sioned. Thus, the value of the skewed Gaussian is always in ordinate units while the value of W (Equation 3) is in abscissa units. The area in Equation 11 has units of ordi, have values nate times abscissa. All the moments ( v ~C(k) in abscissa units to the kth power. The first term on the right-hand-side of Equations 18 and 19 is a constant. The second term may be expanded to the kth power by use of the binomial theorem (9). Final evaluation of Equation 17 depends on evaluating the integral of a sum of k + 1 terms. This problem reduces to the sum of k + 1 integrals of the type in Equation 10. Once again it is recalled that Equation 10 is a general result for any constant b. The result of these calculations is given in Table I. Results are given with sums from 0 to k and from 1 to k + 1. This latter result is more amenable to rapid calculation in computer programs. Units. The asymmetry parameter ( b ) in Equation 2 is a dimensionless constant. Both x o and Ax have the units of the abscissa of the experimental data as, of course, does x . Depending on the type of experimental data being considered, the ordinate parameter Y may or may not be dimen(9) "Handbook of Chemistry and Physics." The Chemical Rubber Publishing Co., Cleveland, Ohio, 1961, p 334.
CONCLUSION Determination of the four parameters ( Y , Ax, xo, b ) of the skewed Gaussian distribution function (6) provides a complete description of an asymmetric peak. Not only can the shape of the peak be reproduced but the moments of the distribution can be calculated. Use of the non-linear least squares method (7) to obtain the four parameters permits the calculation of the errors of the "final values." Errors of the calculated moments of distribution may then also be estimated. This is in marked contrast to the numerical calculation of moments of the distribution from discrete experimental values where no such estimates of the errors are possible although they can be quite large. Received for review July 3, 1972. Accepted January 22, 1973. One of us (PFR) would like to acknowledge the receipt of a NSF-CNRS fellowship which permitted him to study with the research group of the Laboratoire de Chimie-Physique.
Electrophoretic Desalting of Acid Hydrolysates for the Isolation of Amino Acids Mario R. Stevens' Jet Propulsion Laboratory, California institute of Technology, Pasadena, Calif The successful application of the quantitative procedures for the analysis of amino acids, as developed by Gehrke and Stalling ( I ) , Roach and Gehrke ( 2 ) , and Gehrke, Kuo, and Zumwalt ( 3 ) , is predicated upon the elimination of any inorganic salts present in the amino acid-sample matrix. Each of the above cited procedures makes use of gas-liquid chromatography for the separation and identification of the individual amino acids. Since the free amino acid is not volatile enough to permit a direct gas chromatography analysis, one must first convert the amino acid into a volatile derivative. Several such derivatives have been prepared, the most prominent being the N-TFA-n-butyl esters ( I , 4 ) , N-TFA-2-butyl esters ( 5 ) , the N-acetyl-n-butyl esters ( 6 ) .and the N-trimethylsilyl-0-1-butyl esters (7). Generally, the amino acids of interest are those associated with soils, sedimentary rocks, and biological materials. In most procedures, the initial sample processing step involves acid hydrolysis using 6N HCl. High salt concenPresent address, Towne-Paulsen & Co.
Inc., Monrovia, Calif.
(1) C. W. Gehrkeand D. L. Stalling.Separ. Sci., 2, 101 (1967). (2) D. Roach and C. W.Gehrke, J. Chrornatogr., 44, 269 (1969) (3) C. W . Gehrke, K . Kuo, and R . W . Zumwalt, J. Chrornatogr., 57, 209 11971). (4) E. Geipi, W. A. Koenig. J. Gilbert, and J. Oro, J. Chrornatogr. Sci., 7. 604 (1969). ~~. (5) G. E. Pollock, A. K. Mijamoto, and V. I. Oyarna, "Life Science and Space Research," Vol V I I , North Holland Publishing Co., 1970, pp 99-1 07. (6) S. C. J. Fu and D. S. H. Mak, J. Chrornatogr., 54, 205 (1972). (7) 3 . P. Hardy and S. L. Kerrin, Anal. Chern., 44, 1497 (1972).
