The following selection is from: Myers, Victor Carlyl. Practical Chemical Analysis of Blood. 2nd ed. C. V. Mosby Company, St. Louis (1924).

Copyright © 1999 Richard A. Paselk






It is perhaps appropriate that some special remarks be made regarding the very delicate quantitative colorimetric methods which have been developed for the chemical analysis of blood. Owing to, their very great sensitiveness these methods yield a higher degree of accuracy with the small quantities of material available than is possible with the older gravimetric and volumetric methods. Indeed, in many instances they have made possible determinations otherwise quite impossible. Furthermore, these colorimetric methods are very rapid. All of the methods described in the main part of this book (Chapters II to IX) are colorimetric except those for the blood bicarbonate and chlorides. Such extensive use of colorimetric methods is a development of the past fifteen years. In 1904 Folin introduced his accurate and surprisingly simple colorimetric method for the estimation of creatinine in urine and this gave a great impetus to the development of colorimetric methods. For this work Folin employed the Duboscq colorimeter and at the present time there are very few laboratories of biochemistry that do not boast one or more of these instruments. This instrument was first announced in 1854 by Jules Duboscq and is still made in the factory of his successors (Pellin). An improved form of the present French instrument is shown in Fig. 25.
Owing largely to the increase in the number of colorimetric methods and their extensive utilization for both scientific and clinical purposes, the demand for colorimeters has greatly multiplied. Until 1915 the instruments in use in this country were all of foreign manufacture (Pellin, Krüse, Hellige), but since that time a number of American concerns (Klett, Eimer & Amend, Leitz N. Y., Bausch & Lomb, Spencer) have undertaken the manufacture of such instruments.


The various colorimeters may be simply designated as of the (1) plunger, (2) wedge or (3) dilution type. With the plunger type the intensity of the color of either the standard or unknown is varied by changing the depth of solution through which the light passes with the aid of a plunger. The Duboscq in its various forms, the Klett and Bock-Benedict are of this type, although with the last named instrument a plunger is used for the unknown only.
In the case of instruments constructed on the Duboscq pattern, light from some even source of illumination is passed through the two sides of the instrument. The solutions to be compared are placed in cups in these two light paths. Some of the light in passing through the liquids is ab-




sorbed, the amount of absorption depending on the depth of the solution. The two beams of light are now brought to a common axis by means of rhombohedral prisms. Light from one cup illuminates one-half of a circular field, and light from the other cup illuminates the other half. The observing microscope, by which the observer sees both fields with one eye, is focused on the line of separation of the two fields. It is now possible to alter the depths of the columns of liquid until the two halves of the field are identical in intensity. When this condition holds, the concentrations of the two solutions are inversely proportional to their depths, which may then be read on the vernier scales of the instruments.

With a colorimeter of the wedge type such as the Hellige, the standard is placed in a wedge and the unknown in a small cup with sides parallel to the wedge. The wedge is moved up and down until the intensity of color is the same as the unknown. The two solutions are brought together for comparison with the aid of Helmholtz prisms.
With the dilution type of instrument such as the Sahli hemoglobinometer and the inexpensive test tube instrument described by the writer, the unknown is diluted with water or some diluting fluid until it has identically the same color as the standard.

Colorimeters of the Plunger Type

Instruments of the plunger type have been most extensively employed where accuracy and rapidity are required. Of the individual instruments


