V O L U M E 2 3 , NO. 1, J A N U A R Y 1 9 5 1 of thcsc Lmds arc' affectod Iiy the 11)-drosyl Ix~ndabeing partially superimposed on absorptions from the carbolcyl groups, but suficiently valid comparisons should bc possible because the interfercncc's are from the same ratio of carboxyl t o hydroxyl in both caws. SPECTRA OF PLANT-GROWTH REGULATORS
The. solvent mixtures dcscrihed here have been of considerable liclp in obtaining characteristic infrared patterns of 2,4-dichlorophcfinosyacetic acid and some related plant-growth regulating acids, as shown in Figure 3, for which no other suitable infraredtrarisniitting solvent could be found. The lower curve represents two identical curves of the stable and of the radioactive iodine 131-labeled 2,4-dichloro-5-iodophenoxyaceticacids, which wore synthesized in this laboratory ( I O ) . As only a very small proportion of the iodine was in the radioactive form, and this had largely decayed a t the time, no isotopic differences could be expected. The method was able t o prove with niicrosamples that the trvo preparations of the arid wcsre id~nticalexcept for the radioactivity. Thc CII bonds of these acids are massed relatively near the itcid grc~up,and some of the connecting linkages are conjugated. The CII bonds are also comnion to both the triethylaniine and the mmple. Therefore the CII hands are both distorted and t1ecw~:isedin height, and in two cases t.liey extend above the 100% transmission line. The carbon?-l bands appear to have been decrc:1secl i n peak absorption and broadened. The additional Ixintl a t 13.8 microns is prominent. The most characteristic difft~rrncc~s in the spectra of thesr substances appear between 11 :tnd 13 microns. The: esters or isolated triethylamine salts of such acids could
137 have been studied alternatively, but the method described has some special features to recommend it. I t is simple, rapid, and uses the product directly a t hand, without the need to prepare a derivative. The spectrograms represent more esactly the actual state of purity of the sample. Though a frlir distortional effects appear, the systcni of bands is considerably ~iinplific~il by the compensative removal of most of the coniplic!ntionn atltlt.tl l)y the drrivativr nttachment. LITERATURE CITED
(1) Barnes, R . B., Liddel, U., and Williams, V. Z., IND.E K C . CHEM., .ANAL. ED., 15, 659-709 (1943). (2) Bushwell, A. 31.,Rodebush, W. H., and Roy, RI. F., J . A m . Chem. Soc., 60, 223944 (1938). (3) Coggeshall, N. D., ANAL.CHEM.,22, 381-95 (1950). (4) Coggeshall, N. D., J . Am. Chem. Soc., 72, 2836-44 (1950). (5) Gore, R. C., Barnes, R. B., and Peterson, Elizabeth, .Ix.- have been reserved primarily for research, inasmuch ae the analytical results could be obtained only days later, when they had become of academic rather thnri practical value to the clitiician.
By t,he t,echnique of flame phot,oinetry, data on sodium and pot,:tsium concentrations in plasma, urine, or other biological fluids can l)e obtained in a few minute?. I3cJcause important differences h e t w r n norinal antl pathologicxl valiies may tie of small m:ignit,ude, high :tnal!~tic:il precision is required. The method ( 1 ) consiqtq of at,omizing R dilut,e solution of the sample and mising t,he arrosol obtained with the gas utilizcd by a Bunsen burner. A4sthe niist,ure of gas, air, and aerosol is ignited by the burner, light, of characteristic wave length is emitted by the test niaterial. T h e intensity of the emitted light is measured antl compared with that found for known standard solutions. .S more accurate technique is to add lithium as an internal standard to all solutions taken for analysis and then to read the ratio of podium or potassium to lit'hium (,?I. The original publications of Barnes and Berry and their associates (1, 5)set forth these essentials for accurate flame photom-
ANALYTICAL CHEMISTRY
138