trations are characteristic of such hydrolysates. These salts interfere with the preparation of the required derivatives and/or with the subsequent gas chromatography of the derivatives. Gehrke and Leimer (8) made a study of the effects of salts upon the derivatization and chromatography of amino acids. They found that iron(II1) salts interfere with the derivative-chromatographic process. Iron( I n ) salts are most common to geological samples. They demonstrated interference at 1:1 concentration ratios of amino acids to salts whereas, in geological samples, the ratio is more likely to be 1:lo3 or 1:104. Ion exchange and electrolytic desalting techniques are commonly employed. Both are very effective. In particular cases, adsorption dialysis, solvent extraction, and ultrafiltration are also applicable, as shown by Smith (9). Both ion exchange and electrolytic methods may prove to be inadequate when trace quantities of amino acids are present. The former requires controlled elution techniques and subsequent concentrating steps. Losses may take place in each step. This method may prove cumbersome for automated, remote control applications. The latter method will lead to losses with respect to acidic and basic amino acids. These will behave as anions and cations, respectively, and will be removed from the solution under the electrolysis conditions.
I
(8) C. W. Gehrkeand K . Leimer, J. Chrornatogr., 53, 195 (1970). (9) "Chromatographic and Electrophoretic Techniques," lvor Smith, Ed., Vol. 1, Chapter 3, lnterscience Publishers Inc., New York, N.Y., 1960. ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
1543
“7
\
front flows, toward the center of the support, where all flow ceases. Electroendosmosis involves electrolyte flow in the support to compensate for the lack of movement of fixed ionic groups attached to the support material-ie., carboxyl groups of filter paper. In this case, the flow will be toward the cathode, moving the neutral particles a distance which is a function of the run time. Zwitterions will be further influenced by the p H of the media. They may act as anions or cations depending upon the hydrogen ion concentration of the electrolyte.
3
4-y
5’
Schematic of apparatus for high voltage electrophoresis studies
Figure 1.
1, Electrolyte; 2, wick; 3, support medium; 4 , connections for coolant; 5 , copper cooling block; 6 , lower polyethylene insulating plate; and 7, upper polyethylene insulating plate
2
2
Figure 2. Schematic of apparatus employed for electrophoresis studies
low
voltage
1 , Electrolyte: 2, support medium; 3, cover plate; and 4 , support plate
It appears that, these problems may be greatly reduced or avoided completely by means of electrophoretic desalting. This paper discusses the application of electrophoresis to desalting of amino acid hydrolysates wherein the amino acid/salt concentration ratio varies from 10-3 to approximately unity.
THEORETICAL Electrophoresis is the process of causing particles or molecules to move under the influence of an electric field. Depending upon whether or not the particles are charged and the nature of the charge, the particles will migrate to either the anode or cathode of the apparatus. Neutral particles and zwitterions should remain nearly stationary. The rate of migration of ionic material will be, to a first approximation, proportional to the force exerted upon it. This force is the product of the field strength, X,and the net charge, q : F=Xq
(1)
However, the force driving the ion toward an electrode is counterbalanced by resisting forces such that the actual migration rate or ionic mobility is given as: u = F/(Gi~rn)
(2)
where r = ionic radius and n = viscosity of the electrolyte. Therefore, for a given set of operating conditions-ie., temperature and electrolyte concentration-the rate of movement of ionic particles is directly proportional to the field strength and net charge and inversely proportional to the ionic size and viscosity of the electrolyte. While neutral molecules and zwitterions should remain stationary, in practice they too will move under the influence of an electric field. This movement is due to two phenomena: electrolyte evaporation and electroendosmosis. Electrolyte evaporation will increase the flow of electrolyte into the support a t both ends of the support material. This will cause the particles to flow as the flow 1544
ANALYTICPL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
