Choosing columns

This chapter illustrates the improvements in SEC column technology and modern applications of SEC separations. The better understanding of SEC column design and separation parameters described in the theoretical sections of this chapter will help the reader fine-tune his or her own work. The same is true for column performance tests, which should be applied regularly, especially after a column purchase. In order to obtain reproducible results, it is recommended to choose column manufacturers who can assure constant quality and performance and to invest in knowledgeable, well-trained support personnel and experienced application chemists. 

Select the column k of the n by n occurrence matrix that contains the most nonzero entries. For example, in the occurrence matrix of Fig. 12a, choose column 1. Columns 2 or 4 could have been chosen just as well. 

The major concerns that are general to the use of all capillary electrophoresis systems for the separation of proteins and their building-block components are (a) choosing columns (b) buffer solution compatibility with the system and (c) the selection of the hardware. 

To change the width of a cell or column, choose Column from the Format menu, then choose Width from the submenu. You can enter the new width of... 

Chiral HPLC is the method of choice for analysing enantiomers and determining % ee. It can be used preparatively. In either application it is best to consult an expert when choosing columns and solvents. 

In a previous paper [ref. 34], a comparison of the separation behaviors between silica- and acrylate-based SEC columns was reported. Elution profiles of polyethylene glycols with molecular weights of 200 and 400 on TSKgel G2000PW and G2000SW are depicted in Fig.l and 2, respectively. This comparison shows a difference in the separation mechanism between these columns, which have the same pore size. It should be noted, therefore, that base materials should be considered in choosing columns as well as pore size of columns. 

In the following sections, simple methods for choosing column pressure, for calculating the minimum reflux and the minimum number of stages, and for sizing columns will be introduced. 

In the discussion diat follows, the basic criteriem for choosing column-base holdup will be good, or at least adojuate, control of die column. As mentioned earlier, good composition control is favored by smaU holdups. But if the column base serves as a feed vessel for anodier step, its required volume may be influenced by downstream requirements. This is discussed in Chapter 5. If possible, we will avoid the use of separate, intermediate buffer tanlu to feed another process step. 

The selectivity of a chromatographic method is determined by the column stationary phase chemistry and the mobile phase composition consequently, it is advisable to keep the mobile phase and column chemistry the same when the method is transferred between HPLC and UHPLC. This means that in labs where exchange of methods between instrument types is anticipated, chromatographers are advised to choose column brands where both UHPLC and HPLC columns with the same stationary phase chemistry are available. Table 3.7 lists the column brands currently provided by the major manufactures. It can be seen that not all brands carry both columns packed with the conventional 3-5 xm and the sub-2 xm particle sizes. Within each brand, not all stationary phases are available in both platforms. In such situations, column equivalence assessment needs to be performed to find the best alternative column by using tools such as the reversed-phase colunm selectivity charts available from column vendors. 

Distillation. There is a large inventory of boiling liquid, sometimes under pressure, in a distillation column, both in the base and held up in the column. If a sequence of columns is involved, then, as discussed in Chap. 5, the sequence can be chosen to minimize the inventory of hazardous material. If all materials are equally hazardous, then choosing the sequence that tends to minimize the flow rate of nonkey components also will tend to minimize the inventory. Use of the dividing-wall column shown in Fig. 5.17c will reduce considerably the inventory relative to two simple columns. Dividing-wall columns are inherently safer than conventional arrangements because they lower not only the inventory but also the number of items of equipment and hence lower the potential for leaks. 

Having wide and increasing quantity of RP HPLC sorbents in disposal the main question in RP HPLC is their interchangeability. Column chai acteristics that ai e usually described by their manufacturers are not full enough for the analytic to choose a suitable column for the specified resolutions or he ought to choose other similar column used before. In fact, nomenclature of reversed-phase stationai y phases is too unsophisticated and is a source of confusion in their application. 

Table 2.1 ranks materials by their cost per unit weight UK per tonne (i.e. 1000 kg) in the second column, US per tonne in the third. The most expensive materials - diamond, platinum, gold - are at the top. The cheapest - cast iron, wood, cement - are at the bottom. Such data are obviously important in choosing a material. How do we keep informed about materials prices change and what controls them ... 

For reports submitted for calendar years 1987, 1988, and 1989 only, you may take advantage of range reporting for releases to an environmental medium that are less than 1,000 pounds forthe year. If you choose this option, mark one of the three boxes, 0, 1-499, or 500-999, that corresponds to releases of the chemical to the appropriate environmental medium (i.o., any line item). You are not required, however, to use these range check boxes you have the option of providing a specific value in column A.2, as described below. However, gg not mark a range and also enter a specific estimate in A.2. 

Generally, optimizing the selectivity by choosing a gel medium of suitable pore size and pore size distribution is the single most important parameter. Examples of the effect of pore size on the separation of a protein mixture are given in Fig. 2.15. The gain in selectivity may then be traded for speed and/ or sample load. However, if the selectivity is limited, other parameters such as eluent velocity, column length, and sample load need to be optimized to yield the separation required. 

Select the appropriate chromatographic column or columns. Choose a column packing with a pore size that will resolve the molecular size range of the sample. 

Select suitable mobile phase condition. Choose a mobile phase that will solubilize the sample and will be compatible with the column packing material. 

Many researchers choose to buy expensive GPC/SEC columns from one of the major producers because that producer s columns had been used in the past or because of a successful marketing campaign by one particular producer. It should be noted that repacked columns can be obtained for a fraction of the cost of new columns. American Polymer Standards repacked columns are guaranteed to perform just as well as new columns from any company. When a column is repacked the only parts reused are the stainless-steel tube and end caps. This hardware is then repacked using new frits and new ST-DVB gel. Each column is individually tested in a quality control laboratory and shipped in the customer s choice of solvent. American Polymer Standards offers a column repacking service because it is a practical, inexpensive way for customers to acquire state of the art GPC/SEC columns. 

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