Detection Limits for Flame Spectrophotometric Monitoring of High Speed Liquid Chromatographic Effluents David R. Jones IV’ and Stanley E. Manahan* Department of Chemistry, 123 Chemistry Building, University of Missouri-Columbia,
Atomic absorption spectrophotometry (AAS) and flame emisslon can be used as element-specific detectors in hlgh speed liquid chromatography for the detectlon of organometallic compounds or compounds, such as chelating agents, which bind strongly to metals. Species can be monitored which would be obscured by background with conventlonal detectlon methods. A major limitation with this method is the relatively high detection limit resultlng from limits inherent with flame spectrophotometrlc techniques and chromatographic peak spreading. An a priori calculation of detection limit is helpful in determining the feasibillty of flame spectrometry for AAS detection. An approach to this calculation as applled to the HSLC separation of copper chelates of NTA and EDTA is presented and the calculated values are compared to values determlned experlmentally.
Flame atomic absorption spectrophotometry (AAS) has been described in a number of earlier papers (1-5) as a detector system for high speed liquid chromatography (HSLC). In addition, flame emission has been used for HSLC detection (6). The greatest advantage of these detectors applied to HSLC is their extremely high selectivity. Ideally, the detector responds to only one element-normally a metal-so that only compounds containing that metal are “seen” by the detector. It is possible, therefore, to analyze species, such as chelating agents, organometallics, or metalloenzymes, which are either bound to, or can be made to bind to, a particular metal. Such specificity makes particular species stand out in a generally noisy background which might completely obscure the sought-for species with more general detectors. Figure 1 illustrates the very high specificity possible with HSLC detection with AAS. I t is a separation of a sewage effluent spiked with copper chelates of (ethylenedinitri1o)tetraacetate (EDTA) and nitrilotriacetate (NTA). Figure 1(A) shows the chromatogram as recorded by conventional ultraviolet absorbance of the column effluent. As expected for a sewage sample containing a large number of ultraviolet-absorbing species, the chromatogram is quite complicated, with many peaks and a heavy background. Figure 1(B) is a chromatogram of the same sample taken under identical conditions with the substitution of the AAS set to record copper, in place of the ultraviolet detector. In this case, the column effluent was force-fed directly to the AAS flame nebulizer. The resulting chromatogram records only peaks from the copper-containing chelates. None of the other species in the sample interfere. A fringe benefit with this system is that solvents may be used which absorb strongly in the ultraviolet region, and spectroquality solvents are not required. Regarding sensitivity of flame methods as HSLC detectors, a major disadvantage is the very short time which the sample is actually in the flame. Attempts to circumvent this problem have been made through the use of flameless atomizers for HSLC detection (7). With this detector, column effluents are
Present address, FMC Corporation, Princeton, N.J. 08540.
Columbia, Mo. 6520 1
directed into a graphite rod furnace. After a predetermined quantity of column effluent is pumped into the flameless atomizer, the flow is arrested, and a heating-ashing-vaporization cycle is initiated. Under optimum conditions, increased sensitivity is obtained. However, the chromatograms produced are of a stepwise appearance, and resolution is lost. Other disadvantages include long analysis time, the need for a complicated pumping-atomization sequence, and the loss of volatile analytes. In some specialized applications, flame techniques offer unique advantages for HSLC detection. The feasibility of their use requires knowledge of the sensitivity and detection limits of the detector system. This requires simultaneous consideration of chromatographic parameters (e.g., peak spreading) and characteristics (e.g., detection limit) of the AAS system. This paper deals with these considerations in the calculation of the minimum detectable amount of analyte in a specific HSLC-AAS analysis.
