Anal. Chem. 1986, 58, 2571-2576
Brass
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Table I. Absorbance Values at Various Path Lengths Used to Calibrate the Cell drum (L)/mm
A (220 nm)
A (230 nm)
PTFE
0.05
rtz
0.10
0.3489 0.5840 0.8212 1.053
0.2351 0.3926 0.5492 0.7080
6 = 0.0243 mm
6 = 0.0246 inm
Perspex
0.15 0.20 (luartz Windows IQ15x21 Calibration RI
\Inlet
Figure 1. A diagram of variable path length flow-through spectrophotometer cell.
is fabricated so that the quartz windows just about touch when the drum is set to reading zero, resulting in a very small difference between the drum reading and the actual path length. Calibration is therefore necessary for measurements a t very short path lengths and, depending on the degree of accuracy required, also at longer path lengths. One calibration method is to measure absorbances of an aqueous solution (e.g., KNOBor K2Cr04)of suitable concentration against air a t a number of short path settings and a t any convenient wavelength. The stray light error of the spectrophotometer should of course be negligible a t the chosen wavelength (4). The procedure is then repeated with distilled water to obtain the correct absorbance of the solution by subtraction of the blank values. An illustrative set of absorbance values measured a t two wavelengths for each of four different path length settings is given in Table I. These values were used to calibrate the cell for a titration where a path length of 0.2 mm was required. The true path length, b, is deduced as follows from the data: If the reading on the drum is denoted by L, then the path length is given by
b=L+6
(1)
Values for cc and ec6 can be readily obtained as the slope and intercept of the straight line plot of A against L, or by fitting eq 3 to the data by least-squares analysis. The quotient of tc6/cc then gives the path length correction. The agreement between the two 6 values, 0.0246 and 0.0243 mm, calculated from the two sets of independent measurements is satisfactory and implies an uncertainty of less than 0.1% a t path length 0.2 mm. The path length to be used and the accuracy required should dictate the range and number of path length settings chosen for calibration purposes. Since aging of the O-ring can have a measurable effect on the value of 6, occasional calibration is recommended. In fact, calibration can be most conveniently carried out as part of a titration experiment by using the reaction mixture and its blank as described above.
CONCLUSION The variable short path capability of the cell offers advantages in cases where a titration procedure can be utilized to vary conditions of a reaction mixture to be investigated by spectrophotometry (e.g., ref 5). Examples are, end-point determinations in volumetric analysis, the determination of complex stoichiometry by the slope ratio or molar ratio methods, and especially equilibrium investigations in which the concentration of a strongly absorbing component has to be varied over a wide range.
LITERATURE CITED where 6 is the path length correction. From Beer’s law A = ecb one obtains
A = EC(L+ 6 ) = tcL ec6
+
(1) Llnge, H. G.; Jones, A. L. Aust. J . Chem. 1988, 21, 1445. (2) Lyhamn, L.; Pettersson, L. Chem. Scr. 1980, 16, 52. (3) Cruywagen, J. J.; Heyns, J. B. 8.; Rohwer, E. F. C. H. J . Inorg. Nucl. Chem. 1978, 4 0 , 51. (4) Edisbury, J. R. fractlcal Hints on Absorption Spectrometry; Hllger and Watts: London, 1966; Chapter 12. (5) Cruywagen, J. J.; Heyns, J. B. B. Taknta 1983, 30, 197.
RECEIVED for review February 25,1986. Accepted May 8,1986.
