1878
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
reservoir system. A glass syringe ( S ) serves as a variable volume solvent container. Most of the glass syringes commercially available for medical use are accessible for this purpose; a syringe with a capacity of about 500 mL may be good enough for usual analytical HPLC; if necessary, two or more syringes are connected in parallel. The outlet of the syringe is fitted to Teflon tubing (1.5-mm i.d.) with a Durrum ~ 2 4 0 4 4luer adaptor (A). A three-way valve (V) is used to control the delivery of solvent. A filter (F) is attached at the solvent inlet in this system. Prior to being stored in the syringe, the solvent to be applied must be degassed by an appropriate procedure: for example, several minutes stirring of the solvent in a flask under reduced pressure. The degassed solvent is then transferred into the syringe by pulling the plunger of the syringe manually. If air remains in the tubing or syringe, it has to be expelled from the system by pushing the plunger. When the syringe is filled with the solvent, the three-way valve is turned to deliver the solvent from the syringe to a pump. The syringe is then set upside down as shown in Figure 1, and a stand is used only to keep the syringe from upsetting; the cylinder of the syringe must be freely movable in this arrangement. The cylinder moves spontaneously and continuously downward because of its weight while the solvent is delivered from the syringe to the pump. The replenishment of degassed solvent into the syringe during a chromatographic experiment is performed, if necessary, by turning the three-way valve to deliver the solvent from the solvent inlet to both syringe and pump. The degassed solvent which has been once charged in the syringe never comes into contact with atmosphere. The present solvent reservoir is thus effective to keep the degassed solvent for a long period. According to our experience, no bubble formation in a detector was observed with the 9O:lO methanol-water mixture stored in this reservoir over a week a t room temperature.
6-3 I'
J
U
L
Figure 1. Schematic diagram of degassed solvent reservoir. (S)Glass syringe; (A) syringe-tube adaptor: (V) three-way valve; (F) filter
components of te3t samples, the solvent degassing has to be done to keep a column from damage, or to avoid undesirable transformation of the components. Some expensive commercially available instruments for HPLC include built-in degassers which enable continuous delivery of degassed solvent to the column. However, in an experiment with a relatively simple instrument, the solvents which have been degassed previously are stored in simple solvent reservoirs such as glass flasks and bottles. It is not easy to use such a simple solvent reservoir because of redissolution of gases, moistening, and vaporization of the solvent during storage. The solvent reservoir designed in our laboratory is free from the above problems because of its exposure-proof structure to atmosphere. Figure 1 is a schematic representation of the
RECEIVED for review January 3,1979. Accepted May 23,1979.
Application of the Window Analysis Optimization Method to Lanthanide Shift Nuclear Magnetic Resonance Spectra R. J. Laub Department of Chemistry, Ohio State University. Columbus, Ohio 432 10
A. Pelter and J. H. Purnell" University college of Swansea, Singleton Park, Swansea, SA2 8PP Wales The window analysis method of optimizing chromatographic analyses introduced by Laub and Purnell ( I , 2), has proved extremely successful. In chromatography the important separation parameter is the relative retention ( a ) ,and optimization involves ascertaining means to maximize this quantity for the two components presenting the greatest difficulty in separation. Laub and Purnell have shown how cy can be derived, as a function of several relevant chromatographic variables, from readily obtainable experimental information (3, 4). Plotting derived values of cy against the other variable, arranging that cy > 1 a t all times, then yields a diagram normally consisting of overlapping inverted triangles. The areas of the diagrams where overlap does not occur (windows) define a range of conditions in which complete separation of the mixture of interest is possible, the optimum choice, in terms of column length, corresponding to the peak of the highest window, Le., highest minimum cy. 0003-2700/79/0351-1878$01.00/0
