USE OF TIlE SPECTROPHOTOMETER IN CAPILLARY TUBE COLORIMETRY FOR THE DETERMINATION OF REDUCING SUBSTANCES (GLUCOSE), CHLORIDES, AMMONIA NITROGEN, URIC ACID AND CREATININE WALTER C. LOBITZ, JR., M.D. Section on Dermatology AND
ARNOLD E. OSTERBERG, PH.D. Division of Clinical Biochemistry Mayo Clinic, Rochester, Minnesota
Received for publication Feb. 16, 1946
In the study of the chemistry of palmar sweat (1) it was found necessary to make determinations from volumes of sweat of less than 1 cu. mm. The capillary tube colorimetric methods, devised by Richards, Bordley and Walker (2), there-
fore, were adapted so that certain constituents of these volumes of collected sweat might be determined quantitatively. It occurred to us at that time that if it were possible to read the color intensities produced in capillary tube colorimetry with the aid of a spectrophotometer, the efficiency of the methods could be increased both as to time and as to accuracy. This report deals with the design of the cuvette adapters necessary to hold capillary tubes of both 0.35 mm. uniform inside diameter and 0.60 mm. uniform inside diameter in a Coleman, Model 6, Junior Clinical Spectrophotometer. In this article also we are presenting curves showing 1) the percentage of light transmitted at various wavelengths for each chemical reaction and 2) the percentage
of light transmitted with various concentrations of each substance studied. These curves were obtained for 1) reducing substances (glucose), 2) chlorides,
3) ammonia nitrogen, 4) uric acid and 5) creatinine. Hereafter in this paper these two types of curves will be referred to as the spectral transmission curve and the concentration transmission curve respectively. THE ADAPTER
The adapter is made of black plastic to fit snugly into the cuvette well of a Coleman, Model 6, Junior Clinical Spectrophotometer. It differs from the cuvette adapters listed for this instrument only in the following ways (fig. 1): 1. The opening in the adapter facing the monochromatic exit slit of the spectrum is a beveled slit with an opening 14 mm. in length and 0.40 mm. in width for the 0.35 mm. tube, and 12 mm. in length and 0.46 mm. in width for the 0.60 mm. tube. The slit is so beveled that the capillary tube fits firmly into the open-
ing without allowing leakage of light around the tube's edges. The tube is placed in the beveled slit by sliding it in from below. A portion of the capillary 135
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tube lies above and a portion below the slit's opening for the light beam. The tube is held in place by plasticine already present in the adapters The excess tubing that might extend below the adapter is easily cut off by scratching the tube with a carborundum edge. 2. The key on the side of the photoelectric cell of the adapter is adjustable by loosening a screw in the head of the adapter. It is important that the key's position be adjusted as follows and rechecked from time to time. The screw controffing the key'in the adapter head is loosened. The adapter, containing a capillary tube filled with water, is placed in position in the cuvette well so that the key coincides with the keyway of the well. The adapter is then
pushed down into the well without undue force. Once in place, the adapter is rotated gently to right and to left until a maximal percentage of transmission of light is registered on the galvanometric scale. The screw is then tightened careAdjusting
SI
Fia. 1. THE Cuvrrn ADAP!rEn DESIGNED TOII0LD THE CAPILLARY TUBE
The oblique views are above and a cross-section at the slit-level is below
fully so as not to rotate the adapter in the well. Once adjusted properly, the adapter will fit snugly into the cuvette well and key at exactly the proper angle if no undue force is used in its handling. 3. On the side of the adapter are two flat springs so placed as to exert lateral pressure between the adapter and the wall of the cuvette well. This eliminates any play and insures keying at exactly the proper angle. It is important that for each determination at least three readings should be made; the capillary tube should be removed and re-inserted into the slit between readings. If the tube is seated properly in the beveled slit and the adapter is keyed at the proper angle, all readings should be exactly the same. REDUCING SUBSTANCES (GLUCOSE)
The capillary tube colorimetric method of Walker and Reisinger (3) fof the determination of reducing substances in the urine is an ultramicro adaptation of Sunmer's method. The spectral transmission curve in figure 2 was obtained by using 1 volume of a
SPECTROPHOTOMETER AND CAPILLARY TUBE COLOR IMETRY
137
solution containing 40 mg. of glucose per 100 cc. of water, plus 3 volumes of dini-
trosalicylic acid as the reagent in the 0.60 mm. tube. Water was used as a reference. The spectral transmission curve for the same solution in the 0.35 mm. 100
90 80 70 60 50
40 30 z0 QI
10 0
300 350 400 450 500 550 600 650 700 750 800 Wave. length- miilimicvon$ FII. 2. SPECTRAL TRANSMISSION CuRvE FOR REDUCING SUBSTANCES (GLUcoSE)
I 0.60 mm. I.D. tube
10 0 30 40 50 60 70 80 90 100 Glucose solution- mg. pci' 100 c.c.