on the market, the Duboscq, as pointed out above, is the oldest and best known. The instruments on the Duboscq principle are made in several different sizes, i. e., to accommodate cups 20, 40, 50, 65 and 100 mm. in height. The colorimetric methods described for various biochemical analyses seldom necessitate working with very lightly colored solutions. Consequently the large 100 mm. instruments are not necessary, and as a matter of fact, their size makes them quite cumbersome to operate. Many biochemical laboratories appear to have purchased these large colorimeters under the impression that they were securing a superior instrument, but they possess no advantage aside from their ability to accommodate a greater depth of solution. In special instances where dilute solutions need to be matched, taller cups may be employed in the 50 mm. instruments. The author has used such cups with the Klett instrument.
It is difficult to compare the various 50 mm. instruments on the market at the present time, and an attempt will be made simply to point out the special features of the different colorimeters. Although the cups of the Klett instrument are slightly taller than those of the various 50 mm. Duboscq colorimeters, the instrument is essentially the same size and belongs in the same category. The same is true of the B. & L. Biological, and the BockBenedict. Although the cups in these instruments are slightly smaller, they are large enough to be adequate for practically all biochemical determinations. Although the more expensive instruments in this group possess added features and refinements and are larger, it is doubtful if they will yield any more accurate results than some of the smaller and cheaper instruments.
The original French instrument (Pellin) is very substantially constructed (see Fig. 25) and the glass parts are of excellent quality. This latter fact is very important, since good prisms are probably the most essential part of the instrument, otherwise it would not be possible to make the two halves of the field appear the same. Disadvantages of this instrument are the high cost and the fact that the cups and plungers are mounted in balsam and require frequent remounting, (The author has found it advantageous to remount the plungers and cups, when they become loose, in plaster of Paris or sealing wax. In using plaster of Paris, sufficient water should be employed, so that the mixture will just flow. If it is too stiff, it cannot be properly manipulated.) The instrument manufactured by Eimer Amend is practically an exact copy of the French Duboscq, except that cups and plungers are mounted in plaster of Paris and the vernier scale is adjustable. The Krüse model of the Duboscq, available before the war, contained some advantages over the French instrument, notably in the mounting of the cups and plungers, and was more reasonable in price, although in general it was hardly the equal of the Duboscq.
The American made Klett (see 26), originally designed by P. A. Kober, has a number of mechanical advantages over the French Duboscq and is less expensive. It has a somewhat better arrangement for adjusting the stages than the old style rack and pinion and the reflecting mirrors are divided to permit more even adjustment of the light of the two fields.


The cups and plungers are of black glass with, fused-on clear glass bottoms, thus removing all light except that passing from the reflecting mirrors to the prisms. Since the cups are fused and not mounted in balsam, chloroform may be used with impunity. It should be noted, however, that the cups are much more fragile than in the Duboscq. (Metal encased cups may be obtained with the instrument if desired). The prisms are somewhat smaller than those used in the French instrument, and it seems doubtful if the fields are quite as evenly matched. Kober and Klett1 have recently introduced a device containing a reflecting mirror (see Fig. 26) whereby readings may be made without materially changing the position of the eye. They have also designed a similar device for attachment to the standard

Duboscq instrument. This unquestionably saves some time in making readings and is very convenient.
Bausch and Lomb have rather recently entered the colorimeter field, and manufacture a 50 mm. instrument similar to the original Duboscq but designed to meet the most exciting requirements. Into its construction are built such refinements as optically inactive tube bottoms, plungers of optical glass matched for color, adjustments of microscopic precision and a dust-proof housing for the prism system. The verniers are provided with a reading mirror set at forty-five degrees, enabling the observer to make the scale reading with only a slight shift of the eye to the side. The verniers are adjustable. Although the comparative cost of this colorimeter is


rather high, it can hardly be said to be out of proportion to its quality.
Bausch and Lomb also make a smaller instrument, illustrated in Fig 27, which has been designated the Biological Colorimeter. It is appropriately named, as the instrument is well suited to biochemical work. It possesses practically all the advantages of the larger instrument although it sells for less than half the price. The compound prism of this instrument is of the overlapping type, in which the dividing line is a silver edge and practically invisible. The plungers are made of optical glass of high light transmission, free from color and are six-sided in form so that light is deflected and enters the prism only from the bottom. The cups, in metal casings, have plane parallel bottoms and are separable for cleaning. This design was suggested by Dr. Folin. The right-hand cup, containing the "unknown," is moved by rack and pinion, while the left-hand cup, contain-

