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The determination of sodium and potassium in biological fluids with the dual channel ultramicro flame photometer
ANALYTICAL
BIO(‘IIEILIISTRT
The Determination Fluids
1,
17-22
jl%i())
of Sodium with
the Flame
and
Potassium
Dual Channel Photometer”’
in Biological
Ultramicro
The presentt trend of l~iot*hemic~:d and pathologioul inquiry is t,o demand the analysis of smaller and smaller amounts of mat~erial. The newssity for determining sodium and potassium ill very small amounts led us to attempt analyses by ultramicro flame photomet,er. Although our immediate problem dealt with analyses of renal tubule fluid ill amphibia as well as in ~n~mun:ds (l-3), we wished to have an instrument capable of analyzing biological fluids in general. Since the instrument described by Rnmsay, Brown, and Falloon (4) in England was designed and used for the simultaneous dekrmination of micromicromoln (lesst#hanmillimicrogram) amounts of sodium and potassium in hiologkal fluids, it was devided to try a modification of t,his instrument for our work in which, in addition to tulmle fluid, 1)lood serum and urine were to he analyzed. In Ramsay’s procedure an ultramicro sample cf fluid was placed upon :I platinum loop, dried in ZL heated (wil of wire, :tnd t.hc pl:tkinum loop thwt thrust into an air-gw fkmc. The emission from the flame entered a spcctrometer, the spevt,rum fmn which fell upon :I, mask having two slits, one passing the potassium douhM and the ot,her the sodium doublet. These emissionsreached phot~om~~lt,iplicrtub whkh were connwted to integrating circuits for met,er presentation. The maximum deflwtions were maintained sufficiently long t,o obtain readings.
G~~nural. Our of Philadelphia.
modified instrllment was bllilt t)y the American Electronic Laboratories It involws the same principles :ts that dcscribcd hy Ramsay rt rd. (4)
18
BOTT
but differs in some detail. .-\. cotnpletc description of thr inst.rument proper will be pill)lishrd elsewhere by Mr. John Buss& of that rompany. Figllre 1 shows the instrltmrnt, complete cscept for the air pump. To the left are the pressure gaugrs for propane and air which arc kept at 4 + 0.02 in. water tlv a series of regulators. Meters for potassium and sodium readings are at, the right-hand end of the case, and below each meter art’ controls for drift, dark current,, and sensitivity adjustments. :2bovr the% right-hand meter is t.he multiposition switch which controls the operation of the instrument. In Fig. 2 thr mot.or-driven sample-rarrier and cover arms are shown spread apart,, in position to push the platinum loop into the heating coil. On signal from t,he multiposition switch, the arms move together until they reach the flame whcrcx the sample-carrier is stopped and the rover proceeds on, allowing a flash of light, to t)tl rc>cGcd throllgh the yuartz rondensing lens. The arms then reverse their direction under the control of micro switches which work automaticSally oriw motion has been st:LrltBti.
011
FIG. 1. Wide-angle the shelf below.
photograph of dual channel See text for description.
flame
photometer
with
power
supplies
The condensing lens renters the light on the 50-p entrance slit of a spectrograph containing a special grsting from which the spectrum is reflected. Slits in a mask, which is installed in place of the film strip of the original spectrograph, allow the light to reach the two photomultiplier tubes and current from these is lead to the integrating circuits. Plotinrcm Loop and Burning 7’imr. With the Z&gauge platinum wire loop and a l-se< burning time used in our earlier work, there was a pronounced effect of sodium on pot’assium readings. At this stage it was necessary to use an elaborate set of standardization curves in order t,o correct for sodium content (2). Later Dr. Ratnsay informed us that he was using a finer wire and a longer burning time than first described in order to prevent accumulation of salts on the wire. In our experience, changing to a loop (approximately 0.7 mm I.D.) of 31 or S&gauge wire attached to a heavier stem and increasing the burning time to 2 set not, onlv markedly decreased the accumulation of salts on the wire but a Present
address:
Franklin
Institute,
Philadelphia,
Pa.
also eliminated, essentially, t,he cffrct of sodium upon potassium when wires became roughened from long use they l~rgan to ahsorh discarded.