The method is, in fact, widely applicable in analysis in general since its use requires only identification of the parameter to be optimized and a knowledge of its dependence upon various relevant variables. We illustrate here its application to lanthanide shift reagent NMR, now widely used for spectral clarification. It is normal for the associated shifts to be linearly dependent upon the concentration ratio [M]/[S] (where M represents the metal chelate and S the substrate) when [M]/[S] lies in the approximate range 0.1 to 0.7, although linearity sometimes extends outside this range. The data are then normally interpreted by visual inspection, often aided by graphical representation as plots of T (or 6) against [M]/[S]. There is no particular difficulty in locating an optimum value of [MI / [SI when the spectrum is simple, but in complex cases, such diagrams may be difficult even to construct, let alone interpret, because of superimposition of lines, and a value of 0 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
1879
8
AT
UL 0 25
0 35
0 45
3 5'1
0 65
[SI
Figure 2. window diagram constructed from data represented by straight lines drawn in Figure 1. Optimum [Eu]/[S] = 0.313, AT = 0.179
I 0 35
0
0 25
I 0 45 [E
Figure 1. Plots of
obtained with
T
I
0 55
0.65
;I 1[SI
against [Eu]/[S]. Data taken from indivkIual spectra using CHCI, as solvent. Eu = Eu(dpm),; S =
HA100
dimethyldihydroisonorscandenin
[M]/[S] offering the chance of complete resolution may be impossible to locate with any confidence. Application of the window method to lanthanide shift NMR involves obtaining spectra a t four or five values of [M]/[S], plotting values of T against [M]/[S] for every line discernible and then ascertaining the parallelism or otherwise of the plotted lines in order to identify related groups of lines. The central value of T for each group (or singlet) at each value of [MI/ [SI is then calculated and the differences, AT, for all pairs of lines or groups are then determined. These are plotted against [M]/[S]. By treating AT as positive, irrespective of sign, any pair for which AT actually changes sign as a function of [M]/[S] will generate an inverted triangle on the plot. The crossings of the various pairs of lines will then produce the windows. The highest window defines both the optimum value of [M]/[S] and the smallest AT in the optimized spectrum, as well as the corresponding lines or groups of lines generating this value, Le., the most difficult to resolve. If the instrument used can resolve these particular lines, all other lines in the spectrum must obviously also be resolved. As an example, we illustrate data for dimethyldihydroisonorscandenin ( 5 ) (1) CH3%
/
0 CHI
OCH3
'
OCH]
CHj (1)
with an europium shift reagent [Eu(dpm),] in CHC13, obtained with a HA100 spectrometer. The spectra are of only moderate
1 I
,
, , , , , , , , , 2
1' - 6a
, , , , , , I , , , , , , , ~ , 3
"
'i
"
" "
''i
Figure 3. Spectrum obtained at [Eu]/[S] = 0.313. F:rom left to right: doublet, doublet, singlet, singlet, triplet, triplet, triplet, triplet, singlet, singlet
complexity and can, in fact, be interpreted (6). Figure 1 shows plots of T against [Eu]/[S] for compound 1. Those peaks for which the plots are close together and move in a parallel fashion are defined as multiplets and inspection of Figure 1 readily leads to the identification of four singlets, four triplets, and two doublets. It is evidently unnecessary to calculate and plot values of AT for pairs of lines or groups of lines that are very widely separated throughout the concentration range. In this example it is necessary, in fact, to include only six pairs of groups or lines. Figure 2 illustrates the window diagram resulting. Two good windows are seen, the better being at [Eu]/[S] = 0.313, with the minimum separation among the groups corresponding to a pair of triplets having AT = 0.179 between centers. Since each triplet had a composite line width of about 0.18, it should thus be possible, at this value of [Eu]/[S] to obtain a fully resolved spectrum. Figure 3 illustrates the spectrum obtained which confirms the prediction. Further, by reading up Figure 1 at [Eu]/[S] = 0.313 we can predict, the T values of all lines in the resolved spectrum. These agree extremely well with the experimental values and give further confidence in the interpretation of the spectra.
1880
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 (
’ -
