shows that in less than one occasion in twenty would duplicate determinations differ by more than 3 p.p.b. or 2 p.p.b. for copper and lead, respectively. The results shown in Table I1 indicate the drgree of accuracy attainable in the range 10 to 100 p.p.b. The lead results by the colorimetric method (Tables I1 and 111) were obtained on a Hellige visual comparator using a disk having 20-p.p.b. steps and, therefore] a n estimate of the color intensity bctneen strps had to be made. Kevertheless the agreement between this method and the value obtained using the square wave polarograph is considered satisfactory. The copper results (Tables I1 and 111) show reasonable agreement between the two techniques at the lower levels but a n appreciable disparity a t the higher concentrations. Table I V shows the results obtained on up to six determinations of each of the two elements in the blended samples originally used in Table 11. As this program had not been statistically designed i t was not possible to obtain a reliable estimate of the way in which precision changes with roncentration. Consequently such a program was carried out and the results are shown in Table 1ESTIMATES OF PRECISION
Lead. Duplicate determinations should riifler b y 7 p.p.b. or more on only one occasion in twenty. One of the results was excluded from the analysis because of its wide divergence from the other results on the same sample. Copper. I n this case there was evidence t h a t t h e precision varied with thP level of t h e determination.
Table IV.
Determination of Added Copper and Lead in Typical Naphtha from Middle East Crude Oil
Copper, P.P.B. Added Found
10 12 11 9 7
20 19 18
21 17 17
12 13 Table V.
Added 9 .i
19 28.5 38 47.5 57 76 95
.. , .
..
100 104 107 119 117 113 119
50 48 49 46 48 47 46
25 26 26 20 25
10 12 13 12 11
12 12
20 19 20 19 19 18
.
Lead, P.P.B. 50 25 59 24 54 23 52 27 53 25 . 52 .. 52
100 112 111 103 108 107 107
Statistically Designed Program for Determination of Added Copper and Lead in Typical Naphtha from Middle East Crude Oil
Copper P.P.B. Found 6 19 18
34 45 69 88 106
8
14
21 32 38 60 73 93
Lead P.P.B. Found
Added 13 15 20 31 42 57 81
95
7 14 23 29 36 62 76 96
T h e estimate of precision obtained was such t h a t duplicate determinations should differ b y more t h a n 14.8y0 of their mean value ( + 5 . 3 p.p.b.) on only one occasion in twenty . The main advantage of the method described lies in its accuracy for measuring concentrations below 20 p.p.b. and in the fact t h a t both elements may be determined simultaneously. The time required for duplicate determinations of copper and lead by one analyst is approximately 4 hours, of which about one half is manipulative time. However, it is possible for one analyst to complete duplicate determinations on six samples within one working day.
11.5
7
~~
22 34 45 59 71 89 109
23 34.5 46 57.5 69 92 115
8
26
32 42 55 67 92 111
11 ~~
23 33 48
58 71 93 113
~. 10
24 30 48 62 74 93 82
ACKNOWLEDGMENT
The authors thank the Chairman and Directors of The British Petroleum Co. Ltd. for permission to publish this paper. LITERATURE CITED
(1) Barker, G. C., Cockbaine, D. R., Atomic Energy Research Establ. (G. Brit.) Rept. C/R 1404, H. M. Stationery Office, London, 1956. (2) Barker, G. C., Jenkins, I. L., Analyst 77, 685, 1952). (3) Hansen, K. A., Parks, T. D., Lykken, Louis, ANAL.CHEM.22, 1232, (1950). RECEIVEDfor review July 7, 1960. Accepted October 7, 1960. Group Session on Analytical Research, 25th Midyear Meeting of the Division of Refining, American Petroleum Institute, Detroit, IIich., May 9-12, 1960.
Continuous Polarographic Determination of AI pha -Amino Acids W. J. BLAEDEL and J. W. TODD' Chemistry Department! University of Wisconsin, Madison, Wis. A method for measuring a-amino acids continuously in flowing solutions i s applied to ion exchange column effluents. The sample stream is neutralized and buffered, and is then passed through a small copper phosphate tube. Amino acids solubilize the copper phosphate, forming proportional amounts of copper-amino acid chelates. The copper-amino acid chelates are converted to the copper-EDTA
chelate, the stream is deaerated, and the copper is determined polarographically. Standardization may b e performed with any a-amino acid. Amino acid concentrations over a wide range up to 6mM may b e determined with a standard deviation around 5 p M (the limiting detectable concentration) or 3% relative, whichever i s larger. The method i s insensitive to ammonia.
