Injection on column

FIGURE 12.5 Human serum tryptic digest analysis. Fractionation in the first LC dimension was performed using a C18 column at pH 10. Fractions were analyzed using NanoEase 0.3 x 150 mm Atlantis d18 column. Approximately 66 lg (400 pmole of semm albumin peptides) was injected on column. Arrow points to a selected albumin peptide illustrating a local column mass overloading. Ten-5mm wide fractions were collected in 1st LC dimension. 

LC-NMR can be operated in two different modes on-flow and stopped-flow. In the onflow mode, LC-NMR spectra are acquired continuously during the separation. The data are processed as a two-dimensional (2D) NMR experiment. The main drawback is the inherent low sensitivity. The detection limit with a 60 p.1 cell in a 500 MHz instrument for a compound with a molecular weight around 400 amu is 20 pig. Thus, on-flow LC-NMR runs are mainly restricted to the direct measurement of the main constituents of a crude extract and this is often under overloaded HPLC conditions. Typically, 1 to 5 mg of crude plant extract will have to be injected on-column.In the stopped-flow mode, the flow of solvent after HPLC separation is stopped for a certain length of time when the required peak reaches the NMR flow cell. This makes it possible to acquire a large number of transients for a given LC peak and improves the detection limit. In this mode, various 2D correlation experiments (COSY, NOESY, HSQC, HMBC) are possible. 

Split injection Splitless injection On-column injection... 

Column overloading. (Retention times usually decrease as mass of solute injected on column exceeds column capacity.)... 

NG, DNT, TNT, RDX, EGND and tetryl Parallel GC- TE A/HPLC-TE A. No need for sample clean-up before analysis 900°C Low pg level. Sensitivity of 4-5 pg injected on-column [14]... 

Grob Jr, K. (1984) Effect of dirt injected on-column in capillary gas chromatography analysis of the sterol fraction of oils as an example. J. Chrom., 287, 1—14. 

Sensitivity of Detection. Under TSP, a full spectrum (100 - 500 amu) can be obtained on 1 - 50 ng especially where ammonium acetate (0.1 M) has been used as buffer. The sensitivity of detection limit observed can be extremely compound dependent. Blakely and Vestal (15) have demonstrated impressive detection limits in the 50-100 pg range for some compounds. However, under multiple ion detection (MID) a sensitivity of about 1-50 pg can be achieved with optimized TSP conditions (J6). In the case of DLI with a split of 1 100 in the effluent, a full mass spectrum can be obtained on about 1 ng injected on column (13). With micro column LC, a full spectrum can be obtained with 1-10 ng injected. For the MBI a full mass spectrum can be obtained on about 25 ng injected (37). This level can be... 

Retention time 7 (S), 9 (R) (measured after injection on column B)... 

The most common injection methods in the determination of FRs are splitless injection and on column injection. On column injection is suitable especially to thermally labile analytes, and it suits very well to quantitative analysis. However, the sample extract should be clean from nonvolatile matrix components in on column injection. Split injection is not recommended because of its low sensitivity and strong discrimination effects which can occur during the injection. Large volume injection techniques have also been applied in the analysis of FRs. ... 

Figure 5. Separation of amino acids derivatized with o-phthaldialdehyde (OTA) by reverse-phase chromatography. Extracts of nodules were derivatized according to the procedures described by Hill et al, (51, except the final volume was reduced from 5 mL to 0.5 mL or less and the concentrations of OTA and ethanethiol in the derivaUzing solution were increased to 60 mgjmL and 0.1 mglmLy respectively. A sample volume of 20 fiL was injected on column and the derivatized amino acids were eluted with an increasing gradient of acetonitrile from 12% to 100%.

Figure 16 Capacity factors of methotrexate (MTX), leucovorin (Leu), and folic acid (Fol) injected on columns packed with p-2-VPY MIP and NPs (mobile phase CH3CN/CH3COOH/H2O 92.5/5/2.5, 10 nmol injection).

Figure 15.47 illustrates the detection and identification, by ion trap mass spectrometry, of PCB isomer 2,3-dichlorobiphenyl at a concentration of 0.05 ppb (1.75 pg injected on-column). 

The retardation factor can also be used to explain how on-column injections work. When B is injected on-column, 90% of it sorbs into the stationary phase and only 10% goes into the vapor state. These numbers show that it is not necessary to evaporate all of the injected material in fact, most of the solute goes directly into the stationary phase. Similarly, in Chapter 9, R will aid in our understanding of programmed temperature GC. 

It will become clear in the following that it is necessary to ensure that the sample extract to be injected on column uses a solvent that is either the HPLC mobile phase itself (the t = 0 composition in the case of gradient elution) or a solvent of lower elution strength (Section 4.4.2a) otherwise, the chromatographic peak is broadened by the interference with the intended separation by partitioning between mobile and stationary phases, since now refers to the equilibrium distribution of A between the stationary phase and a stronger solvent than the mobile phase. 

It is also necessary to ensure that the sample extract to be injected on column uses a solvent that is either the LC mobile phase itself or a solvent of lower elution strength (Section 4.4.2a). 

Figure 5.27 (a) Pneumatically assisted electrospray source and API interface designed for LC/MS applications, (b) Total ion current (m/z 200-450) chromatogram observed in LC/MS analysis of a mixture of five monosulfonated azo dyes ( 20 ng of each injected on-column), and ESI mass spectrum of the last-eluting component. Separation was achieved using a 1 x 100 mm C,g column, operated in reverse phase isocratic mode with 40 jiL.min of a 30 70 mixture of acetonitrile and aqueous ammonium acetate (10 mol.L ). Reproduced from Bruins, Anal Chem. 59, 2642 (1987), copyright (1987), with permission of the American Chemical Society. 

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