Column HPLC

The heart of an HPLC system is the column it is the choice of packing upon which the success or failure of any separation will ultimately depend. 

The columns are made of stainless steel (to withstand the high back pressures generated) and designed and packed to reduce side and edge effects during chromatography. They are available prepacked or empty in a variety of sizes for both analytical and preparative purposes. 

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Giovanni Boocaletti is gratefully acknowledged for the large number of experiments that paved the way to enantioselective Lewis-acid catalysis in water. Furthermore, we kindly thank the Syncom company for the use of the chiral HPLC column. 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

The nebulization and evaporation processes used for the particle-beam interface have closely similar parallels with atmospheric-pressure ionization (API), thermospray (TS), plasmaspray (PS), and electrospray (ES) combined inlet/ionization systems (see Chapters 8, 9, and 11). In all of these systems, a stream of liquid, usually but not necessarily from an HPLC column, is first nebulized... 

Practical inlet systems for attaching a high-pressure liquid chromatography (HPLC) column to a mass spectrometer utilize atmospheric-pressure ionization (see Chapters 8 and 11). 

Fig. 3. The chiral separation obtained for oxa2epam on a sulfated cyclodextrin hplc column (4.6 mm ID x 25 cm) using a 10% acetonitrile/buffer (25 mM...

If the hGH is exported to the culture medium the product can easily be collected by removal of the cells from the culture medium by centrifiigation. Purification of hGH from the culture medium is faciUtated by low amounts of contaminating proteins present. In fact, it has been shown that hGH can be purified on a laboratory scale by a single purification step on a reversed-phase hplc column (43). Mammalian cells growing in tissue culture have also been used as hosts to produce hGH, which is exported into the culture media (44). 

Chromatographic Method. Progress in the development of chromatographic techniques (55), especially, in high performance Hquid chromatography, or hplc, is remarkable (56). Today, chiral separations are mainly carried out by three hplc methods chiral hplc columns, achiral hplc columns together with chiral mobile phases, and derivatization with optical reagents and separation on achiral columns. All three methods are usehil but none provides universal appHcation. 

Chiral Hplc Columns. There are about 40 commercially available chiral columns which are suitable for analytical and preparative purposes (57). In spite of the large number of commercially available chiral stationary phases, it is difficult and time-consuming to obtain good chiral separation. In order to try a specific resolution meaninghilly, a battery of chiral hplc columns is necessary and this is quite expensive. 

Derivatization with Optically Active Reagents and Separation on Achiral Columns. This method has been reviewed (65) a great number of homochiral derivatizing agents (HD A) are described together with many appHcations. An important group is the chloroformate HD As. The reaction of chloroformate HD As with racemic, amino-containing compounds yields carbamates, which are easily separated on conventional hplc columns, eg (66),... 

Resolution of Optical Isomers), Gakkai Shuppan Senta, Tokyo, Japan, 1989, Chapt. 11, pp. 132—143 Chiral HPLC columns are available from Regis, Spelco, and AST in the United States Merck, Nagel, and Serva in Germany LKB in Sweden and Daisem, Sumitomo, and Toso in Japan. 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

The first observation of the enantioselective properties of an albumin was made in 1958 (28) when it was discovered that the affinity for L-tryptophan exceeded that of the D-enantiomer by a factor of approximately 100. This led to more studies in 1973 of the separation of DL-tryptophan [54-12-6] C22H22N2O2, on BSA immobilized to Sepharose (29). After extensive investigation of the chromatographic behavior of numerous racemic compounds under different mobile-phase conditions, a BSA-SILICA hplc column (Resolvosil-R-BSA, Macherey-Nagel GmvH, Duren, Germany) was... 

A high performance Hquid chromotography (hplc) method to determine citric acid and other organic acids has been developed (46). The method is an isocratic system using sulfuric acid to elute organic acids onto a specific hplc column. The method is sensitive for citric acid down to ppm levels and is capable of quantifying citric acid in clear aqueous systems. 

In this work the state-of-the-art and perspectives of column characterization and compai ison have been presented and discussed. All information about physico-chemical properties of RP HPLC Cl8 and C8 columns as porosity, average surface area, free silanol concentration, binding ligand density and others, were summarized. The points of views about column classifications, its advantages and disadvantages were discussed. It was shown that Cl8 and C8 HPLC column classification processes do not allow selecting the column with the same or preai range selectivity. 