EXPERIMENTAL Apparatus. All high voltage electrophoresis data were obtained using a Desaga/Heidelberg Migration Chamber. The system was powered by a John Fluke Power Supply, Model 407DR. This power supply is a 600-V, 300-mA unit. The electrophoresis was carried out using the enclosed strip method. A schematic of the experimental arrangement is shown by Figure 1. An LKB Migration Chamber was used for the Low Voltage Electrophoresis runs. The unit was powered by an LKB Power Supply, Type 329B. This power supply is a 400-V, 40-mA unit. Electrophoresis was carried out by the open supported strip method. In this case, the electrophoretic support medium rests upon a plastic plate, with a small air space between it and the top cover plate. The support plate is cooled by convective air flow. Figure 2 presents a schematic of this arrangement. Reagents. Test solutions of amino acid hydrolysates were prepared using one or more of the following amino acids dissolved in 6N HCI (J. T. Baker Chemical Co., Phillipsburg, N.J.): alanine, glycine, proline, phenylalanine, leucine (Cal BioChem, La Jolla, Calif.), valine, isoleucine, beta alanine (Pflatz and Bauer, Flushing, N.Y.), arginine, glutamic acid, and aspartic acid (MCB Chemicals, Los Angeles, Calif.). Whatman Chromatographic Paper, No. 1 (VWR, Los Angeles, Calif.), served as the electrophoresis support medium for both high and low voltage experiments. Development of the electropherograms (with regard to amino acids only) was accomplished by use of Kinhydrin Reagent (50 mg ninhydrin, Eastman Organic Chemicals, Rochester, N.Y.) dissolved in 75 ml of absolute ethanol (US1 Chemicals, New York, S . Y . ) and 25 ml of 2 N acetic acid (Allied Chemical, Morristown, K.J.). The electrolyte, in all cases except where noted, is a solution consisting of 20 grams of pyridine and 9.5 grams of glacial acetic acid (Allied Chemicals, New York, X.Y.) diluted to 1 liter with distilled water. The pH of the resulting solution is 5.2. Technique. The support medium, for the high voltage runs, was cut into 1-inch strips (2.54 cm) by 14 inches (35.56 cm). In either case, the strips were washed in 6N HCI, thoroughly rinsed with distilled water and air dried. When dry, the strips were placed on the supporting plate of the appropriate apparatus. For high voltage runs, the plate was precooled by circulating ice water (4 “C) through the copper cooling block. The ends of the strip were then immersed in the electrolyte. The strips were spotted using 3-pl portions of the particular amino acid solution. To minimize spot spreading, the solutions were applied 1 p1 at a time. The spot zone was a t the center of the strip and was, on the average, 17 mm in diameter. The cover plate was placed into position and the run started when the electrolyte, flowing into the strip by capillary attraction, met a t the center of the spot zone. In each occasion, the voltage was set and maintained a t the desired value for the duration of the run. The current was allowed to seek its maximum at the equilibrium established. Run times varied but generally lasted from 4 hr (high voltage runs) to 6 hr (low voltage runs). At the completion of a run, the strips were removed from the apparatus and suspended in air until dry. They were sprayed with ninhydrin reagent, and incubated at 60 “C for 10 min. The resulting bands served as the basis for analysis of the data. The strip was divided into “cuts” relative to the spot zone. Usually eight cuts were defined. Each cut was subjected to atomic adsorption analysis for the determination of the metal concentration. In this way, the purity of the amino acid bands, with regard to the metals, was determined. The detection limits for the metal ions used in the test solutions were 2 pg for iron and 6 pg for aluminum.
Table I . Test Solution Composition, gram/cm3 Composition Soin 5,
Soin 6,
x
x 10-4
Components
3.58 2.23 5.69 2.85 2.50 1.52 1.07 1.17 4.8 0.67 1.53 25.5
Alanine Valine Arginine Isoleucine Leucine Glycine Proline Aspartic acid Methionine Phenylalanine Glutamic acid @-Alanine Total amino
Soin 3, 10-5
x
10-6
Soin 2,
10-4
10-4
x
...
5.89 3.71 9.33 4.7 4.1 2.5 1.78 1.91 8.3 1.1 2.55 42.5
Soh A,
x
6.0 ... 24.0
... 23.0 ...
...
7.0 30.0 19.0 ...
...
, . .
... 61 .O ...
6.5 181.0
9.7
...
AIC13.6H20 Total salts Ratio: salts/acids
... 126.0 ... 22.0 191 .o 77.0 ...
...
106.7 118.0 ...
...
10-4
34.0
... ...
53.1 1 88.27 93.0 273.5 0.2834 X 10' 0.3118 X 100 0.2153 X 10" 0.2162 X 0.1412 0.1554 , . . 0.1 571 0.4246 0.4672 0.21 53 0.3733 80.0 5290 230 13.7
acids FeCI3.6H20
x
198.0 ... 34.3 ... 34.0 46.1 35.0
...
