912
Anal. Chem. 11989,61. 912-914
i
1 .
1
A
B
C
I
Figure 2. Chromatogram resulting from the injection of 23 pg of decanoic acid (A), 20 pg of lauric acid (e),and 20 pg of myristic acid (C). The top chromatogram is the indirect TL detected signal and the bottom is the result of integrating the base-line-adjustedchromatogram.
tection method will be readily adaptable to microcolumn conditions. Optimization of other experimental variables such as background absorbance or the use of visualization reagents with similar hydrophilic properties but larger molar absorptivities will also result in improvements in the LOD by increasing the DR. This is reasonable because the system is presently operating below the background limitation by 2 orders of magnitude. In conclusion, this communication shows that indirect absorbance detection is feasible for microcolumn chromatography when photothermal methods of detection are applied. This is a result of the large dynamic reserve available in the differential thermal lens spectrometer described as well as the reduced detection volume available with laser probes. While more studies are needed to optimize the experimental parameters both in the chromatography and in the spectrometry, a universal mass LOD of 15 ng of detected and- is a marked improvement for a simple, universal HPLC detection scheme.
LITERATURE CITED Halgunset, J.; Lund, E. W.; Sunde, A. J . Chromafogr. 1982, 237(3),
jection variability, to the ratio of the amount of material injected. In contrast, the signal observed in previously described indirect methods is a function not only of the amount injected but also of the retention time. If the signal arises only from the dilution of the visualization reagent, response of this detector should not rely on any physical or chemical property of the eluent other than injected mass. This universal response would be quite useful for quantitative measurements in that a standard response curve for one analyte could be used for any other analyte detected in a given solvent system. This statement would be valid as long as the analyte and solvent behave as an ideal solution. The response of the detector has been determined to be linear over at least 2 orders of magnitude above the detection limit. The plot of normalized peak height response versus the normalized mass of dodecanoic acid injected had a slope of 0.98, an intercept of 0.06, and a correlation coefficient of 0.990 (5 points). The signals measured in several chromatograms were used to determine an average limit of detection (LOD) of 15 ng detectable in the cell corresponding to 500 ng of injected acid. In terms of the actual probed volume of 32 nL, the amount of decanoic acid detectable in the detection volume a t the detection limit is 90 pg. It should be stressed that the detection volumes demonstrated here are best suited to microcolumn techniques rather than the large diameter columns used in this study. Smaller column sizes can reduce the LOD by a factor of 20 (1 mm i.d. column) due to the reduced peak volumes (9) characteristic of microcolumn chromatography. Preliminary results using 0.5-pL detection cells (1.3 mm path length) have demonstrated that this de-
496-499.
Miyaguchi, K.; Honda, K.; Imai, K. J .
Chromafogr.
1984, 316,
501-505.
Watanabe, Y.; Imai. K. Anal. Biochem. 1981, 116(2), 471-472. Hancock, D. 0.; Synovec. R. E. Anal. Chem. 1088, 60, 1915-1920. Bornhop, D. J.; Dovichi, N. J. Anal. Chem. 1986. 58, 504-505. Small, H.; Miller, T. E. Anal. Chem. 1982, 54, 462-469. Downey, B. P.; Jenke, D. R. J . Chromatcgr. Sci. 1987, 25(11), 5 19-524.
Takeuchi, T.; Ishii, D. J . Chromatogr. 1987, 393, 419-425. Takeuchi, T.; Yeung, E. S. J . Chromatogr. 1988, 366, 145-152.
Banerjee, S. Anal. Chem. 1985. 57, 2590-2592. Takeuchi, T.; Ishii, D. J . chrometogr. 1987, 403, 324-330. Dovichi, N. J.; Harris, J. M. Anal. Chem. 1980, 52, 2338-2342. Erskine, S. R.; Bobbitt, D. R. Awl. Spectrosc., in press. Synovec, R. E.; Yeung, E. S. Anal. Chem. 1985, 57, 2162-2167. Synovec, R. E.; Yeung, E. S. Anal. Chem. 1986, 58, 2093-2095. Stranahan, J. J.; Deming, S. N. Anal. Chem. 1982, 54, 1540-1546. Banerjee, S.; Pack, E. J. Anal. Chem. 1982, 54, 324-326. Woodruff, S. D.; Yeung, E. S. J . Chromatcgr. 1983, 260, 363-370.
Steven R. Erskine Donald R. Bobbitt* Department of Chemistry and Biochemistry University of Arkansas Fayetteville, Arkansas 72701 RECEIVED for review October 18, 1988. Accepted January 24, 1989. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this work. S.R.E. thanks the Analytical division of the American Chemical Society for support through a fellowship sponsored by DOW Chemical Co. D.R.B. acknowledges the support of the Camille and Henry Dreyfus Foundation through a teacher-scholar fellowship. This work was presented in part a t the 27th Annual Eastern Analytical Symposium, Oct 2-7, 1988.
