Columns mass overload

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. 

Volume overload results from too large a volume of sample being placed on the column, and this effect will be discussed later. It will be seen that volume overload does not, in itself, produce asymmetric peaks unless accompanied by mass overload, but it does broaden the peak. Mass overload, however, frequently results in a nonlinear adsorption isotherm. However, the isotherm is quite different from the Langmuir isotherm and is caused by an entirely different phenomenon. 

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... 

Preparative chromatography involves the collection of individual solutes as they are eluted from the column for further use, but does not necessarily entail the separation of large samples. Special columns can be designed and fabricated for preparative use, but for small samples the analytical column can often be overloaded for preparative purposes. Columns can be either volume overloaded or mass overloaded. Volume overload causes the peak to broaden, but the retention time of the front of the peak... 

The easiest way for an analyst to obtain small quantities of a component of a mixture is to overload an analytical column. In order to exercise this technique, the solute of interest must be well separated from its closest neighbor. The column can then be overloaded with sample until the peak dispersion resulting from the overload, causes the two peaks to touch at their base. There are two types of column overload, volume overload and mass overload. In practice, it is often advantageous to employ a combination of both methods and a simple procedure for doing this will be given overleaf. 

Volume overload can be treated in a simple way by the plate theory (8,9). In contrast, the theory of mass overload is complicated (10-12) and requires a considerable amount of basic physical chemical data, such as the adsorption isotherms of the solutes, before it can be applied to a practical problem. Volume overload is useful where the solutes of interest are relatively insoluble in the mobile phase and thus, to apply a sample of sufficient size onto the column, a large sample volume is necessary. If the sample is very soluble in the mobile phase then mass overload might be appropriate. 

Volume overload employing a solution of the material in the mobile phase at a level of about 5% w/v is a recommended method of sampling for preparative columns if the system is not optimized. However, a combination of volume overload and mass overload has also been suggested as an alternative procedure by Knox (13). 

In practice the value of (w) will vary between about 2 and 5 ( i.e sample concentrations will lie between 2%w/v and 5%w/v) before mass overload becomes a significant factor In band dispersion. A numerical value for (g>) of 5 will be taken In subsequent calculations. The correct value of ( ), for the particular solute concerned, can be experimentally determined on an analytical column carrying the same phase system If so required. 

Band dispersion from sample mass overload is a direct result of the chromatographic process proceeding under conditions, where the adsorption isotherm of the solute on the stationary phase, is no longer linear. The development of an equation that describes the extent, of band spreadinn as a function of mass of sample placed on the column, is complex. This problem has been elegantly approached by 6uiochon and his co-workers (15-18) from the basis of the adsorption isotherm of the solute on the stationary phase. 

Figure 2.23 Mass overload. Top 2 mg each of acetophenone (first peak) and veratrole bottom 2ixg each. Column, 25cmx3.2mm i.d. stationary phase, LiChrosorb SI 60 5ixm mobile phase, hexane-diethyl ether 9 1, 1 ml min UV detector, 290 nm, with preparative or analytical cell, respectively.

The amount of sample that may be injected has already been discussed in Section 2.7 and the volume of solvent required for dissolution may vary. The sample volume should be kept as smaU as possible, thereby not increasing the inevitable band broadening. On the other hand, it may be preferable to dissolve the sample in a relatively large volume to prevent mass overload at the column inlet. Obviously there... 

Inductively coupled plasma mass spectrometry has several inherent advantages over conventional detection techniques, including its sensitivity and element specificity. However, IC with inductively coupled plasma mass spectrometry was not used for the analysis of iodide and iodate in seawater, except in a very recent report (Chen et al.y 2007). This could be because the high concentration of matrices in seawater led to loss of sensitivity from the build-up of salts on the sampler and skimmer cones of the mass spectrometer. In addition, chloride interfered with eluting very near to iodate because the column was overloaded when the chloride concentration was very high in seawater. Recently, a nonsuppressed IC with inductively coupled plasma mass spectrometry was developed for simultaneous determination of iodate and iodide in seawater (Chen et al., 2007). An anion-exchange column... 

In the previous section we assumed that the dispersion of the column was negligible. However, dispersion does exist, and the factors that govern it are the same as in linear chromatography, namely, without mass overload. Therefore they can be assessed by the same equations as those derived in Section 2.2. Using the van Deemter equation, we can derive the variance due to dispersion effects as follows ... 

In the following we win examine the combined effects of the injection volume, the mass overload, and the column dispersion. A commonly used gnq>hic... 

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 ... 

In a similar manner, 2, 4 and 6 ml of a solution each containing 9.0 mg of naphthalene was injected onto the column and the chromatograms obtained are shown on the right hand side of figure 12.3. It is seen that the experimental sample volume of 6 ml (6.1 ml calculated from equation 2) just permits the separation of naphthalene and anthracene. Destefano and Beachel [4], investigated the effect of volume and mass overload on resolution and concluded that it is better to overload a column with a large volume of a dilute solution of sample, than to... 

These complex effects can be best illustrated by experiment. The same column was used for mass overload experiments as that employed in the volume overload experiment. In this case, the sample volume was kept... 

Preparative Chiral Chromatography The Loading Capacity of a Column The Maximum Sample Volume Sample Volume Overload Sample Mass Overload Preparative Chromatography Apparatus Solvent Reservoirs Pumps... 

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