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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Having ascertained the exact structure of the basic spectrum, if further data for the range [Eu]/[S] = 0 to 0.1 are available, it becomes possible to trace these back with confidence, even though the plots may be curved, so as to elucidate T values a t [Eu]/[S] = 0. Further, of course, the slopes of the lines of Figure 1 provide additional, useful information. The situation for which this method seems to us to offer the greatest value is where spectra obtained with an instrument of medium resolving power are so complex that even where the lanthanide reagent produces shifts, the resulting spectra cannot be properly interpreted because of inadequate resolution. Knowing then the line widths from the original spectra, the increase in resolution needed for full resolution can be calculated precisely. Since we also know the optimum [M]/[S], only a single run on the appropriate more sophisticated machine need be made, with consequent savings in time and expense. If the calculation indicates that full resolution cannot be achieved with any available spectrometer,
window analysis may still be applied since it is not necessary to deal only with the full spectrum. Window diagrams may be constructed for particular sections of the spectrum presenting especial difficulty. Thus a number of precisely defined values of [M]/[S] can be derived and will indicate the experiments needed to provide a set of spectra within which all lines or groups may be somewhere resolved.
LITERATURE CITED (1) R. J. Laub and J. H. Purnell, J . Chromatogr., 112, 71 (1975). (2) R. J. Laub and J. H. Purnell, Anal. Chem., 48, 799 (1976). (3) R. J. Laub and J. H. Purnell, Anal. Chem.. 48, 1720 (1976). (4) R . J. Laub and J. H. Purnell, J . Chromatogr.. 181. 49 (1978). (5) A. Peker, A. P. Johnson, and P. Stainton, J . Chem. Soc. C , 192 (1966). (6) A. Pelter and A. Albert, unpublished observations.
RECEIVED for review December 26, 1978. Accepted June 5 , 1979. We thank the Science Research Council for support of R.J.L.
Liquid Chromatogram Peak Storage and Analysis by Atomic Absorption Spectrometry Thomas M. Vickrey,’ Huston E. Howell, and Michael T. Paradise Department of Chemistry, Texas A & M University,
College Station, Texas 77843
The need for, and application of, element specific detectors to liquid chromatography has been described in the literature (1-3). The use of graphite furnace atomic absorption spectrometers as liquid chromatographic detectors (LCGFAA) shows high sensitivity and elemental selectivity ( I , 2). The mode of stream sampling which has been described by our laboratory and others involves removing an aliquot of the flowing eluate stream and dispensing the aliquot into the graphite furnace using a modified auto sampler ( 2 ) or a stream sampling valve and movable dispensing rack ( I ) . This is termed the “pulsed” mode of sampling because the AA data is not continuous in terms of the eluate stream. Because the sampling rate depends on the rate of graphite furnace analysis and subsequent cool-down time, only broad chromatographic peaks can be analyzed, or low flow rates used. Narrow peaks a t high flow rates can be totally missed by the AA analysis system. We have developed a sampling procedure which stores the peak containing eluate during the chromatographic run and then the capillary tube containing the peak is subsequently analyzed off line incrementally by the atomic absorption spectrometer. Only the chromatographic peak is, therefore, consumed in the analysis. This yields mdre atomic absorption analyses per chromatographic peak. With the increased number of analysis, the amodnt of signal obtained for each peak increased relative to the noise level. This signal to noise increase improves the accuracy of the LCGFAA technique over the “pulsed” mode of sampling. The additional sampling of each peak can, in the limit of totally consuming the peak, yield a nearly continuous single element concentration profile. This method of sampling should be directly applicable to routine sets of samples containing the same organometallic compounds. This work described the application of our microprocessor controlled Liquid Chromatograph-Graphite Furnace Atomic Absorption (LCGFAA) system to this mode of LCGFAA analysis. The benefit of this sampling method over other methods is described as are the limitations of this technique 0003-2700/79/035 1- 1880$0 1 OO/O
_-.
and the necessary loss of chromatographic resolution.