Table I. Comparison of Flame Photometers Aeroaol Internal Type of Air SUPPLY Centristandard Atormzer to Burner fuge Fuel Photocells a n d Galvanometer, No External Current Supply Required 1. B a r c l h ~ Yea Hvoodermic Atmomhetie NO Proo~ne ikedles air 2. Bowman (6) Yes Adjustable Compressed No Propane sprayer air 3. For ( 8 ) Yes Adiusthble Atmospheric ' Yes Propane or SP We' am illurn. gas Electronic Phototubes and Amplifier, Stbbiliaed Extornal Current Required Compressed No Propane or 4. Beakman (14) No Glass" DrYge" , illurn. gP.8 CapiIiaW 5. Perliin-Elmer 18 No Glass: Atmospheric No Propane (4 IB 16. 16) earX11sry 2.m 6. P&kibElmer 52A (17) Yes Adjustable Atmowherim No Propane 01 acetylene *prayer all Compressed No Propane or 7. Weiohselbanm (29) No Oxygen", oxygen ill"rn. gas Venturi Model
'
Cabinet Metal Metal Wood
Metal
Metal Metal Metal
S a m D h are Doured in: this avoids transfirring ssmple, to small beakers from which they are drawn u p by suction. The threaded needle valve is graduated for regulating velocity of sample consumption, and is easily removed for cleaning by running a fine wire through t h e i n t e r n a l orifice. The atomizer sprays into the periphery of a centrifugal separator chamber ( 0 1 , Figure 2 lower) a d a p t e d f r o m a dedign of Bartholomew (8). This chamber collects and condenses large liquid droplets or particles, passing only very minute particles. The aerosol pro-
the base of the Meker burner. These parts are made of copper rather than glass to eliminate the danger of breakage and are securelv iastened in oosition. Cornnrewwl ir. . rlp~ m _.__ nlid ~~~~~~. .....sir -... to the sprayer-through a i r e s s u x regulator valve (such as the 40A line regulator made by the Harris Calorific Co.). When the compressed air is hewily contaminated with oil, z most effective method of removal is by means of the Turbo-Rotor air purifier (Model A l . Goodver Industries. 224 S0nt.h Mirhisnn AVO .~.~~. --.. . . . -. Chicago 3, Ill.). The propane gas fuel supply is controlled by 8: multistago regulator (92 SC, Harris Calorific Ca.). Recently, however, these instruments have been operated satisfactorily directly from the city gas in 10 different laboratories. The dual optical system and partitions are shown in Figure 2, upper. It is important to focus the fronesurfnced mirrors and ~
~
~~,
~
~~~~~
~
~~~~~~~.~~ ._........._.
the procedure d6scribed'below for making direct readings to obta& a pair that gives maximum and approximately equal galvanom-
JITHIUM FILTER
Figure 1.
Flame Photometer
Ownings in chimney are 1.8 X 5.0 cm. with oenter 5 cm. above burner
grid.
Compressed air pressure gage mounted on front panel
etry: a steady burning flame a t r&ively low temperature to avoid exciting other elements, obtained preferably with room air, gas, or propane, and a Meker-type Bunsen burner; elimins, tion of drafts, use of only the central part of the flame, and steady rate of atomization; avoidance of photoelectric devices (employing an external power supply), which me subject to drift and fatigue with an output that falls off for several hours and itre not itvailahle for immediate u8e a t d l times; utilization of the sup* rior internal standard technique; and almost exact linearity of the curve of closely spaced standards plotted against readings. As shown in Table I, which compares the available flame photometers, sufficient attention has not been paid to these important features in designing instruments for general use. DESCRIPTION OF INTERNAL STANDARD FLAME PHOTOMETER
The instrument (Figure 1) (built by J m k e Aircraft Engine Test Equipment Co., 38 Railroad Ave., Hackensack N. J.) is enclosed in a wooden box with asbestos board lining f h n g the flame and with the burner side oDen a t the bottom. The chimnev is made of coDDer and is set &th a louvre. This tvue of con-
1, air 64, fluid 2050, made by the'water Cooling Carp., Ne;, York, PIT. Y.) used upside down and fitted with a 4 e m . funnel.
SP~KhY-NOLFLL
Figure 2.
Schematic Diagram of Photometer
Upper. Center line of optioal path is 5 cm. above t o p of burner. Othe, dimenaions depend u ~ o nmountings of lenses mirrors s o d photocells. Lower. Chamber C1 is aDDrorimateiy 5 om.'in diamkter and 10 am. high. center outflow t u b e pmieots 6 cm. into ohsmber. Condensed liquid i; drained offa t bottom: B S C ~ Pof~ &em801VBDOI is prevented by having drain under 2 cm. of water. Chamber C2 is approximately sire and shape of 125-ml. Erlenmeyer Bask with neck expanded t o encircle base of burner. Aerosol inflow tube from C1 enters C2 tanpentially. 80 vapor 18 spiraled toward sir intake openings at base of burner.