EXPERIMENTAL The liquid chromatographic pump employed was a Waters, Inc. (Milford, Mass.) Model M-6000A. This pump was found to perform satisfactorily in regard to reproducibility and even flow rate, which can be especially important with the AAS detector system. The column used was 5 cm long, 2.1-mm i.d. stainless steel packed with 20-pm diameter Aminex A-14 anion exchange resin (Bio-Rad Laboratories, Richmond, Calif.). The solvent system was identical to that previously reported ( 5 ) for the separation of the copper chelates of aminocarboxylate ions. It consisted of aqueous 0.05 M (NH&S04. Copper chelates of EDTA and NTA were separated. A flow rate of 2.0 ml/min was employed. A Perkin-Elmer Model 403 atomic absorption spectrophotometer set to record absorbance due to copper was used as the detector. A 4-inch single-slot burner using a standard air-acetylene flame was employed. The column and AAS aspirator inlet were connected by small diameter flexible tubing. Connection to the column outlet was made by way of Teflon heat-shrink tubing &-inch 0.d. before shrinking). This tubing was joined to a 3-cm length of 0.023inch i.d. polyethylene tubing attached to the AAS aspirator inlet. This is the standard tubing supplied by the manufacturer for drawing sample from containers into the aspirator. This HSLC-AAS interface was found to minimize peak spreading due to dead volumes in the connections. For these studies, minimum damping of the AAS was employed.
THEORETICAL CONSIDERATIONS In the course of a chromatographic separation, two major processeq occur simultaneously (8-11). The first of these is zone separation, whereby two solutes are separated on the column because of their differing interactions with the stationary phase. The second process is zone spreading which results in the dilution of an individual solute within a discrete internal column volume as a function of time spent on the column. In brief, the first of these favors good chromatograms because it is the process by which separation is achieved. The second process is unfavorable because the amount of solute per unit volume of effluent is constantly decreasing due to dilution as the solute traverses the column. This has an adverse effect upon detector sensitivity. The effect of zone spreading upon detector sensitivity is especially crucial with flame HSLC detectors because the parameter being measured is the concentration of metal per unit volume of liquid intro-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
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Figure 2. Representationof absorbance due to copper with flame AAS from aspiration of 626 pI of copper solution at detection limit concentration
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ATOMIC ABSORPTION DETECTION OF COPPER
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Figure 3. Representation of chromatographic peak from CU-NTA- in which the copper concentration at its maximum value reaches the detection limit for copper as measured by AAS I
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Figure 1. Chromatograms of sewage spiked with copper chelates of NTA and EDTA Ultraviolet detector ( A ) and copper AAS detector (B);Aminex A-I4 anion exchange resin, 0.05 M (“.,)*SO4 column, 2.0 ml/min flow rate
duced into the flame at a specific instant. Decreased zone spreading increases the sensitivity of AAS flame detection. Practically, this can be accomplished by using as short a column as is possible while still achieving separation, because the diffusion of the peak increases with the square root of column distance travelled. The calculation of the minimum amount of analyte detectable in a peak from HSLC separation using flame AAS detection is difficult because of the number of independent variables affecting it. A knowledge of the AAS system parameters is required. As discussed in a previous paper ( 4 ) ,the nebulization efficiency of the burner is one such important parameter. It is a function of the mobile phase composition and of the flow rate of column effluent into the AAS burner. Of course, the inherent sensitivity and detection limit of AAS for the particular metal being assayed are factors. In general, detection limits published for the determination of metals by flame AAS are enhanced through the use of long time constants available in modern amplifier circuits which serve to decrease background noise and lower the detection limit. Since the chromatographic peaks monitored in HSLC are transient, this sort of signal damping is not acceptable for decreasing the detection limit. The calculation of the minimum amount of analyte in a particular HSLC peak which can be detected by the AAS detector is simplified by several assumptions regarding chromatographic performance. The first assumption is that the column behaves in a linear and reproducible fashion. Plate height is assumed to be uniform throughout the length of the column. It is assumed that plate height does not vary with the particular metal-containing species, e.g., copper-NTA vs. copper-EDTA. In addition, a knowledge of AAS sensitivities and detection limits for the instrument when not interfaced to the HSLC must be available. The number of theoretical plates, N , is given by the relationship,