Fm. 3. CONCENTRATION TRANSMISSION CURVES FOR REDUCING SUBSTANCES (GLUCOSE) WHEN A WAVELENGTH OF 490 rn/h Is USED
tube is of the same type. From the curve it can be seen that minimal percentage of transmission of light is at a wavelength of 440 mp. However, not enough light was transmitted at 440 rn/L when a reagent plus water was used as a reference, hence in preparing the concentration transmission curve a wavelength of 490 mj
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was used. It must be emphasized that when a reading is made on the slope of the spectral transmission curve, the wavelength dial must be set exactly at the selected wavelength. The concentration transmission curves shown in figure 3 were read then at a wavelength of 490 m with a dinitrosalicylic acid reagent blank as a reference. With the 0.35 mm. tube solutions containing 50, 75 and 100 mg. of glucose per 100 cc. of water were used to obtain the concentration transmission curve. With the 0.60 mm. tube, solutions containing 40, 60 and 100 mg. of glucose per 100 cc. of water were used.
Reci5ent + NaC1 50 mg. per'
U
I
100
c.c.
40 30 £0 10
0
300 350 400 450 500 550 600 650 700 50
Wa.ve 1enth- millimicpons
Fxo. 4. SPECTRAL TRtxsMIssIoN CURVE USING POTASSIUM CHROMATE SOLUTION EQUIVALENT TO 50 MG. OF SODIUM CHLORIDE PER 100 CC. PLUS REAGENT CHLORIDES
The method of Westfall, Findley and Richards (4) for the capillary colonmetric determination of chloride is a modification of Isaac's method. The spectral transmission curves for both 0.60 and 0.35 mm. tubes were similar. With water used as a reference, the spectral transmission curve in figure 4 was obtained by taking 1 volume of a potassium chromate solution equivalent to 50 mg. of sodium chloride per 100 cc. plus 14 volumes of a 10 per cent acetic acid solution of diphenylcarbazide. The 10 per cent acetic acid was used as solvent instead of alcohol because the color, although slower to develop, did not fade as rapidly as with an alcoholic solution. As can be seen from the curve, the minimal percentage of transmission of light was obtained at 550 m and this wavelength was used in determining the concentration transmission curves.
SPECTROPHOTOMETER AND CAPILLARY PUllS COLORIMRTRY
139
The concentration transmission curve (fig. 5) was obtained from the 0.35 mm. tube containing 1 volume of potassium chromate solutions equivalent to 25, 50
and 100 mg. of sodium chloride per 100 cc. plus 14 volumes of the acetic aci4 dliphenylcarbazide reagent. The concentration transmission curve (fig. 5) for the 0.60 mm. tube was obtained with potassium chromate solutions equivalent to 10, 25 and 60 mg. of sodium chloride per 100 cc. plus the acetic acid diphenylcarbazide reagent. AMMONIA NITROGEN
The ultramicro determination of ammonia nitrogen by capillary tube colorimetry as described by Walker (5) is an ultramicro modification of direct nessleri-
NaC1 - m. pet' 100 cc. Fxa. 5. CONCENTRATION TRANSMISSION CURVE FOR SODIUM CrnoRIDE WREN A WAVELENGTH OF 550 mji WAS USED
zation. The only modification which was made in this method was to omit the use of gum ghatti, since with the low concentrations of ammonia nitrogen which we encountered, stable colors could be developed without the use of that colloid. The spectral transmission curve in figure 6 was obtained by using a nesslerized solution of ammonium chloride (1 volume of Nessler's solution to 2 volumes of the ammonium chloride) containing 5 mg. of nitrogen per 100 cc. of solution in
the 0.60 mm. tube. Water was used as a reference. A similar curve was obtained when the same solution was used in the 0.35 mm. tube. The wavelength of minimal transmission of light was 400 m.i and the concentration transmission curves were made with light of this wavelength. The concentration transmission curves (fig. 7) were determined with nesslerized solutions (two parts of the ammonia solutions to one part of Nessler's solution)
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of ammonium chloride containing 0.2, 0.4, 0.8 and 1.0 mg. of nitrogen per 100 cc. in both the 0.60 and 0.35 mm. tubes. The reagent-water blank solution was used as a reference. 100
90 80 'TO
-
-
60 -
40
30 -
Ra.nt + Ammonia nitvoen 5 m. per 100 c.c.