Rejection of Spike Nolse from Size Exclusion Chromatography/Low-Angle Laser Light Scattering Experiments Steven A. Berkowitz* Celanese Research Company, 86 Morris Avenue, Summit, New Jersey 07901 The direct coupling of a low-angle laser light scattering detector, LALLS, with a high-performance size exclusion chromatography, SEC, unit ( I , 2) has provided a unique and attractive technique for obtaining absolute molecular weight information and other physicochemical information about macromolecular systems (3-7). The feasibility of this coupling has been made possible by the use of a laser light source that *Present address: J. T. Baker Chemical Co., 222 Red School Ln., Phillipsburg, NJ 08865. 0003-2700/86/0358-2571$01.50/0
permits the scattering volume to be greatly reduced without degrading the signal to noise ratio (this is due to the laser’s high-power density). This reduction in the scattering volume along with the spatial coherent nature of the laser source and novel instrumentation design (8-1 0) has permitted intensity light scattering measurements to be made at very low angles (3-7O). Hence, intraparticle interference effects are negligible for most studies allowing molecular weights to be obtained without the need of extrapolation procedures to Oo (1.2). In addition, the high level to which particulate material (e.g., 0 1988 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
dust) must be removed from both the solvent and polymer solution is reduced with this LALLS detector since the small scattering volume (which is on the order of 20000 times smaller than conventional light scattering instruments) minimizes the effect of this problem (12). Nevertheless some sample and solvent clarification is still required in order to visualize the true scattering intensity level. This is especially important where computers are being utilized to gather SEC-LALLS data since the present ability of computer data acquisition systems to discern the true light scattering chromatogram is inferior in comparison to the visual ability of the experimenter. In our use of SEC-LALLS we have frequently encountered polymer-solvents systems which have proved to be very difficult to clarify (7). Hence, we have had to tolerate fairly high levels of spike noise. This noise has presented serious problems for commercially available data acquisition systems that use computer software (which uses bunching (13,141 and Savitzky and Golay (15) digital smoothing algorithms) and hardware (which uses integrating type analog to digital converters and low-pass electronic filtering) to obtain undistorted data. Hence, we have had to resort to manual digitization and processing of these data. In order to eliminate this timeconsuming procedure, we have investigated alternate approaches for dealing with this problem. The subject of this paper thus concerns our success in creating a computer data acquisition system capable of extracting undistorted light scattering data from chromatograms heavily contaminated with spike noise.
Flgure 1. Schematic hardware configuration of our computer data acquisition system.
n
EXPERIMENTAL SECTION Materials. The polystyrene samples 706 and 705 were obtained from the National Bureau of Standards (NBS). All polymer solutions were made on a weight/volume basis in tetrahydrofuran, THF (obtained from Burdick and Jackson Laboratories), and filtered through a 5.0-pm fluoropore filter (obtained from Millipore Corp.). Instrumentation. Size exclusion chromatography runs were made on a Waters Associates 201 chromatography unit equipped with an M-6000A pump, U6K injector and an R401 differential refractive index (DRI) detector at a flow rate of 1.2 mL/min. Two SEC column banks were used during this study. They included a set of two linear ultragel columns from Analytical Sciences, Inc. (MI),and a set of two p-Styragel (lo38, and 1068,)columns from Waters Associates. In-line light scattering measurements of SEC column effluent were made with a Chromatix KMX-6 light scattering photometer equipped with a 5 mm stainless steel flow-through cell. Light scattering measurements were made at a forward scattering angle of 6-7' (in air) using an instrumental time constant of 30 ms, with or without a ScientificSystems, Inc. (SSI), high pressure, low dead volume, in-line filter (0.5 pm) between the column outlet and the inlet on the KMX-6 flow cell. The analog signals from both the DRI and LALLS detector were sent to both a dual pen strip-chart recorder and a data acquisition system. Two data acquisition systems were used throughout this work. One system consisted of a CMX-10 interface unit (from Chromatix) coupled to a Digital Equipment Corp. (DEC), PDP11/23 MINC computer while the other system employed the use of specially constructed hardware linked to the DEC laboratory interface modules (which included a successive approximation A/D converter and clock input modules) on the MINC computer. Storing, processing, and analysis of all data were conducted by use of either the MOLWT I1 software (developed by Chromatix) in the case of the former system or with specially written software developed by the author in the case of the latter system. Molecular weight calculations performed by both programs were conducted by using the equations outlined by McConnell (16).The value used for the specific refractive index, at 633 nm, and the average second virial coefficient for polystyrene in THF was 0.1845 cm3 g-I (17) and 6 X cm3 mol g-2, respectively. Operation of Computer Hardware and Software. A schematic diagram of the hardware configuration of our data acquisition system (which included the despiking hardware) is