V O L U M E 23, NO. 1, J A N U A R Y 1 9 5 1
\loo,
139
OUM
considerable sodium and some potassium, and because of deliquescence, the same batch of stock lithium nitrate solution is used in preparing the working standards shown in Table I1 and the unknowns for photometry. I n these solutions the lithium concentration is 500 p.p.m., which gives maximum precision and optimal sensitivity when sodium and potassium range up to 2 me. per liter. Stock solutions of sodium or potassium containing 50 me. per liter are made up from their C.P. chlorides, thoroughly dried ar 110" C. for several days or to constant weight, using w-ater free of these ions. More highly purified salts are obtainable ( 4 ) . T o prevent leaching sodium from the glass, a Xosolvit glass bottle (manufactured by T. C. Wheaton Co., Millville, N. J.) is used for the sodium stock Isohtion; borosilicate glass is used for the xvorking standard sodium solutions, which are renewed frequently.
POTENT IOMh? CI1
(COARSE)
PREPARATION OF SAMPLES FOR PHOTOMETRY
PHOTO CELL
Samples of plasma, ascitic or pleural fluid, or gastrointestinal drainage are prepared for photometry by pipetting 0.5 ml. of the sample (centriFigure 3. Wiring Diagram fuged if there is sediment) into a 100-ml. voluGalranoineter is spot light-type (such as Rubicon 3518), coil resistance 1800 ohms, C D R X metric flask freshly rinsed to remove any traces of 40,000 ohms, sensitivity 0.0007 microampere per mm. scale division, period 4 seconds. sodium, adding 10 ml. of stock lithium solution P-1 is General Radio 471A potentiometer equipped with 907L.4 gear drive 6-inch dial. Other components are standard radio parts from Lafayette Radio Co., Kew York, K.Y. from a buret, and then making up to volume with distilled w-ater. The utmost care in pipetting samDles and adding the lithium solution is essential: the final volume i i f a r less critical. Plasma potassium can be deterPter deflections behind the appropriate filters v,ith the 1.0 mined more easily by making lorrer dilutions (1to 50) if more samme. per liter standard and with the coarse adjustment a t 35,000 ple is available and if low values are suspected. For this purpose ohms (No. 7 on the coarse dial). The deflections are recorded 0.5 ml. of plasma and 2.5 ml. of stock lithium solution can be for future reference if deterioration is suspected. The author's used with a 25-mI. volumetric flask. (In one series of experidata indicate that the photocells employed do not fatigue; ments, to compare the relative accuracy of 1 to 25 and 1 to 100 the same pair has been in service in one instrument for 3 years dilutions for plasma potassium, the average deviation found for aithout signs of fatigue. No light must be allowed to leak seven samples was *0.013 and *0.005, respectively.) Depro around the filter or otherwise reach the photocells. I n the comteinization of plasma is neither necessary nor desirable, because pleted instrument the readings of a loiv concentration of either the internal standard compensates for changes in viscosity, pressodium or potassium must be unaffected by high concentrations ence of other substances, etc. ( S ) , and thus dilution errors, or of the other-e.g., the potassium reading of a 0.5 me. per liter those due to protein binding, are avoided. Urine samples are potassium solution must be the same as the potassium reading diluted 1 to 100 or 1 to 200, depending on their source. When of a solution containing 0.5 me. per liter of potassium and 15 very low concentrations have been found, more accuracy is obof sodium. This corresponds to a 1 to 10 dilution of plasma. tained by repeating x i t h lower dilutions, but in general 1 to 200 The 2-inch-square polished Corning glass filters for sodiuni dilution will avoid samples too concentrated to read within the (3482 and 9780) and for potassium (5850 and 2403) are mounted range of standards recommended. I t may lead to considerable 111 a bracket like a water wheel, facilitating the change to any error to read samples which fall above the highest standard by one of four different combinations by rotating the wheel. The merely extending the line nithout actually testing a standard filters fo- lithium (Corning 2404 and a liquid filter consisting of above the sample. 