EXPERIMENTAL LCGFAA Interface, Instrumentation. The description of the LCGFAA interface has been previously reported ( I ) . The current system employs a Motorola 6800 based Heathkit microprocessor trainer ( 4 ) in lieu of the hard wired timer system previously used ( I ) . (The configuration of the H-174 trainer with the peripheral interface adapter is derived from that given in the user’s manual.) The peripheral interface adapter chip generates an eight bit control pattern which activates (1) the motor to lower and raise the dispenser rack, (2) the pneumatic valve which controls the sampling valve position, (3) the modified Sargent Welch automatic buret which forces the stored sample out of the capillary tube at a rate of 11.0 pL/s += 0.1, and (4) the remote switch to initiate the Hitachi 170-70 Zeeman effect atomic absorption spectrometer analysis cycle (see Figure 1). The sequence of events in sampling is to apply the sample to the column, store the eluate which contains the sample (this is determined by the UV detector response and/or knowledge of the retention time), manually transfer the sample tube to the syringe pump and to the LCAA interface, and begin the analysis by starting microprocessor program. The liquid chromatograph system used was an Altex (110 A Pumping system) isocratic system using a fixed wavelength (8-pL cell volume) 254-nm UV detector. The mobile phase used was MeOH:H20 (9O:lO); the column was a 0.46-cm id., 25-cm Lichrosphere (10 pm), C-18 reversed phase stationary phase. A Hitachi Model 170-70 Zeeman Effect Atomic Absorption Spectrometer was used for the lead determinations using the 283-nm analytical wavelength of Pb. In all cases the graphite cup type (internal volume, 50 /*L) furnace was used. The analysis sequence was: Dry, 25 s (60 “C); Ash, 12 s (500 “C) and Atomization, 5 s, (2400 OC). The Ar carrier gas was interrupted during the atomization. Sample Storage. The eluate stream containing the alkyllead compound was diverted after passing through the UV detector using a 6-way stream switching valve. The storage tube was a 10-ft,0.05-cm i d . Teflon capillary tubing (Altex) and was found to contain ca. 20 kL/in. After diverting the sample peak, the normal flow pattern was resumed and the tube containing the peak was attached to a syringe pump (modified Sargeant Welch 0 1979 American Chemical Society . _ 1 1 (
’ -
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
+-A
PEAK STORAGE
valve
collect-
//
storage tube
Storage Tube Manually Transfered
IYCREWEYTAL
ANALYSIS
,q5
B
storage tube
syringe oumo
7/
t o waste
Jdispenser
1881
the sample-loop dispenser and the syringe pump were determined. These determinations were performed monitoring a 0.5 ppb Cd solution. For 20 replicate samplings, the sample loop was found to deliver 37 1.7 pL. Similarly, the rate of delivery of the syringe was determined to be 11.0 f 0.1 pL/s of operation. Tetraphenyllead Analysis. Tetraphenyllead (PbPh4)was chromatographed and analyzed in the on-line pulsed mode and in the peak storage-pulsed mode. The comparative AA responses are given in Figure 2 along with thct UV detector output for the same peak. For a series of tetraphenyl lead solutions, the sensitivity of the peak storage method was determined. The area (block integration) plotted against ng of PbPh, chromatographed is a straight line (Corr. Coeff. = 0.9995). The detection limit for PbF’h4 we have determined to be 0.480 ng in a 20-pL sample injected onto the column. Figure 3 depicts the plots of the LCGFAA data obtained for tetraphenyllead. Peak Area Calculations. From the peak area one may determine the amount of sample present. The area of each peak was determined by block integration of the incremental peaks. The rectangular blocks were corrected for the overrun volume. The formula was:
*
Hitachi 170-70 wicroorocessor Flgure 1. (A) Schematic diagram of the peak storage device. (B) The AA and sampling apparatus
automatic buret) and then displaced from the tube with distilled water from the syringe pump. The eluate was displaced from the tube into the dispensing valve. Typically, 100 or 220 pL were pumped through the 37-pL sampling loop and the overrun gave high reproducibility of the volume injected (37 p L f 1.7) into the AA. Sample Solutions. Stock solutions of tetraphenyllead, PbPh4, were made fresh just prior to analysis. The solid was dissolved in a minimum amount of benzene and diluted to volume with methanol. RESULTS Precision of Sampling Method. The precision of both
where h, = The height in millivolts of the ith injection of a segment of the peak being analyzed. V,, = Volume between loop injections (overrun volume). S = Sensitivity in ng/mV of the AA detector. Extension of Analysis Range. The previous report on the pulsed mode LCGFAA analysis for tetraphenyllead ( 2 ) gave the detection limit for the LCGFAA method roughly equivalent to the detection limit by a LC-UV detector operating a t 254 nm. This value of ca. 30 ng Pb2Fh8in a 20-k~L sample injected onto the chromatograph does not reflect the obtainable sensitivity of the AA detector. In this study we found a useful lower limit of detection by the UV detector to be roughly the same level (2.0 ppm, 20-pL Injection) for the molecular PbPh4. However, with the addition of a short C.
