Volume-based injection

Injection mode Inlet liner Injection volume Base temperature Transfer rate Transfer temperature Qeaning step Closed until Open Split flow Start Ramp 1 Ramp 2... 

GC assay of the organic layer showed no EIN(TMS)2 remaining after 15 min of stirring (GC conditions Restek RTX-1 column (30 m x 0.53 mm, 1 m film thickness), 2.53 mL/min, initial temperature 50°C, final temperature 300°C, rate 20 deg/min, injection temperature 200°C, detector temperature 350°C, injection volume 1 pL, inject sample neat retention times fert-butyl alcohol 1.4 min, THF 1.7 min, heptane 2.1 min, HN(TMS)2 2.6 min, ethylbenzene (present in commercial LHS) 3.1 min, te/ t-butyl acetate 4.0 min). Volume percents were determined based on standard solution counts. 

Thus, the column diameters chosen for the two dimensions are determined by the amount of sample available and will dictate the flow rate ranges available to use. In split-flow systems, where only a portion of the first-dimension effluent is injected into the second dimension, the choice of column size is unlimited and the two methods can be developed independently. In comprehensive systems where the entire sample from the first dimension is injected into the second dimension, the flow rates are generally lower in the first dimension to accommodate the lower injection volumes into the second dimension. For example, for a 1-mm ID column in the first dimension with a flow rate of 50 (tL/min and a sampling rate of 1 min, 50 pL could be injected onto the second dimension. A 50-(lL injection onto a4.6-mm ID column flowing at 1 mL/min should be accommodated fairly well based upon its composition. In Chapter 6, the first dimension column diameters are estimated based upon the injection volume and sampling rate into the second dimension. 

FIGURE 16.2 Representative base peak electropherograms from CZE runs of RPLC fractions, (a) Fraction 15 (5 peptide identifications) and (b) fraction 20 (19 peptide identifications). Column, bare fused silica capillary, 60 cm x 180 pm ODx30pm i.d. separation voltage, 15 kV observed CZE current, 1.91 p.A running electrolyte, 200 mm acetic acid + 10% isopropanol temperature, 22°C injection time, 10 s at 2 psi ( 4 nL total injection volume) supplementary pressure, 2 psi flow rate, 25nL/min spray voltage, 1.5 kV (reprinted with permission from Electrophoresis). 

The concept of a theoretical plate is based on the number of equilibria that may have taken place during the separation process and this number is related to the number of times the effective volume of a column is greater than the peak volume. The variance (a2) for the peak is a measure of the broadening of the injection volume while the square of the retention time (fR2) is a measure of the effective column volume for that compound. Hence the number of theoretical plates (AO may be calculated from the following equation ... 

FIGURE 1.30 Micro-HPLC separation of all 4 stereoisomers of the dipeptide alanyl-alanine as FMOC derivatives (a) and DNP-derivatives (b), respectively, on a 0-9-(tert-butylcarbamoyl)quinine-based CSP. Experimental conditions Column dimension, 150 X 0.5 mm ID mobile phase (a) acetonitrile-methanol (80 20 v/v) containing 400 mM acetic acid and 4 mM triethylamine, and (b) methanol-0.5 M ammonium acetate buffer (80 20 v/v) (pHa 6.0) flow rate, 10 ixLmin temperature, 25 C injection volume, 250 nL detection, UV at 250 nm. (Reproduced fromC. Czerwenka et al., J. Pharm. Biomed. Anal., 30 1789 (2003). With permission.)... 

Figure 3.5 Measurement of the chiral purity of commercially available Jacobson s catalyst using a cyclodextrin-based CSP. (a) Lower trace / ,/ -enantiomer product upper trace / ,/ -enantiomer product artificially enriched with S -enantiomer and (b) lower trace S. S -enantiomer product upper trace S. S -enantiomer product artificially enriched with / ,/ -enantiomer. (Conditions CYCLOBOND 1 2000RSP 25 cm X 0.46 cm i.d. mobile phase acetonitrile triethylamine glacial acetic acid [1000 0.5 2.5, v/v] flow rate 1 ml/min temperature ambient detection UV at 240 nm sample preparation 1 mg/ml in acetonitrile injection volume 10 fxl). Reprinted from [19], copyright 1998, with permission of Wiley-Liss, Inc., a subsidiary of John Wiley and Sons, Inc.