0.55 to 0.56 gram of cupric chloride dihydrate in 100 ml. of saturated sodium chloride sealed in a Corning flask 4480) are mounted permanently in front of the back photocell. The circuit (Figure 3) is designed to use a high sensitivity galvanometer and barrier layer photocells yhich require no elecTable 11. Working Standards for Photometer tronic amplifier. The simplicity of the circuit and its independ50 Millimolar Stock ence of external current sources contribute importantly to Final Concn. Sample Equivalent, (Including 100 1\11, of Stock operating stability. When used as an internal standard instruof Pia or K Diluted 1/200 Li Solution per liter) ment, the lithium emission is constant. Readings are made b y Me./l. Me./Z. Me./l. balancing a fraction of the lithium photocell output against the 1.0 20 200 sodium-potassium photocell output. For this balance the 0.75a 150 15 0.7" accurate linearly wound potentiometer, P-1, is used. P-2 140 14 0 . 6 5 = 13 130 adjusts the sensitivity by shunting current generated by the 0.60" 12 120 sodium-potassium photocell; this makes it possible to read more 0.5 100 10 0.25 concentrated solutions of sodium or potassium without changing 5 50 2 0 . l b 20 the concentration of lithium used. P-3, a small resistor in series 1 0.056 10 with P-1 across the lithium photocell, facilitates bringing the 0.25b 5 0.5 reading for the 0 me. per liter sodium-potassium solution near 0 0 0 zero on the balance dial, P-1. The instrument can also be a Solutions used for reading sodium in plasma diluted 1/?00. utilized for direct readings by employing the switches shown in b Solutions used for resding potassiuni in pla3nin diluted 1/200 t o 1 '50. the wiring diagram.
3-K
STANDARD SOLUTIOhS
The standard solutions used provide the absolute basis for all measurements, because the instrument determines the ratios of sodium or potassium to lithium. For this reason the greatest possible accuracy in preparing standards is mandatory. The lithium stock solution for internal standard measurements (5000 p.p.m., 49.85 grams of lithium nitrate per liter) is made up in large volume in 10 times the strength to be used in the samples. Because most samples of C.P. lithium salts are contaminated with
Tissues are analyzed after extracting either the fresh or the dried sample with ten or more times their weight of 0.75 N nitric acid a t room temperature (8, I S ) . (Khen nonprotein or protein nitrogen is to be determined, the wet tissue should be extracted with 3% trichloroacetic or 5% sulfosalicylic acids. Dried tissues are not as readily extracted with these organic acids.) The protein-free supernatant liquid is diluted 5 to 20 times with inclusion of one tenth the final volume of stock lithium solution. The internal standard will compensate for the acid and other ions present in the final solutions (3). From preliminary studies it arould appear that stools, diets, etc., can be prepared similarly,
ANALYTICAL CHEMISTRY
140 Table 111. Sodium Concentration, JIe./L. Flame Gravimetrica photometer
Comparative Plasma Sodium Analyses
Sodium Concentration, M e / L Flame Difference, Gravimetric" photomet,er % +1.2 138.2 137 -0.9 139.5 142.2 +0.7 +l.9 -4.2 142 140 -1.4 -2.8 157.1 153 -2.5 -0.9 169.5 162.5 -4.1 -4.4 131 132 +0.8 -3.7 133 134 -0.7 0 -2.2 diililicates + 2 . fi -1.1 -0.7 138 133 -3.8 T h e following are comparative plasma sodium analyses performed wlth .Janke Co inbtrument in lahoratory of Department of Pediatrics. New York Hospital-Cornel1 Medlcal Center +3.8 137 3 136.0 -0.9 -4.0 138.6 137.6 -0.7 14.7 139.2 138.6 -0.4 130.4 +1.8 139.0 -0.3 -2.2 139.8 138.1 -1.2 +1.7 140.7 140.0 -0.5 +3.1 141.3 143.6 +l.6 12.1 142.9 143.6 fO.5 143.1 -2.3 145.6 f1.7 -0.5 143.1 138.6 +3.1 +2.4 143.5 144.2 +0.5 +1.5 144.7 139.5 -3.6 +1.3 145.5 -0.3 145.0 +5.5 146.0 145.6 -0.3 0 146.9 140.4 -4.4 +2.3 149.1 148.2 -0.6 +1.6 183.4 182.0 -0.8 a .40 !.O-samples of plasma dry-ashed in Vycor crucibles with concd. HnSOd, as advised by Consolaaio and Dill in their modifioation (6) of ButlerTuthill zinc uranyl acetate method. Difference,
70
;!:;,
lii,
thereby avoiding the tedious ashing procrss ( 4 , fa); more detailed instructions will soon be published by Wallace et al. (f8). MEASURE.MENT OF SAMPLES
