Method development in the laboratory

After we have discussed the development of chromatographic methods, this final section of chapter 1 will discuss the role of method development in the modem laboratory. We have seen that in developing methods we should aim at simple, rapid analyses. Programmed analysis in a routine situation should be avoided whenever possible and a high degree of automation should be feasible. 

To achieve this goal, method developers should ideally find themselves in the opposite situation, being equipped with flexible, advanced instrumentation, including a variety of possible injectors and detectors, facilities for temperature or solvent programming, etc.. Multichannel detectors are very useful, as they may be of assistance in recognizing the different sample components when they move about in the chromatogram during the selectivity optimization process (section 5.6). 

It should be the aim of the method developer to try and develop methods which allow the use of level 3 instruments instead of level 2 ones as much as possible. If this can be achieved, the money spent on sophisticated instrumentation for method development will not be wasted. 

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NIR-CI instrumentation is rugged and flexible, suitable for both the laboratory and manufacturing environment. Therefore analysis methods developed in the laboratory can often be tailored for implementation near-line or at-line. NIR-CI is also a massively parallel approach to NIR spectroscopy, making the technique well suited for high throughput applications. 

This review emphasizes TLC methods developed in the laboratories of the Food and Drug Administration (FDA) for mycotoxins and plant toxins. Summaries of literature data are presented and are intended to serve as references both for known compounds and for possible extension to additional compounds of similar structures. 

Based on the analytical method developed in the laboratory, SMB separations can be implemented easily and straightforwardly. That stationary phase and mobile phase remain the same is one of the main reasons for the success of the SMB methodology and allows a speeding up of the development process for obtaining a fine chemical or a pharmaceutically active compound [31]. 

For the preparation of nitrocymene a method developed in the Colour Laboratory and described in the Jour, of Ind. and Eng. Chem. in 1918, p. 453, was used. The nitro group enters in the ortho position with respect to the methyl group. The reduction of this compound to aminocymene or cymidine was accomplished by means of iron powder and hydrochloric acid in exactly the same way as nitrobenzene is reduced to aniline. 

Fine and specialty chemicals can be obtained from renewable resonrces via multi-step catalytic conversion from platform molecules obtained by fermentation. An alternative method decreasing the processing cost is to carry out one-pot catalytic conversion to final product without intermediate product recovery. This latter option is illustrated by an iimovative oxidation method developed in our laboratory to oxidize native polysaccharides to obtain valuable hydrophilic end-products useful for various technical applications. 

The experiments initially conducted were designed to test the performance of the apparatus by determination of the effect of pressure on the viseosity/quality spectrum of a typical foaming agent when it flows through a straight capillary tube. The method used is based on a technique recently developed in the laboratories(7) for this purpose, at atmospheric pressure,... 

An alternative method (developed in the Eli Lilly research laboratories), entails inserting a nucleotide sequence coding for human proinsulin into recombinant E. coli. This is followed by purification of the expressed proinsulin and subsequent proteolytic excision of the C peptide in vitro. This approach has become more popular, largely due to the requirement for a single fermentation and subsequent purification scheme. Such preparations have been termed human insulin prb ... 

The potential applications of NIR OFCD determination of metal ions are numerous. The detection of metal contaminants can be accomplished in real-time by using a portable fiber optical metal sensor (OFMD). Metal probe applications developed in the laboratory can be directly transferred to portable environmental applications with minimal effort. The response time of the NIR probe is comparable to its visible counterparts and is much faster than the traditional methods of metal analysis such as atomic absorption spectroscopy, polarography, and ion chromatography. With the use of OFMD results can be monitored on-site resulting in a significant reduction in labor cost and analysis time. 

The effort required to get the necessary information can be minimized with careful planning. This is because a particular set of experiments often yields information on several parameters. Some of the parameters may have been determined during the method development stage. The laboratory makes the appropriate decision as to the degree of validation reqtrired taking into accormt the customer s requirements, existing experience in the use of the method and the need for compatibihty with other similar methods already in use within the laboratory or being used by other laboratories. 

The environment in which a method is used changes significantly when the method is transferred to a quality control laboratory at the manufacturing site. The method may be replicated in several laboratories, multiple analysts may use it, and the method may be one of many methods used in the laboratory daily. The developing laboratory must therefore be aware of the needs of the receiving... 

Method development is vastly simplified by computer simulations using commercial software. With input from a small number of real experiments, a program can predict the effects of solvent composition and temperature in isocratic or gradient separations. You can select optimum conditions in minutes with the computer instead of days in the lab. Of course, you must verify the prediction by a real experiment. Commercial software saves huge expenses in method development in industrial laboratories. 