Fro. 2. Close condensing Icns
view of motor assembly and is in upper left-hand eorn(‘r.
burner.
Drying
unit
readings. However, salt and had to 1~.
is at left
and quartz
/Mizwry oJ‘&mp~rs to Ihe Loop. Samples of standard solutions or diluted serum, tubule fluid, or urine were delivered to the loop from Wigglesworth type pipets (5) made of quartz antI delivering, usdly, npprosimately 0.2 JAI. The piprt. was :tt.tached to a. mirrosyringe which was in turn fastened to a micromarlipl~lator. It was tilled by rapill:wit>., eit.her by dipping into a solution or from a xtorsgcl pip& Delivery of the snmplc was made either to a dry wire or into a larger drop of water or solution. When the material t)o be analyzed contained much prokin, best results were obtained 1)~ pulling some of t.hc mixture back into the pipet in order to rinse it. This, of course, newssitatcd the use of the same technique for all st,andnrds and unknown fluids used in the series. When substances which interfered wit.h the readings for sodium and/or potassium were prcaent~ in the biological solutions 1 mmolnr diahasir ammonium phosphate sohtt,ion was substituted for the water drop. This technique was found neces-
4 These
items
may
be obtained
from
the Bishop
Platinum
Works,
~Ialvwn,
Pa.
20
BOTT
sary in analysis of fluid from mammals even when these WWP wnsidrrably though separ:ttc standardizations were rrwess:try because of thts interfering phosphahc, errors tl~w t,o phosphatr, sulfate, and othrr sul)st:rnws werr “swampc’d” in this wry as suggested by Ramsny ct ccl. (4). RESIV,TS
AND
diluted. -\Ieffect, of t.hcs found to br
DISCUSSION
On standard solutions of pure potassium and sodium chloride, standardization curves were linear over ranges varying almost, 1000fold, although ranges usually used in analysis of biological fluids required curves with only I-loo-fold variat’ion in concentration. In order to t,est t’he reproducibility of readings at8lowest Icvelr, C ‘1 number of trials were made with solutions just distinguish:lble from wtJer5 in the amounts of pipetted to the bare wire. Figures for a typical trial seriesof seven wnsecutive readings on a solution containing 0.01 mmole each of potassium and sodium per liter were obtained using a pipet delivering approximately 0.2 ~1, t,he total amount per sample therefore being approximately 2 ~~moles each of potassium and sodium or 8 X 10es mg potassium and 4.6 X 1OP mg sodium. Readings for potassium and sodium were made simultaneously. For the potassium meter readings a mean of 3.3 was obtained, with a standard deviation &0.27 amounting to about Sa/c. The mean for distilled water was 0.5, and the difference which was due t’o potassium alone, 2.8. Putting the figures in a form similar to that, used by Kamsay for comparisolr t.his indicates that the st,andard derint,ion in t,erms of potassium was 8 x 10--y x g
= h7.7 x IO-”
mg.
In the case of sodium, alt,hough the difference btltween readings on water (mean 3.2) and a 0.01 mmole per lit,er solution (mean 37.0) was much greater than that for pot8assium,the serieswas marred by one anomalous reading. Even with t,his the standard deviation (43.10) in terms of milliErams was 4.6 x 1OV x 2
= h4.2 x 1OY’.