Figure 2. Each data point, y,, in the LALLS or DRI chromatogram, shown in part "A", is computed by averaging N (where N is a programmable parameter set prior to the start of run and is typically equal to 256) separate A/D readings, y,', as shown in part "B". shown in Figure 1. The analog signals from the DRI and LALLS detectors are initially signal conditioned and sent to the A/D converter, interfaced to the MINC computer. The latter signal is also sent to a high-pass filter. The analog signal from the high-pass filter (which is a simple RC network, having a variable time constant from 0.5 to 50 ms, buffered by an operational amplifier having a variable gain from 1 to 50) is then fed into a third channel on the A/D converter. Digitization of data from these three channels occurs effectively at the same time. The digitized data point readings, yj', for the DRI and LALLS detectors are then separately averaged (see Figure 2) in real-time according to eq 1 to obtain a series of mean value data points, yi,for each detector
where N (a programmable parameter) is the number of stated data point readings over which the averaging process is conducted. Data from the high-pass filter are used to determine whether a dust particle is in the light scattering volume element of the LALLS detector during acquisition of each y; data point used to calculate jii. This is achieved in two stages. In the first stage a discrimination voltage is established for both negative and positive transitions from the high pass filter which results from the transient appearance of "dust" particles within the light scattering volume element of this detector; see Figure 3. This voltage level is implemented with software. Hence, it can be readily adjusted to optimize discrimination between inherent electronic noise and the presence of "dust" scattering spikes.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
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B)
’
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Figure 4. Part A of this figure shows a hypothetical LALLS output noise spike from a single “dust” particle. The corresponding output from the high-pass filter is shown In part B along with appropriate discrimination voltage levels (- - -). The rectangular shaded region indicates where our spke detector would fail to function effectively’. This region is shown in part A to correspond to peak maximum (for readings above the indicated dash line, - -) of the light scattering noise.
-
same as Figure 2A,B, but depicts the sltuation where “dust” particles are present. The addltional trace shown in part “C” is the analog output from the high-passfleer. When the indicated voltage discrimination levels (- - -) are exceeded by the digltized readings from the high-pass filter, the corresponding y/ readings from the LALLS detector are not used to calculate y,. Figure 3. This figure is the
Whenever the voltage level from the high-pass filter exceeds this discrimination level, the corresponding data point y i , collected from the LALLS detector, is not used in, calculating yi. Hence, the actual total number of data points, yj’, used to calculate each yi is equal to or less than the number of data points stated to be averaged, N , prior to the start of the actual experiment. However, if the total number of good data points should be less than some initially stated programmable value (which was 3 for this work), the corresponding j$value is calculated by using the same procedures discussed below for replacing bad data points found during the second stage of spike detection. This method of detecting particles within the scattering volume of the flow-through cell fails, unfortunately, during a defined short time window when the rate of change in the voltage signal falls within the stated discrimination voltage levels for the high-pass fiiter output. This occurs at the peak maximum region of the light scattering spike from the particle; see Figure 4. In this situation a high false amplitude reading will be averaged into the calculation of the LALLS reading. In order to circumvent this problem, a second stage of spike detection is used to determine if a signifcant amount of spike noise had passed undetected through the data acquisition system. This is achieved by calculating the standard deviation
(in real time) associated with each mean value data point, yi. If one or more noise spikes passed undetected through the f i t stage of detection, one or more high readings will have been incorporated into the calculation of yi. This will result in an unusually high standard deviation relative to the situation where these spikes have been effectively removed. By using this principle, one can establish a discrimination value for the computed standard deviation so that if it is exceeded, the associated yi,biased by the presence of “dust“ spike readings, could be identified. These biased yi data points are then eliminated and replaced with calculated values obtained from a least-squares quadratic fit to three yi data points (which have associated standard deviations below the set discrimination level) on each side of the bad data point. It should be noted that this second stage of spike detection is conducted by a separate program after all data acquisition has been completed.
