Quantitative Thin Layer Chromatography with X-Ray Emission Spectrometry R. A. Libby' Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio 45239 The application of X-ray emission spectrometry to the in situ quantitative analysis of solute zones after separation by thin layer chromatography is described. This new technique has been used to measure microgram amounts of phosphates, phospholipids, bromosalicylamides, and organosulfur compounds, in cellulose or silica gel thin layer adsorbents. Three calibration techniques are described and discussed. The intensity of emitted X-rays depends, in part, on the mass absorption coefficient, density, and thickness of the adsorbent. The variation in the absorption of the X-rays by the adsorbent, the chromatographic resolution, and the statistical counting error are the principal sources of the 5% relative error in this technique. This technique is also applicable to the analysis of paper chromatograms and, in principle, to other TLC systems.
THIN LAYER CHROMATOGRAPHY (TLC) is widely used as a qualitative technique for the separation of chemical species. Techniques for quantitative TLC are available, but these are not of general applicability for the following reasons. The techniques of aspirating or eluting the solute zone followed by the appropriate measurement technique have been reported for several classes of compounds. This technique is arduous and, in principle, is limited to those systems where a sensitive chemical or instrumental method is available. Volatilization or chemical change during handling may cause this technique to be unsatisfactory. Klaus has evaluated the photometric measurement of colored and fluorescent spots on TLC plates ( I ) . Schettino concludes that photodensitometric evaluation of chromatograms does not provide data so precise as that obtained by radiochromatographic or spectrophotometric methods (2). Gordon reports that the variation in dispersion of the fluorescent compound in TLC adsorbents causes a 10-20% deviation in triplicate analyses of urethanes of sterols by fluorescence scanning (3). Spectral reflectance has been reported for direct measurement on the TLC surface ( 4 ) and a planchet technique to remove the solute zone has been reported to improve this technique (5). Jork reports the detection of 0.5 to 10 pg of material, with an accuracy of 3-4z, by measuring the absorption spectra, based on directional reflectance (6). These techniques are applicable to those species having a usable absorption or fluorescence spectra. Spot shape is also known to be important. Bernhart and Chess measured several condensed phosphates on paper chromatograms by 1 Present address, Procter & Gamble Co., Winton Hill Technical Center, 6000 Center Hill Road, Cincinnati, Ohio 45224 ~~
(1) R. Klaus, J. Chromatogr., 16,311 (1964). (2) 0. Schettino, Farmaco, Ed. Prat., 20, 40 (1965).
Anal Abstr.,
13, 5278 (1966).
using densitometric measurement of the blue phosphomolybdate spots (7). Complete hydrolysis of the condensed phosphates and quantitative color development were important in obtaining accurate measurements. The charring technique is useful for carbonaceous solute zones on noncombustible adsorbents, but the errors associated with nonuniform and incomplete charring usually limit the precision to 5 % . The combination of volatility and low carbon content of some molecules also limits this technique. Charring cannot be used with cellulosic adsorbents or, in some cases, with inorganic adsorbents having organic binders or plastic supports. Radioactive solute zones on paper or thin layer chromatograms are analyzed by a variety of radioautographic methods. In theory, this technique is applicable to approximately 60% of the elements, but is limited in practice by the availability of suitable radioactive compounds. The radiochemical technique is applicable to some low atomic number species-e.g., gram of 3 2 P 2 0 ~ I4Cand 3H and is sensitive to as little as (8). Neutron activation has been used to measure the Na/P ratio of sodium orthophosphate zones on paper chromatograms (9). In this paper the application of X-ray emission spectrometry (XES) to the quantitative analysis of solute zones of thin layer chromatograms is reported. This TLC-XES combination was used to determine the amount of phosphorus, bromine, or sulfur in solute zones after separation of phosphates, phospholipids, bromosalicylamides, or organosulfur compounds. This new technique has been evaluated for cellulose and silica gel matrices and for paper chromatograms. In principle, the technique is applicable to the determination of any element having an accessible X-ray spectrum, and is generally not sensitive to the chemical binding of that element. Therefore, the technique can be easily adapted to the quantitative analysis of a wide variety of organometallic and inorganic species on thin layer or paper chromatograms without further chemical or physical modification. EXPERIMENTAL
Apparatus. TLC and XES measurements for those mixtures in Table I were made on Eastman K 301RzChromagram (silica gel), Brinkmann MN-Polygram Cel 300 cellulose sheets (Brinkmann Instruments, Inc., Westbury, N. Y . ) ,and Eastman 6064 Chromagram (microcrystalline cellulose) as received. Whatman No. 1 paper was used for paper chromatography. TLC adsorbent layers of different thicknesses were prepared as follows. Blank polyethyleneterephthalate sheets were prepared by floating the silica adsorbent from an Eastman K 301R2 Chromagram sheet (Distillation Products Industries, Rochester, N. Y.)and rinsing with deionized distilled water. The adsorbent spreader used to apply cellulose layers of varying thickness (10-215 p) is illustrated in Figure 1. The 3-mm
(3) H. T. Gordon, J. Chromatogr., 22, 60 (1966).