T
HE CONTINCOUS DETERMINATION O f
amino acids in ion exchange column effluents has been established by Moore, Spackman, and Stein (4). Commercial equipment is available for this
1 Present address, Central Research, Minnesota Mining and Manufacturing Co., Saint Paul 19, Minn.
VOL. 33, NO. 2, FEBRUARY 1961
205
Chromatographic Column I
\/ Neutralizer-
J \
Buffer
Reagent
Figure 2. Continuous record of amino acids in ion exchange column effluent
\I
\/
1
E D T A Reagent
$/
I
Continuous Polarographic Anabzer
1
Figure 1. Continuous polarographic determination of amino acids in ion exchange column effluents
method, which is based on reaction n-ith ninhydrin (1, 6). I n the present paper, an indirect method for polarographic analysis of amino acids (5) is adapted to the continuous analysis of amino acids in an ion eschange column effluent. The amino acids are not themselves polarographically reducible, but their reaction n i t h copper phosphate liberates a n equivalent amount of cupric ion, which is polarographically determinable. A study of the copper phosphate method (3) has shown it to be nonspecific for all of the naturally occurring a-amino acids, which may be determined a t concentrations around O.lmJ1, and with a standard error around 37,. Any single amino acid may be used for standardization. PROCEDURE
The continuous polarographic determination of amino acids is outlined scheniatically in Figure 1. The ion eschange effluent is first neutralized and buffered, and then is passed through a small tube of copper phosphate. The effluent from the copper phosphate tube is joined with an EDTA stream, to convert the copper-amino acid chelates to a copper-EDTA chelate, which is polarographed continuously in a previously described apparatus ( 2 ) . The equipment and steps represented in Figure 1 are detailed in the follon-ing sections of this paper. Operation of Ion Exchange Column. T h e chromatographic column (15 cm. long, 10 mm. in inner diameter) was filled with Dowex 5OW-XS resin (100- to 200-mesh). T h e eluent flow
206
ANALYTICAL CHEMISTRY
Polarographic data: Sargent XXI at 0.003 pa./mm. sensitivity in all regions except for aspartic acid and glycine peaks, where a sensitivity of 0.004 pa./mm. was used Dropping mercury electrode: h = 51 cm., m = 2.24 mg./sec., f = 4.0 sec., applied potential, -0.8 v. (vs. S.C.E.) A. Column ( 1 5 cm. X 10 mm. i. d., Dowex 50W-X8, 100- to 200-mesh) equilibrated to 0.08M formic acid (pH 3.61 A-6. Standardization with 0.6mM glycine in O.08M formic acid (pH 3.6) 6-C. 0.08M formic acid [pH 3.6) C-D. Introduction of sample in 5 ml. of 0.04M formic acid (pH 2-61; 10 pmoles aspartic acid, 15 pmoles glycine, 2 pmoles leucine, 10 pmoles arginine, and 100 pmoles ammonium nitrate D-E. 1 2 5 ml. 0.08M formic acid (pH 3.6) E-F. 8 ml. 0.07M formic acid (pH 4.0) F-G. 6 0 ml. 0.2M formic acid with 0.4M N a N O J (pH 4.25) After G. 80 ml. 0 . 2 M acetic acid with 1.OM N O N O SIpH 5.25)
n a s metered a t 0.9 nil. pci minute through the first Tygon tube of a multichannel peristaltic pump ( 5 ) . The Tygon pumping tube nas: 0.056 inch in inner diameter arid was obtairied from Technicon Instruments Corp., Chauncey, r\l. Y. A 0.030-1nch inner dianieter Tygon tube used subsequently n as also from this source. The sample containing sevcral amino acids was adsorbed on thc tolumn and eluted according to the k w n d of Figure 2, 11-hirh i.; a typical elution rccord. Elution conditions n ('re not ncarly so stringent as those of l l o o i c and coivorkers, and conseqwntly did not permit as good a resolution of amino acids. However, the 0bjectiT.c of our nork n a s simply to illustrate the continuous measurmwnt of amino acid concentrations in the efflucnt stream, and not to obtain separation of complex mistures. The chromatographic colunin served mainly as the saniple source, a simple synthetio niixture of four amino acids in 2- to 13-pmole aniounts (Figure 2 ) being used to dcmonstrate the method. Addition of Neutralizer-Buffer Solution. Before reacting with copper phosphate, t h e colunin effluent was adjusted t o pH 9 n i t h neutralizerbuffer solution, n hich c o n t a i n 4 0.1M hoiic acid, plu. 0.0tOM sodium hydroxide in exceqs of t h a t iequired t o neutralize the boiic acid to p H 9.0. The neutralizer-buffer solution TT as metered a t 0.9 ml. Inin. through the second channel of the peristaltic pump. For elution purposes alone. considerable latitude is permissible in composition of the eluent solutions. However, to make the same neutralizer-buffer solution suffice for all eluents, the free acid ronceiitration in all eluents was set a t 0.04051, salt of the arid bcing added to each eluent to give the required pH. When reacted with an equal volume of neutralizer-buffer, the resultant solution had a pH ot 9.0, which mas optimum for the copper phosphate reaction. Com-
