b
hand, it is unlikely that nitrate would interfere in other colorimetric determinations of formaldehyde. The Schiff s Reagent Method, for example, developed by Deniges (9) or the Hantzsch Reaction Method by Nash (10) may be suitable, although these methods are not as simple and rapid as the method of P. W. West and B. Sen. Experimenters who attempt to detect the presence of formaldehyde by the violet color development with chromotropic acid should be aware that nitrate- or nitrite-containing compounds may mask the expected violet color with an orange-brown background. Other nitrate-containing compounds such as nitrosamines may cause interference similar to nitrate in the chromotropic acid procedure, but none of these were examined. It is interesting to note that NOz gas apparently has no significant effect on formaldehyde determinations (11) while both nitrate and nitrite ions do have significant influences as reported here. No other ions in our culture medium interfered in the assay.
LITERATURE CITED
r
0
1
I
1
1
I
IO
20
30
40
50
TIME, hr.
Figure 4. Time stability of interference of A570due to nitrate. Samples contained either (a) 15 Kg/mL formaldehyde or (b) 5 pgImL formaldehyde and NaN03 in concentrations of (A)1.0 mg/mL, (0)0.5 mg/mL, (A) 0.05 mg/mL, (0)0.005 mg/mL, or ( 0 )none. The data represent t h e averages of duplicate determinations. Percent was calculated by determining the ratio X 100 of the A570of the sample containing formaldehyde and/or nitrate at a particular time to the A,,, of the corresponding formaldehyde standard (which contained no nitrate) at that same time
desirable and nitrate cannot be removed, dilution of samples to obtain low nitrate concentration will overcome the interference since the assay is sensitive to very low formaldehyde concentrations. Otherwise, nitrate interference will exaggerate or mask formaldehyde levels in samples depending upon the true concentration of each, the ratio of the two, and the time the absorbance is read after adding the reagents. On the other
(1) J. F. Walker, “Formaldehyde”, ACS Monograph, 159, 3rd ed., (1964). (2) P. W. West and 8.Sen, Fresenius’ 2.Anal. Ctwm., 153, 177-183 (1956). (3) F. Feigi, “Spot Tests in Organic Analysis”, 6th ed., Elsevler Publlshing Company, New York, N.Y. 1960. (4) P. W . West and T. P. Ramachandran, Anal. Chim. Acta, 3 5 , 317-324 (1966). ( 5 ) Y. Chalfan and R. I . Mateies, Appl. Microbiol., 2 3 , 135-140 (1972). (6) P. Pilat and A. Prokop, Blotechnol. Bfoeng., 17, 1717-1728 (1975). (7) R. Whittenbury, K . C. Phillips, and J. F. Wilkinson, J , Gen. Microbiol., 61, 205-216 (1970). (8) P. W. West and P. L. Sarma, Mikrochim. Acta, 4 , 506-509 (1957). (9) G. Deniges, J . Pharm. Chim., 6, (4) 193 (1896). (10) T. Nash, Biochem. J . , 55, 416-421 (1953). (11) A . P. Altshuller, D. L. Miller, and S.F. SIeva, Anal. Chem., 33, 621-625 (1961).
Elizabeth L. R. Krug* William E. Hirt School of Chemical Engineering Purdue University West Lafayette, Indiana 47907
RECEIVED for review May 2, 1977. Accepted July 11, 1977. This work was supported by a grant from the National Science Foundation, Grant No. E N G 75-17796.
Consistent Terminology for Efficiency in High Performance Thin-Layer Chromatography Sir: T h e t e r m “high performance thin-layer chromatography” (HPTLC) has begun to appear in the chromatographic literature within the past year (1-4). Its use in various contexts has given rise to some confusion concerning the definition of “high performance”. Part of this confusion stems from the lack of a widely applicable terminology for the description of efficiency. Although many measures of chromatographic efficiency have been used, those most easily related to fundamental system parameters are the theoretical plate number, n, and the plate height, h (5,6). In liquid-solid chromatography, however, n and h are unequivocally defined only for isocratic, isothermal chromatographic systems in which the mobile phase velocity a t any instant is identical throughout the system. The use of theoretical plate number and plate height, without further qualification, to describe gradient chromatographic systems (specifically, in the present context, radial (7), evaporative (8), or multiple development (9) TLC, all of which produce or mimic solvent velocity gradients) can be confusing and misleading.
