Automated analyses

There are many instruments designed for either enzyme assays or substrate assays using enzymes. Information on the analytical capabilities of these instruments will be supplied by the manufacturers. This will often include protocols for specified assays using kits of commercially available pre-prepared reagents. These may be in liquid or dry form and may, for substrate assays, include immobilized enzymes. The facility to be able to develop additional automated methods on a particular instrument will depend upon its design and some instruments are dedicated solely to specified analyses. 

Many automated instruments measure enzyme activity using fixed time colorimetric methods. Some, however, can be classed as reaction rate analysers, e.g. the centrifugal analysers, and these instruments determine the reaction rate from either the initial slope of the reaction curve or from repeat measurements at fixed intervals. In both methods the slope of the line is taken to represent the activity of the enzyme. 

The presence of a lag period in many coupled assays and difficulties in determining the linear portion of a curve present the main problems in the calculation of enzyme activity using reaction rate analysers. In the simplest instruments the slope of the curve in the first few seconds of the reaction is extrapolated into a straight line or, if the reaction is known to show a lag period, the rate of reaction after a defined period of time can be measured. The more sophisticated instruments use microcomputers to determine the linear portion of the curve and calculate the enzyme activity directly from the slope. The second derivative of the reaction progress curve (rate of change of the slope) can be monitored by the computer and when a value of zero is held for a period of time (10—15 seconds) this indicates a linear section of the graph. From the value for the slope, the enzyme activity can be calculated. 

An alternative approach involves the measurement of the time taken for small fixed changes in absorbance and the calculation of the rate for each single measurement. The computer stores and compares all the values, finally selecting the slope of the linear section of the curve and again calculating the enzyme activity. 

It is possible to bind enzymes to an insoluble matrix by a variety of methods and still retain their catalytic activity. The reusable nature of immobilized enzymes can significantly reduce costs and provides a convenient source of enzymes for performing substrate assays. Such preparations often show a greater stability and reduced inhibition effects than do soluble enzymes, although occasionally optimum pH values may be altered slightly. 

Nuclear Magnetic Resonance, Volume 34 The Royal Society of Chemistry, 2005 

Residue type and sequential assignments obtained from specifically labeled samples, when combined with 3D heteronuclear data, can significantly increase the efficiency and accuracy of the assignment process, the first step in structure determination by NMR. A protocol for the design of specifically labeled samples with high information content has been developed by Shi et aV. In vitro protein synthesis methods were used to produce four specifically labeled samples of the 23.5 kDa protein phosphoserine phosphatase (PSP) from Methanoccous jannas-chii. Each sample contained two C/ N-labeled amino acids and one N- 

The algorithm was demonstrated on NMR data from a 76- residue protein, human ubiquitin, matched to four structures, including one mutant (homolog), determined either by X-ray crystallography or NMR experiments (without RDCs). NVR achieves an average assignment accuracy of over 99%.  

The increased use of RDCs in high-resolution NMR studies of macro- 

Recent advancements in RDCs as a means of structure validation and elucidation have demonstrated the need for, not only a more user friendly, but more powerful RDC analysis tools. Valafar and Prestegard have introduced a software package, named REsidual Dipolar Coupling Analysis Tool (REDCAT) that 

---

Time, Cost, and Equipment Controlled-potential coulometry is a relatively time-consuming analysis, with a typical analysis requiring 30-60 min. Coulometric titrations, on the other hand, require only a few minutes and are easily adapted for automated analysis. Commercial instrumentation for both controlled-potential and controlled-current coulometry is available and is relatively inexpensive. Low-cost potentiostats and constant-current sources are available for less than 1000. 

Time, Cost, and Equipment Automated chemical kinetic methods of analysis provide a rapid means for analyzing samples, with throughputs ranging from several hundred to several thousand determinations per hour. The initial start-up costs, however, may be fairly high because an automated analysis requires a dedicated instrument designed to meet the specific needs of the analysis. When handled manually, chemical kinetic methods can be accomplished using equipment and instrumentation routinely available in most laboratories. Sample throughput, however, is much lower than with automated methods. 

Chemical kinetic methods are particularly useful for reactions that are too slow for a convenient analysis by other analytical methods. In addition, chemical kinetic methods are often easily adapted to an automated analysis. For reactions with fast kinetics, automation allows hundreds (or more) of samples to be analyzed per hour. Another important application of chemical kinetic... 

T. H. M. Noij, M. E. Margo and M. E. van der Kooi, Automated analysis of polar pesticides in water by on-line solid phase extr action and gas cliromatography using the cosolvent effect , 7. High Resolut. Chromatogr. 18 535-539 (1995). 