Separation of Two Components of Horse Myoglobin by Isoelectric Focusing Field-Flow Fractionation Sir: In 1984 a theoretical analysis of the focusing field-flow fractionation in channels of various cross-sections was published (I). The focusing principle was originally described by one of the authors (2) in 1982 and approved experimentally in 1986 for a special case of the sedimentation-flotation focusing field-flow fractionation (3). Other methods of focusing field-flow fractionation were suggested but none was realized (4-6). 0003-2700/89/0361-0912$01.50/0
In this communication the first experimental implementation of the isoelectric focusing field-flow fractionation (IEFFFF) is described. In addition to the electric field and pH gradient (3, IEFFFF employs a third active separationaffecting factor, viz., the flow of the liquid through the separation channel with the direction of the flow perpendicular to that of the electric field. The shape of the flow velocity profile formed is influenced by the geometry of the fraction0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 8 , APRIL 15, 1989
913
t
INJECTION PORT
Flgure 1.
M E M B R A N E S ELECTRODES FRACTION COLLECTOR
Schematic diagram of IEFFFF apparatus.
ation channel (8). An amphoteric solute is transported under the influence of the electric field and pH gradient to the position where the solute possesses no net overall charge. As soon as the dynamic equilibrium b e h e e n the concentrating (focusing) and dispersive (diffusion) processes is reached, the narrow focused solute zone with nearly Gaussian concentration distribution is formed. Provided that different solutes exhibit different isoelectric points, they are focused in different positions across the fractionation channel where the local values of pH are the same as the isoelectric points of the solutes. The velocity profile formed in the liquid flow causes the migration of focused zones along the channel a t different velocities, so that the solutes are longitudinally separated.
EXPERIMENTAL SECTION Materials. Horse heart myoglobin (Sigma Chemical Co.) was dissolved in an ampholyte solution prepared by mixing Ampholine 3-6, Ampholine 7-10 (LKB), and water at a ratio of 1:1:38. Apparatus and Procedure. The use of the fractionation channel with a trapezoidal cross-section was described in detail earlier (1,3, 8). The modification of the channel for IEFFFF consisted of situating the chambers for platinum wire (diameter 0.05 mm) electrodes on the opposite sides of the trapezoidal channel. These chambers were separated from the free space of the fractionation channel by membranes (Kalle, Wiesbaden). The dimensions of the Plexiglas channel were as follows: length, 25 cm; height, 0.5 cm; width of the opposite walls of the trapezoid, 0.045 and 0.095 cm. The apparatus is shown schematically in Figure 1. A freshly prepared solution of myoglobin was injected into the channel by using a syringe. The injected volume was 50 pL and M. A linear disthe myoglobin concentration was 2.0 X placement feeder was used to pump the ampholyte solution through the fractionation channel. The flow rate of the liquid was 25 pL/min. A HP 1040A diode array spectrophotometric detector with a 4.5-pL flow cell was used. The fractograms were recorded at 410 nm. A linear displacement feeder with two syringes was used to pump electrolyte solutions through the electrode chambers: 0.05 M acetic acid was an anodic solution, 0.01 M NaOH was a cathodic one, and flow rates of both solutions were 500 pL/min. RESULTS AND DISCUSSION The experiments were intended to verify the separation capability of IEFFFF. As apparent in Figure 2, no separation w8s reached by performing the experiment without the electric field. On the other hand (see Figure 3),the separation of acidic and basic myoglobin was observed in the presence of the electric field (V = 70 V, I = 50 mA). The result of the separation was similar to that performed by using isoelectric focusing in polyacrylamide gel rods with the same sample. The first peak in Figure 3 corresponds to the acidic myoglobin (PI 6.85) and the second one corresponds to the basic myoglobin (9) (PI7.35). The orientation of the pH gradient in these experiments caused the focusing of acidic solutes in the broader part of the channel where solutes migrate at higher velocities than in the narrower part of channel. The deter-
0
Flgure 2. Fractogram (absorbance at 410 nm, A,,,,, vs retention volume, V,) of myoglobin without electric field applied: injected volume, 50 pL; flow rate, 25 pL/min. 0.10
A,,O
0.05
I
I
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Fractogram of myoglobin with electric field applied. Vottage, current, 50 mA. For other experimental conditions see Figure
Figure 3.