...
Soin A - I ,
26.0 317.0
...
572.6 794.0 0.1854 X IOo 0.0561 X l o o ... 0.0671 0.1854 0.1232 3.2 1.55
loo
Table I I . Electrophoretic Desalting of Amino Acid Solutions, High-Voltage Data
% Fe
No. Metal left of in neutral basic amino % A I acids % Fe %AI
No. Metal left of in basic acidic amino acids % Fe % Ai
...
1
0
...
1
0
...
0
5 5
.O
0 0 0 7.0 0 0
1
0
0
1
0
0
1 1 1 1
0 0 0 0
0 0 0 0
Metal left in neutrals Run No.
Volts
mA
Time, hrs
Soln No.
1
550
3.0
4.5
3
0
2 3 4 5 6 7
550 550 550 550 550 550
2.7 3.3 3.7 3.7 2.7 2.7
4.0 4.0 5.0 5.0 5.0 5.0
A A
9.0 0 0 8.8 7.7 6.7
No. of acidic amino acids
Ratio, salt/ acid
Comments
1
230.0
No aluminum present in solution
5 5 6 6
0 0 0 0 0
,3.7 0 4.5 2.8 4.0
9 9 9 9
13.7 13.7 5290.0 5290.0 80.0 80.0
Table I I I . Electrophoretic Desalting of Amino Acid Solutions, Low-Voltage Data Metal left in neutrals
Metai left in basic
Metal left in No. acidic of acids % Fe % A I
No.
Volts
mA
hrs
Soln No.
%AI
No. of acids
1
260
1.2
5.16
3
0
...
1
9.0
..
,
1
0
.
..
1
230.0
No aluminum pres-
2
350
2.6
5.75
Z
16.4
...
5
5.2
..,
1
0
...
1
3.2
No aluminum pres-
3 4 5
350 350 350
1.1 1.0 1.55
5.67 6.00 6.00
A A
3.0 5.8 30.0
0 0 10.0
5 5
9.0 0 12.0
77.0 15.4 53.0
1 1 1
0 0 0
0 0 0
1 1 2
13.7 13.7 80.0
Run
Time
% Fe
% Fe
%AI
-
No. of acids
Ratio, salt/ acid
Comments ent in solution ent in solution
6
9
Table IV. Reproducibility of Electrophoretic Desalting of Amino Acid Solutions Metal left in neutrals Run No.
Volts
mA
Time hrs
1 2 3 4
550 550 550 550 550
3.1 3.1 3.1 3.1 3.1
5 5 5 4 4
5
Soln No.
% Fe
%AI
A-1
0 0
0 0
0
0 '0
A-1 A- 1 A-1 A- 1
0 0
0
No. of neutral amino acids
5
5 5 5 5
Metal left in basic % Fe
0 0 0 8 9
No. Of
%AI
0
0 0 36 34
basic amino acids
1 1 1 1 1
Metal left in acidic %Fe
0
%AI
No. of
acidic amino acids
0 0
0 0 0
1 1
0
0
0
0
1 1
1
ANALYTICAL CHEMISTRY, VOL. 45, NO. 8 , JULY 1973
Ratio, salt/ acid
1.55 1.55 1.55 1.55 1.55 1545
RESULTS AND DISCUSSION Table I presents the composition of the six solutions used in this study. These solutions were all 6N in HC1. They varied in complexity from 4 to 14 components. The salt/amino acid ratio ranged from 1.55 to 5290. Both low and high voltage electrophoresis caused the amino acids to be separated into three groups., two migrated toward the cathode and one migrated toward the anode-ie., neutral, basic, and acidic amino acids, respectively. The basic amino acids were displaced as much as 10 cm toward the cathode while the neutrals migrated 1 to 3 cm toward the cathode. The metal cations were displaced toward the cathode. In the case of the high voltage runs, the basic amino acids were displaced further toward the cathode than the metal cations present. It is noted that under high voltage conditions, solutions 5 and 6 give slightly different results from the others with regard to amino acid migration. In these instances, the neutral amino acids were d i t into two bands. One remained verv close to the spot zohe, while the other was displaced about 2 to 3 cm from the spot zone, Since these solutions differed from the others by containing /3-alanine, methionine, aspartic acid, isoleucine, and valine, one or all of these materials are responsible for the band in the vicinity of the spot zone. No attempt was made to ascertain which of these amino acids were involved.