RESULTS AND DISCUSSION A SEC-LALLS chromatogram, recorded using a strip-chart recorder, for NBS polystyrene 706 standard in THF is shown in Figure 5a for a situation where the “dust” problem is minimal. In this case the commercial data acquisition system does a good job in removing occasional noise spikes; see Figure 5B. The situation, however, is very different when the frequency of spike noise is very high; see Figure 6. By visual inspection, one can clearly see that the digital data output from the commercial system has been severely distorted by the high frequency of spike noise; see Figure 6B. Quantitative data analysis shows these data are useless since computed molecular weight values are approximately a fador of 2 greater than reported NBS values; see Table I. The key property of our spike detector is the ability of its high-pass filter to discriminate between the slow changing polymer peak signal and the very fast changing noise spikes. As discussed in the Experimental Section, this discrimination is used with the appropriate threshold voltage level settings to inform the computer of the presence of “dust” in the scattering volume element. Data gathered using this technique
2574
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
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Figure 5. (A) Skip-chart recordhg of the analog LALLS chromatogram of NBS 706 run under conditions when the level of spike noise was
low. (B) Computer dgitized output (of the same experiment shown in part A) obtained with the commercial data acquisition system. Table I. Molecular Weight Data for SEC-LALLS Chromatograms Shown in This Paper sample
NBS 706" NBS 705" Figure 5 Figure 6 Figure 7A-D Figure 7E-H
M"
MW
137 000 171 000 168OOOc 266 OOOc 157 800' 175000'
273 OOOb 185 OOOb 281 000 446 000 278 000 191000
a Molecular weight information obtained from the National Bureau of Standards. The stated weight-average molecular weights, M w , represent the average value obtained by light scattering and sedimentation equilibrium. The number-average molecular weight, M,,, calculated from SEC-LALLS data is known to be biased toward values higher than the true M,.
for NBS polystyrene 706 is shown in Figure 7A-D. A similar set of results is also shown in Figure 7E-H for NBS polystyrene standard 705. This sample, which is highly monodisperse, gives a much narrower polymer peak. Hence it places greater demands on the discrimination ability of our spike removal system. In addition to the good visional quality of the resulting chromatograms obtained with out data acquisition system, quantitative data reduction of these chromatograms gave molecular weight values that are in good agreement with stated NBS values; see Table I. Light scattering measurements are inherently susceptible to the occurrence of high intensity readings due to the passage of particulate material (e.g., dust) through the scattering volume (12). When liquids are forced to flow through the light
I
..
, 50
100
160
200
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om mni Flgure 6. LAUS chromatogram of NBS 706 recorded under condiiins when the level of spike noise was fairly high as indicated by the trace obtained from the stripchart recorder shown in part A. Part B shows
the distorted data obtained from the commercial data acquisition system. scattering cell, the relatively erratic and slow changing light scattering readings from the particulate material suspended in this liquid (which results from normal Brownian motion) is transformed into very sharp light scattering spikes. If the level of dust is not too great so that the time period between the last and next incoming dust particle is long enough for the detection system to record the true light scattering reading for the dissolved polymer, than the correct data can be obtained with our data acquisition system. Clearly, the most obvious and direct way to handle the "dust" problem in light scattering measurements is to completely remove all particulate matter from both the solvent and polymer solution. Needless to say this is a real challenge and at times an impossible task,e.g., when the equilibration time to achieve "dust-free" liquids is too long (especially if the mobile phase in expensive (7)) or in-line filters rapidly become clogged (especially with new columns (3)) or if the solute of interest is too large to permit small enough filters to be used (since fractionation or degradation via shearing effects are possible). Hence, in various situations some level of spike noise will have to be tolerated. When the frequency of spike noise is too high, simple computer data acquisition systems and software are not adequate and manual data reduction is necessary. In order to eliminate the latter situation, a data acquisition system has been developed that has been shown to be capable of approaching the ability of the experimenter to discriminate between noise and signal. Al-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
2575
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Figure 7. (A) Stripehart recording of LALLS output for NBS 706 in the presence of high levels of "dust" spike noise. (B) Resulting output from our data acquisition system after the first level of spike detection. (C) Corresponding plot of the standard deviations for each data point plotted in B. (Dash line indicates discrimination level used in the second level of spike detection; see discussion in the Experimental Section under the title Operation of Computer Hardware and Software for explanation.) (D) Final LALLS output obtained after passing data through the second level of spike detection. (E-H) Corresponds to the same information given In A-D but the results are for NBS 705, a highly monodisperse polystyrene sample.
