Ultramicro methods

Natelson, S Routine use of ultramicro methods in the clinical laboratory Am J Clin Path (1951),... 

H. L. "Ultramicro Method for Determination of Iron In Serum with the Graphite Furnace". Clin. Chem. (1973), 19, 326-329. 

Applications of EELS for Chemical Analysis. Apart from the fact that it is an absolute technique requiring no standard for calibration the special advantage possessed by EELS as a means of chemical analysis is that it combines sensitivity with spatial resolution. It is, in a literal sense, an ultramicro method with... 

Novak, M. 1965. Colorimetric ultramicro method for the determination of free fatty acids. J. Lipid Res. 6, 431-433. 

Knights, E. M., R. P. MacDonald, and J. Ploompun Ultramicro Methods for Clinical Laboratories. New York Grune Stratton 1957. 

Petrikova, M. N., and I. P. Alimarin The Ultramicro Method of Chemical Analysis. Amperometric Titration. Zh. Analit. Khim. (Russ.) 12, 462 (1957) J. Anal. Chem. USSR (English Transl.) 12, 481 (1957). 

The activity of the lipase has also been assayed with the ultramicro method of Novak [171] to determine net free fatty acid release from endogenous substrate [172]. Incubation of rat adipose tissue homogenate was carried out in 40 mM phosphate buffer, pH 6.8, in the presence of 30 mM EDTA and 2% bovine serum albumin. 

One micro-Kjeldahl method (10-20 mg protein) requires a minimum heating time of 4 hours with HgO as catalyst (S57), while another (Jl)— carried out in a sealed tube at 460-480°C for 30 minutes without a catalyst — has been proposed. An ultramicro method (0.02-0.1 gg N) can be completed in 1 hour using Se as catalyst (E6). 

The monocarboxylic acid connected in amide conjugation to the amino group of the sphingosine residue proved to consist mainly of the Ci8-fatty acid. The gangliosides of Niemann-Pick disease and metachromatic leucodystrophy differ from those of normal, infant and adult brains, in that C24-acids were not detected. The development of new ultramicro methods led to the exact (differential) determination of the constituents of these gangliosides215 of disease. 

It is obvious that the development of ultramicro methods depends almost entirely on the development of apparatus which can handle minute quantities of samples, solutions, etc. In most cases the methods already in use in microanalysis can be adapted to an ultramicro scale. 

The ultramicro methods of Scholander and co-workers are very ingenious and very small samples of gas can be analyzed (K3). For the routine laboratory, however, the method is not suited as too much depends on the personal skill of the operator. 

As only 10-50 fil of serum is required for this purpose, paper electrophoresis is a real ultramicro method. 

Sanz, M. C., Ultramicro methods and standardization of equipment. Clin. Chem. 8, 406-419 (1957). 

Generally, ultramicro methods are used as a system, not for one special test only. Therefore publications in which ultramicro methods are described usually contain advice on and a description of the apparatus which are used by the author. However, in the first publications on the application in the clinical laboratory only special tests were de-... 

As it is impossible to deal in detail with these articles only a list will be given. Natelson (Nl) published methods which he used for a number of years. He described in detail the construction and use of his apparatus and compared standard deviations of macro and ultramicro modifications of 8 important tests. Sobel and Hanok (S2) give an article on the method of scaling down from micro to ultramicro methods in which they also make a comparison of the errors. Caraway and Fan-ger (Cl) give details about their equipment and describe 16 tests, the most of which are performed on 10-(d samples. Here too a comparison is made with conventional methods. Kaplan and del Carmen (Kl) give a description and details about equipment and performance of 14 tests. Apart from these publications much valuable advice and methods may be found in several books on ultramicro methods (K3, K4, N2). 

The results of these authors prove that ultramicro methods have a distinct advantage over conventional methods and that the majority of the most important tests can be performed with very good results. This no doubt has stimulated the development of complete sets of apparatus with reagents for several tests, which are now commercially available. 

The publications on ultramicro methods of recent years generally concern application of newer methods, e.g., enzymatic and catalytic determinations. Some examples will be given. 

For instance, the normal method for the determination of protein-bound iodine was transformed to an ultramicro method by Sanz et al. (SI). This method is based on the well-known catalytic action of iodine on the reduction of ceric ions by arsenious add and requires only 50 d serum. 

In the latest decennium, methods in clinical chemistry have improved a great deal with regards to the following aspects accuracy specificity sample size speed number of determinations per man-hour costs per determination. The introduction of ultramicro methods offers advantages on most of these aspects. 

While specificity of a test is nearly independent of the equipment (not completely, e.g., spectral purity of colorimeter filters ), the introduction of more or less automatic apparatus makes accuracy more and more dependent on the accuracy of these apparatus. Some authors state that ultramicro methods are more accurate than macrotechniques. In our opinion and experience this is not true, the accuracy of most ultramicro methods being the same or somewhat less. The quantity of sample generally used is 10-20 [il. The reproducibility of pipets in this range is very good. It is quite possible to perform determinations on much smaller samples [see, for instance, the outstanding work of Lowry (LI) on enzymes in single nerve cells and also the methods of Ames and Nesbett (Al)], but either the speed or the accuracy diminishes when using pipets of less than 10 [Jil. 

C2. Close, J. R., and Street, H. V., An ultramicro method for the determination of amylase activity in blood serum. Clin. Chim. Acta 3, 476-479 (1958). 

The easiest and fastest method involves the direct injection of a diluted sample with an internal standard solution into a GL chromatograph. We recommend an ultramicro-method utilizing direct injection. Aliquots of a body fluid and an internal standard, such as n-propanol, are mixed together in equal volumes. A 1 pi aliquot of the mixture is injected into a GL chromatograph equipped with a glass column (2 m long by 0.3 cm ID) packed with 0.2% Carbowax 1500 on 80/100 Carbopak C and an... 

Nisselbaum JS, Green S (1969) A simple ultramicro method for determination of pyridine nucleotides in tissues. Anal Biochem 27 212-217... 

If the material is a liquid, the boiling point is determined by the ultramicro method. If sufficient material is on hand and the boiling point reveals that the material is relatively pure (narrow boiling point range), the density and the re- <-(www) fractive index can provide valuable information for identification purposes. 

Karlmark, B. An Ultramicro Method for the Separate Titration of Hydrogen and Ammonium Ions, Pflugers Arch. 323 361-365 (1971). 

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