Set switches 1, 2, 3 in position for internal standard readings. Bring the galvanometer light spot to null (zwo in thc center of its scale). Light the burner and adjust the needle valve in its base to obtain the steadiest flame, approximately 10 cm. in height, with the small blue cones pulled tightly down to the burner grid. Turn on the compressed air to the atomizer to 10 pounds' pressure; this should not disturb the flame. Allow 0.5 hour warm-up. Atomize some standard solution slowly for another 0.5 hour to excite the photocells before starting readings. (Some of these instruments become stable with less warm-up. ) Set the balance dial which has 300 divisions, P-1, a t 210. Run the 1.0 me. per liter standard through the atomizer and bring the galvanometer to null by adjusting the sensitivity control, P-2. Set the balance dial a t 10. Run the zero standard through the atomizer and bring the galvanometer to null by adjusting the zero control, P-3. Reset the balance dial a t 210. Run the 1.0 standard again and, if necessary, readjust the sensitivity control. Overcareful adjustment of these controls initially is unneceseary, as their settings are only approximate. Having set the sensitivity and zero controls, obtain balance dial readings corresponding to each working standard listed in Table 11. These readings are most accurately made by rotating the balance dial back and forth so that the galvanometer light spot crosses and recrosses the middle zero line, then gradually diminishing the dial rotation until the galvanometer spot is held at zero. Readings are made to a precision of 1% of the number of dial divisions. When facility of operation is achieved, several independent dial readings can be made with a funnel of fluid (10 ml.) nithin 1 to 3 minutes. Readings should he made while the level of liquid in the funnel is more than 1 cm. above the stem. After the galvanometer is brought to the null point and the operator's hand removed from the balance dial, record the reading. The precision of the method is lost if the operator attempts to read the P-1 dial while balancing the galvanometer. Plot these dial readings against concentration of working standard on millimeter graph paper. (2 mm = 1 dial division or 0.01 me.) Failure to obtain smooth, almost linear curves results from error in preparing working standards, contamination berause of the ubiquity of sodium, or improper operation of the instrument, especially partial clogging of the atomizer. When the working standard solutions (Table 11) are found to give reproducible readings and the graph of readings against concentration is linear for potassium and practically linear for sodium, read unkno-m solutions in a similar manner. The dial
readings obtained arc approximately the milliequivalents per liter in the samples when the above procedures are followed and the samples diluted 1 to 200. Significant error may be introduced, however, if too few standards are used to construct the curves. For maximum accuracy, bracket each unknown by standards that read near the dial setting found for the unknown. Should changes ocrur in the readings of these standards, reset the 0 and 1.0 me. per liter (or maximal) solutions with zero and sensitivity controls. This procedure usually restores the readings of intermediary solutions to their former values. OPERATION 4s DIRECT RE4DING INSTRUMENT
Set the switches for direct readings. Move the lever on the galvanometer box to bring the spot to zero (at the left of its scale) with distilled water running through the atomizer. Run the 1.0 me. per liter standard through the atomizer and adjust the coarse and fine controls so that the galvanometer spot light deflects to 100 on its scale. Run water through again and readjust the galvanometer lever to bring the spot back to zero. Recheck the 1.0 solution. Olitain galvanometer deflections corresponding to intermediate standards. Note that sodium standards containing lithium will give erroneous direct-reading values for sodium because of sodium contamination of lithium salts. Detailed instructions regarding this method have been published by Hald (12).
Table IV.
-4nalyses of Weighed Samples by Flame Photometer
KC1 Weighed O u t M g /1. 80 70 60 50 40 30
.
-
Found 1.07 0,945 0.81 0.68 0.54 0.40
Concentration Calculated Difference M e . per Mer 1.07 0.938 +0:607 0.804 fO.007 0.671 fO.007 0.536 +0.004 0.402 -0,002
Error
% 0 0.75 0.87 1.04 0.74 0.50
This technique is primarily useful for verifying the amount of lithium added to all internal standard solutions and for other lithium analyses, and for estimating the composition of unknown samples prior to their preparation for internal standard photometry. This method is not further recommended now because it is subject to many sources of error.
V O L U M E 23, NO. 1, J A N U A R Y 1 9 5 1 Table V.