(4) M. M. Frodyma, R. W. Frei, and D. J. Williams, [bid.,13, 61 (1 964). (5) V. T. Lieu, R. W. Frei, M. M. Frodyma, and I. T. Fukui, Anal. Chim. Acta, 33, 639 (1965). (6) H. Jork, 2.Anal. Chem., 221, 17 (1966).
(7) D. N. Bernhart and W. B. Chess, ANAL.CHEM., 31, 1027 (1959). (8) J. Schroeder and S. Zielinski, Chemi. Anal., (Warsaw), 10, 335 (1965). (9) M. A. Rommel and R. A. Keller, J. Chromatogr., 18, 349 (1965). VOL. 40, NO. 10, AUGUST 1968
1507
Sample Sample wt 1. Na5P3OI06H20 Na4P207. 10HzO NaH2P04.H 2 0 75 Pg 2. Sameas 1 31 Pcg a
Table I. Results of TLC-XES Analyses of Mixtures Components in Each Mixture Are Listed in Increasing Order of Rf Cal procedure Sensitivity, Recovery Theory, Adsorbent" ratio CPSIK3 1 42.1 0.87 39.6 0.86 27.6 300 0.85 32.9 2 300
37.4
3. Same as 2 62 Pg
2 -
37.4
4. Same as 2 31 pg
2 6064
27.1
5. Same as 3 62 fig
2 6064
27.1
6. Sameas 1 75 P g
3 300
33.8
3
15.2
7. Na5P3OI06H20 NaaPn0710H20 NaH1P04.H 2 0 64 Pg 8. Ph. choline Ph. ethanolamine
-
Found,
z
Relative error,
40.0b 27.6 32.4
1.0 0.0
0.54 0.59 0.54 0.94 1.08 1.03 0.98 1.14 0.91
49.0 33.2 17.8 49.0 33.2 17.8 49.0 33.2 17.8 49.0 33.2 17.8 39.6 27.6 32.9 50.6 33.0 16.3
49.1 32.8 18.2 45.5 36.4 18.1 47.1 34.1 18.7 47.5 35.2 17.3 37.4 29.9 34.0 48.5 36.8 14.6
0.2 1.5 2.2 7.1 9.3 1.6 4.3 2.4 5.1 3. i 5.7 2.8 8.3 3.2 4.1 11.4 10.4
1.14 0.98
50.6 49.4
54.4 45.6
7.5 7.7
x
300
1.03 1.01 1.05 0.89 1.05 0.97 0.50
0.53 0.55
7.05 12.9
1.5
5.5
9. (CH&SO CioHziSOzCH3
1 14.0 0.72 50.8 52.7 3.7 K 301R2 0.67 49.2 47.3 3.8 3 27.4d 1.06 48.7 49.1 0.82 10. 3,5-DBR' 38.8d 1.06 51.3 50.9 0.78 5-BRC 300 a 300 = MN-Polygram Cel 300, 6064 = Eastman Microcrystalline Cellulose Chromagram, K 301R2 = Eastman Silica Gel Chromagram. Duplicate analyses. c DBR = dibromosalicylamide, BR = bromosalicylamide. * Not optimum.
r \
30
Figure 1. Plexiglas spreader for preparation of adsorbent layers of different thickness flange remaining on the guide block serves to guide the spreader along a 200- X 200-mm glass plate supporting the polyethyleneterephthalate sheet. Strips of Dymo embossing tape (Dymo Industries, Inc., Berkley, Calif.) or Scotch tape (#810), or combinations thereof are placed along the inner machined surface and up the sides of the guide (the shaded portions in Figure 1) to control layer thickness. The leading edges of the guide blocks are rounded to prevent gouging of 1508
ANALYTICAL CHEMISTRY
the supporting sheet. It is important to have another glass plate and plastic sheet at each end of the plastic sheet being covered. A small pool of adsorbent slurry is placed across one of the adjacent plastic sheets and the spreader is pulled through the slurry and across the wet plastic sheet being coated. The thickness measurements of the cellulose layers were taken with a Peacock #25 Upright Dial gauge (Lux Scientific Instrument Corp., New York, N. Y.)and the thickness reported is the average of 20 measurements of the difference in thickness of the support and the support with cellulose adsorbent. The X-ray emission measurements were made on a Norelco PW-1212 automatic spectrometer or on a General Electric XRD-6 spectrometer with emission attachments, both with proportional flow counters. The size of the rectangular TLC section (li2 x "4 inch) was chosen to ensure uniform coverage of the surface by the incident Cr K, radiation (60 kV, 24 mA for the PW-1212 and 75 kV, 40 mA for the XRD-6). Reagents. Decylmethylsulfide (Wateree Chem. Co., Lugoff, s. C.) was used to prepare the sulfone by conventional oxidation procedures. The bromosalicylamides were commercial materials (K and K Laboratories, Plainview, N. Y.) and were recrystallized from an alcohol-water solvent. The molybdenum spray reagents for inorganic phosphates (IO), and phospholipids ( I I ) , and the spray reagents and solvents (10) F. C. Charalampous and G. C. Mueller, J. Biol. Chew., 201, 161 (1953). (11) J. C . Dittmer and R. L. Lester, J. LipidRes., 5, 126 (1964).