10 I
I
I
I
I
I
O0 350 400 450 500 550 600 650 'TOO '150 800 Wave 1enSth - millimicx'ons FIG. 6. SPECTRAL TRANSMISSION CtrRvE FOR AMMONIA NITROGEN
70 60
50• 40 3G
0
I
0.1
0.2
0.3
0.4
I
I
0.5
0.6
Ammonia. nitPoiEn -
I
0.8 0.9 0.? mt pci-' 100 cc.
1.0
FIG. 7. CONCENTRATION TRANSMISSION CURVES FOR AMMONIA NITROGEN WHEN A WAVELENGTH OF 400 mM WAS USED
URIC ACID
The capillary tube colorimetric method for the determination of uric acid de-
scribed by Bordley and Richards (6) is an ultramicro adaptation of Folin's niethod. The spectral transmission curve in figure 8 was obtained by using 5 volumes of
SPECTROPHOTOMETER AND CAPILLARY TUBE COLORIMETRY
141
a solution of 2 mg. of uric acid per 100 cc. plus 5 volumes of the cyanide-urea solution, plus 1 volume of the uric acid reagent in the 0.60 mm. tube. Water was used as a reference. A similar curve was obtained when the 0.35 mm. tube was
90 t)
80 70
I
60 50
1.,
40
Rea5ent + Ur'ic acid. 2. 1T19. )Cr' 100 C.C.
30 20
10 I
I
0O 350 400 450 500 550 600 650 '100 '150 800 Wave length - millirnicvons FIG. 8. SPECTRAL TEArsrrIssIoN CURVE FOR URIC Acm
bc 90 .4-.,
80 70 60 50
40
00.1 02 0.3040.5 0.6
0.7 0.8 09 1.0
Uric o.cid. solution -
mgi.
1.1 i2iL4
per 100 cc.
Fia. 9. CoNcENTRATIoN TRANSMISSION CURVES FOR URIC ACID WHEN A WAVELENGTH
or 685 mi WAS USED
edus. The wavelength of minimal transmission of light was 6 85 mp. The concentration transmission curve was developed at this wavelength. The concentration transmission curve (fig. 9) for the 0.35 mm. tube was obtained with solutions of 0.2,0.8 and 1.0 mg. uric acid per 100 cc., plus the reagents
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in the proportions just given. The curve obtained when the 0.60mm. used was
tube was
obtained with solutions of 0.2,0.8, 1.0 and 1.4 mg. of uric acid per 100 cc.
plus the reagents. A reagent-water blank solution was used as a reference. The color produced by concentrations of uric acid of more than 1.0 mg. per 100 cc. in the smaller capillary tube and of more than 1.4 mg. per 100 cc. in the larger capillary tube no longer followed the Lambert-Beer law since a greater percentage of light was transmitted with a resultant flattening of the concentration transmission curve. 1OC
90
80 -
I
70
-
60
-
50 -
40 30 R'zaent + Cpeatinine 1 mg. pw 100 c.c.