In addition to automated analysis, ISEs can be used to detect ionic species in chromatographic effluents. Particularly powerful is the coupling of modem ion... 

An Automated Analysis System for a Tensile Tester," Tyson T. Gill, in Computer Applications in the Polvmer Laboratory. ACS Symposium Series 313, 1986. 

As an example, consider the separation of the creatine kinase isoenzymes, MM, MB, and BB. Mercer has used classical ion-exchange chromatography (DEAE - Sephadex - A50) for the resolution of these three isoenzymes (44) To speed up the separation and ultimately to allow an automated analysis,... 

Villanueva, V. R. and Adlakha, R., Automated analysis of common basic amino acids, mono-, di-, and polyamine phenolic amines, and indoleamines in crude biological samples, Anal. Biochem., 91, 264, 1978. 

Table 5.9 summarises the main features of FTIR spectroscopy as applied to extracts (separated or not). Since many additives have quite different absorbance profiles FTIR is an excellent tool for recognition. Qualitative identification is relatively straightforward for the different classes of additives. Library searching entails a sequential, point-by-point, statistical correlation analysis of the unknown spectrum with each of the spectra in the library. Fully automated analysis of... 

The transfer of an automated analysis from the laboratory to the plant will often require special precautions for instance, while turbidities in a process stream can cause a loss of selective absorptivity in a spectrophotometric measurement, in potentiometric methods fouling of the electrodes, potential leakage in metal containers or tubing and loss of signal in remote control may occur (see later). 

The character and the degree of automation in chemical control may have been covered in the above treatment of semi-automatic or completely automatic, and of discontinuous or continuous analysis, but something more should be said about the means by which automation proper has been performed in recent times. Whereas in the past automated analysis involved the use of merely, mechanical robots, to-day s automation is preferably based on computerization in a way which can best be explained with a few specific examples. Adjustment knobs have been increasingly replaced with push-buttons that activate an enclosed fully dedicated microcomputer or microprocessor in line with the measuring instrument the term microcomputer is applicable if, apart from the microprocessor as the central processing unit (CPU), it contains additional, albeit limited, memory (e.g., 4K), control logics and input and output lines, by means of which it can act as satellite of a larger computer system (e.g., in laboratory computerization) if not enclosed, the microcomputer is called on-line. 

GILL AND KOEHLER Automated Analysis for a Tensile Tester... 

As shown by the authors, the almost exclusivity of two-bond correlations in H2BC makes it possible to extract INADEQUA TE-type connectivity information by overlaying H2BC and HSQC spectra. Also, the complementarity of HSQC, H2BC, and HMBC spectra could be used for a semi-automated analysis of this package of spectra based on the H2BC-HMBC rule of thumb mentioned above and the unique one-bond correlations in HSQC. 

Various approaches to the analysis of dissolved silicon have been tried. Most of them are based on the formation of /J-molybdosilic acid [ 199-203 ]. Dissolved silicon exists in seawater almost entirely as undissociated orthosilicic acid. This form and its dimer, termed reactive silicate , combine with molybdosilicic acid to form a- and /I-molybdosilicic acid [180]. The molybdosilicic acid can be reduced to molybdenum blue, which is determined photometrically [206]. The photometric determination of silicate as molybdenum blue is sufficiently sensitive for most seawater samples. It is amenable to automated analysis by segmented continuous flow analysers [206-208]. Most recent analyses of silicate in seawater have, therefore, used this chemistry. Furthermore, reactive silicate is probably the only silicon species in seawater that can be used by siliceous organisms [204]. 

Most workers now use a colorimetric method based on the reaction of urea with biacetylmonoxime [302,303]. The method has been adapted for automated analysis by De Marche et al. [304],... 

It is obvious that the simpler a method of analysis, the easier it will be to automate. Non-destructive methods which involve a minimum of sample treatment are the most attractive. X-ray fluorescence, for example, has been successfully applied to the continuous monitoring and control of process streams. However, the scope of automated analysis is wide and methods have been designed with a basis in nonspecific properties (pH, conductance, viscosity, density) as well as those characteristic of the che-... 

Moody, G.C. et al. 1999. Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes. Xenobiotica. 29 53. 

Egle, H. et al. 2005. Fast, fully automated analysis of voriconazole from serum by LC-LC-ESI-MS-MS with parallel column switching technique. J Chromatogr B. 814 361. 

AUTOMATED ANALYSIS OF STABLE ISOTOPES OF H, C, N, O AND S BY ISOTOPE RATIO MASS SPECTROMETRY 151... 

---