for organosulfur compounds ( 1 2 ) were used without modification. In the solvent system for separating the inorganic phosphates on Brinkmann MN-Polygram Cel 300, isopropanol was substituted for dioxane in the solvent described by Clesceri and Lee (13). The solvent system described by Frei and Ryan (14) was used for chromatographing cupric nitrate salts on cellulose and a solvent composed of acetic acid/methanolland water, 15/35/50 by volume, was used for the bromosalicylamides. In the paper chromatographic separation of the inorganic phosphates, the solvent described by Smith ( 1 5 ) was used. The phospholipids were supplied by Dr. E. S. Lutton and had been isolated from egg yolk using conventional chromatographic techniques and the purity confirmed by elemental analyses and TLC. The samples were dissolved in toluene, stored under refrigeration, and separated on silica gel with a solvent of 65/25 ml chloroformiabsolute methanol. All other reagents were reagent grade chemicals and were used without further purification. Procedure. The adsorbent layer was scored with a narrow stylus to remove the adsorbent and leave seven or eight strips of adsorbent each ”14 inch wide and numbered 1 through 7 (or 8) from left to right. Three procedures for separating the mixture and calibrating the XES count intensity were used. In all three calibration procedures, the net X-ray count intensity (sample minus blank) was used for the calibration curve and analysis of the sample. PROCEDURE 1. The sample(s) (30-100 pg of material) was (were) applied 3 cm from the bottom of a seven-strip TLC sheet on strips 1, 2, 6, and 7. Strips 3 and 5 do not contain the sample and are used as blank sections to measure the Xray count intensity of the adsorbent after TLC. Strip 4 was cut from the chromatogram before chromatographing the remainder of the chromatogram with the TLC solvent. This strip was divided into 14 sections li2 X “14 inch and alternate sections were spotted with 2 4 samples of a concentration H 2 0 or Na2S04. The remaining sections series of NaH2PO4* of strip 4 served as blank strips for the X-ray measurement. After chromatography, strips 1 and 7 are cut from the chromatograms and sprayed with a visualizing reagent. By placing these strips adjacent to strips 2 and 6, respectively, the position of the solute zones can be indicated and the strips inch sections. Four samples can be run on a cut into single chromatogram with this procedure (on strips 1, 2, 6, and 7) if the R, values of the sample are accurately known or if the spots can be located by some technique other than spraying. PROCEDURE 2. In this case, 30 p1 of the appropriate TLC solvent was added to all the sections of strip 4 after preparing the calibration strip described in Procedure 1. In all other respects, Procedure 2 is identical to Procedure 1. PROCEDURE 3. A concentration series of the individual compounds to be separated and measured was chromatographed on the same chromatogram as the mixture being analyzed. The placement of these compounds on the chromatogram can be arranged to suit the requirements of the mixture being analyzed. For example, in the analysis of binary mixtures of bromosalicylamides, three different concentrations of the 5-bromosalicylamide were chromatographed on three strips of an 8-strip chromatogram and three concentrations of the 3,5-dibromosalicylamide were chromatographed on three other strips. The remaining two strips were used for the mixture and blanks. XES. For use in the PW-1212, each TLC section was fastened to a cellulose acetate disk 30 mm in diameter with a drop of rubber cement. The sample chamber of the PW-1212 (12) (13) (14) (15)
L. Fishbein and J. Fawkes, J . Chrornatogr., 22,323 (1966). N. L. Clesceri and G. F. Lee, ANAL.CHEM., 36,2207 (1964). R. W. Frei and D. E. Ryan, Anaf. Chim. Acta, 37, 187 (1967). M. Joyce Smith, ANAL.CHEM., 31,1023 (1959).