position of t h r ncutralizer-l,uffcr \\-asiiot critical, sinre pH fluctuations hrtiveen 8.7 and 9.3 were tolerable (3). TOprovide good mising of the, volumn effluent and neutralizer-buffer solutions with little holdup, the vibrating miser of Figurr 3 was used. Kithout the miser, the solution floniiig into the copper phosphate coluniri \vas nonhomogeneous, various portions rmging from acidic to basic. Th(. acaitl portions dissolved caopper pliosphatc. giying a high and fluctuating blank. 11 it11 the miser. the blank was uniform and 1017. The holdup of the miser wts ahout' 0.4 nil. The Tygon tubing ronaectors prevented transmission of Tillration to other parts of tlie apparatw. l'j-gon tubing was connected to tlie glass capiilaq- tubing by heating the glass tubing and reaming i t out n-ith a tungsten ncwlle until the Tj-gon tube could l x siiiigly insc'rt'ed. K h e n the eluent composition is changed abruptly. spurious or artifact peaks may be produced. For example, the resin equilibrated to 0.0831 formic acid a t pH 3.6 sbill contains considerable residual acid (point E in Figure 2). If the rirst eluent containing 0.2M formic acid and 0.4M sodium nitrate a t pH 3.23 n-ere introduced a t this point, the first portions would contain a pulse of acid in excess of the neutralization capacity of the ncutralizerbuffer solution. Copper phosphate n-odd dissolve in this acid solution. and would give a polarographic peak of the same sort as that given by a n amino acid. The purpose of the resin conditioning solution (0.07M formic acid a t pH 4.0, passed between points E and F in Figure 2) is to liberate moat of the residual acid slot\-ly, so that the buffer capacit'y is not exceeded, and so that a severe acid pulse is not produced. The acid pulse is diminished partly through slower liberation of the acid
Column
i/loll
Pyrex
Neutralizeruffer Reagent t
lamp To ibratool
d k I 2 / i
To
rnm. B a l l
Joint
+ Copper
Reaction
Figure 3.
Phosphate Tube
Vibrating mixer
from the resin, but mainly by using a (soncentration of free acid belolv 0.04.11 in the resin conditioning solution. the borate buffer capacity is eried and no spurious peak is produced. After equilibration to pH 4, t,lie rcxsin cvntains so little residual acid that even large changes in eluent pH producc no artifacts. Bpiirious pcaks could probably be c~liniiiiatctlvery effectively and n-ith less troublr 1 1 ~ -gradient elution, since acid would bc slon-ly and uniformly liberated from the rcsin. without tlie acid pulses that arc produced when cilucnts are c~hallgcd:1bruptIy* Copper Phosphate Reaction Tube. ('opj)ei, phosphate reagent was pre-
5-inch Biicliiier pared 3 s follows: fiinnel w:ts filled u.ith a slurry of i'oppc'r pliospliate stock (3) and suction \viis a p l ) l i d until the mother liquor was just, n-ithtiraivn from above the residue, hut without packing the residue tightly. Thc rositluc on the filter was washed witli a litcar of distilled water. again taking c m c . not to pack the rc.sidue tightly. 'I'he residue was then removed from they funncl, resuspcndod in a litcr of n.:itcsr, fikererl, and washed twicac mor('. After thc third nash, suction \vas :ippliecl to pack the residue into a tight mat. and the mat was removed c d tlriecl for 2 hours a t 105' C. -1fter mat was cut into thin strips ors, t,hen broken into small chunks in a mortar, and finally sieved to ohtaiii a 50- to 60-mesh fraction. (Thc 40- to 100-mesh fraction also provccl satisfactory.) I3c.fore us(', the copper phosphate r c y q i t \vas boiled about 5 minutes in tiistillivl n-at(Jr and thc fines were clecanted. The resulting slurry was storctl nnrl used as needed. Fines foriiicd 011 standing were also decanted h(.fore use. S o deterioration was observcxd in either the dry or wet copper phosphate reagent. l h e copper phosphate tube is shown