141
Sodium Analysis of a Series of Unknowns and Standards to Indicate Reproducibility
Standards, hle./L. 2.0 1.75 1.25 1.0 0.75 0.70 0.60 0.50 0.30 0.10 0 Unknowns A1 2 B1 2
c1
2 D1 2 El 2 F1 2 x1 2
Dial Readings0 359,360 322,322 242,242 200,200 155,154 142,142 122,121 107, 108 68. 67 27, 25 5, 5 140.149 30, 30 181, 183 27, 29 113,115 21, 24 141,143 32. 33 165, 168 127, 127 77, 78 17, 19 98, 96 62, 62
F i w t rradirirs m a d e eerially and then srrond Prt of t h e v riins. ('
Unknowns K1 2 3
Dial Readings" 174,175 287,291 171,170 210,212 4 235,236 5 122,125 6 152, 152 7 181. 182 8 125,126 9 175, 173 10 153, 154 L1 201.202 2 181, 182 3 71, 71 4 212,212 5 123, 123 0 229.227 7 235,235 8 259,260 9 292,295 10 113, 116 Y1 138, 140 2 185, 185 3 195,198 4 233.234 5 188.189 6 240,244 7 238, 2.52 8 9 1,1,173 readings made after rechecking standards. No adjustments of inrtriinient were made during
Tahle VI. ,ACCUR.ACY
Effect of Variation in Pressure of Compressed 4ir on Analvses
C.'ompnrieoris from two laboratories of the analytical results ot)tnined on samples of plasma by a gravimetric method and by flame photometry are presented in Table ITT. The micromethod involved precipitation of sodium as sodium uran;vl zinc acetate, uftcr preliminary dry ashinx to remove protein and phosphate
Compressed .4ir Pressrire. L h .
(6). >lore rigorous evaluation of the accurary of this instrument \viis ohtairied by analyses of known amounts of salts. Samples of
8 7 6 5 4 3 11 I2 13
pot,assinm chloride rvere weighed out and transferred to 500-ml. volumetric flasks. -4 large batch of n 1 to 10 dilution of tht. stock lit,hium solution was then made u p and used to dilute thew weighed samples to volume. These solutions were then re:d :is unknowns against t,he working standards, as prepared in Tul~le11. The results given in Tahle TI' show significantly greal vu a.ccuracy than has Ix=w at,tained with direct-reading instri1ment.s ( 1 , .$, 14-16, 19). In addition, mnny such analyses of 1cnon.n salts have hcen performcd by Be et a ( . ( J ) , who reported 311 (srror of * 1 %. Simulntetl l)ioIngi(~:ilwlutiorw of known salts u i ~:i,n:ilyzed i ~ ~ ~ with an errnr of = t l . O ~1)y Ka1l:tc.e pf al. (18), \ v h o iisc:d :in c:trliclr niotlel of this inst rrini~rit. 111view of the emphasis that has been placed on stability of oper:i tion, freedom from drift, and reproducibility of r e s u h , the data in Table V show how the readings of a long series of unknown solutions and standards repeat themwlves under actual opc,rnting conditions. The range of variation in these duplicate annlyses is significantly less than that ohtainahle with instrumentg of very different design. as reportrtl srvernl investigators C.P.
( I , 4 , 14-16). SENSITIVITY
One of the factors that enables good reproducibility to be obtained with this instrument is the alert response of the galvanometer when the balance dial is moved away from the correct reading. Under optimal operating conditions, a 1% change in the halnnce dial setting causes the galvanometer to deflect approximately 0.3 mm. from zero. Measured another way, a 1-mm. galvanometer deflection from null is produced by turning the balance dial 6 t o 8 divisions off balance. SOURCES OF ERROR
Effect of Fluctuation in Air Pressure. "sing the internal standard instrument described, dial readings were taken on a
10" 9
loa 9 8
7 11 1" 13
?odium .Me. per l i f e r
Pornspirim
.4. Internal Standard Instrunirnt 1 00 0 75 0 50 0 25 0.75 0.25 0.74 0.98 0.74 0.49 0.72 0.74 0.49 0.2ti 0.98 0.70 0.74 0.97 0.48 0.27 0.67 0.97 0.75 0.49 0.28 0.64 0.30 0.76 0.99 0.49 0.61 0.99 0.81 0.55 0.38 1.10 0.90 0.63 9.65 ... 0 . 25 0.77 1.01 0.50 0 7.5 0 20 0 76 0.78 0.51 I .02 0.79 0.77 1.04 0.52 0 27 B. 1.00 0.83 0.79 0.64 1.07 1.10 1.14
Pressure Burner, iYa
0 50 0.47 0.42 0.45 , . .