With these extremely small amounts t,he deviations were similar t)o those of Ramsay et nl. for sodium and about, half as great in t,he caseof pot)assiunt. In thin low range the factor producing the largest errors was the signal-tonoise ratio. Our experimental work on renal tubule fluid did not’ require t’hr determination of t#heseextremely small amounts. With the larger amounts usually used in experiments (often 0.1-3 mpg potassium and l-50 mkg sodium) this ratio improved and the standard i This greater.
limit
:~pplies
t,o pot:rssium
:ts thtx wrlsitivity
to sodium
could
have
been made
deviat,ion of individual readings for solutions of potSassium and sodium chloride was approximately 3yG. Since readings were made at least in duplicat,e, the errors were less than this. Tklivcry of the sample t’n t’hc xire was here the largest source of error. 111t,hc wse of pl’ot~?itI-(:oIlt:l.illillg solut,iolls like serum t.his was wpecially trouhksume and rinsing te~huiciue WH used. Ta.hle 1 show a comparison of rwults ol)tjnincd OIL \wiolls biological fluids hy t,he ultramicro and m:wro met hods. ;1I:\(~ro (lctc~I’nlill:~t,iolls ww~ made hy tr~hnioal assistSants using t#hc instrument and method tlesc:rih~~tl by Fox (6). For thr ultramicro analysis ~r~ioo-~~il~tc~l 0.2+1 s:tnqAes wc’w used. The dilut8ion for amphibian (Sc~t~~cs) serum RW 30 tSinw :~nd t*h:tt# for pooled mammalian (rat) swum \v:ls 20 t’imes or more. ~laddor urillc of AVxturus wa,sso low in salt wntentS t,h:rt’ it \~a:: used \vithout dilution. The fig~lrw prrsented are t,he ;~~cwgcs uf two or thwc wadilrgs. Samples of
pooled serum from Xectrcms were analyzed using a 31- or 36-gauge wire loop and a 2-set burning time. For rat serum the fine \vire loop plus t,hc ammonium phosphate rinring t~echniquowrc used. On serum the greatest difference hebveen rnbults of macro and ultramicro methods was 5(,7! fol potassium and 37; for sodium X sodium det,ermilu~t,iollmade on rat, serum #3 by Dr. Judith Litohfield of this depa.r*tment.x?th the clectxocle dtwrihed by Eisenman et al. (7) yielded a value of 125 mmole per liter. 011urine the percentage difference was larger hut, the :lhsolut,c difference was eswedingl~ small (0.04 mmole per lit,er for potassium and 0.2 for sodium). Itnmsny and his co-workers did not show c*ompsrisonsof macro and ultmmicro analyses nor did they deal with solut8ionscontB:rining as much proteiu as mammalian serum. For this reason a comparison of result,scannot, 1~ made completely, but, it is probable t,hat, stahilitjy and control have hcw~improved in the modified insbwncnt. The :rpparat8us offers no ad\-antage over standard flame photometers for routine work iu which material is abundant.
22
BOTT
However, in those cases where only minute amounts of material are available, the dual channel ultramicro flame photometer should be generally useful. It has been used in scores of determinations of renal t,ubule fluid and serum and should lend itself equally well to the analysis of other body fluids limited in amount. This applies also to extracts of bits of tissue such as are obtained in biopsies. ACKNOWLEDGMENTS The author wishes t,o arknowledge Mills who preparrd many solutions
the assistance and ran macro
of Miss analyses
Jane Cohen as controls.
and
Miss
Sarah
A modification of a dual-channel ultramicro flame photometer is described. It was built for analysis of very small amounts of biological fluids including that from renal tubules and determines potassium and sodium simultaneously in amounts as low as 2 pq~oles. The author’s experience in the use of the modified instrument is described. It should be generally useful for analysis wherever the sample supply is very limited. REFERENCES 1. BoTT, P. A., P&ration Pi-or. 14, 185 (1955). 2. BOTT, P. A., Fe&ration Proc. 16, 223 (1956). 3. BOTT, P. A., in “8th Ann. Conf. on the Nephrotic Syndrome, 1956” (J. M&off, p. 39. National Nephrosis Foundation, New York, 1957. 4. RAMSAY, J. A., BROWN, R. H. J., ANU F~r,~oos, S. W. H. W., .J. Exptl. Biol. (1953). .5. WIGGLESWORTH, V. B., Biochfm. J. 31, 1719 (1937). 6. Fox, C. L. JR., Anal. Chem. 23, 137 (1951). 7. EISENMAN, G., RUIIIS, D. (I., ANI) CASBY, J. U., Science 126, X31 (1957).