Attempts have been made to tackle this problem in describing Programmed Multiple Development (PMD) (10) by using the term “equivalent theoretical plates”, described as: “the number of theoretical plates a conventional development would have to produce to generate the spot characteristics being measured” (11). This definition is inadequate. Very different numbers (all nominally representing equivalent plates) can result from the measurement of different characteristics on the same chromatogram. This is because a common effect of HPTLC techniques is a reduction in both average spot top-to-bottom width and separation (8). The result is almost always an increase in sensitivity (sample zones are more concentrated for a given migration distance). This may, however, be accompanied by either an increase or a decrease in resolution, R,. Resolution is here defined as the ratio of center-to-center separation between adjacent spots (AX) to the average width (W): R, = A X / W . A clear and consistent distinction should be made in HPTLC between plate numbers calculated on the basis of spot width (11) and resolution (10). T o that end, I propose that ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
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use of the word “equivalent” be restricted to that found in the earlier of the above references ( I O ) : to describe efficiency calculated from a measurement of resolution. I also propose that the word “apparent” be used to describe efficiency calculated from a measurement of spot width and migration distance. We thus have the following definitions. For two sample spots whose capacity factors (12) (kl’ and k2’) in a given system are known, and whose resolution (R,) and average migration distance ( X ) are measured, we define the equivalent plate number, ne as: ne = 16R,2((cy/(cy-1))((1 + k 2 ’ ) / k 2 ’ )where cy = k l ’ / k 2 ’ . The equivalent plate height, he, may then be defined by: he = X / n e . For a single sample spot whose top-to-bottom width (W) and migration distance (X)are measured, we define the apparent plate number, n, by: n, = 16(X/ W)’. The apparent plate height, h,, may then be defined by: h , = X / n , . In the absence of gradients, the apparent plate number and the equivalent plate number are numerically equal, and may be referred to simply as the “plate number”. The same is true for plate height. T h e terms “apparent” and “equivalent” can be extended t o other measures of efficiency or performance (e.g., the separation number (13)) in order to clearly distinguish
measurements based on resolution from those based on spot width. What are in fact different criteria for high performance in TLC can in this way become comparable.
LITERATURE CITED (1) J. Riphahn and H. Halpaap, J , Chromatogr., 112, 81 (1976). (2) S. Ebei and J. Hocke, J . Chromatogr.. 126. 449 (1976). (3) T. H. Jupille and J. A. Perry, Science, 194, 288 (1976). (4) T. H. Jupiiie, J . A m . Oi/ Chem. Soc., in press. (5) A. J. P. Martin and R. L. M. Synge, Biochem. J . , 35, 91 (1941). (6) L. S. Ettre, Chromatographia, 8, 291 (1975); 8, 355 (1975). (7) N. V. Rachinskii, J . Chromatogr., 33, 234 (1968). (8) G. H. Stewart and T. D. Gierke, J . Chromatogr. Sci., 8, 129 (1970). (9) J. A. Thorna, Anal. Chem., 35, 214 (1963). (10) T. t i Jupille and J. A . Perry, J . Chromatogr., 99, 231 (1974). (11) J. A. Perry, J . Chromatogr., 113, 267 (1975). (12) B. L. Karger, L. R. Snyder. and C.Horvath, “An Introduction to Separation Science‘ , Wiley-Interscience. New York, N.Y., 1973, pp 30-31. (13) R. E. Kaiser in “High Perfofmance Thin Layer Chromatography”, A. Zhtkis and R. E. Kaiser, Ed., Eisevier, Amsterdam, 1977.