2576
Anal. Chem. 1086, 58, 2576-2578
though we have only demonstrated its ability in terms of SEC-LALLS experiments this procedure can also be used to obtain static light scattering readings and is especially useful when static readings are being obtained using an autoinjector to measure a large number of samples without the attention of the experimenter. Registry No. Polystyrene, 9003-53-6. LITERATURE C I T E D Ouano, A. C.; Kaye, W. J. Polym. Sci. 1974, 12, 1151-1162 Ouano, A. C. Rubber Chem. Techno/. 1982, 5 4 , 535-575. Hjertbert, T.; Kulin, L. I.; Sorvik, E. folym. Test. 1983, 3, 267-289. Ouano, A. C., J . ColloM Interface Sci. 1978, 83, 275-281. Hayashi, Y.; Takagi, T.; Maezawa, S.;Matsui, H. Biochem. Biophys. Acta 1983, 748, 153-167. (6) Jenkins, R., Porter, R. S., J. Poly. S c i , folym. Len. Ed. 1980, 18, 743-750.
(1) (2) (3) (4) (5)
(7) Berkowitz, S.J . Appl. Polym. Sci. 1984, 29, 4353-4361. (8) Kaye, W.; Havlik, A. J.; McDanlei, J. B. Polym. Len. 1971, 9 , 695-699. (9) Kaye, W.: Havlik, A. J. Appl. Opt. 1973, 12,541-550. (10) Kaye, W. Anal. Chem. 1973, 45, 221A-225A. (11) Zimm. B. H. J . Chem. f h y s . 1948, 76, 1093-1099. (12) Tabor, B. E. I n Light Scattering from f o / y m r Solutions; Hughln, M. B., Ed.; Academic Press: New York, 1972; pp 1-22. (13) Brown, H. C., 111; Wallace, D. L.; Burce, G. L.; Mathes, S. I n Liquid Chromatogfaphy Detectors; Vickery, T. M., Ed.; Marcel Dekker: New York, 1984; pp 335-411. (14) Woerlee, E.;Mol. J. J . Chromatogf. Sci. 1980, 18,258-266. (15) Savitsky, A.; Goby, M. J. E. Anal. Chem. 1984, 36, 1627-1638. (16) McConnell, M. L. Am. Lab. (FairfieM, Conn.) 1978, 10, 63-70. (17) Chromatix KMX-16 Application Note LS7. (18) Kotaka, T. J . Appl. Polym. Sci. 1977, 21, 501-518
RECEIVED for review December 9, 1985. Resubmitted May 30, 1986. Accepted June 1, 1986.
