High performance columns

Figure 7 Separation of amino acids by high performance ion exchange Beckman Model 6300/7300 Amino Acid Analyzer. Column 20 cm sodium high performance column. Mobile phase Na-E, Na-F, Na-D, and Na-R regenerant, with buffer changes occurring at ABj and AB2. Flow rate 20 ml/hr. Detection Ninhydrin post-column (absorbance at 440 nm and 570 nm). Temperature 49°C rising to 79°C at AT . Note that the separation requires about 40 min. Compare to Figure 6. (Application Note A6300-AN007, reproduced with permission from Beckman Instruments Fullerton, CA.)...

Apparatus Pumping systems used in these studies for high-performance columns were a Varian 8500 syringe pump and a Varian 5000 isocratic pump. An Altex IlOA was employed for the con-trolled-pore glass (CPG) columns. Waters Associates model 401 refractometers were used on all instruments. Stagnant mobile phase was kept in the reference side of the refractometer. Samples were injected with a Rheodyne 70-10 injection valve using a 20yl loop (lOOyl for CPG columns). 

With the introduction of high performance columns, in which... 

The advantages of HPLC over classical chromatographic methods stem from the employment of a precision instrument that utilizes high-performance columns with concomitantly high analytical speed and resolution and affords total control over the chromatographic process and sensitivity of analysis. In a way, the recent emergence of capillary electrophoresis (CE) follows the same patterns electrophoresis, a well-established and widely used method of biopolymer analysis, is carried out... 

Grob, K., and Grob, G. (1972). Techniques of capillary gas chromatography. Possibilities of the full utilisation of high performance columns. Part I Direct sample injection. Chromatographic 5, 3-12. 

Figure 12.3. Reduced plate height plot showing the band in which h versus v curves lie for high performance columns.

For difficult separations, high performance columns are required, which means small particle columns and instrumentation. The use of an instru-... 

Columns. High performance columns characterized by minimum band broadening comprise the most crucial component of a high performance liquid chromatographic system. 

Models for the interactions of solutes in adsorption chromatography have been discussed extensively in the literature [7-9]. Only the interactions with silica and alumina will be considered here. However, various modifications to the models for the previous two adsorbents have been applied to modern high-performance columns (e.g., amino-silica and cyano-silica). The interactions in adsorption chromatography can be very complex. The model that has emerged which describes many of the interactions is the displacement model developed by Snyder [1,3,4,7,8], Generally, retention is assumed to occur by a displacement process. For ex-... 

The column is arguably the most important component in HPLC separations. The availability of a stable, high-performance column is essential for developing a rugged, reproducible analytical method. Performance of columns from different vendors can vary widely. Separation selectivity, resolution, and efficiency depend on the type and quality of the column. Proper column maintenance is the key to ensure optimum column performance as well as an extended column lifetime. It ensures stability of column plate number, band symmetry, retention, and resolution. The major issues related to column performance and maintenance are discussed here. 

Combining different separation methods, governed by different separation mechanisms, to multidimensional methods is suitable for multiplying the potential of the individual techniques. Reversed-phase chromatography high-performance columns (RP-HPLC) can be coupled with normal phase TLC [9,10]. 

A preparative high-performance column packed with 5, 7 or 10 pm material and available from various manufactures is used after the analytical separation optimization stage (columns with coarser particles of around 40 pm are more suitable for easy separations requiring less than 100 theoretical plates). A 10 mm i.d. column is designed for 10-100 mg samples, rising to 21.5 mm (o.d. 1 in) for samples between 100 mg and 1 g. The transfer from analytical to preparative conditions works best if both columns are packed with the same stationary phase. 

Particles below 20 pm in diameter cannot be packed in dry form as they tend to become lumpy, thus precluding the preparation of high-performance columns. The material must be suspended in a liquid to give a slurry. A high-pressure pump then conveys the slurry into the column at great speed. There are almost as many methods for wet packing as there are column manufacturers. Proponents are convinced that their own ways and means are the best, making it difficult for the uninitiated to choose between them. Slurry production itself is based on a few essential principles ... 

Extraction and separation of fluorescent protein. The soft coral 5. gracillima Kuekenthal was first fractionated by a gel filtration, then the fluorescent protein was separated by anion exchange column chromatography on an anion Q-Sepharose High Performance column (Amersham Biosciences) after ultra-filtration with a USY-1 membrane (Advantec). All the fractions were analyzed by spectrofluorophotometer (RF-5300 PC Shimadzu). 

Quantitation based on visual assessment of spot size compared to standards can be accurate within 10-30%, which may be adequate for many applications of TLC and PC. Quantitative results with accuracy and precision rivaling those in gas chromatography and high performance column liquid chromatography can be obtained by zone elution and microanalysis or by in situ densitometry. 

Application of radiometric detection in high-performance column liquid chromatography 158... 

There is a large variety of analytical problems that require the separation of radioactive species by column liquid chromatography. This need arises, for instance, in activation analyses, the separation of fission products or the separation of radioactively labelled compounds. Examples of special importance come from the biosciences, where labelled molecules are used in research on metabolism. Whenever a sample contains radioactive species, it is possible to follow their elution from a separation column specifically by radiometric detection. This review outlines briefly the principles of radiometric detection and radiometric detectors, concentrating on problems that arise from the combination of separations by high-performance column liquid chromatography with radiometric detection. 

---