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Anal. Chem. 1985, 57, 1477-1479
Table I. Indirect Analysis of Chloride Ion with Radioactive Eluents chloride, pg
cpma
A cpm
(Acpm/B) X cBvx 103
System I* 0 0.1
0.4 1.0
5581 f 75 5414 f 56 5088 f 83 4635 f 134
0 167 493 946
6.46 19.1 36.7
System 2c 0 1.0 5.0 20.0
11435 f 48 11247 f 51 10933 f 24 9480 zk 259
0
188 502 1955
41.4 111 431
Average of five replicate injections; counting efficiency, 87%. *Column, 4.6 X 30 mm Vydac 302IC; mobile phase, 0.09 mM KHP at pH 4.5 containing 3.4 nM Na2S04 [35S, 363 mCi/mmol]; injection volume, 100 pL; collection volume, 2.4 mL. ‘Column, 4.6 X 250 mm Vydac 302IC; mobile phase, 0.9 mM KHP at pH 5.4 con445 mCi/mmol]; injection volume, 100 taining 4.8 nM Na2S04 [35S, pL; collection volume, 2.8 mL. (I
analyte can be quantitated by monitoring the radioactivity of the medium. The detection limit of the method can be estimated from the following calculation. Defining CT as tracer ion concentration (mM), SAas tracer specific activity (mCi/mmol), V as volume of eluate counted (mL), and E as counting efficiency, the background counts B (cpm) in the eluate are given by eq 2.
B = (2.22
X
106)CTS~VE
Now, if the detection limit is assumed to be twice the uncertainty in the background, and since the buffer anion is the predominant one in the system, then the minimum detectable quantity Cmin(Fmol) is given by eq 3
(3) which upon substitution yields eq 4.
CB = mM, = 2*4mL, SA = 363 In system in mCi/mmol. E = 0.87. and CT = 3.4 nM. If T i s set at 1 min, then from eq 4, Cmincorrespbnds to about 0.2 Fg of chloride ion, a result which is consistent with experiment. Of the variables in eq 4, CB and CT can be most easily adjusted to improve sensitivity. Decrease of C B below 0.1 mM is impractical with presently available columns (4). Increasing CT improves detectability, but only a t the cost of increasing the amount of radioactivity that must be processed. However, a level of 1 mCi/L of a weak emitter such as 35Sis well within the constraints of most licensed laboratories, and requires only routine handling precautions. This order of activity would allow the detection limit quoted above to drop to the low nanogram region, which is comparable to the best sensitivity obtained with indirect photometry (2,3). In cation work a suitable nuclide might be 45Ca. A potential advantage of the radiochemical method over other indirect techniques is that the effect of interferences is likely to be smaller. A drawback of all indirect methods is that they are more susceptible to interferences than direct techniques, since the property of the eluent being monitored may be affected by both direct and indirect means. For example, in ion chromatography with indirect UV detection, an UV absorbing species would directly interfere with the analysis, whereas a transparent ion would do so indirectly. Radiochemical analysis would be essentially free from direct interferences, unless of course, the samples themselves contained radioactivity. Registry No. Chloride, 16887-00-6. Oao9
LITERATURE CITED Small, H.; Miller, T. E., Jr. Anal. Chem. 1982, 5 4 , 462-469. (2) Cochrane, R. A.; Hillman, D. E. J . Chromatogr. 1982, 247, 392-394. (3) Heckenberg, A. L.; Haddad, P. R . J . Chromafogr. 1984, 2 9 9 , 301 -305. (4) Haddad, P. R.; Heckenberg, A. L. J . Chromatogr. 1984, 300, (1)
357-394.
Sujit Banerjee* Joseph R. Steimers Safety and Environmental Protection Division Brookhaven National Laboratory Upton, New York 11973 RECEIVED for review December 13, 1984. Resubmitted and accepted February 28,1985. Research carried out under the auspices of the U S . Department of Energy under Contract NO. DE-AC02-76CH00016.