was loaded with three TLC sections and a reference sample and then evacuated. The time required to accumulate l o 5 counts on a reference sample was measured and then the three TLC sections were counted for the same period of time, The average count level from three complete cycles of the ratio mode counting procedure was used for the calibration curve and analyses. A typical counting time for one complete cycle of three samples and the reference is about 160 seconds. A germanium analyzing crystal was used for phosphorus K , and sulfur K, lines, a lithium fluoride crystal for the Cu K , and Br K , lines. On the XRD-6, the TLC section was held in a X inch window of a nylon mask on the sample drawer. The X-ray path was swept with helium for 30 seconds before taking three 40-second counts of the phosphorus radiation. THEORY
The intensity of the emitted X-ray beam that reaches the surface of the matrix is described by the equation (16). dI2t = Q ~ O exp A
1-b csc $1
+ P’ csc 42) pxldx
(1)
relating the emitted intensity dl,, to an excitation constant Q f , the weight per unit volume of the element in the layer dx ( p J , the density of the matrix ( p ) , the incident X-ray intensity Iox, the distance x beneath the surface, and the mass absorption coefficients of the matrix p and p’ for the incident and emitted X-rays, respectively. The angles dl and c $ ~ between the sample surface and the incident and emitted beams on the PW-1212 are 65” and 36”, respectively. The value for p csc $ J ~ p’ csc $2 was calculated using values of 14.7, 37.0, and 0.5 cm2/g ( 1 7 ) for the mass absorption coefficients for carbon, oxygen: and hydrogen, respectively, for Cr K , incident radiation (2.3 A) and values of 269, 620, and 3.5 cm2/g, by graphical extrapolation, for the same elements at 6.16 A (18). The integrated form of Equation 1 (16) is shown below
+
+
where p” replaces the p csc 41 p ’ csc 42 term in Equation 1. This equation was rearranged to
QJox
l-exp(-p”px) (3)
where pi was written as a term for element weight per area ( g r cm-2) in a layer of thickness x(cm). The values of the exponential term in Equations 1 and 3 at different layer thicknesses were calculated and these calculated values are compared to the experimental data in Figure 2. The dashed portion of the curve in Figure 2 is an extrapolation of the experimental data, using an experimental point for a 10 p layer as a guide. This extrapolated value of count intensity at zero thickness was used to establish the point where either exponential function on the right ordinate has a value of unity. Curve 2 in Figure 2 is that calculated by Equation 1 for a point source of phosphorus one-fourth the distance into the cellulose layer. Curve 1 in Figure 2 was calculated by Equation 3 for an even distribution of phosphorus in the cellulose layer. Curve 1 was normalized to the (16) L. S. Birks, “X-Ray Spectrochemical Analysis,” Interscience, New York, N. Y., 1959, p 59. (1 7) International Tables for X-Ray Crystallography, Vol. 111, C.H. Macgillavry and G. D. Rieck, Eds., Kynoch Press, Birmingham, England, 1962, p 164. (18) H. A. Liebhafsky, H. G. Pfeiffer, E. H. Winslow, and P. D. Zemany, “X-Ray Absorption and Emission in Analytical Chemistry,” Wiley, New York, N. Y.,1960, p 315. VOL. 40, NO. 10, AUGUST 1968
1509
,
11
70
-
x
-
-5
- 0.4 -3F.a - 0.3
-
0
4
\
l.83 0
1.0
e
4
1.2-
0.6
- 0.2
-
O
-0.1 !
0
l I l I I~ I , , , , I 40 80 120 160 200 240 280 Cellulose Layer Thickness, Microns I
I
I
2
Figure 2. Layer thickness-intensity profile. Solid lines calculated from X-ray absorption laws; Curve I calculated from Equation 3, normalized to experimental curves at 85 p, Curve 2 calculated from Equation 1 for point source one-fourth the distance into the adsorbent layer. Experimental points from data using calibration procedure 1. Dashed line is extrapolation to zero thickness for experimental points using calibration procedure 1
experimental data at 85 p. Calculated data for point sources one-half the distance or more into the cellulose layer and for even distributions normalized to the experimental point at other thicknesses do not fit the experimental data as well. RESULTS
Effect of Solute Penetration and Layer Thickness on X-Ray Intensity. The decrease in the net counts with an increase in cellulose layer thickness for the experimental points in Figure 2 indicates that the phosphorus solute zone penetrates into the cellulose adsorbent. The similarity between the experimental data and the curves calculated from two forms of the X-ray absorption law suggests that matrix absorption of the incident and emitted X-rays is important in applying this technique to the quantitation of thin layer chromatograms. In this system, matrix absorption is more important for the emitted X-ray than for the incident X-ray. The comparison of the experimental and calculated values for the layer thickness-intensity relationship shown in Figure 2 will not unequivocally distinguish between an even distribution or a point source locus of the solute zone one fourth the distance into the layer. Some equivocation is due to approximations in the exponential term and some is due to the relative nature of the calculations imposed by unknowns in the preexponential term. It does appear that the position of the phosphorus solute zone in the cellulose layer depends on the thickness of the cellulose layer. For thin layers (