in Figure 1. The 3-inch length was optimum. For murh shorter lengths, reaction between the amino aeids and copper phosphate became rather incomplete, owing to the short residence of the solution in the reaction tube, and differences among the individual amino acids became significant'. For greater lengths, small but significant amounts of aniino acids w r e adsorbed by the copper phosphate. and were only slowly washcd out. Suc*li retrntion, if too great. would decrensc> the resolution of chromato$ral,hic~ally separated amino acids. Khilr both of these effects nere still present in the 3-inch tube 01 Figure 4, good accuracy !vas achieved in the deterniination of tliffcrent amino acids (see Results). The life of the couuer nhosuhate bed mas good. -10.5m-iI giyciie solution passed for 10 hours a t 1.8 nil. per minute decreased the reactivity of the tube in Figure 4 by only 570. To prevent destruct)ion of the bed, it was necessary to reject acidic start-up column effluents through thc waste outlet of Figure 3, until proper equilibration to p H 9 was achieved. Polarographic Determination of Liberated Copper. T h e various copper-aniino acid chelates have different diffusion coefficients. Therefore, t o make tlie polarographic response nonspecific for t h e different amino acids, the ropper-amino acid chelates were all converted to t h e more stable copper-EDT.1 chelates. A 0.75-1f EDTAi solution (disodium salt) \vas metered into the copper phosphatc. column effluc,nt a t 0.27 ml. per minute through t h r third channel of the perijtaltic pump. The rrsultant stream was then fed into the continuous deaerator and polarographic analyzer rlescrih(v1 prel-iously ( 2 ) . RESULTS
The follon ing conclusions may be dran n from Figure 2, 1%hich is a typical record of the separation of a mixture of four amino acids. Areas under pcaks arr proportional to the amounts of tlie amino acids, n i t h a standard deviation of 37,. Since the flow rates are exceedingly uniform, an ordinate height a t any instant must be proportional to amino acid concentration a t that instant u-ith the same relative standard deviation of 3%. However, becausp of variable peak shapes, a peak height cannot be accurately taken as proportional to the total amount of an amino acid elutcd in that peak. This standard deviation of 3y0 does not include the arginine elution, in which a much higher sodium nitrate concentration \\as present than in the case of the other amino acids. Because of the high sodium nitrate concentration, the sensitivity toward arginine was about 10% less than toward the other
[ 1%
rnm S o c k e t J o i n t
?
Filled 3rnrn. IW . Di .t h T u5b0e--6 0 3 " LMoens gh
'.'
Copper
Phosphate
;
VI,'
L o n g Glass Wool P l u g
I m m . Capillary T u b e - 2 " L o n g '*/I
mm. B a l l Joint
Figure 4. Copper reaction tube
phosphate
amino acids. Greater discrepancies were noted nhen salts other than sodium nitrate w r e used-NaC1, iYanSOd. Khenever the composition of an eluent changes greatly, a recalibration with a standard amount of a n amino acid is necessary for good accuracy. T o recalibrate. the column efluent is diverted for a few minutes and the standard solution is passed instead. The polarographic background current is significantly above zero, but its variation during the \\ hole elution docs not excwd 0.010 pa. This variation corresponds to an amino acid concentration of 5 ptM in the column effluent, which may be regarded as the limit of detection. The sensitivity of thc niethod for ammonia is only about one t n o-hundredth of that for amino acids. I n most cases, this is an advantage over the ninhydrin method, for which XHB constitutes an interference. I n general, the copper phosphate method i i insensitivc for amino atids other than the alpha type (3). K i t h the 11-ide range of polai ographic sensitivities, a nide range of amino acid concentrations (up to 6 m X ) can be detrrmincd, and tail con(-rntrations can be folloned as easily as pcnk ( oncentrations. LITERATURE CITED
(1) Beckman Instruments, Inc., "Beck-
man/Spinco Model 120 .kmino '4cid
Analyzer," Palo Alto, Calif., 1959. (2) Blaeclel, \T-. J., Todd, J. \Y.,ANAL. CHEX 30,1821 (1958). (3) Ibid., 32, 1018 (1960). (4) Moore, S., Ypackman, D. T I . , Stein, R. H., Ibid., 30, 1185, 1190 (1958). ( 5 ) Skegge, I,. T., Airi. J . Clin. Pathol.
28,311 ( 1 9 5 i ) . (6) ,Technicon Instriiments Cork)., "Technicon .-\utoAnalyzer," Channcey, S . Y., 19.57.
RECEIVED for review August 12, 1960. A4ccepted October 28, 1960. Work supported by grants from the U. S. Atomic Energy Commission.
VOL. 33, NO. 2, FEBRUARY 1961
207
