Sample-injection effects

The effective use of column volume overload for preparative separations was experimentally demonstrated by Scott and Kucera [1]. These authors used a column 25 cm long, 4.6 mm I.D. packed with Partisil silica gel 10 mm particle diameter and employed n-heptane as the mobile phase. The total mass of sample injected was kept constant at 176 mg, 8 mg and 0.3 mg of benzene, naphthalene and anthracene, respectively, but the sample volumes used which contained the same mixture of solutes were 1 pi, 1 ml, 2 ml and 3 ml. The chromatograms of each separation are... 

The simplest mode of IGC is the infinite dilution mode , effected when the adsorbing species is present at very low concentration in a non-adsorbing carrier gas. Under such conditions, the adsorption may be assumed to be sub-monolayer, and if one assumes in addition that the surface is energetically homogeneous with respect to the adsorption (often an acceptable assumption for dispersion-force-only adsorbates), the isotherm will be linear (Henry s Law), i.e. the amount adsorbed will be linearly dependent on the partial saturation of the gas. The proportionality factor is the adsorption equilibrium constant, which is the ratio of the volume of gas adsorbed per unit area of solid to its relative saturation in the carrier. The quantity measured experimentally is the relative retention volume, Vn, for a gas sample injected into the column. It is the volume of carrier gas required to completely elute the sample, relative to the amount required to elute a non-adsorbing probe, i.e. 

Figure 13.7 Selectivity effected by employing different step gradients in the coupled-column RPLC analysis of a surface water containing 0.40 p-g 1 bentazone, by using direct sample injection (2.00 ml). Clean-up volumes, (a), (c) and (d) 4.65 ml of M-1, and (b) 3.75 ml of M-1 transfer volumes, (a), (c) and (d), 0.50 ml of M-1, and (b), 0.40 ml of M-1. The displayed cliromatograms start after clean-up on the first column. Reprinted from Journal of Chromatography, A 644, E. A. Hogendoom et al, Coupled-column reversed-phase liquid chromatography-UV analyser for the determination of polar pesticides in water , pp. 307-314, copyright 1993, with permission from Elsevier Science.

Programmed temperature vaporization (PTV) Most versatile inlet Allows large volume injection Little-no sample degradation Effective trace (to sub-ppb) analysis Expensive Requires optimization of many parameters Not well-known... 

Table 9.11 shows the effect of this concentration on the responses of the other pesticides. In every instance the peak height was increased while the peak area remained constant. All of the columns used for this study were aged by repeated sample injections, but had not deteriorated to the point where they would normally be replaced. No values are included in Table 9.11 where the chromatographic system was not suitable for the pesticide concerned. 

TSDC experiments are customarily analyzed assuming the sample behaves ohmic, i.e., the contacts do not introduce an inhomogeneous distfibution of the electric field or carrier density and a uniform bulk density of carriers extend through the entire sample. Experiments were carried out in such a way as to minimize injection effects. Contact configuration was typical for TSDC experiments. Because the currents through the sample are, in almost aU cases, extremely small, we have used a sensitive DC ammeter (model Ul-15, detection limit <10 A) with a hnear output signal. The simplest way to obtain a record of TSDC is an X-Y recorder that displays I(T) and the temperature. The equipment for the extraction of trap-spectroscopic information may be connected with devices for electronic data processing. The experimental errors in determination are less than 2%. 

Carryover. Small amounts of analyte may get carried over from the previous injection and contaminate the next sample to be injected [10]. The carryover will affect the accurate quantitation of the subsequent sample. The problem is more serious when a dilute sample is injected after a concentrated sample. To avoid cross-contamination from the preceding sample injection, all the parts in the injector that come into contact with the sample (the injection loop, the injection needle, and the needle seat) have to be cleaned effectively after the injection. The carryover can be evaluated by injecting a blank after a sample that contains a high concentration of analyte. The response of the analyte found in the blank sample expressed as a percentage of the response of the concentrated sample can be used to determine the level of carryover. Caffeine can be used for the system carryover test for assessing the performance of an injector and serves as a common standard for comparing the performance of different injectors. 

Figure 24-16 shows effects of operating parameters in split and splitless injections. Experiment A is a standard split injection with brisk flow through the split vent in Figure 24-15. The column was kept at 75"C. The injection liner was purged rapidly by carrier gas, and peaks are quite sharp. Experiment B shows the same sample injected in the same way, except the split vent was closed. Then the injection liner was purged slowly, and sample was applied to the column over a long time. Peaks are broad, and they tail badly because fresh carrier gas continuously mixes with vapor in the injector, making it more and more dilute but never completely flushing the sample from the injector. Peak areas in B are much greater than those in A because the entire sample reaches the column in B, whereas only a small fraction of sample reaches the column in A.

Fig. 15. Memory effect. Gradient elution of 500 pg phosphorylase kinase on a RP 18 column (250 x 4.6 mm dP = 5 pm). Mobile phase A 0.1% TFA in water B 0.08% TFA in acetonitrile gradient program 0 % B (0-8 min), 46 % (9 min) 68 % (24 min), 75% (33 min) flow rate 1 ml/min. The lower chromatogram was obtained upon sample injection the five blank gradients were performed immediately after the initial separation. Molar mass of the subunits a — 132,000 daltons P — 113,000 y - 43,000 5 - 16,680. (From Ref. 66> with permission)...

The determination of polyphenolics may result in interference due to co-elution of phenolic acids and procyanidins. This problem can be eliminated by fractionation of polyphenolics into acidic and neutral polyphenolics prior to sample injection into the HPLC system. Because the fractionation techniques effectively improve the resolution of many polyphenolic peaks in the reversed-phase HPLC system, it is suggested that further characterization and identification of unknown peaks be conducted by additional methods such as mass spectrometry and nuclear magnetic resonance. 

Determination of pesticide residues in fatty samples by GC requires the elimination of interfering compounds, mainly lipids, from the extracts before sample injection into the chromatographic system. Even small amounts of lipids can cause damage to the column and contaminate the detector. The effectiveness of HPLC techniques for the separation of different molecules makes this technique adequate for the cleanup of this type of samples. 

R Szucs, J Vindevogel, P Sandra, LC Verhagen. Sample stacking effects and large injection volumes in micellar electrokinetic chromatography of ionic compounds direct determination of iso-a-acids in beer. Chromatographia 36 323-329, 1993. 

Detailed analysis of the results published by Casper and Schulz 2) and measurements with the new chromatograph mentioned above 3) have shown that irrevesible thermodynamics, including two different kinetic effects, has to be applied to explain the resolution of the PDC-column 4 5 9) and to obtain the MWD of narrowly distributed polystyrene samples 6 8). In this way, not only the MWD is obtained, but also kinetic constants and thermodynamic functions of the polymer transfer between sol and gel, as well as hydrodynamic and kinetic spreading parameters of the system investigated, can be calculated from PDC-measurements performed at different constant column temperatures, with the same sample injected. The usual static quantities (such as the exponent of the partition function, ratio of the gel/sol volumes, etc.) proposed by Casper and Schulz can then be obtained by extrapolating the results to the theta temperature of the system. In addition, spreading phenomena alone can directly be... 

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