Column selectivity, HPLC development

The same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187], Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189],... 

The determination of endogenous compounds and drugs in biological matrices has always presented a formidable challenge as one has to consider various factors before attempting to develop a suitable HPLC assay. These include the physicochemical properties of the compound such as the pKa value, solubility, volatility, particular functional groups (e.g., possessing chromophores, fluorophores, or electroactive characteristics), potential metabolites, and the required sensitivity and specificity. All these aspects will determine the type of extraction processes, analytical column selection, and suitable detector systems to be used as part of the HPLC apparatus. 

SOURCE C. S. Young and R. J. Weigand, An Efficient Approach to Column Selection in HPLC Method Development," LCGC 2002, 20, 464. 

Figure 25-29 Separation of six compounds on (a) phenyl- and (b) Cle-silica columns with 3-p.m particle size using 35 65 (vol/vol) acelonitrile/0.2% aqueous trifluoroacetic acid. Column size 7 x 53 mm flow rate = 2.5 mL/min. [From C. S. Young and R. J. Weigand, "An Efficient Approach to Column Selection In HPLC Method Development." LCGC 2002,20.464. Courtesy Alltech Associates.]...

In common with other application areas of chromatographic separation, a considerable amount of effort has been expended recently on the development of different elution conditions and types of stationary phases for peptide separations in attempts to maximize column selectivities without adversely affecting column efficiences. Peptide retention will invariably be mediated by the participation of electrostatic, hydrogen bonding, and hydrophobic interactions in the distribution phenomenon. The nature of the predominant distribution mechanism will be dependent on the physical and chemical characteristics of the stationary phase as well as the nature of the molecular forces which hold the solute molecules within the mobile and stationary zones. The retention of the solute in all HPLC modes can be described by the equation... 

The column oven tends to be a cross between a GC and an HPLC oven. It is now common to mount a column selection valve in the oven with 6 or even 12 columns, particularly when performing chiral method development. 

Packed columns. The techniques for packed columns for HPLC have been extensively developed during recent decades. Thus very small particles with narrow size distributions are available. With such particles, reasonable plate counts can be obtained in short column lengths. The optimal mobile phase velocities are often in the range of 1-2 mm s in such columns, and thus they need to be short in order to avoid excessive analysis times. Compared with the plate numbers obtained in GC, the plate numbers of HPLC are quite low. The classical remedy in chromatography in such cases is to opt for selectivity, and that is precisely what makes HPLC such a versatile technique. Selectivity profits from intensive partitioning of the analytes between the mobile phase and the stationary phase. Thus the ratio / , the volume of mobile phase, ymobiie the volume of stationary phase, Vstationary should be... 

Brucine is used in a similar manner and the carboxyl unit of the amino acid is coordinated to the tertiary amine unit in brucine rather than the poorly basic amide nitrogen. Selective crystallization of these salts leads to their separation, and basic hydrolysis leads to an enantiopure amino acid. It is now possible to separate many racemic mixtures into their enantiomeric components by using high-pressure liquid chromatography (HPLC) fitted with a column that contains a chiral compound bound to an adsorbent (known as chiral HPLC columns). Such columns have been developed by William H. Pirkle (United States 1934-). The chiral HPLC column is prepared by coating a chiral chemical compound on an inert material when a solution of the racemic mixture passes through this column, one enantiomer is adsorbed to the column material better than the other. These are sometimes called Pirkle columns. 

Choosing the best column is the most important decision in HPLC. Reviews have discussed this in detail [45-48]. Marchylo [45] noted that column selection can be bewildering due to the many columns available but that many columns offer satisfactory performance, with some differences in resolution and selectivity. Verevacek and Huyghebaert [47] also reviewed commercial columns for lE-HPLC, SE-HPLC, and RP-HPLC of food proteins, along with column characteristics and manufacturers. Here I will briefly review the status and recent developments in columns for RP-, IE-, and SE-HPLC. 

The selectivity of reversed-phase liquid chromatography (RP-LC) columns is known to vary, even columns with the same ligand (e.g., Cjg). Column selectivity can also vary from batch to batch for columns claimed to be equivalent by the manufacturer. For different reasons, it is sometimes necessary to locate a replacement column for a given assay that will provide the same separation as the previous column. In other cases, as in HPLC method development, a column of very different selectivity may be needed - in order to separate peaks that overlap on the original column. For each of these situations, means for measuring and comparing column selectivity are required. Until recently, no such characterization of column selectivity was able to guarantee that two different columns can provide equivalent separation for any sample or separation conditions. 

Of particular importance in HPLC development has been the availability of specialized chromatographic column packings and sensitive on-line detection systems for continuous monitoring of the separations being carried out. These developments have led to systems which in favorable instances can on the one hand detect parts per billion (1 in 10 ) levels of organic compounds, and on the other hand be used for collecting gram quantities of pure chemicals by preparative HPLC. The lack of truly universal detectors has resulted in the development of several selective detectors as described in Table I. 

Sugar analysis by hplc has advanced greatly as a result of the development of columns specifically designed for carbohydrate separation. These columns fall into several categories. (/) Aminopropyl-bonded siHca used in reverse-phase mode with acetonitrile—water as the eluent. (2) Ion-moderated cation-exchange resins using water as the eluent. Efficiency of these columns is enhanced at elevated temperature, ca 80—90°C. Calcium is the usual counterion for carbohydrate analysis, but lead, silver, hydrogen, sodium, and potassium are used to confer specific selectivities for mono-, di-, and... 

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. 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

Another important issue that must be considered in the development of CSPs for preparative separations is the solubility of enantiomers in the mobile phase. For example, the mixtures of hexane and polar solvents such as tetrahydrofuran, ethyl acetate, and 2-propanol typically used for normal-phase HPLC may not dissolve enough compound to overload the column. Since the selectivity of chiral recognition is strongly mobile phase-dependent, the development and optimization of the selector must be carried out in such a solvent that is well suited for the analytes. In contrast to analytical separations, separations on process scale do not require selectivity for a broad variety of racemates, since the unit often separates only a unique mixture of enantiomers. Therefore, a very high key-and-lock type selectivity, well known in the recognition of biosystems, would be most advantageous for the separation of a specific pair of enantiomers in large-scale production. 

Detection of the PSP toxins has proven to be one of the largest hurdles in the development of analytical methods. The traditional means, and still in wide use today, is determination of mouse death times for a 1 mL injection of the test solution. There are a variety of drawbacks to utilization of this technique in routine analytical methods, that have prompted the search for replacements. In 1975 Bates and Rapoport (3) reported the development of a fluorescence technique that has proven to be highly selective for the PSP toxins, and very sensitive for many of them. This detection technique has formed the basis for analytical methods involving TLC (77), electrophoresis (72), column chromatography (7J), autoanalyzers (7 ), and HPLC (5,6,7). 

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