cd.), 30, 1
BIO(‘IIEILIISTRT
The Determination Fluids
1,
17-22
jl%i())
of Sodium with
the Flame
and
Potassium
Dual Channel Photometer”’
in Biological
Ultramicro
The presentt trend of l~iot*hemic~:d and pathologioul inquiry is t,o demand the analysis of smaller and smaller amounts of mat~erial. The newssity for determining sodium and potassium ill very small amounts led us to attempt analyses by ultramicro flame photomet,er. Although our immediate problem dealt with analyses of renal tubule fluid ill amphibia as well as in ~n~mun:ds (l-3), we wished to have an instrument capable of analyzing biological fluids in general. Since the instrument described by Rnmsay, Brown, and Falloon (4) in England was designed and used for the simultaneous dekrmination of micromicromoln (lesst#hanmillimicrogram) amounts of sodium and potassium in hiologkal fluids, it was devided to try a modification of t,his instrument for our work in which, in addition to tulmle fluid, 1)lood serum and urine were to he analyzed. In Ramsay’s procedure an ultramicro sample cf fluid was placed upon :I platinum loop, dried in ZL heated (wil of wire, :tnd t.hc pl:tkinum loop thwt thrust into an air-gw fkmc. The emission from the flame entered a spcctrometer, the spevt,rum fmn which fell upon :I, mask having two slits, one passing the potassium douhM and the ot,her the sodium doublet. These emissionsreached phot~om~~lt,iplicrtub whkh were connwted to integrating circuits for met,er presentation. The maximum deflwtions were maintained sufficiently long t,o obtain readings.
G~~nural. Our of Philadelphia.
modified instrllment was bllilt t)y the American Electronic Laboratories It involws the same principles :ts that dcscribcd hy Ramsay rt rd. (4)
18
BOTT
but differs in some detail. .-\. cotnpletc description of thr inst.rument proper will be pill)lishrd elsewhere by Mr. John Buss& of that rompany. Figllre 1 shows the instrltmrnt, complete cscept for the air pump. To the left are the pressure gaugrs for propane and air which arc kept at 4 + 0.02 in. water tlv a series of regulators. Meters for potassium and sodium readings are at, the right-hand end of the case, and below each meter art’ controls for drift, dark current,, and sensitivity adjustments. :2bovr the% right-hand meter is t.he multiposition switch which controls the operation of the instrument. In Fig. 2 thr mot.or-driven sample-rarrier and cover arms are shown spread apart,, in position to push the platinum loop into the heating coil. On signal from t,he multiposition switch, the arms move together until they reach the flame whcrcx the sample-carrier is stopped and the rover proceeds on, allowing a flash of light, to t)tl rc>cGcd throllgh the yuartz rondensing lens. The arms then reverse their direction under the control of micro switches which work automaticSally oriw motion has been st:LrltBti.
011
FIG. 1. Wide-angle the shelf below.
photograph of dual channel See text for description.
flame
photometer
with
power
supplies
The condensing lens renters the light on the 50-p entrance slit of a spectrograph containing a special grsting from which the spectrum is reflected. Slits in a mask, which is installed in place of the film strip of the original spectrograph, allow the light to reach the two photomultiplier tubes and current from these is lead to the integrating circuits. Plotinrcm Loop and Burning 7’imr. With the Z&gauge platinum wire loop and a l-se< burning time used in our earlier work, there was a pronounced effect of sodium on pot’assium readings. At this stage it was necessary to use an elaborate set of standardization curves in order t,o correct for sodium content (2). Later Dr. Ratnsay informed us that he was using a finer wire and a longer burning time than first described in order to prevent accumulation of salts on the wire. In our experience, changing to a loop (approximately 0.7 mm I.D.) of 31 or S&gauge wire attached to a heavier stem and increasing the burning time to 2 set not, onlv markedly decreased the accumulation of salts on the wire but a Present
address:
Franklin
Institute,
Philadelphia,
Pa.
also eliminated, essentially, t,he cffrct of sodium upon potassium when wires became roughened from long use they l~rgan to ahsorh discarded.