ing the "standard," is held at any desired position by means of a thumb screw. Although the omission of the rack and pinion on the left-hand cup was made as an economy, and may be supplied if desired, it has the important advantage that when the standard is once set in place, it cannot be jarred out of position. The field in this instrument is large and, owing chiefly to the excellent prisms, is very evenly matched, making accurate readings comparatively easy. Since the stand of this instrument is very low, readings can conveniently be made from a sitting position. As pointed out on page 167, the insertion of a colored glass plate on the top of the left hand prism, following Newcomer's suggestion, converts the instrument into an accurate hemoglobinometer.
Very recently the Spencer Lens Co. has put out a colorimeter on the Duboscq pattern (illustrated in Fig. 28), which accommodates 50 mm. cups and still is comparatively small in size and quite reasonable in price.


The cups are mounted in a metal case with plaster of Paris, and with a slight turn lock in place so that they cannot fall out if the instrument is tipped. The positions of both cup[s are adjustable by independent rack and

pinion movements, the rack and pinion being very accurately constructed. The plungers of solid glass are round. It possibly would have been an advantage to have made hexagonal to deflect light coming from the


side. The stand is sufficiently low to permit use of the instrument in a sitting position.
For several years E. Leitz, N. Y., has manufactured an instrument on the Duboscq pattern which they have called the EIei colorimeter (see Fig. 29). This is a comparatively small and compact instrument, accommodating 50 mm. cups. The cylinders of the plungers and cups are of opaque glass with clear glass bottoms. The bottoms of the cups are fused on and then mounted in a metal base with plaster of Paris. The usual prism system is employed. For the customary rack and pinion movement, a quickacting but extremely sensitive lever device has been substituted. Owing possibly to this economy the price of the EIei colorimeter is below that of the various instruments mentioned above.

A simple and relatively inexpensive colorimeter has recently been introduced by Bock and Benedict,2 and is now made by Klett Mfg. Co. (see Fig. 30). In this instrument small reflecting mirrors have been substituted for the expensive prisms. The standard is put in a cell of known dimensions through which the rays of light pass longitudinally, thus removing the necessity for more than one moving part. Colorimetric estimations may be made very rapidly with this instrument, and it is possible, when the mirrors are properly matched to make very accurate color comparisons. To some the position of the eyepiece may not appear convenient, but we have found that when the instrument is placed on a shelf level with the eye facing the light (north), it can be very easily manipulated. The cup of the instrument is small and when selected to closely fit the plunger, satisfactory comparisons may be made with 2 c.c. of fluid, as small an amount as can be handled in the so-called microcolorimeters. Although the colorimeter gives very satisfactory service when carefully handled. it will not


stand hard and careless use. Fortunately the mirrors, cup and cell can be easily and cheaply replaced. However, when carelessly used, it is difficult to keep the instrument in proper adjustment.
The Duboscq biological colorimeter (Pellin) with 20 mm. cups seems to fall just short of being large enough to be useful in biochemical work.

Hellige Wedge Colorimeter

The Autenrieth-Königsberger colorimeter of Hellige the advantage that permanent standards may be kept in individual wedges, but the disadvantages that few of these standards are really permanent and that the wedges need to be empirically standardized. When a wedge is carefully calibrated for a given purpose by the individual using the instrument, very satisfactory results may be obtained. However, just the individuals to whom this instrument especially appeals are the ones who will not insure the accurate calibration of the standard wedges. The instrument is very well suited for certain special micro-determinations, but is not as satisfactory for general biochemical use as instruments made on the Duboscq pattern.