Multivariate calibration methods are in general not analyte specific. Calibration models are built based on correlations in the data, which may be owing to the analyte or to systematic or spurious effects. One way to effectively boost the model specificity is through incorporation of additional analyte-specific information such as its pure spectrum. Hybrid methods merge additional spectral information with calibration data in an implicit calibration scheme. In the following section, we present two of these methods developed in our laboratory. 

In the following sections, we begin with a description of photolithography, then focus on a number of methods developed in our laboratory and conclude with some other non-traditional techniques. More extensive descriptions of traditional approaches are reviewed elsewhere [78]. 

If the activities of the laboratory in this field are said to be at the borders of quantum chemistry and statistical thermodynamics, these two disciplines are declared to be techniques." The problems raised by molecular liquids and solvent effects can be solved, or at least simplified by these techniques. This is firmly stated everywhere the method of calculation of molecular orbitals for the o-bonds was developed in the laboratory (Rinaldi, 1969), for instance, by giving some indications about the configuration of a molecule. The value and direction of a dipolar moment constitutes a properly quantum chemistry method to be applied to the advancing of the essential problems in the laboratory. In the same way, statistical mechanics or statistical thermodynamics constitute methods that were elaborated to render an account of the systems studied by chemists and physicists. In Elements de Mecanique Statistique, these methods are well said to constitute the second step, the first step being taken by quantum chemistry that studies the stuctures and properties of the constitutive particles. [53]... 

Thus, the methods described above, both of which functionally require the same tasting facilities and time as the official ASTA method, are capable of yielding reliable values which are statistically acceptable and useful for correlation work. The method developed in our laboratory with the simpler, prescribed, dilution series has been successfully used for testing pungency of pepper and ginger also, and has been adopted as an official method for pungency determination by the Indian Standards Institution (14). 

The method developed in the BCR project (Franz and Rijk 1997) to determine butadiene in all of the official food simulants and probably also in real foodstuffs was pre-validated by a collaborative trial with three laboratories. It was found appropriate in principle for the quantitative determination of butadiene at a range of 0.01 to 0.1 mg/kg in food simulants. Indeed the limit of detection was found to be in the range 4 to 9 (J-g/kg, thus being even in the worst case significantly lower than originally presumed when establishing the Plastics Directive limit of 0.02 mg/kg. 

The analytical method developed in the BCR project (Franz and Rijk, 1997) to determine residual carbonyl chloride monomer in polymers was pre-validated by two laboratories and found appropriate for the quantitative determination of carbonyl chloride with a LOD = 0.3 mg/kg below and in the range of the restriction criterion of 1 mg/kg polymer, with observed repeatability values of r = 0.23 and 0.32 mg carbonyl chloride/kg polymer, respectively. The method is applicable to polycarbonate as well as to other polymers and copolymers where these are soluble in methylene chloride. 

Acetylcholine. Acetylcholine was measured by a new gas chromatographic method developed in this laboratory (10). Use of this method in the measurement of tissue levels of acetylcholine has been described in detail (9). Briefly, the tissue is frozen in a dry ice-ether bath immediately after excision, weighed, crushed, and extracted by a modification of the method of Crossland (4) with an acetic acid-alcohol mixture. After centrifugation and washing of the residue, the supemates are combined and evaporated to dryness under reduced pressure. The residue from the evaporation is taken up in water acidified with acetic acid and the product is centrifuged. Again, the supernate is evaporated to dryness. The residue is dissolved in a small volume of water, and solid potassium borohydride and calcium chloride are added. The subsequent reaction converts the acetate moiety of acetylcholine to ethanol, which is then assayed by means of a gas chromatograph. 

All samples were assayed according to the method developed in our laboratories (6). After saponification of the fat, the pesticides were extracted into hexane. Since DDT is quantitatively dehydrohalogenated to DDE during alkaline hydrolysis, all results reported as DDE are the sum of DDT plus DDE. The hexane extracts were resolved and assayed in gas chromatographs equipped with electron capture detectors. In addition to columns packed with Dow 11 plus Epon coated on Fluoropak 80, at least one replicate from each sample was assayed on a column packed with 3% QF-1 on anakrom ABS. This latter column showed excellent resolution for dieldrin and DDE, as shown in Figure 1. 

We will now discuss some of the reasons why mold allergens are more difficult to standardize than other aeroallergens such as pollen allergens and why some of the commercially available extracts are very poor when analyzed for the presence of major allergens. These commercial extracts were also inferior to pure recombinant allergens when compared in clinical tests. Methods developed in our laboratory for optimal production and extraction of mold allergens will be described. 

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