0.41
... ... ...
... ...
Direct-Reading Instrument 0.75 0.50 0.25 0.65 0.39 0.20 0 . 15 0.31 0.58 0.24 0 51 0.10 0 81 0.56 0.20 0 86 0.60 0.31 0.02 0 91 0.33
a Initial readinga on standards made a t 10-lb. pressure: readings a t other pressures referred t o these base line values. Below 5 Ih. indirect readings are sluggish; rate of atoriiization is slowed considrrahly.
series of solutions at 10 pounds of air pressure, illuminating gas, and Kith the sample atomizer flow a t maximum speed. The air pressure was then reduced by 1-pound increments and the same solutions were read again. The changes in the readings are listed in Table V1,A. For comparison with the pressure burner and atomizing system used in some other instruments-e.g., 2 in Table I-the burner and aerosol expansion chamber, C-2, were then removed and a propane pressure burner was substituted. This type of burner receives most of its air supply under pressure from the atomizer: it Kill not burn properly unless compressed air is supplied to the atomizer. The 0.5 me. sodium solution was read a t each reduced pressure and t h e results are shown in the last column of Table VI,A. With the pressure burner there is considerable error n-ith small changes in air pressure (1 t o 2 pounds): in contrast, the burner and expansion chamber employed in this instrument minimize the error from air pressure fluctuations. This has been pointed out previously ( 1 ) : i t is emphasized because of the practical difficulties of absolute regulation of pressure and velocity of a n air supply. I n places where tho room air is contaminated with the test elements, a cloeed system with purified compressed air ivould be essential.
142
These tests were also made with the regular atomizer and burner, but with the direct-reading circuit. The considerable error introduced is shown in the data in Table V1,B. This illustrates one of the most serious defects of photometers (4,5,7in Table I) which do not utilize ( 1 4 ) the internal standard. Similar results were obtained with propane. Effect of Changes in Gas Pressure. The practical independence of the internal standard method to changes in gas pressure has been described (3). I n this instrument negligible change in internal standard readings resulted from 10 to 20% changes in propane pressure and even extreme reduction from 4 pounds to 1 pound caused only a 1% change in the readings. For this reason the city gas supply has been used without intervening regulators and is yielding satisfactory results in ten laboratories. Direct readings, however, changed 2 to 5 % with 7.5% reduction in propane pressure. With other types of burner and atomizer. more marked effects were observed (3,14). Effect of Change of Rate of Flow of Sample. Reducing the flow v.-ith the atomizer needle valve from maximum (25 ml. per minute) almost to the minimum (3 to 5 ml. per minute), which still gave color to the flame and satisfactory readings, caused from 0 to +2’% difference in the dial readings on the internal standard instrument. On the direcbreading instrument, reduction in rate of flow resulted in up to 22% change in the galvanometer readings. These data demonstrate another important advantage of the internal standard method, especially since biological samples frequently cause partial clogging and unnoticed changes in rate of flow through the narrow atomizing orifice. .itomizers constructed of hypodermic needles or glass capillaries have been found unsatisfactory because of clogging, change in droplet size, and difficulty in cleaning. Effects of Other Substances. The presence of other ions or substances which do not themselves emit light in the spectral region of the alkali metals affects their emission spectrum under the excitation conditions used in the flame photometer (3). The chief effect is to depress the amount of light emitted by the ion being measured, although copper and some organic substances may augment the emission. (Other exciting conditions-e.g., air or oxygen-acetylene or gas flames-may produce different interfering effects.) The magnitude of the effect is influenced by the temperature of the flame, rate of atomization, concentration of both the interfering substances and the ions being measured, and other unknown factors. All these variables are held constant by the internal standard technique (S), which measures the emissions of the unknown ion (sodium or potassium) and the reference ion (lithium) simultaneously in the same solution. Because the emission spectra of the alkali metals within