Comments on Least-Squares Polynomial Filters for Initial Point and Slope Estimation Sir: In a 1964 paper published in this journal, Savitzky and Golay (I) proposed a technique for smoothing, and obtaining smoothed derivatives of, data arrays containing uniformly spaced data points. The technique involved fitting a small subset of the data to a polynomial, in order to define smoothed or derivative values at the center of the fitting interval, and repeating the process by moving the fitting interval within the bounds of the total array. This represented a convolution procedure, which could be represented mathematically as i=m
Yj* =
c ciYj+i
i=-m
where the index j represents the running index of the data in the original data array (Y). They published sets of coeffor various filter widths and polynomials of various ficients (Ci) degrees. The original tables contained a number of numerical and typographical errors, which were corrected in the paper by Steinier, Termonia, and Deltour ( 2 ) . The Savitzky-Golay technique has received widespread application, as pointed out by Madden (3)on the basis of citations listed in the “Citation Index”. In a recent paper, Leach, Carter, and Harris ( 4 ) point out that in some applications it may be desirable to compute fitted values and derivative estimates a t the initial point in the fitting interval, rather than at the central channel (as in
This article not subject to US. Copyright. Published 1985 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
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Anal. Chem. 1985, 57, 1479-1480
the Savitzky-Golay procedure): i=2m
Yj* =
c CiYj+i
i=O
They published two tables of coefficients for deriving fitted values of initial value and intitial slope by means of a quadratic fit with fitting intervals ranging from 5 to 21 data points. In attempting to apply their coefficients, I noticed that although they reported their coefficients to 7 places, their calculations contained occasional errors in the 5th place. A simple test of accuracy is that the initial point coefficients should sum to 1.000 000 0 and the initial slope coefficients should sum to 0.000 000 0. In attempting to duplicate the authors’ tables, I prepared a Fortran computer program to compute the fitting coefficients for either initial-point or central-point estimation. I found that I was generally able to reproduce the authors’ coefficients to five decimal places except when filter widths of 7 and 9 data points were used. In these two cases, the authors tabulated values appear to be incorrect. Coefficients
for initial-point slope and value estimation are tabulated in Tables I and 11. The computer program developed is quite general and can be used to compute fitting coefficients for higher derivatives and for fitting with higher order polynomials. A copy of the program is available on request.
LITERATURE CITED ( I ) Savitzky, A,; Golay, M. J. E. Anal. Chem. 1984, 36, 1627-1639. (2) Steinier, J.; Termonia, Y.; Deltour, J. Anal. Chem. 1872, 4 4 , 1906- 1900. (3) Madden, H. H. Anal. Chem. 1978, 50, 1383-1386. (4) Leach, R. A,; Carter, C. A,; Harris, J. M. Anal. Chem. 1984, 56, 2304-2307.
Philip A. Baedecker Mail Stop 923 Branch of Analytical Chemistry U S . Geological Survey Reston, Virginia 22092
RECEIVED for review February 4, 1985. Accepted March 20, 1985.
A I D S FOR ANALYTICAL CHEMISTS Wide Mass Range Scanning for the Fast Atom Bombardment Mass Spectrometry of Very Large Compounds R. J. Cotter,* B, S. Larsen, D. N. Heller, J. E. Campana,’ and Catherine Fenselau
The Middle Atlantic Mass Spectrometry Facility, The Johns Hopkins University, Baltimore, Maryland 21205 We note that many publications on the FAB mass spectra of compounds weighing more than 5000 amu report only the molecular ion region (1,2). There are a number of reasons for this. First, the very large compounds studied thus far have had known molecular weights so that it has been possible to select a priori the region which will be scanned. Secondly, low signal levels often demand that signal averaging techniques be used which, when applied over a large mass range and involving multiple scans, may be cumbersome for many data systems and may exceed the sample lifetime or demand multiple sample loadings. For samples of unknown and high molecular weight, it may be useful to rapidly survey a wide mass range for molecular ion and major fragment ion regions. For high-resolution double-focusing sector instruments, we have used a time binning technique which is useful for accentuating the major features of the spectrum.
EXPERIMENTAL SECTION Mass spectra were acquired on a Kratos MS-50 double focusing mass spectrometer equipped with a 23-kG high field magnet, using a Kratos DS-55 data system. Wide mass scans consisting of single magnet field scans from 8000 to 800 amu were acquired at 100 s/decade and 2000 resolution. The ion current was sampled at constant time intervals during the scan, with several of these measurements added to form approximately 7500 time data points. The average mass of the resulting unresolved mass peaks appearing in the spectrum was assigned by comparison with time data for a CsI sample acquired in the same manner. The spectra, plotted on a Versatec plotter, represent approximately one data
Present address: Naval Research Laboratory, Washington, DC
20375.
point for every 25 amu, so that the entire mass range may be observed as a single trace.
RESULTS AND DISCUSSION By use of the wide mass scan, the singly, doubly, and triply charged molecular ions of porcine insulin (Figure 1)are easily identified, as well as ions corresponding to the A and B chains present as impurities in the sample. The wide mass scan has the effect of accentuating these general features while deemphasizing the detailed relative abundances of the molecular ion isotope cluster. Since the mass of the molecular ion can be identified from this spectrum, it is then possible to acquire a new spectrum at unit resolution for the molecular ion region (Figure 2). This approach has been used to determine the molecular weights of mouse epidermal growth factor (EGF). This factor is reported (3,4to contain two peptides with similar primary sequences and the spectrum in Figure 3 reveals two constituents with a difference in molecular weight of 114 amu. Four scans were averaged in this case. Other aspects of these wide scanned spectra deserve comment. The intensity of the low mass ions reveals that fragmentation is indeed extensive for this “soft ionization” technique. Field and Chait (5)have pointed out that much of this fragmentation is the result of metastable decomposition and in TOF spectra may be observed as “tailing” on the low mass side of the molecular ion cluster. In sector instruments metastable peaks m* resulting from fragmentation in the field free region between the electric and magnetic sectors are observed a t
m * = md2 I m p
0003-2700/85/0357-1479$01.50/0 0 1985 American Chemical Society
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