REPORT FOR ANALYTICAL CHEMISTS
Microanalysis in the M o d e r n Analytical Laboratory of Clinical Chemistry THE modern clinical chemist has many and varied responsibilities. These include super vising an analytical laboratory for the determination of certain constituents in biological materials, acting as consultant to the medical staff in clinical chemical problems, and carrying on a research program on problems related to health and disease. This article concerns only activities related to the analytical l a b o r a t o r y . Specifically, it is an attempt to outline the present status of microchemistry in the analytical l a b o r a t o r y of clinical chemistry. It is concerned also with certain problems of vital interest to the clini cal chemist, whose solution may well come from newer knowledge in other disciplines. Improved understanding of the significance of levels of numerous components in body fluids and tis sues in the diagnosis and treatment of disease has resulted in a demand t h a t the clinical chemistry labora tory be prepared to determine these components. This has stimulated a rapid development in analytical techniques and upgrading quality of personnel to supervise and per form laboratory procedures (17). The problem of the clinical chem ist can best be appreciated if it is pointed out t h a t he m a y be called upon to determine approximately 40 of the elements in the periodic table. I n addition, the laboratory must be prepared to determine the numerous organic compounds, in cluding various proteins, t h a t occur naturally in the body fluids or tis sues or have been administered to the patient or taken by accident. M a n y of these elements and com pounds exist in amounts of 0.1 p.p.m. or less in the fluids or tissues. The problem is further compli
cated by the fact t h a t the amount of material for analysis—e.g., blood— is limited. I t is not uncommon to assay as m a n y as 20 components in a single blood sample, which m a y have come from an infant from whom it is not safe to t a k e more than a few milliliters for analysis. Often, in these cases the total sam ple comprises 0.1 or 0.2 ml. Another limitation put on the laboratory is t h a t results must be made available rapidly enough to help in the treatment. This re quires t h a t procedures be direct, and require a minimum number of transfers. Further, analytical pro cedures chosen must be applicable to large numbers of samples. T h u s a procedure which requires sequen tial analysis is undesirable. A gasometric method, for example, which requires one at a time processing in the instrument is not as desirable as a colorimetric procedure where the samples m a y be prepared simul taneously in test tubes for reading in the colorimeter.
S a m u e l N a t e l s o n , head of the Department of Biochemistry, Roosevelt Hospital in New York City, received his Ph.D. degree in organic chemistry from New York University in 1931. For the next year he was associated with the New York Testing Laboratories working on plastics and resins. Since then his career has been as sociated with hospital work. In 1932 he joined the staff of the Jewish Hospital of Brooklyn. He went from there in 1949 to tlye Rockford Memorial Hospital, Rockford, Illinois, as head of the Biochemistry Department. In 1957 he returned to New York as head of the Biochemistry Department, St. Vincent's Hospital. In 1958 he joined the staff of the Roosevelt Hospital. His interest in microanalysis dates back to 1929 when he studied in the organic microanalysis laboratory at New York University. H e has prepared more than 100 papers in organic synthesis, micro-techniques and clinical chemistry. He is the author of "Microtechniques of Clini cal Chemistry," (Thomas, Springfield, Illinois, 1957). Natelson is currently president of the New York section of the American Asso ciation of Clinical Chemists.