Fro. 2. Close condensing Icns
view of motor assembly and is in upper left-hand eorn(‘r.
burner.
Drying
unit
readings. However, salt and had to 1~.
is at left
and quartz
/Mizwry oJ‘&mp~rs to Ihe Loop. Samples of standard solutions or diluted serum, tubule fluid, or urine were delivered to the loop from Wigglesworth type pipets (5) made of quartz antI delivering, usdly, npprosimately 0.2 JAI. The piprt. was :tt.tached to a. mirrosyringe which was in turn fastened to a micromarlipl~lator. It was tilled by rapill:wit>., eit.her by dipping into a solution or from a xtorsgcl pip& Delivery of the snmplc was made either to a dry wire or into a larger drop of water or solution. When the material t)o be analyzed contained much prokin, best results were obtained 1)~ pulling some of t.hc mixture back into the pipet in order to rinse it. This, of course, newssitatcd the use of the same technique for all st,andnrds and unknown fluids used in the series. When substances which interfered wit.h the readings for sodium and/or potassium were prcaent~ in the biological solutions 1 mmolnr diahasir ammonium phosphate sohtt,ion was substituted for the water drop. This technique was found neces-
4 These
items
may
be obtained
from
the Bishop
Platinum
Works,
~Ialvwn,
Pa.
20
BOTT
sary in analysis of fluid from mammals even when these WWP wnsidrrably though separ:ttc standardizations were rrwess:try because of thts interfering phosphahc, errors tl~w t,o phosphatr, sulfate, and othrr sul)st:rnws werr “swampc’d” in this wry as suggested by Ramsny ct ccl. (4). RESIV,TS
AND
diluted. -\Ieffect, of t.hcs found to br
DISCUSSION
On standard solutions of pure potassium and sodium chloride, standardization curves were linear over ranges varying almost, 1000fold, although ranges usually used in analysis of biological fluids required curves with only I-loo-fold variat’ion in concentration. In order to t,est t’he reproducibility of readings at8lowest Icvelr, C ‘1 number of trials were made with solutions just distinguish:lble from wtJer5 in the amounts of pipetted to the bare wire. Figures for a typical trial seriesof seven wnsecutive readings on a solution containing 0.01 mmole each of potassium and sodium per liter were obtained using a pipet delivering approximately 0.2 ~1, t,he total amount per sample therefore being approximately 2 ~~moles each of potassium and sodium or 8 X 10es mg potassium and 4.6 X 1OP mg sodium. Readings for potassium and sodium were made simultaneously. For the potassium meter readings a mean of 3.3 was obtained, with a standard deviation &0.27 amounting to about Sa/c. The mean for distilled water was 0.5, and the difference which was due t’o potassium alone, 2.8. Putting the figures in a form similar to that, used by Kamsay for comparisolr t.his indicates that the st,andard derint,ion in t,erms of potassium was 8 x 10--y x g
= h7.7 x IO-”
mg.
In the case of sodium, alt,hough the difference btltween readings on water (mean 3.2) and a 0.01 mmole per lit,er solution (mean 37.0) was much greater than that for pot8assium,the serieswas marred by one anomalous reading. Even with t,his the standard deviation (43.10) in terms of milliErams was 4.6 x 1OV x 2
= h4.2 x 1OY’.
With these extremely small amounts t,he deviations were similar t)o those of Ramsay et nl. for sodium and about, half as great in t,he caseof pot)assiunt. In thin low range the factor producing the largest errors was the signal-tonoise ratio. Our experimental work on renal tubule fluid did not’ require t’hr determination of t#heseextremely small amounts. With the larger amounts usually used in experiments (often 0.1-3 mpg potassium and l-50 mkg sodium) this ratio improved and the standard i This greater.