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where V,,, is the total volume eluted a t the peak maximum, and p is the peak width at a fraction of l/e times the maximum peak height (e = base of natural logarithms). The value of N for the column used in this study was exactly 100. With ideal column behavior, this figure should not change from solute to solute. It was found to be reasonably constant for the 5-cm column used. With a knowledge of N and the retention volume of a specific solute, it is possible to calculate the /3 value for that solute. The ,R value should remain constant, independent of solute concetration. For simplicity in the geometric calculations which follow, “volume” will be expressed as millimeters of chart paper traversed at a rate of 10 mm/min. This can be converted to actual volumes with a knowledge of the flow rate, 2.0 ml/min. The copper chelates of NTA and EDTA used for this study had retention times of 0.7 and 1.5 min, respectively. From the equation,
the value of p for the copper-NTA complex is 1.98 mm and that for the copper-EDTA complex is 4.25 mm. The sensitivity for copper of the Perkin-Elmer Model 403 AAS instrument used in this study is given as 0.1 mgh. for 1% absorption (22).The same source gives the detection limit as 0.001 mg/l. However, this very low detection limit is attained taking advantage of relatively large time constants in the instrument electronics, thus maximizing the signal-to-noise ratio a t the detection limit. This system is not operative for the detection of a chromatographic peak in which the species concentration gets as high as the detection limit for only an instant. With the instrument, flame, and solvent conditions employed in this study, a detection limit of 0.01 mg/l. of copper was determined for continuous aspiration of copper solution. This value is used here for the detection limit. It corresponds to an absorbance of 0.0004 absorbance unit. Expansion of the recorder range to 0.0436 absorbance unit a t full-scale deflection yields a signal of 3 mm height (on a chart which is 300 mm full-scale) for copper at its detection limit. If a chromatographic peak is approximated as a triangle, the height of the peak for copper-NTA is 3 mm at the detection limit. As calculated from chromatographic parameters, the width of this peak at l/e times its height is 1.98 mm. Geometrical considerations show that the base of the peak is 3.13 mm. At a flow rate of 2.0 ml/min and a chart speed of 10
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
mm/min, this corresponds to 0.2 ml/mm X 3.13 mm = 0.626 ml = 626 pl. Aspiration of a 0.01 mg/l. (Cu) copper-NTA SOlution a t a rate of 2.00 ml/min for 0.313 min corresponds to aspiration of a total of 626 pl of copper-containing solution. This should yield the "square-wave" signal in Figure 2. The amount of copper which would elute during this period would equal 626 p1 X 0.01 ng/pl = 6.26 ng. Assuming a triangular peak, the area of a chromatographic peak of copper-NTA which would reach the detection limit value at maximum peak height is half of the area of the "square-wave" peak. This is shown in Figure 3. Therefore, the minimum detectable amount of copper-NTA corresponds to 3.13 ng of copper. A similar calculation for copper-EDTA on the column used in this study predicts a detection limit of 6.72 ng of copper for this peak.
RESULTS AND CONCLUSIONS The validity of the above calculations was tested by comparison to actual detection limits from known amounts of the copper chelates of NTA and EDTA. Following the arguments advanced above, the minimum detectable amount of copper-NTA would generate a triangular peak of 30 mm2 area and the area of the copper-EDTA peak would be 68 mm2. Experimentally it was found that 100 p1 of a copper-NTA solution gave a peak with an area of 280 mm2. The area of a corresponding copper-EDTA peak was 288 mm2. By proportion, the copper content of the copper-EDTA peak at the detection limit is, 100 ng 30 mm2 x -- 10.7 ng Cu for copper-NTA 280 mm2 and the copper content of the copper-EDTA peak at the detection limit is the following: 100 ng 68 mm2 X = 23.6 ng Cu for copper-EDTA 288 mm2 Comparison to the calculated values of 3.13 ng of copper for ~
copper-NTA and 6.72 ng for copper-EDTA shows that the experimentally determined detection limits are 3.4 and 3.5 times as high as the calculated values for the NTA and EDTA chelates, respectively. The consistency of these two ratios lends credibility to the approach. The differences between calculated and experimental values could be due to less than ideal behavior near the detection limit for copper. In addition, the assumption that the detection limit for a metal at the height of a chromatographic peak is as low as that for continuous aspiration is likely to be too optimistic. In summary, flame spectrometric methods of detection for HSLC have unique applications for the determination of certain types of species. Unfavorable detection limits can be a major handicap for this application. A method has been presented for the calculation of these limits from known chromatographic and spectrophotometric parameters.