Measuring the Sample
For the clinical chemist, the term "micro" refers only to the size of the initial sample [16, Ιβ, 52—5^, 78). Once the sample—e.g. serum —has been measured out, subse quent measurements are usually in the macro range. For example, in the estimation of total protein, 10 μ\. is washed into 1 ml. of water, 1 ml. of biuret reagent is added, and the color produced is read in a colorimeter. As another example, 10 ju.1. of serum is washed into 0.2 ml. of water, a drop of diphenylcarbazone indicator is added, and the chloride is estimated by titration with standardized mercuric nitrate. I t is evident t h a t to the clinical chemist, who is concerned primarily with the analysis of liquids, accu rate and precise methods of measur ing small volumes are of particular interest. At first, the micropipet was, in effect, a narrow bore capillary. This was introduced about 50 years ago to measure hemoglobin and to sample blood for the counting of cells (Sahli pipet). This pipet is approximately 80 mm. from tip to m a r k for 20 μ\. Because it is used as a washout pipet, and thus cali brated to contain, reproducibility easily achieved with this pipet is to 1 mm. in length, and thus 1 part in 80 or ± 1 . 2 5 % . This compares fa vorably with the conventional 1-ml. transfer pipet used to deliver. To increase precision, micropipets were introduced with an automatic stop for the calibrated volume. These VOL. 3 1 , N O . 3, MARCH 1959
·
17
A
Single Optical Surface Provides Better A n a l y s i s
B a i r d - A t o m i c Spectrographs - w i t h the Eagle M o u n t design - are the only commercially available high-sen sitivity instruments using the modern concave diffrac tion grating as the only optical surface. The Baird-Atomic 3-Meter Grating Spectrograph with but a single reflect ing surface minimizes light loss and light scattering, particularly in the important far ultraviolet region. This assures excellent identification of extremely low concentration elements. In many cases, analyses of these elements by spectrographs with 3, 4 or 5 reflecting surfaces is extremely difficult if not impossible, since too much light is lost at each reflecting surface, especially in the far ultraviolet region where the reflectivity of aluminum drops off. In such situations, the Baird-Atomic Spectrograph with only one reflecting surface provides the high line-to-background ratio required for extreme sensitivity. The Baird-Atomic 3-Meter Spectrograph is a precalibrated instrument and permits all adjustments to be made from front control panel. Versatil ity and flexibility of application and high resolution across the entire photographic plate are inherent in B-A Spectrographs. Write for more detailed information.
Baird-Atomic,
Inc.
33 U N I V E R S I T Y RD., CAMBRIDGE 30, M A S S .
Instrumentation for Better Analysis Circle No. 126 on Readers' Service Card
18
A
·
ANALYTICAL CHEMISTRY
REPORT included the constriction pipet (44), the overflow pipet (3), and pipets based on variations of these princi ples (55, 56). These pipets had narrow tips, so t h a t t h e y could be inserted into capillaries holding the sample. By narrowing the bore of the Sahli pipet to about half its di ameter, and blowing a bulb in t h e tube to avoid increase in length, an inexpensive pipet was produced in which for 20 μ\. 1-mm. length repre sented only 1 p a r t in 320 (55). This is more t h a n adequate for usual clinical laboratory proce dures. This pipet has a blunt tip which is not easily broken. The blunt tip has no disadvantage over the narrow tipped pipets, as it was soon learned t h a t aspiration of the sample, when both ends of the capil lary were open, made the narrow tip unnecessary (59). Further, the ease with which the sample flow could be controlled and brought to the m a r k b y tilting the pipet and capillary made these automatic stops unnecessary. Accuracy is achieved by using the same pipet for both unknown and standard. Careful calibration of the pipet is therefore unnecessary. T h u s the clinical microanalyst has completed the cycle and returned to, essen tially, the original simple design. Separation of the Substance Sought I n general it is highly desirable to be able to test samples directly without preliminary separation. This is the general procedure in m a n y determinations in micro analysis in the clinical laboratory. However, because this is not always possible, certain techniques have found favor in isolating the sub stance sought or in removing inter fering substances. For numerous components of serum, a direct test can be made for the substance sought if protein is first removed. This is done by pre cipitating the proteins by such sub stances as tungstic acid, phosphomolybdic acid, trichloroacetic acid, zinc hydroxide, or cadmium hydrox ide. With perchloric acid a separa tion of mucoproteins from other proteinaceous material is accomp lished (85). I n every case, the sample is diluted, reducing the con centration further from t h a t of the
REPORT FOR ANALYTICAL CHEMISTS
original sample. While this proce dure serves for materials like sugar, urea, creatine, creatinine, and phos phate, and m a n y other materials, methods of concentrating the sub stance sought must be used. Often, this eliminates need to precipitate the original protein. For volatile materials like am monia, steam micro distillation was first resorted to (65). This process is still used by some, particularly for protein bound iodine estimations (70). This has the weakness t h a t only one sample can be processed at a time. This was followed by de velopment of micro aeration setups (79) and finally by the use of diffu sion techniques (20). At first, mi cro diffusion setups consisted of a small container holding absorbant, suspended in a flask containing the sample to be diffused (29, 83). This evolved to a special dish with concentric chambers, the so called Conway diffusion dish (21). These dishes presented problems in clean ing, especially because the cover plate had to be greased to avoid leakage. A movement back to the original setup then followed. W h a t evolved was a glass rod held in a one-hole rubber stopper (see Figure 1). T h e rod, after dipping in the absorbant, is p u t into a bottle containing the sample. After diffu sion takes place, the rod is then washed into the color reagent—e.g. Nessler's reagent for ammonia. This permits control of the final sample volume. Amounts of am monia of the order of fractions of micrograms can be assayed (63). Evolution toward simplicity and convenience has resulted in return ing to essentially the original de sign. In the modern clinical chem istry laboratory ammonia, carbon monoxide (34), volatile alcohols (23), aldehydes (formaldehyde, acetaldehyde), and acetone (84) are determined by microdiffusion. Gases such as carbon dioxide and oxygen are estimated by the microgasometer (see Figure 1) capable of measuring from 5 to 15 μ\. of gas (42, 60). Arsenic is separated by volatilizing it as either the chloride or arsine, and determined by stain or colorimetrically. Amounts of the order of 1 7 or less m a y be deter mined (8).