limit
:~pplies
t,o pot:rssium
:ts thtx wrlsitivity
to sodium
could
have
been made
deviat,ion of individual readings for solutions of potSassium and sodium chloride was approximately 3yG. Since readings were made at least in duplicat,e, the errors were less than this. Tklivcry of the sample t’n t’hc xire was here the largest source of error. 111t,hc wse of pl’ot~?itI-(:oIlt:l.illillg solut,iolls like serum t.his was wpecially trouhksume and rinsing te~huiciue WH used. Ta.hle 1 show a comparison of rwults ol)tjnincd OIL \wiolls biological fluids hy t,he ultramicro and m:wro met hods. ;1I:\(~ro (lctc~I’nlill:~t,iolls ww~ made hy tr~hnioal assistSants using t#hc instrument and method tlesc:rih~~tl by Fox (6). For thr ultramicro analysis ~r~ioo-~~il~tc~l 0.2+1 s:tnqAes wc’w used. The dilut8ion for amphibian (Sc~t~~cs) serum RW 30 tSinw :~nd t*h:tt# for pooled mammalian (rat) swum \v:ls 20 t’imes or more. ~laddor urillc of AVxturus wa,sso low in salt wntentS t,h:rt’ it \~a:: used \vithout dilution. The fig~lrw prrsented are t,he ;~~cwgcs uf two or thwc wadilrgs. Samples of
pooled serum from Xectrcms were analyzed using a 31- or 36-gauge wire loop and a 2-set burning time. For rat serum the fine \vire loop plus t,hc ammonium phosphate rinring t~echniquowrc used. On serum the greatest difference hebveen rnbults of macro and ultramicro methods was 5(,7! fol potassium and 37; for sodium X sodium det,ermilu~t,iollmade on rat, serum #3 by Dr. Judith Litohfield of this depa.r*tment.x?th the clectxocle dtwrihed by Eisenman et al. (7) yielded a value of 125 mmole per liter. 011urine the percentage difference was larger hut, the :lhsolut,c difference was eswedingl~ small (0.04 mmole per lit,er for potassium and 0.2 for sodium). Itnmsny and his co-workers did not show c*ompsrisonsof macro and ultmmicro analyses nor did they deal with solut8ionscontB:rining as much proteiu as mammalian serum. For this reason a comparison of result,scannot, 1~ made completely, but, it is probable t,hat, stahilitjy and control have hcw~improved in the modified insbwncnt. The :rpparat8us offers no ad\-antage over standard flame photometers for routine work iu which material is abundant.
22
BOTT
However, in those cases where only minute amounts of material are available, the dual channel ultramicro flame photometer should be generally useful. It has been used in scores of determinations of renal t,ubule fluid and serum and should lend itself equally well to the analysis of other body fluids limited in amount. This applies also to extracts of bits of tissue such as are obtained in biopsies. ACKNOWLEDGMENTS The author wishes t,o arknowledge Mills who preparrd many solutions
the assistance and ran macro
of Miss analyses
Jane Cohen as controls.
and
Miss
Sarah
A modification of a dual-channel ultramicro flame photometer is described. It was built for analysis of very small amounts of biological fluids including that from renal tubules and determines potassium and sodium simultaneously in amounts as low as 2 pq~oles. The author’s experience in the use of the modified instrument is described. It should be generally useful for analysis wherever the sample supply is very limited. REFERENCES 1. BoTT, P. A., P&ration Pi-or. 14, 185 (1955). 2. BOTT, P. A., Fe&ration Proc. 16, 223 (1956). 3. BOTT, P. A., in “8th Ann. Conf. on the Nephrotic Syndrome, 1956” (J. M&off, p. 39. National Nephrosis Foundation, New York, 1957. 4. RAMSAY, J. A., BROWN, R. H. J., ANU F~r,~oos, S. W. H. W., .J. Exptl. Biol. (1953). .5. WIGGLESWORTH, V. B., Biochfm. J. 31, 1719 (1937). 6. Fox, C. L. JR., Anal. Chem. 23, 137 (1951). 7. EISENMAN, G., RUIIIS, D. (I., ANI) CASBY, J. U., Science 126, X31 (1957).
cd.), 30, 1