fairly a i d e limits are similarly affected by the interfering substances in biological samples, the ratio of lithium emission to sodium or potassium emission is essentially unchanged (3); thus it is unnecessary to prepare standards with added glucose or phosphate as suggested (14). Because of the variable effects of other ions which may depress or enhance readings ( 3 ) , substances present in significant concentration in samples other than plasma, blood, urine, or tissue eytracts should be studied for possible interference. Substances which emit light in the region of sodium or potassium will appear to enhance the readings; substances which emit light in the region of lithium will appear to depress the readings. These interference effects must be carefully distinguished from light leaks or other defects in the optical system resulting from faulty construction or inadequate filters. These are usually the cause for enhancement of potassium readings by high concentrations of sodium. The opposite sides of the flame are conveniently used for energy measurements. The slots in the chimney admit only the central part of the flame. It is not important that the two sides be equal but their ratio should remain constant. The data in Table V indicate that during operation this ratio remains constant. In practice, partial obstruction of the gas orifice or salt deposits on
ANALYTICAL CHEMISTRY the burner grid cause fluctuations in readings. This difficulty is readily corrected by periodic cleaning of these parts. SUMMARY
A description is given of the design, construction, and operation of an internal standard flame photometer evolved from the original instrument (3)over a period of 4 years. This instrument has been used for a variety of electrolyte investigations (7-11 ). Data are given to indicate the accuracy, precision, and stability of the photometer. Fifteen copies have been constructed and are now operating satisfactorily in other research and hospital laboratories. ACKYOWLEDGMENT
Grateful acknowledgment is made to R. B. Barnes, V. Z. Killiams, and particularly John Berry of the American Cyanamid Co., Stamford Research Laboratories, for their advice in building the first instruments; to John Kieffer for help in constructing the first instruments, to JacquelineIsola for her cooperation inperforming many experimental tests, and to Samuel Raymond for valuable assistance in designing the aerosol centrifuge and in the preparation of this manuscript. Special acknowledgment is made to Betty Freeman for the gravimetric analyses and for patiently conducting the many tests with various instruments and modifications leading to the development of this model. Sincere thanks are expressed to Henry Barnett of the Sew Tork Hospital-Cornell Medical Crnter for contributing the comparative analyses in Table 111. LITERATURE CITED
Barnes, R. B., Richardson, O., Berry, J. W.,and Hood, R. L . , IND.ENG.CHEM.,ANAL.ED., 17, 605 (1945). Bartholomew, W.H., ANAL.CHEM.,21, 527 (1949). Berry, J. W.,Chappell, D. G., and Barnes, R. B., IND.E m . CHEM.,ANAL.ED., 18, 19 (1946). Bills, C. E., McDonald, F. G., Niedermeier, W., and Schwarte, M. C., ANAL.CHEM.,21, 1076 (1949). Bowman, R. L., and Berliner, R. W.,Federation Proc., 8 , 14 (1949).
Consolazio, W. V., and Dill, D. B., J . Biol. Chem., 137, 587 (1941).
Fox, C . L., Jr., J . Am. Med. Assoc., 124, 207 (1944). Fox, C. L., Jr., and Baer, H., Am. J . Physiol., 151, 155 (1947). Fox, L., Jr., and Freeman, E. B., Federation Proc., 8, 19 (1949). Fox, C. L., Jr., Friedberg, C. K., and White, 4 . G., J . Clin. Innest., 28, 781 (1949).
Fox, C. L., JF., and McCune, D. J., Am. J . Med. Sci., 216, 1 (1948); Pediatrics, in press. Hald, P. If.,J . Bid. Chem., 167, 499 (1947). Lowry, 0. H., and Hastings, A. B., J . Bid. Chem., 143, 257 (1942).
Mosher, R. E., Boyle, A. J., Bird, E. J., Jacobson, S.D., Batchelor, T. M., Iseri, L. T., and Myers, G. B., Am. J . Clin.Path., 19, 461 (1949); ANAL.CHEM.,22, 782 (1950). Overman, R. R., and David, A. K., J. Bid. Chem., 168, 2 (1947).
Parks, T. D., Johnson, H. O., and Lykken, L., ANAL.CHEM., 20, 822 (1948).
Toth, S. J., Prince, A. L., Wallace, A , , and Mikkelsen, D. S., SoilSci., 66, 459 (1948).
Wallace, W. M., Holliday, M.,Cushman, M., and Elbinton, J. R., J . Lab. Clin. Med., in press. Weichselbaum, T. E., and Varney, P. L., PTOC. SOC.Exptl. Biol. Med., 71, 470 (1949). RECEIVED April 10, 1950. Aided by a grant from the Division of Research G r a n t s and Fellowships, National Institutes of Health, U. 8. Publio Health Service.