Estimation of Degree of Methylation of Pectin by Pyrolysis-Gas Chromatography R. A. Barford,* P. Magidman, J. G . Phillips, a n d M. L. Fishman
U.S. Department of Agriculture, 600 East Mermaid Lane, Philadelphia, Pennsylvania 19118 The physicochemical properties of pectin, as related to its function as food fiber, cell wall component in plants, and thickening agents in foods, are determined to a great extent by the degree of methylation of carboxylic acid groups ( I ) . A pectin molecule is represented schematically in Figure 1. Basically pectin is comprised of a-1-4 linked D-galacturonic acid with varying degrees of methylated (DM) carboxyl groups. At points along the chain, neutral sugars, predominantly rhamnose, are attached to form T-junctions (2). The number average degree of polymerization of unaggregated citrus pectins is 50-85 galacturonic units (3). Differences in DM are caused by natural variations in the methyl ester content of pectins as they occur in nature and by the extent of demethylation induced during extraction and purification (4). Traditionally, DM has been determined by titration of pectin carboxyl groups before and after basic hydrolysis ( 5 ) , though GC has been proposed more recently to measure methanol release by pectin hydrolysis (6, 7). The ratio of carbomethoxy to carboxyl also may be determined ( 1 )from the ratio of 13C NMR resonances a t 171.3 and 172.8 ppm. However, the need exists for a rapid means to determine structural information of pectin. Analytical pyrolysis offers such potential. Because of their importance in foodstuffs, the thermal decomposition of polysaccharides has been studied extensively. Primarily, these studies have involved slow heating in the presence of air and off-line analysis (8). Reports of polysaccharide decomposition under the anaerobic and almost instantaneous conditions of analytical pyrolysis have suggested that the principal reactions are (1)depolymerization, (2) flelimination of water, (3) decarbonylation, and (4) retroaldolization (9). In a preliminary study, the products of pectin pyrolysis were surveyed to ascertain whether structural features could be determined. Some correlations with DM were found but the need for more thorough investigation was recognized (10). The purpose of this research is to further study pectin pyrolysis to develop useful relationships between pyrograms and structure. INSTRUMENTATION Pyrolysis-gas chromatography (PY-GC) was carried out in the CDS Model 122 Pyroprobe connected directly to the
capillary injection port of a Varian Model 3700 gas chromatograph. Detection was by flame ionization and resulting data were processed by a Varian CDS 111 data system. The pyrolyzer was equipped with both a ribbon probe and a coiled probe. The former is used with liquid, soluble, or meltable samples and the latter for samples that are insoluble and will not melt. In these cases, samples were contained in a small quartz tube placed within the hollow core of the coiled heating element. For pyrolysis-gas chromatography-mass spectrometry the pyrolyzer was interfaced directly to the injection port of a Hewlett-Packard Model 5995 gas chromatography-mass spectrometer. Two chromatographic columns were used: a 50-m fused silica capillary (0.2 mm i.d.1 coated with free fatty acid phase (FFAP)obtained from Scientific Glass Engineering, Ltd., and a 60-m glass capillary (0.75 mm i.d.) coated with SP-1000 obtained from Supelco, Inc. The former was used for all determinations reported here except for chromatograms shown in Figure 3. MATERIALS Commercial citrus pectins with degrees of methyl esterification (DM) of 35, 58-60, and 70 were gifts from Bulmers, Ltd., Hereford, England. Two other pectin samples with DM 37 and 73 were manufactured by Bulmer but were gifts from E. R. Morris and M. J. Gidley at Unilever Co., England. The DM 57 pectin was a gift from Sunkist Growers, Corona, CA. The DM 75 pectin was obtained from the General Foods (GF) Corp. Another DM 73 pectin was obtained by extraction of fresh grapefruit albedo, according to standard procedures (11). Polygalacturonic acid (>98% pure) was obtained from Sigma Chemical Co., St. Louis, MO. Percentage of methyl esterification was determined by the colorimetric method of Wood and Siddiqui (12)and 13C NMR (I). Samples to be neutralized with NaOH were dissolved in 0.01 M phosphate buffer (pH 6.1) containing 0.1 M EDTA, titrated to pH 7 with 0.1 M NaOH, dialyzed against four changes of water over 48 h, centrifuged for 1h at 3oooOg to remove insoluble matter, and lyophilized. Dialysis bags were Spectrapor with a molecular weight cutoff of 12 000. To increase the data base, four mixtures were made using sodium polygalacturonate (DM 0) and GF pectin Na+ salt
This article not subject to US. Copyright. Published 1986 by the American Chemical Society
