( )-coumachIor [13]. The chiral CEC was performed in capillaries packed with 5.0 pm (3R, 4S)-Whelk-01 chiral stationary phase. AcetonitriIe/5 mM ammonium acetate (pH 4.0) (70 30, vA ) and methanoI/5 mM anuno-nium acetate (pH 8.5) (70 30, v/v) were used as mobile phase and sheath liquid, respectively. It was found that the externally tapered colunm showed much more reproducible retention time compared to the untapered colunm. However, because of the fragile outlet end of the external... 

Figure 3.9 Headspace gas chromatography of light-exposed oils. Peak with retention time of 11.2 min is 2 E), 4( )-decadienal. Chromatographic conditions 50 m x 0.25 mm fused silica column coated with Carbowax , 50°C (7 min), then 6°Cmin to 160°C. Sample thermo-statted at 160°C in air for 40 min. Reproduced from Marsili, R. T., Measuring light-induced chemical changes in soybean oil by capillary headspace gas chromatography, Journal of Chromatographic Science, 22, 61-7, 1984.

In summary, a gas chromatograph functions as follows. An inert carrier gas (like helium) flows continuously from a large gas cylinder through the injection port, the column, and the detector. The flow rate of the carrier gas is carefully controlled to ensure reproducible retention times and to minimize detector drift and noise. The sample is injected (usually with a microsyringe) into the heated injection port where it is vaporized and carried into the column, typically a capillary column 15 to 30 m long, coated on the inside with a thin (0.2 fim) film of high boiling liquid (the stationary phase). The sample partitions between the mobile and stationary phases, and is separated into individual components based on relative solubility in the liquid phase and relative vapor pressures. 

Retention-time locking (RTL) is a unique feature available in some GC equipment which has revolutionized gas chromatography by reproducing retention times within hundredths of a minute from one instrument to another. This capability is possible thanks to highly reliable electronic pneumatic control (EPC), (pressure and flow control), good temperature control and high reproducibility of capillary columns. The result is ultimately retention time stability between any GC systems with EPC. 

Recent developments in capillary GC, i.e., electronic pneumatic control (EPC) of the carrier gas, improved oven temperature stability, and excellent reproducibility in column making have led to the concept of retention time locking (RTL) [77], [78]. With retention time locked data bases, absolute retention times instead of retention indices can be used to elucidate the structures of eluting solutes. Moreover, retention time locking can be u.sed in combination with different injectors and detectors. Exact scaling of capillary GC-FID, capillary GC-MS, and capillary GC-AED chromatograms is feasible. 

The oven of an SFC system should meet the same requirements as a normal GC oven. A constant temperature (variation 0.1 °C) must prevail in the entire oven at any time of a positive or negative temperature gradient. This is very important for reproducible capillary column SFC analysis. These columns are very sensitive to even slight variations in temperature, which can result in peak shape deformation, peak splitting, or irre-producible retention times. 

Capillary colunms provide reproducible retention times due to their high resolving power. This is helpful in computer assisted pattern recognition of GC profiles and subsequent identifications based on Kovats indices [39,41], The fused silica columns offer exceptional durability and chemical inertness. A typical fused silica column can detect components in less than 100 ng of concentration in a volatile mixture. 

The flow rate through a capillary column whose inner diameter is less than 0.53 mm is difficult to measure accurately and reproducibly by a conventional soap-bubble meter. Instead, the flow of carrier gas through a capillary column is usually expressed as a linear velocity rather than as a volumetric flow rate. Linear velocity may be calculated by injecting a volatile nonretained solute and noting its retention time, tM (seconds). For a capillary column of length L in centimeters. 

Despite the many advances in capillary gas chromatography instrumentation and the remarkable resolution achievable, it has proven difficult to standardize a test method for the analysis of a mixture as complex as petroleum naphtha. Because of the proliferation of numerous, similar columns and the endless choices of phase thickness, column internal diameter, length, etc., as well as instrument operating parameters, many laboratories use similar but not identical methods for the capillary GC analysis of petroleum naphthas. Even minute differences in column polarity or column oven temperature, for example, can change resolution or elution order of components and make their identiflcation an individual interpretive process rather than the desirable, objective application of standard retention data. To avoid this, stringent column specifications and temperature and flow conditions have been adopted in this test method to ensure consistent elution order and resolution and reproducible retention times. Strict adherence to the specified conditions is essential to the successful application of this test method. 

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