Two-column system

The large pore structure of the TSK-GEL G6000PW allows it to separate large molecules such as pBR322 plasmid from contaminating RNAs and proteins in a much shorter time frame than other methods (23). A two column system of G6000PW (7.5 mm i.d. X 60 cm) was used to separate the cleared lysate and phenol extract of the plasmid as shown in Fig. 4.34 (page 130). The plasmid... 

Figure 8.25 Schematic arrangement ot a two-column system for separation of a sample comprised of co xinents spanning a wide rage of capacity factors (A) and heartcutting of a group of analytes of similar retention to an analytical column for separation (B).

In 1995, when HPLC/MS/MS was becoming the premier tool for PK assays, chromatographic sample cycle times were typically 10 to 12 min. At 10 min per sample, 16 hr were required to process 96 samples. By 2000, scientists used shorter HPLC columns and per-sample cycle times decreased to 5 to 6 min. At 5 min per sample, it takes about 8 hr to assay one 96-well plate of samples. As a result, parallel HPLC became popular Korfmacher et al.154 described a two-column system and an MS vendor produced a triple quadrupole system designed to work with four HPLC columns.16155-158 Advances in fast chromatography continued and by 2005, sample cycle times of 1 to 2 min became common.21 87 159-161 At 2 min per sample, 3 hr are required to assay one 96-well plate of samples. 

In considering multicomponent systems, a feed of say A, B and C has been fed to a single column and the top product, mainly pure A, is obtained with bottoms of mainly B and C. If each component is required pure, then a two-column system is required in which the bottom stream is fed to the second column to separate B and C. With more components in the feed, more columns will be required and their arrangement becomes more complex. This is a typical problem in the petrochemical industry and a paper by Eliceche and Sargent 42) offers particularly helpful advice. 

The HPLC separation previously made using 0.3-in. ID diameter columns was scaled up to 0.9-in. ID columns. The scaleup was straightforward and permitted 1.0 g to be injected on the 24 ft of 0.9-in. ID columns. The amount of sample injected was increased by the ratio of the cross-sectional areas of the two column systems. The flow rate was increased by the ratio of the cross-sectional areas of the 0.3- to 0.9-in. ID columns to 30 mL/min. The linear velocity and hence the separation time remained essentially the same. 

Figure 13-8. SimpMed schematic of two-valve/two-column system (buUt ia-house) used in the turbulent flow LC experiment [47]. The first column (e.g.. Cyclone P, 50 x 0.5 mm) is nsed for the extraction step. In our laboratory, this design has been nsed in conjunction with chiral, standard narrow bore (e.g., 50 x 2 mm), and monohthic analytical colnmns.

There has been a recent trend towards the use of high pressure systems and reagents other than ninhydrin to increase sensitivity and speed (e.g. see Moore 1972). Although not yet in general use, such systems hold considerable promise for the future. Expanded scales and one-column systems of analysis have also been used to boost sensitivity. However, in most of these more sensitive systems, the baseline is usually not as straight and free of drift as in the two-column systems, and there are often more problems encountered with interfering substances. At present we would recommend the use of the common commercial analyzers without special modifications (except for the possible inclusion of expanded scale modes) for the greatest accuracy in routine work. However, for situations where samples are obtained in limited amounts or where many samples need to be analyzed quickly, some of the newer systems may well be worthy of consideration. The application of fluorescamine to the quantitative fluorimetric determination of picomole quantities of amino acids, peptides and proteins is of particular interest in this context (Udenfriend et al. 1972). 

In most analyzer systems O-phosphoserine and O-phospho-threonine elute at the same volume as cysteic acid (the unretarded position) and can, therefore, interfere with analyses for cysteic acid (and vice versa). The color value for O-phosphoserine is 1.04 times the average value calculated for the amino acids (except proline and cystine) eluted from a 60 cm column in a two-column system (Beckman manuals). It should be noted that O-serine sulfate and O-threonine sulfate, which can be formed during preparation of hydrolysates in the presence of sulfate (Moore 1963 Murray and Milstein 1967), also elute in this position. Electrophoresis at pH 3.5 is an effective means of separating O-phosphoserine from other amino acids, since it migrates... 

The two column system is operated in closed mode (without continuous product withdrawal) with a single decanter. The two components are simultaneously produced as bottom residues. 

In the usual two-column scheme, the primary column depends on recovered heat for vapor formation a few refiners use a fired heater reboiler. The main crude column runs at low pressure compared to the single-column situation. The main column in two-column systems is often called the secondary column or the atmospheric column. ... 

Hewlett-Packard 6890 gas chromatograph equipped with two column systems, a 6 ft HayeSep Q connected to a TCD for CO2 determination, and an HP-INNOWax 30 m x 0.32 mm i.d. column connected to an FID for MEK analyses. 

Figure 6-9 Use of a two-column system to separate a heterogeneous minimum-boiling azeotrope. (M. Van Winkle, Distillation, 1967, by courtesy McGraw-Hill Book Company.)...

Figure 5.8. Schematic diagram of a two-column system for the separation of a sample containing components with a wide range of retention factors.

Studies of heat economy made by Faldix and Stage [176a] have resulted in optimum column connexions in two-column systems. Flow diagram variants for the multicomponent separation in columns with intermediate take-off were developed by Muller [176b]. 

Numerous methods are described in the literature for effecting the separation of amino acid components from complex mixtures. Most methods are derived from the two-column system of Spackman, et al (1958), wherein sodium citrate buffer solutions of both increasing pH and ionic strength are used. Benson (1972, 1975) described a single-column method in which amino acids are eluted by solutions with essentially constant pH but in which the dominant driving force is the increasing cation molarity. The chief benefit of this system is that the baselines are quieter and more stable. 

By using a two column system on a commercially available analyser, amino-acids and the a- and j3-anomers of 2-amino-2-deoxy-D-glucose and 2-amino-2-... 

A two-column system always consists of a light ends column, which is sometimes also termed an extraction column, and a refining column. The former is used to expel the light ends overhead, or sometimes only the dimethyl ether, which can be conveniently marketed. If this is the case, the other low boilers are withdrawn from a side outlet above the inlet. Methanol, water and high boilers are withdrawn from the bottoms and fed to the refining column in which as a rule the pure methanol is obtained at the column top, the high boilers below the feed nozzle, and the water in the column bottoms. 

Table 7.7 Molecular properties of some acidic compounds, logn represents the Human Serum Albumin (HSA)-drug binding affinity, from ref. 13. lognSf represents the HSA-drug binding affinity measured using a two-column system, from ref. 13. pKl are reference values, from ref. 13 TpKl values were measured by ion-exchange liquid chromatography. Mlm represents the molecular interaction eneigy calculated for the molecular-form compound, and Mlj the molecular interaction energy calculated for the ionic-form compound (kcal moP ).

The steady-state design of this two-column system with recycle was achieved by a sequential approach. First, the flow rates of the distillates from the two columns were set equal to the molar flow rates of DME and MeOH in the feed. The RR in the main column was set at 2. The flow rate of the vapor sidestream 51 was set at three times the MeOH product rate, which gives an RR in the rectifier of 2. Note that there is only one degree of freedom in the rectifier, so setting the distillate flow rate completely specifies the column. 

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