Operation of a Chromatographic Column

Sometimes it is necessary to pack a column under pressure (5 to 10 psi). This leads to a tightly packed bed that yields more reproducible results, especially with gradient elution (see below). 

The sample to be analyzed by chromatography should be applied to the top of the column in a concentrated form. If the sample is solid, it is dissolved in a minimum amount of solvent if already in solution, it may be concentrated by ultrafiltration as described in Chapter 2. After the sample is loaded onto the column with a graduated or disposable pipet, it is allowed to percolate into the adsorbent. A few milliliters of solvent are then carefully added to wash the sample into the column material. The column is then filled with eluting solvent. 

The chromatography column is developed by continuous flow of a solvent. Maintaining the appropriate flow rate is important for effective separation. 

The completion of a chromatographic experiment calls for a means to detect the presence of solutes in the collected fractions. The detection method used will depend on the nature of the solutes. Smaller molecules such as lipids, amino acids, and carbohydrates can be detected by spotting fractions 

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Isothermal operation of a chromatographic column has a number of drawbacks, as illustrated in the scenario depicted for the separation of lime oil in Figure 3.62. If the selected isothermal column temperature is too low, the early-eluting peaks will... 

Proper consideration of all of these variables is important if successful results are to be obtained. Shortcuts taken to save time in selection, preparation, or operation of the column may only result in inadequate results or delays. The reader is encouraged to study the few references cited to obtain a better understanding of the workings of a chromatographic column. 

Isocratic. By isocratic is meant the operation of the chromatographic column, by allowing a solvent mixture of unvarying composition to run through the column until separation is complete. The importance of mobile phase flow-rate has previously been discussed. 

The simplest equilibrium model of a chromatographic column assumes isothermal operation, plug fluid flow, infinitely fast mass transfer between fluid and sohd phases (instantaneous equilibrium at the interface), negligible pressure drop and trace system. The model equations are the mass balance in a bed volume element and the equilibrium law at fluid/solid interface ... 

In active applications only part of the computers time is devoted to data collection, and the rest is used for data processing and control. Thus, active applications are real-time operations. Most modern instrumenis contain one or more microprocessors that perform control functions. Examples include adjustment of (1) the slit width and wavelength settings of a monochromator, (2) the temperature of a chromatographic column, (3) the potential applied to an electrode. 

Column efficiency is best given in terms of the reduced plate height, h, and the reduced velocity, v, at which the column Ls operated. The simplest form of the equation governing the efficiency of a chromatographic column (as measured in these terms) is the dimensionless version proposed by Knox (4) summarized below. 

Each of the PLgel individual pore sizes is produced hy suspension polymerization, which yields a fairly diverse range of particle sizes. For optimum performance in a chromatographic column the particle size distribution of the beads should be narrow this is achieved by air classification after the cross-linked beads have been washed and dried thoroughly. Similarly, for consistent column performance, the particle size distribution is critical and is another quality control aspect where both the median particle size and the width of the distribution are specified. The efficiency of the packed column is extremely sensitive to the median particle size, as predicted by the van Deemter equation (4), whereas the width of the particle size distribution can affect column operating pressure and packed bed stability. 

Xt is very difficult to provide an optimum set of conditions for operation of a liquid chromatograph under overload conditions due to the coiq>lex interactions among a large number of parameters [591,592,595,608,620,625]. The following general observations seem to be applicable in most cases. The column efficiency should be as high as possible and separatimis should be carried out using concentration overload conditions. The production rate of a... 

Residue chemists would be most interested in comparable confidence bands or confidence bandwidths. These values become a performance characteristic for any detection system. The values indicate the precision of not only the prepared standards but also the precision of the overall operating detection system. It is ultimately envisioned that a given system of a separation column in a chromatograph with a certain type of detector should give bands of standardized values. If a chemist finds he has not met... 

The principles underlying the operation of the chromatograph and its use in a commercial separation process are discussed in Volume 2, Chapter 19. In this section emphasis is placed on its function as an on-line process analyser in which form it consists of three major subdivisions (apart from any electronic readout system), viz. the sampling assembly, the chromatograph column and the detector. All three are generally contained within the same temperature-controlled environment (Fig. 6.50). 

Let us consider some of the special problems encountered in the operation of a radioisotope detector and the compromises that must be considered. Like any chromatographic detector, a carbon-14 detector should have a small volume and a short hold-up time in order to minimize band spreading and loss of resolution. Unfortunately radioisotopes are measured with an inherent time factor - disintegrations per minute. Therefore, the smaller the cell and the shorter the hold-up, the lower will be the sensitivity, a circumstance which is totally at odds with the first requirement. In practice, we have found that a U-tube with a cross-section diameter of 2mm is generally satisfactory. This gives a cell with a void volume of 200-300 yl, which is high compared to the 2-10 yl volumes of many UV flow cells, and may introduce some band spreading when used with the best new HPLC columns. 

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