Comparison of Tables I1 and I11 indicates clearly that high voltage electrophoretic desalting is more effective than low voltage applications. Even in the case of the simplest solution. No. 3, 9% of the iron cation remained with the basic amino acid band after the low voltage run was completed. The variations noted between high voltage runs 4 and 5 and between runs 6 and 7 are probably due to slight changes in experimental conditions during the course of the runs involved. The data shown in Table IV made this a plausible explanation. In this case, the runs were closely monitored specifically with respect to temperature. Not: only were the results very consistant, but they also demonstrated the effect of varying the run time. These data clearly show that electrophoretic desalting is practical for the preparation of amino acid concentrates prior to derivatization. The potential of this technique as a step in a fully automated procedure for the analysis of amino acids in geological sample is under further investigation. Received for review December 14, 1972. Accepted February 26, 1973. This paper presents the results of one phase of research carried out a t the J e t Propulsion Laboratory, California Institute of Technology, under Contract No. NAS 7-100, sponsored by the National Aeronautics and Space Administration.
Curie Point Pyrolysis in Direct Combination with Low Voltage Electron Impact Ionization Mass Spectrometry New Method for the Analysis of Nonvolatile Organic Materials H. L. C. Meuzelaar, M. A. Posthumus, P. G. Kistemaker, and J. Kistemaker FOM-lnstituut voor Atoom- en Molecuulfysica, Kruislaan 407, Amsterdam/ Wgm., The Netherlands
As early as 1948, pyrolysis mass spectrometry (Py-MS) was introduced as a promising technique for the study of polymers ( I , 2 ) and in 1952 Zemany (3) reported the use of Py-MS for the identification of complex organic materials, including proteins such as albumin and pepsin. The field of Py-MS developed only slowly thereafter and its potentials were soon more or less obscured by the success of pyrolysis gas-liquid chromatography (Py-GLC) for the routine identification of nonvolatile organic materials. Meuzelaar and Kistemaker ( 4 ) recently reported the combination of a Curie point pyrolysis unit and a small quadrupole mass spectrometer for the reproducible fingerprinting of complex biological samples. The advantages of their Py-MS method in comparison with Py-GLC are the high analysis speed, less than 1 min per sample seems perfectly feasible, and the comparative ease of automatic data processing afforded by the stable and linear mass scale and the uniform resolution. The apparatus described
would perhaps cost twice as much as an average Py-GLC system. A crucial role in these experiments is played by a 1024 channel signal averager which allows the summing of a series of rapid mass scans. The resulting final summed mass spectrum is more representative for the overall composition of the pyrolyzate than any single mass spectrum and also less dependent on temporary variations in the experimental conditions. In spite of the above mentioned strong advantages over Py-GLC methods, the extensive fragmentation suffered by most pyrolysis products a t the ion source of the quadrupole is a serious drawback since this makes chemical identification of individual pyrolysis products virtually impossible. Though unambiguous identification is also difficult to obtain by GLC methods alone if complex mixtures of pyrolysis products are involved, the combination of GLC with mass spectrometry provides a powerful method for chemical identification of individual pyrolysis products in many cases (5, 6 ) .
(1) S L Madorsky and S Strauss Ind Eng Chem 40,848 (1948) (2) L A Wall J Res Nat Bur Stand 41, 315 (1948) (3) P D Zernany Anai Chem 24, 1709 (1952) (4) H L C Meuzelaar and P G Kisternaker, Ana/ Chem 45, 587 (1973)
(5) W . Simon, P. Kriemler, J. A . Voellrnin, and H . Steiner, J . Gas Chrornatogr.. 5 , 53 (1967). (6) P. G. Sirnrnonds. G. P. Shulrnann, and G. H . Sternbridge, J. Chrornatogr. Sci., 7, 36 (1969)
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ANALYTICAL CHEMISTRY, VOL. 4 5 , NO. 8, JULY 1973
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