Thomas Jupille Chemical Division BioRad Laboratories 32nd and Griffin Avenue Richmond, California 94804
RECEIVED for review February 9, 1977. Accepted June 24, 1977.
Ligand Interference Preventive Buffer in Determination of Copper(l1) by a Cupric Ion Selective Electrode Sir: In the determination of copper(I1) by means of a cupric ion selective electrode, the problems encountered are variation of activities caused by complexation of copper(II1 with ligands in samples and the abnormal response of the electrode in the presence of some ligands such as NTA and EDTA ( I ) . In addition, interferences of halide ions limit its application or practical analysis (2, 3). I n this communication, we propose to use a ligand interference preventive buffer (LIPB) to mask such interferents. In copper(I1) buffer solution consisting of polyamines and copper(I1)) the electrode shows an ideal behavior down to 10”” M of free copper(I1) or to further lower levels ( I ) . This indicates that interferences caused by complexing agents may be eliminated by adding an excess of one of the polyamines, which forms a more stable complex than any other ligand contained in the samples originally, This is a principle of
LIPB’
to the Of (4)for the determination of fluoride, the use of LIPB is based on a 1:l dilution of both standards and samdes with a solution which simultaneously has the following functions: (1)A sufficiently high level of non-interfering electrolyte is contained to fix the total ionic strength of samples by adding the solution. (2) The polyamine in the solution forms a very stable complex with copper(I1) to displace any bound ligand in samples, and total polyamine concentration is higher than that of copper(I1) in samples. (3) The solution is buffered at an appropriate pH t o complete the complexation with the polyamine.
EXPERIMENTAL The LIPB consisted of 0.40 M triethylenetetramine, 0.20 M nitric acid, and 2.0 M potassium nitrate. It was confirmed by atomic absorption analysis that copper was not contained by an amount higher than lo4 M in the LIPB. An Orion 94-29A cupric ion selective electrode was used with an Orion Model 801A Digital Ion Analyzer and a SCE with 10% potassium nitrate salt bridge. All measurements were made under ambient conditions. To 10.0 1868
A N A L Y T I C A L CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
Table I. Removal of Troubles Caused by NTA and EDTA with the LIPB Electrode potential, mV Total concn of Cu(II), Ma NTA~,~ EDTA~,~ 1.0 X - 3 6 7 . 3 (-366.9)‘ 1.0X -400.9 (-400.6) - 4 0 0 . 3 (-400.3)c 1.0X -433.3 (-432.7) -432.2 ( - 4 3 2 . 6 ) 1.0X lo-’ 1.0X 10.’
-455.3 (-451.1) - 4 6 0 . 2 (-457.8)
- 4 5 7 . 2 (-456.4) -465.7 ( - 4 5 9 . 4 )
a All concentrations were final ones after addition of the LIPB. The concentration of each ligand was 1.0 X M. ‘Figures in parentheses are potentials for standards.
mL of standards Or t o 10.0 mL of samples containing an aPpropriate interferent, 10.0 mL of the LIPB was added. The solutions were stirred with a magnetic stirrer, and the electrode ootential was _ _read ~ after min,
RESULTS AND DISCUSSION Although the electrode showed an abnormal response such as a potential drift for a long time or a potential variation dependent on a rate of stirring in the presence of NTA and EDTA, such a problem was successfully removed by addition of the LIPB. As shown in Table I, the potential values in the presence and in the absence of NTA or EDTA were in good agreement within 1.0 mV above 1.0 X M of copper(I1). A slope of calibration curves was slightly higher than a theoretical one. However, this does not present any problem for measurements, since the reproducibility of potentials is high. As copper(I1) complexes easily with many of the anions and others, the electrode determination of copper(I1) is difficult without an appropriate pretreatment of samples in which complexing agents coexist. The results suggest that a single calibration curve can be obtained in the presence of