LITERATURE CITED (1) S. E. Manahan and D. R. Jones IV, Anal. Lett., 6,745 (1973). (2)M. Unebayashi and K. Kitagishi, 5th International Conference on Atomic Spectroscopy, Monash University, Melbourne, Australia, Aug. 25-29, 1975. (3) D. R. Jones iV and S.E. Manahan, Anal. Lett., 8, 569 (1975). (4)D. R. Jones IV, H. C. Tung, and S. E. Manahan, Anal. Chem., 48, 7 (1976). (5) D. R . Jones IV and S. E. Manahan, Anal. Chem., 48, 502 (1976). (6)D. J. Freed, Anal. Chem., 47, 186 (1975). (7)A. Y. Cantilb and D. A. Segar, Proceedings of the InternationalConference On Heavy Metals in Environment, Toronto, Canada, 1976. ( 8 ) A. J. P. Martin and R. L. M. Synge, Biochem. J., 35, 1358 (1941). (9)H. C. Thomas, J. Am. Chem. Soc., 66,1664 (1944). (IO)C. E. Boyd, L. S.Meyers, and A. W. Adamson, J. Am. Chem. Soc., 6% 2849 (1947). (11)I. M. Kolthoff, E. B. Sandell, E. J. Meehan, and Stanley Bruckenstein, "Quantitative Chemical Analysis", 4th ed., The Macmillan Company, New York, N.Y., 1969. (12) Perkin-Elmer Corp.. "Anal. Methods for AAS," "Reference Manual" for Perkin-Elmer Model 403 AAS, March 1971.
RECEIVEDfor review July 7,1976. Accepted August 13,1976. This research was supported by National Science Foundation Grant No. MPS75-03330 and USDI OWRT Matching Grant B-095-MO.
Autocorrelation Analysis of Noisy Periodic Signals Utilizing a Serial Analog Memory K. R. Betty and Gary Horlick" Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2E1
The nature of autocorrelation analysis is briefly reviewed and then a new type of Integrated circuit Is described that is uniquely suited to the development of autocorrelationinstrumentation. This integrated circuit can be generally classified as a discrete time analog signal processlng device and is called a serlal analog memory. The serlal analog memory can temporarily store 128 consecutivesignal samples directly as analog levels. An autocorrelator has been constructed using the serial analog memory as a variable analog delay line. The operation of the autocorrelator is illustrated using several periodic signals. A noisy sine wave signal ( S I N < 0.2) can easily be recovered using this autocorrelator.
Correlation techniques have long been used to measure and process signals in the chemical, biological, physical, and engineering fields. Lee (1)discussed the application of correlation analysis to the detection of periodic communication sig-
nals rather early and later reviewed the topic in some detail ( 2 ) .Correlation techniques, again applied primarily to the communication field, have also been discussed by Lange ( 3 ) and were applied at a relatively early stage to the analysis of electroencephalographic data ( 4 , 5 ) .Correlation techniques have been applied to the measurement and processing of spectrochemical data (6, 7) and the application of correlation methods to chemical data measurement has recently been reviewed (8). Although correlation techniques have been employed in these and other fields with considerable advantage and success, their use has unfortunately been somewhat limited primarily by a lack of effective methods and instrumentation for the rapid, automatic evaluation of correlation functions. Present developments in certain large scale integrated circuits such as diode arrays, charge coupled devices, and bucket brigade devices are now beginning to provide very inexpensive instrumentation capable of sophisticated real time correlation operations (9). These integrated circuits can be generally classified as discrete time analog signal processing
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