One of the advantages of CE, which is also a disadvantage, is the very small volume needed for analysis. Typical injection volumes are 1-10 nL. This ability to inject small volumes has been shown to be useful in the analysis of single cells and microdialysis samples. Although the concentration-based detection limits of LCEC and CEEC are similar, detection limits based on actual mass analyzed are much lower for CEEC. Typical mass limits of detection for an electroactive compound are in the attomole range. 

For strong UV absorbers such as isoflavones, total injected amounts of <20 pg and injection volumes of 10 to 30pi are typical. Larger injection volumes can cause broadening of peaks. The flow rate was developed based on the column inner diameter and mass sensitivity. 

Jiminez de Bias et al. [32] have reported a method for the determination of total arsenic in soils based on hydride generation atomic absorption spectrometry and flow injection analysis. The method gave good recoveries and had a detection limit below 1 ig/l for an injection volume of 160 pi... 

High performance liquid chromatography techniques may be successfiilly applied to analyze phthalate esters. A 15 or 25 cm column filled with 5 or 10 pm silica-based packings is suitable. Short columns (3.3 cm x 4.6 mm), commonly called 3x3 columns, offer sufficient efficiency and reduce analysis time and solvent consumption Phthalate esters resolve rapidly on a 3 x 3 Supelcosil LC-8 column (3 pm packing) at 35°C and detected by a UV detector at 254 nm. Acetonitrile-water is used as mobile phase (flow rate 2 ml/min injection volume 1 mL). Other equivalent columns under optimized conditions may be used. 

Figure 8-40. Analysis of Product M (free base) as neutral species using two types of volatile buffers. Chromatographic conditions Column Luna C8(2) 150 x 4.6 mm. Mobile phase Aqueous (see A and B for exact conditions), acetonitrile. Wavelength, 247 nm column temperature, 40°C flow, 1 mL/min injection volume, 10 pL. Linear gradient from 5% acetonitrile to 95% acetonitrile over 20 min, with 3-min hold at 95% acetonitrile.

Figure 13-13. Comparison of choice of sample preparation on MRM signal intensity of an investigation compound. The injection volume was 40pL.The count per second (CPS) signifies the detector (an electron multiplier) response. Protein precipitation (PPT), hydrophilic-lipophilic balanced co-polymer-based SPE (Oasis HLB- copolymer of styrene, divinylbenzene and -vinylpirrolidone monomers the hydrophihc refers to the NVP monomer, and the lipophilic refers to the SDVB monomers), and strong anion exchange SPE (Max) (all in 96-well plate format) were used in control rat plasma (unpublished data).

Analysis of Phenolic Compounds. A Hewlett-Packard (Palo Alto, CA) Model 1090 HPLC System, was used to determine the levels of specific phenolic components. The HPLC system was equipped with a ternary solvent delivery system, a diode array UV-VIS detector, and HP ChemStation software for data collection and analysis. Full chromatographic traces were collected at 280, 520, 316, and 365 nm, and spectra were collected on peaks. The stationary phase was a Hewlett-Packard LiChrosphere C-18 coliram, 4mm X 250 mm, with 5 pM particle size packing. Operating conditions include an oven temperature of 40 C, injection volume of 25 pL, and flow rate of 0.5 mL/minute. The mefriod was based on a previously published method for phenolic components in wine (30) and used the modified solvent gradient shown in Table II. Solvent A was 50 mM dihydrogen ammonium phosphate, adjusted to pH 2.6 with orthophosphoric acid. Solvent... 

In the experiments described here the volume of liquid adsorbate which was injected was sufficient to saturate the intra-granular mesopores. This volume was calculated from a knowledge of the sample meiss in the cell and the mesopore volume, derived from the nitrogen adsorption isotherms, (eg for the kinetic measurements with the S2M silica, this mass was 42mg for each of the two samples and the injected volume was 40 pi.) This volume was injected via the hypodermic needle, to the base of the cell which was below the zone of the incident neutron beam. The transmissions for the different samples was in a range of 0.7 to 0.9, being typieal for such porous silicas in these standard quartz neutron scattering cells. 

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