70 V; 2.
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0.5
1 X
Figure 4. pH gradient inside the channel in the absence of myoglobin. The pH values at the relative positions x = 0 and x = 1 show the pH values of cathodic and anodic electrolytes.
mination of the pH gradient was performed in the continuous preparative channel with modiied output (I). The ampholyte solutions from three outlet capillaries were collected and pH
Anal. Chem. 1989. 67,914-917
914 0.1
c
300
Basic lorm
500
900
A, n m Flgure 5. Absorption spectra measured in the maxima of peaks in
Figure 3. values were measured. The results are shown together with pH values of the anodic and cathodic solutions in Figure 4. It follows from the comparison of Figure 2 and Figure 3 that both peaks in Figure 3 correspond to retained species. The peaks in Figure 3 were identified with the aid of absorption spectra in the Soret region. It can be seen from Figure 5 that spectra of both peaks are characteristic of horse myoglobin (10). Although the molar absorption coefficients of myoglobins are pH dependent, the difference between pH values of 6.85 and 7.35 is negligible (11). Thus the quantitative proportions of the two fractogram peaks agree with the well-known fact that myoglobin contains a higher amount of the basic form. The difference between the integral of the curve in Figure 2 and the sum of the integrals in Figure 3 can be explained either by the presence of low molecular ionic species of iron in the myoglobin preparate, which are separated from the channel through membranes via electrophoresis, or by changes in the molar absorption coefficient of myoglobin in the wide pH range in the absence of the electric field. In this case the pH gradient is formed by diffusion of H+ and OH- ions from electrolyte solutions into the channel through membranes and
myoglobin is spread across the channel. The molar absorption coefficients of myoglobin in the alkaline region are considerably higher than those in the neutral and acidic regions (11). Isoelectric focusing is a frequently used technique with an exceptional resolution; nevertheless it is relatively time-consuming and requires a high voltage and an efficient cooling. The addition of the solvent flow as the third separation-affecting factor can reduce these disadvantages. The dimension of the channel in the direction of the electric field (perpendicularly to the direction of the liquid flow) can be reduced to the millimeter range (in our case 5 mm). This permitted the decrease in the absolute voltage values while keeping a high field strength and resulted in a lower Joule heat production and a shorter focusing time. Moreover, the laminar flow of the solvent stabilizes pH gradient against convection. The successful separation of two components of myoglobin in relatively short time (- 1h) proved the viability of IEFFFF. Although IEFFFF was invented as an analytical technique, it can also be suggested as a continuous preparative technique (1).
LITERATURE CITED (1) Jan&, J.; Chmeik, J. Anal. Chem. 1984,56, 2481-2484. (2) Jan&, J. Makromol. Chem., RapM Commun. 1982,3 , 887-889. (3)Chmeiik, J.; JanEa, J. J . Lip. Chromatogr. 1988,9 , 55-66. (4) Giddings, J. C. Sep. Sci. Techno/. 1983, 18, 765-773. (5) Chmellk, J.; Jan&, J. National Meeting of the Czechoslovak Chemical Society, Proceedings Section 1, 7; Banski Stiavnica, July 1984 p 38. (6) Semenov, S. N.; Kuznetsov, A. A.; Zolotarev, P. P. J . Chromatogr. 1988,364, 389-396. (7) Svensson, H. Acta Chem. S c a d . lS81, 15, 325-341. (8) Jan&, J.; Jahnovi, V. J . Lip. Chromatop. 1983,6 , 1559-1576. (9) Radola, B. J. Biochim. Blophys. Acta 1973,295, 412-428. (10) Sono, M.; Smlth, P. D.; Mc Cray, J. A.; Asakura, T. J . Bioi. Chem. 1978, 251, 1418-1426. (11) Sono, M.; Asakura, T. J . Bioi. Chem. 1976,251, 2684-2670.
J. Chmelik* M. Deml J. Janira Institute of Analytical Chemistry Czechoslovak Academy of Sciences 611 42 Brno. Czechoslovakia
TECHNICAL NOTES New Way To Mount Particulate Materlal for Laser Microprobe Mass Analysis R. A. Fletcher
Center for Analytical Chemistry, National Institute of Standards and Technology, Building 222, Room A121, Gaithersburg, Maryland 20899 INTRODUCTION When particles are analyzed by a variety of microanalytical techniques, there is often a problem with transferring the particles of interest from the collector medium to the appropriate instrument-compatible support for analysis. This is especially true for mounting samples in the laser microprobe mass analyzer (LAMMA 500). The normal procedure for mounting particles is to first suspend them in a liquid and then evaporate an aliquot of this liquid onto a thin organic support film (such as collodian). Analysis is then performed by ablating the particles in or on the film, and at the same time puncturing and ablating part of the film. If the particles are initially caught on a membrane type filter, a small portion
of the filter can be cut, mounted on a support grid, and dissolved away leaving the insoluble particles. In both approaches, there is a question of particle loss in the transfer procedure and problems with dissolving particles and particle contamination from the solution. We wish to report a new way of mounting particles and microstructures on the LAMMA 500 which may also be useful for other microanalytical techniques. The mounting technique involves removing the top fiber layer from a high-purity quartz fiber filter that contains a collected particle sample. The small particles ( 1 to 10 pm diameter) remain attached to the fibers by van der Waals forces and possibly static charge. The particles are analyzed directly on the fiber by the laser microprobe.
This article not subject to US. Copyright. Published 1989 by the American Chemical Society
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