Axis of a chromatogram

Time-dominated processes inherently govern chromatography. The horizontal axis of a chromatogram is time (and not energy as in spectroscopy). To describe the quality of a chromatographic system the concepts of the height equivalent to a theoretical plate, HETP or H, and the number of theoretical plates N are used (Equation 4.1) ... 

Chromatography is based on the interaction of molecules with molecules (not with a field) and it is inherently governed by time-dominated processes. The x-axis of a chromatogram is time (and not energy as in spectroscopy). Chromatography and spectroscopy are totally orthogonal techniques, and consequently their online combination (spectroscopic investigation of the separated sample components) is a most powerful approach. 

Let us now consider how the elution volume axis of a chromatogram, such as shown in Fig. 4.19, can be translated into a molecular weight scale. This necessitates calibration of the particular GPC column using monodisperse polymer samples. The main problem encormtered in this task is... 

Most chromatographs are operated with a constant flow (F) of mobile phase unless the flow is intentionally being changed or programmed. Consequently, the x axis of the chromatogram can be labeled as time (t) or... 

Make sure you know the direction of the horizontal axis of the chromatogram (usually, either volume or time) - it may run from right to left or vice versa - and make a note of the detector sensitivity on the vertical axis. Ideally, the base line should be flat between peaks, but it may drift up or down owing to a number of factors including ... 

We now consider how the elution volume axis of a raw chromatogram, such as shown in Fig. 4.25, can be translated into a molecular weight scale. This necessitates a calibration of the particular GPC column for the particular polymer-solvent system used. Such a calibration requires the establishment of a relationship between the volume of solution eluted (or, equivalently, the elution time for a given flow rate of solution) and molecular weight of monodisperse fractions of the same polymer. The main problem encountered in this task is that monodisperse or very narrow distribution samples of most polymers are not generally available. However, such samples are available for a few specific polymers. A notable example is polystyrene for which anionically polymerized samples of narrow mole-... 

Mass Overload Mass overload is encountered, when the mass injected onto the column exceeds a certain limit. For most HPLC packings and for low-molecular-weight analytes, this limit is somewhere around 10 to 10 g/mL column volume. If you use a UV detector, this translates for a compound with a typical extinction coefficient to a peak hei t of about 0.1 AUFS. Thus a quick glance at the y axis of the chromatogram can tell you immediately whether the observed peak distortion may be due to mass overload. Also, simply injecting a sm r amount will allow you to pinpoint the problem. See Figure 17.5 for an example of mass overload ... 

Retention Behavior. On a chromatogram the distance on the time axis from the point of sample injection to the peak of an eluted component is called the uncorrected retention time The corresponding retention volume is the product of retention time and flow rate, expressed as volume of mobile phase per unit time ... 

Typical (a) gas and (b) liquid chromatograms. The charts show amounts (y-axis) of substance emerging from a column versus time (x-axis). The time taken (measured at the top of a peak) for a substance to elute is called a retention time. 

In GC X GC, a sample is separated into a large number of small fractions and each of these is subsequently quantitatively transferred to a secondary column to be further separated. The second separation is very much faster than the first separation, so that the fractions can be narrow and the separation obtained on the first column can be maintained. The collection of the fractions from the first column is achieved by focusing, rather than by valve switching, and the entire sample reaches the detector. The consequence is a chromatogram, with a two-dimensional plane, rather than a one-dimensional axis, as the time domain. One dimension of this plane represents the retention time on the first column, while the second dimension represents the retention time on the second column. Every separated peak can be presented as a... 

Figure 2.3. Capillary gas chromatogram of Si(OCH3)4 (3M) (a) after hydrolysis/ condensation with H20 (1.8 M) and HC1 (0.05 M) showing assignments of molecular formulas and structures and (b) with H20 (1.5 M) and catalysis conditions shown. For (a), linear and cyclical structures are indicated along the x-axis. All plots illustrate relative concentrations of species (y-axis) as a function of GC column retention time (x-axis). Higher mass species (e.g., hexamers (Si6) and pentamers (Si5)) demonstrate longer retention times. [Reprinted from Ref. 72, with permission.]...

Figure 4.1 Separation of H3 9-17 trimethylated subsequentlydigested with trypsin. The resulting at l<9 from H3 9-17 monoacetylated. Histone peptides were analyzed by LC-MS/MSemploying H3 isolated from Drosophila melanogaster was LTQ-Orbitrap (Thermo Scientific) as detector, acylated with deuterated acetic anhydride and (a) Chromatogram of the analysis. The y axis...

FIGURE 10.18 Illustration of the different types of possible peaks (1) the perturbation peak, (2) the mass peak, and (3) the plateau perturbation peaks, on three concentration plateaus. A single Langmnir model was assumed with a=2.0 and b=0.100. (a) A linear plateau, C=0.05mM. (b) A weakly nonlinear platean, C=0.5mM. (c) A clearly nonlinear plateau, C=5mM. The chromatogram shows the result of an analytical injection of a mixture of labeled and unlabeled molecules on a concentration plateau of unlabeled molecules. The solid line shows the perturbation peak (left scale), the dashed-dotted line shows the plateau perturbation peaks (left scale), and the dotted line shows the mass peak (right scale). Here (mM) is the concentration of unlabeled molecules, Q is the concentration of labeled molecules, and the x axis is time. The mean retention times,, and calculated... 

The problem to be solved in this paragraph is to determine the rate of spread of the chromatogram under the following conditions. The gas and liquid phases flow in the annular space between two coaxial cylinders of radii ro and r2, the interface being a cylinder with the same axis and radius rx (0 r0 < r < r2). Both phases may be in motion with linear velocity a function of radial distance from the axis, r, and the solute diffuses in both phases with a diffusion coefficient which may also be a function of r. At equilibrium the concentration of solute in the liquid, c2, is a constant multiple of that in the gas, ci(c2 = acj) and at any instant the rate of transfer across the interface is proportional to the distance from equilibrium there, i.e. the value of (c2 - aci). The dispersion of the solute is due to three processes (i) the combined effect of diffusion and convection in the gas phase, (ii) the finite rate of transfer at the interface, (iii) the combined effect of diffusion and convection in the liquid phase. In what follows the equations will often be in sets of five, labelled (a),..., (e) the differential equations expression the three processes (i), (ii) (iii) above are always (b), (c) and (d), respectively equations (a) and (e) represent the condition that there is no flow over the boundaries at r = r0 and r = r2. 

Figure G1.8.4 An FID chromatogram of concentrated extract of Niagara grape juice drawn to display the data on a linear retention index scale where the y axis is flame ionization response (upper trace). Below it is the charm chromatogram, where the / axis is dilution value. By simply comparing the index of a peak with the data listed in the Flavornet (see Internet Resource), it is possible to determine which odorants have similar retention indices. Notice how large the methyl anthranilate peak is, whereas there is no convincing peak for p-damascenone in the FID chromatogram, even though both compounds have the same potency in the charm chromatogram.

---