1
I
Abs.
Abs.
5:r 10-
5~10‘~
H . 7 ml
w 2 . 1 ml
k-4 2.0 ml
Figure 2. (A) UV (254 nm) detector response for 20 pL of 2.3 ppm PbPh,. The HPLC flow rate was 0.5 mL min-’. (B) Peak storage mode for the same peak as in A. (C) Normal LCGFAA chromatogram for 20 pL of 5.7 ppm PbPh,. The HPLC flow rate was 0.2 mL min-‘
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
C
B
7 . 2 ng Ph Pb
36 n g P h q P b
Ilhl
I+----+1 . 5 ml
2 . 1 rnl
1.44 ng Bh4Pb
4
II 1
ft-------icl 4 . 0 ml
D
* IIII,
I
1 . 2 ml
Figure 3. LCGFAA peak profiles for the storage-mode analysis for a series of PbPh, solutions. (A) and (6)37-pL samples taken at 220-pL intervals. (C)
and (D) 37-pL samples taken at
100-pL
intervals
term storage of the liquid eluate, we have been able to obtain complete peak shape profiles for solutions containing as little as 0.024 ppm PbPh4. The analysis level is, therefore, improved by two orders of magnitude. Further, the increased number of AA analysis data points per chromatographic peak greatly improves the precision and accuracy of the analysis for total lead components. Figure 2 demonstrates this by comparing the peak storage off-line analysis to the pulsed on-line analysis method. Both sampling methods were discontinous in terms of the eluate stream; however, it is clear that the storage mode gives a more complete peak profile than the on-line analysis method. The theoretical limit for detection of peaks using this storage-off line mode is determined by the peak shape. This is related to the chromatographed sample detection limits by the decline in the maximum concentration of the peak. This decline is given by Equation 2 for Gaussian peak shapes. hmax =
total mass
(2)
or&
where the total mass and h,, are in picograms of PbPh4 and the standard deviation of the peak is in units relative to the injection volume. Because some broadening of the peak is going to occur as a result of the chromatographic process, the detection limit of the LCGFAA technique will be at least 2~’’’ times the detection limit of the AA method. The limits we have found are roughly an order of magnitude higher than this theoretical limit. The detection limit for PbPh4 which we have determined is ca. 20 pg, for solutions of PbPh4 directly analyzed by the GFAA, and 480 pg of PbPh4 subjected to LCGFAA analysis.
DISCUSSION One of the drawbacks of “pulsed’ LCGFAA as an analytical tool is the analysis time of the graphite furnace. The eluate stream may only be sampled when the furnace is cool. This leads to a very incomplete description of the actual concentration profiles eluting from the liquid chromatograph. This problem becomes very serious when the time between the analysis becomes equal to the width (in time units) of the chromatographic peak. Under these conditions the detector could conceivably miss a peak of interest. To overcome this problem, the eluate containing the chromatographic peak of interest is diverted (Figure 1) to a capillary tube of about the same volume as the volume of the analyte peak. The chromatographic peak is therefore available for off-line analysis. The incremented analysis in this way greatly improves the description of the chromatographic peak from the AA detector. The peak area can be more accurately calculated from the greater number of samples taken from the same peak. The concentration vs. response plot shown in Figure 3 demonstrates a typical data obtained for PbPh4. A direct comparison of the pulsed method of sampling and the storage method is shown in Figure 2. The trace concentration profile is given by the continuous detector (UV) response A. The PbPh4 peak tails only slightly in A. The tube storage analysis method (B) demonstrates the increased sensitivity of the GFAA detector for the same peak. Again the peak shape is slightly skewed. The pulsed mode analysis of PbPhl (on a higher concentration injected) in C, however, does not allow the peak shape (or area) to be determined accurately. The flow rate of the mobile phase had to be decreased by a factor of 2.5 to acquire this pulsed mode data,
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
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the tube a t the rate of delivery to the graphite furnace. The results are shown in Figure 4. The broadening is significant, but follows the classical broadening equations fcir laminar flow in a dead volume (5). For narrow peaks this might pose a problem because of loss in chromatographic resolution. This can be avoided by designing the storage tubes to be only slightly larger in interval volume than the peak volume. Likewise the resolution of two peaks can be maintained if the peaks are physically separated by storing each in a separate tube; then even though there is some peak broadening, the peak overlap will not occur.