Dilution Type of Colorimeter

With the dilution type of instrument such as the Salhi hemoglobinometer and the inexpensive test tube instrument (see Fig. 32) described by the writer,3 the unknown is diluted with water or some diluting fluid until it has identically the same color as the standard. Theoretically the principle of diluting the unknown to the same intensity of color as the standard is excellent, but practically, it requires considerable care in execution and fairly large volumes of fluid in order to secure accuracy, since


near the end point the diluting fluid must be added a drop or a few drops at a time. Where the volume of fluid is large enough that one or two drops of fluid more or less do not impair the accuracy of the test, as in the test tube instrument mentioned above, the method is perfectly satisfactory, especially for an occasional estimation, but in such very small instruments as the Sahli and Kuttner a large error is necessarily introduced.


There are many purposes for which a colorimeter employing a single color standard is inadequate. One of the best illustrations is, the color-

imetric determination of the hydrogen ion concentration. As ordinarily carried out this determination is cumbersome and rather crude, since it has been necessary to employ a series of tubes with graduated pH values as standards, owing to the fact that here one is dealing with combinations of two colors instead of a single color.


It has been pointed out by the author4 that with the use of two (Hellige) wedges in a special colorimeter, it is possible to match all shades of color in a given indicator from the acid to the alkaline side, when one wedge is filled with an acid solution of the dye and the other with the alkaline solution, thus reducing the number of standards employed with a single indicator to two.
A new wedge colorimeter, designed especially for bicolorimetric work has been described by the author (see Fig 33). This instrument comprises a brass box with heavy metal base, and contains a rack and pinion arrangement for three wedges, the movement of the wedges being entirely within the closed box. Readings are taken from 100 mm. (adjustable) scales which emerge from the top of the instrument as the wedges are raised. The same type of rhombohedral prisms employed in the colorimeters constructed on the Duboscq pattern have been employed to bring the light passing through the wedges and the cup containing the unknown into the same observation field for comparison. A milk-glass plate (shown by A in Fig. 33) in back allows for the entrance of light. A small lamp box may be substituted for this. Only reflected light is used, which passes through a thick daylight glass. The light obtained compares favorably with daylight in quality although somewhat greater in intensity. A door at the side of the instrument gives access to the wedges and to the cup for the unknown which is mounted on it. Two wedges provide for bicolorimetric work as in the pH determination. However, to obtain a perfect match with unknown solutions which are slightly turbid or colored a third wedge may be used. To secure an even field it is sometimes necessary to counterbalance the greater thickness of glass on the wedge side of the field. (The wedges used by the author were constructed by E. Leitz, N. Y., of somewhat thinner optical glass than ordinarily employed in the Hellige wedges.). Provision has been made for this by attaching to the rack carrying the cup for the unknown, a clip which will hold one or more glass plates.
With the aid of this instrument very accurate estimations of the hydrogen ion concentration of blood may be made. It is also well suited for pH determinations in urine, gastric contents and bacteriological culture media. Although the instrument was not designed for ordinary colorimetric work, very accurate colorimetric comparisons may be made when only the first wedge is used. Obviously the same criticisms apply to its use as a colorimeter as in the case of the Hellige instrument.


Nephelometric attachments may be obtained for the Duboscq (Pellin) and are constructed for the Klett, B. & L. 50 mm., Duboscq and Biological and the Elei colorimeters. In some cases the lamp employed for the nephelometric work may also be utilized as the artificial illumination for colorimetric work.
For the convenience of those who may be in need of a colorimeter, the latest retail prices of various instruments together with the names of the


manufacturers are given below. The instruments may be secured from practically any dealer in laboratory supplies.



1 Kober, P. A., and Klett, R. E.: Further Improvements in the Nephelometer-Colorimeter. Jour. Biol. Chem., 1921, xlvii, 19-25.
2 Bock, J. C., and Benedict, S. R.: A New Form of Colorimeter. Jour. Biol. Chem., 1918, xxxv, 227-230.
3 Myers, V. C.: A Simple Colorimeter for Clinical Purposes. Jour. Lab. and Clin. Med., 1915-16, i, 760-761.
4 Myers, V. C.: A Colorimeter for Bicolorimetric Work. Jour. Biol. Chem., 1922, liv, 675-682.