U. D. Neue, HPLC Columns Theory, Technology, and Practice, Wiley-VCH, New York, 1997. ISBN 0471190373. 

The structure of a natural product is shown without any specification of stereochem-istiy. It is a pure substance which gives no indication of being a mixture of stereoisomers and has zero optical rotation. It is not a racemic mixture because it does not yield separate peaks on a chiral HPLC column. When the material is completely hydrolyzed, it gives a racemic sample of the product shown. Deduce the complete stereochemical structure of the natural product fiom this information. 

Zorbax GF-250 and GF-450 Size Exclusion Columns. (1996). Rockland Technologies, Inc. Zorbax HPLC Column Catalog. (1988). MAC-MOD Analytical, Inc., Chadds Ford, PA. 

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

Electrostatic effects have long been recognized in commercial HPLC columns for SEC of proteins (15,21,22). The usual remedy is to add 100 mM salt to the mobile phase. This works here too the Lys and Asp peaks collapse into the Gly peak with 100 mM salt (Eig. 8.8). High concentrations of sodium sulfate were added to determine the role played in SEC by hydrophobic interactions (sodium sulfate, a structure-forming salt, strengthens such interactions). Sodium sulfate increased the retention only of the most hydrophobic amino acids to any extent, and then only when the concentration approached 1 M. Clearly, hydrophobic interaction cannot account for the elution order of amino acids on PolyHEA. 

Additional factors influencing column performance are the type and quality of the packing process, which mainly determines the theoretical plate count (N) of the column. In contrast to HPLC columns the efficiency of the separation itself is determined predominantly by the quality of the sorbent alone (pore... 

Macfarlane, J. D., Tokiida, T., Mori, K., Yamada, T., and Morigiichi, S. Enhanced Organic GPC Analysis Employing Aqueous GPC HPLC Column, presented at the 1995 Pittsburgh Conference. 

An LC-LC coupling experiment system can be performed by employing a commercially available HPLC apparatus and involving various combinations of HPLC columns, eluents, additives, switching devices and detectors. 

Figure 9.3 Schematic illustration of the electrophoretic transfer of proteins in the chromatophoresis process. After being eluted from the HPLC column, the proteins were reduced with /3-mercaptoethanol in the protein reaction system (PRS), and then deposited onto the polyacrylamide gradient gel. (PRC, protein reaction cocktail). Reprinted from Journal of Chromatography, 443, W. G. Button et al., Separation of proteins by reversed-phase Mgh-performance liquid cliromatography , pp 363-379, copyright 1988, with permission from Elsevier Science.

Yamamoto et al. also coupled gel permeation HPLC and CE in an on-line fashion in 1990, where capillary isotachophoresis was again used in the second dimension. This technique was also not comprehensive due to the loss of resolution between the techniques. It was also not particularly fast, with a 23 min CE cycle, which was repeated 90 times throughout the HPLC run (14). Volume incompatibility between HPLC and CE was one problem not addressed in this study, in which a large HPLC column was coupled to an electrophoresis capillary. 

In 1993, Jorgenson s group improved upon then earlier reverse phase HPLC-CZE system. Instead of the six-port valve, they used an eight-port electrically actuated valve that utilized two 10-p.L loops. While the effluent from the HPLC column filled one loop, the contents of the other loop were injected onto the CZE capillary. The entii e effluent from the HPLC column was collected and sampled by CZE, making this too a comprehensive technique, this time with enhanced resolving power. Having the two-loop valve made it possible to overlap the CZE runs. The total CZE run time was 15 s, with peaks occurring between 7.5 and 14.8 s. In order to save separation space, an injection was made into the CZE capillary every 7.5s,... 

In 1995, Moore and Jorgenson used the optically gated CZE system to obtain extremely rapid separations with HPLC coupled to CZE. The rapid CZE analysis made possible more frequent sampling of the HPLC column, thus increasing the comprehensive resolving power. Complete two-dimensional analyses were performed in less than 10 min, with the CZE analyses requiring only 2.5s. A peak... 

Several applications involve the removal of large amounts of triglicerides, including the determination of wax esters in olive oil (39), sterols and other minor components in oils and fats (40, 41), PCBs in fish (42), lactones in food products (43, 44), pesticides (45), and mineral oil products in food (46,47). Grob et al. (47) studied the capacity of silica gel HPLC columns for retaining fats, and concluded that the capacity of such columns is proportional to their size, although the fractions of the volumes that are then transferred to the GC system grow proportionally with the column capacity. For these reasons, 2-3 mm i.d. LC columns are to be preferred for LC-GC applications. 

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

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