Have you tried these Time-Saving
FILTRATION AIDS? s*s FILTER PULP No.
Figure I. Compact instrumentation is used t o analyze volatile materials in the clinical laboratory. The rota tor (left) turns at 100 r.p.m. t o mix reagents and expose the surface for microdiffusion. The shaft is bent so that a rocking motion adds to the ro tary motion. The hand holds the rod which is dipped in the absorbent. This is inserted into the bottle prior to rotation. The microgasometer (right) is used for analysis of such gases as carbon dioxide and oxygen. (Scientific Industries, Springfield, Mass.)
289, Ash-Free
Helps to mini mize clogging of the filter paper, and maintain fil t r a t i o n speed. Helps obtain better retention of precipitate, insure more accurate r e s u l t s . Dry-Dispersed form replaces moist pulp and brittle tablets. Send f o r f r e e b u l l e t i n
s*s ANTI-CREEP Reagent Fluid
A few drops added to the wash solution prevents the "creep" of precipitates up the filter paper, facili tates the transfer of insolubles from the precipitation vessel, reduces the problem of film deposit on the walls of the beaker. Send f o r f r e e f o l d e r
s*s Lipides are separated by extrac tion into an organic solvent. Ex traction into organic solvents is also used to concentrate certain elements for assay. Practical examples are lead as dithizone (10), calcium as the alizarinate (62), and iron as the thiocyanate (60). I n the examples cited, amounts of the order of 1 to 2 γ are determined. Extraction into . organic solvents is of particular value in t h a t it permits concentra tion into a small final volume so t h a t reasonably high absorbances are obtained with a small amount of material being analyzed. Paper electrophoresis (2) is rou tine in m a n y clinical chemistry laboratories. With amounts of serum or blood of the order of 2 to 10 μ\., partition of the proteins, lipide- and carbohydrate-carrying proteins, and hemoglobins (47, 50) is carried out routinely. I n some laboratories, electrophoresis on starch blocks is also utilized for this purpose (31).
FILTER P A P E R SUPPORTS
No. 123 With these Supports even soft grades of Filter Paper can be submitted to high suction without danger of bursting at the apex. Special pure, porous textile material will not retard filtration. Inexpensive. Send f o r f r e e f o l d e r
All three of these S&S- Filtration Aids are time savers. They are available, with S&S High Quality Analytical Filter Papers, from your laboratory supply house. MAIL
COUPON
TODAY!
CARL SCHLEICHER & SCHUELL CO.
Dept. AC-39 Keene, Ν. Η. Send Free data on S&S •
FILTER SUPPORTS
Π FILTER PULP
D ANTI-CREEP
NameCompanyAddress City
StateCircle No. 77 on Readers' Service Card
VOL. 3 1 , NO. 3, MARCH 1959
·
19
A
REPORT
Analyze innumerable compounds by their PHOSPHORESCENCE Qualities
Λ'*
> . 1 Λ:' ΐ > * S » J
. î ." Ί
Ι I » J I , , « "^ * ' î '