Abs.
0.008
CONCLUSION The technique of Liquid Chromatography-Graphite Furnace Atomic Absorption spectroscopy (LCGFAA) has been applied to the study of environmental samples. In these samples the technique, LCGFAA, aids in the study of metal biotransport and trace organometallic determination a t low levels with concurrent molecular identification. The on-line pulsed mode of sampling gives a single element concentration as a function of retention time. The modification of this technique to an off-line procedure greatly increases the precision of the technique. The off-line storage of individual peaks allows a much higher number of AA data points to be obtained per peak. These additional data more completely describe the concentration profile of the organometallic compound present in the mobile phase. For a sample which contains multiple components, and the identity and retention time of each component is known. The separate component may be stored in different tubes. Each tube may then be analyzed automatically and the peak area for each component determined. For samples in which several organometallics are to he monitored routinely, this technique could be used. We are currently investigating the application of the temporary peak storage mode of sampling for multicomponent mixtures of organometallics of the same metal, and for inultielement multicomponent mixtures.
Abs,
0.008
LITERATURE CITED (1) Vickrey, T. M.; Buren. M. S.; Howell, H. E. Anal. Lett 1978, A l l ,
Figure 4. The UV (254 nm) responses for toluene (upper) and PbPh, (lower) in the peak broadening study
thus lengthening the time required for the experiment. Peak Broadening. The additional AA data for each analyte peak is accompanied by peak broadening due to the dead volume of the storage tube. The possible loss of chromatographic resolution was investigated using toluene and PbPh4. To analyze the effect of analyte storage the samples were chromatographed, passed through the sample site of the UV detector, stored in the tube, and pumped back through
1075-1095. (2) Brinckman, F. E.;Bhir, W. R.; Jewett. D. L.; Iverson, W. F', J. Chromtcgr. Sci. 1977, 15, 495-503. (3) Fernandez, F. J. A f . Absorpt. News/. 1977, 16, 33-37, and references therein. (4) "Heathkit Microprocessor Trainer Manual", Part 2, Sections 7, and 8; Heath Company: Benton Harbor, Mich., 1977. (5) Snyder! L. R.; Kirkland, J. J. "Modern Liquid Chromatography"; Wiley Interscience; New York, 1974; p 34.
RECEIVED for review March 13,1979 Accepted May 14, 1979. We thank the Robert A Welch Foundation For financial support of this work (Grant No. A-694), and the Texas A&M College of Science for the organized Research Funds (1977) to purchase the liquid chromatograph.
Automated Vapor Pressure Osmometer for Determining the Molecular Weight of Polymers Mark E. Myers, Jr.,' Stephen J. Swarln,"' and Byron L. Nellis' General Motors Research Laboratories, Warren, Michigan 48090
The vapor pressure osmometer (VPO) instrument is us_ed for determining the number average molecular weight (M,) 'Analytical Chemistry Department. Instrumentation Department. 0003-2700/79/0351-1883$01 O O i O
of organic and inorganic molecules in the molecular weight range of 50 to 20000 ( I , 2). In our experience it is used pJimarily for determinations on synthesized polymers having M , = 1000 to 10000. Such values of M , are too low to be determined by membrane osmometry because the solute IE 1979 American Chemical Society