20 -
10 I
I
I
I
I
I
I
00 350 400 450 500 550 600 650 '?OO 750 800 Waw 1nith - millimicr'ons FIG. 10. SPECTRAL TRANSMISsIoN CURYR FoR CRRATININR
CREATININE
Bordley, Hendrix and Richards (7) described the capillary tube colorimetric method for the determination of creatinine. This is an ultramicro adaptation of the method of Folin and Wu. The spectral transmission curves obtained for both the 0.60 and 0.35 mm. tubes were similar. Figure 10 shows the spectral transmission curve when the 0.60 mm. tube contains 2 volumes of a solution of 1 mg. of creatinine per 100 cc. in 0.01 normal hydrochloric acid, plus 1 volume of the alkaline picrate reagent and water is used as a reference. The minimal percentage of transmission of light occurred at 425 mp. However, when the reagent-water blank was used as a reference, the
light transmitted was not sufficient to allow preparation of the concentration transmission curve. The wavelength of 515 mt was found to be most satis-
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SPECTROPITOTOMETER AND CAPILLARY TUBE COLORIMETRY
factory for the development of the concentration transmission curves. Again we emphasize the importance of a very accurate setting of the wavelength dial when reading on the slope of the spectral transmission curve. These concentration transmission curves when both capillary tubes are used are shown in figure 11. They were developed by using two volumes of solutions containing 2.0, 4.0 and 6.0 mg. of creatinine per 100 cc. in 0.01 normal hydrochloric acid solution, plus 1 volume of the alkaline picrate solution. SUMMARY
The spectral transmission curves and the concentration transmission curves were determined for the analyses of 1) reducing substances (glucose), 2) chlorides, 3) ammonia nitrogen, 4) uric acid and 5) creatinine by using the spectrophotorneter in capillary tube colorimetry.
60
0.60 mm. ID. tube
50
40 3C
0
I
I
0.5
1.0
I
25 3.0 3.5 4.0 4.5 5.0 Crtinine - mc. pet' 100 c.c. 1.5
2.0
I
5.5
6.0
Fia. 11. CONCENTRATION TRANSMISSION CURVES FOR CREATININE WHEN A WAVELENGTH
OF 515 m WAS USED
The adapter designed to hold the capillary tubes of a uniform inside diameter of 0.60 mm. and 0.35 mm. permitted use of the spectrophotometer. The over-all resultant volumes needed, after dilution of the unknown solution and addition of reagents are 1.68 cu. mm. in the capillary tubes with an inside
diameter of 0.35 mm. and 4.24 cu. mm. in the capillary tubes with an inside diameter of 0.60 mm. REFERENCES 1. LOBITZ, W. C., JR., AND OSTERBEEG, A. E.: Chemistry of palmar sweat. Preliminary
report: apparatus and technics. J. Invest. Dermat. 6: 63 (Feb.) 1945.
2. RICHARDS, A. N., BORDLEY, JAMES, 3RD, AND WALKER, A. M.: Quantitative studies of the
composition of glomerular urine. VII. Manipulative technique of capillary tube colorimetry. J. Biol. Chem. 101: 179 (June) 1933. 3. WALKER, A. M., AND REISINGER, J. A.: Quantitative studies of the composition of gb-
merular urine. IX. The concentration of reducing substances in gbomerular urine from frogs and necturi determined by an ultramicroadaptation of the method of Sumner. Observations on the action of phborhizin. J. Biol. Chem. 101: 223 (June) 1033.
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4. WHSTFALL, B. B., FINDLEY, THOMAS, AND RIcHAiths, A. N.: Quantitative studies of the
composition of glomerular urine. XII. The concentration of chloride in glomerular urine of frogs and necturi. J. Biol. Chem. 107: 661 (Dec.) 1934. 5. WALHER, A. M.: Ammonia formation in the amphibian kidney. Am. J. Physiol. 181: 187 (Nov.) 1940. 6. BORDLEY, JAMEs, 3RD, AND RiCHARDS, A. N.: Quantitative studies of the composition of
glomerular urine. VIII. The concentration of uric acid in glomerular urine of snakes and frogs, determined by an iltramicroadaptation of Folin's method. J. Biol. Chem. 7.
101: 193 (June) 1933. 3RD, HENDRIX, J. P.,rn RIcHARDS, A. N.: Quantitative studies of the
BORDLEY, JAMEs,
composition of glomerular urine. XI. The concentration of creatinine in glomerular urine from frogs determined by an ultramicroadaptation of the Folin method. J. Biol. Chem. 101: 255 (June) 1933.
