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It is usually difficult to obtain narrow molecular weight distribution samples of a given polymer for calibrating the gel permeation chromatographic instruments in the conventional way. The literature reports several methods which have been used to obtain instrument calibrations with samples of broad distribution polymer. However, these methods generally must assume a linear calibration of the logarithm of molecular weight versus elution volume. This assumption may lead to significant errors in precise molecular weight distribution computations. It is possible to avoid this assumption by making use of the universal calibration curve proposed by Benoit without the necessity of defining this curve by any mathematical function. A number of methods have been developed for combining the universal calibration curve with measurable parameters of the polymeric material (such as intrinsic viscosity, number average molecular weight and weight average molecular weight) to calculate molecular weight distributions. Results will be reported on a careful evaluation of the accuracy and reproducibility of several of these methods.

The requirements that have to be met in the processing of calibration data by the linear regression method in quantitative gas chromatography are discussed and documented on four typical variants of the absolute calibration technique. A mean slope method has been suggested for the precise processing of the data in cases where the statistically dependent variable displays a constant relative error. The effect of the systematic error on the course of the calibration line has been shown.

A method for calibration of tightly cross-linked gel filtration chromatography media based on the elution characteristics of a series of selected sugars and alcohols is described. The elution position of all molecules is determined by the column effluent refractive index change and is correlated with both the molecular weight and the unhydrated radius estimated from molecular models.

The mass detector was used to confirm the predictability of response of the Martin gas density balance, to calibrate a katharometer, and to measure limits of detection. The mass detector was operated in parallel with two destructive detectors, the flame thermocouple and flame ionisation detectors, and response curves were constructed for several different compounds.

Various procedures for universal calibration in gel permeation chromatography with polystyrene gels are examined for polystyrene and poly(dimethylsiloxane) fractions. For o-dichlorobenzene at 138°, experimental intrinsic viscosity-molecular weight data show that the Mark-Houwink exponents are 0.70 and 0.57 for polystyrene and poly(dimethylsiloxane), respectively. In principle, this difference permits a distinction between the various polymer size parameters proposed for universal calibration. An interpretation of the experimental poly(dimethylsiloxane) calibration for o-dichlorobenzene at 138° requires a consideration of errors in average molecular weights and errors arising from the use of average molecular weight instead of peak molecular weight. When hydrodynamic volume and unperturbed dimensions calibration procedures are examined, the difference between them is comparable with experimental error. If the Flory-Fox viscosity expression is employed, the perturbed end-to-end distance (or radius of gyration) gives an equivalent universal calibration to the hydrodynamic volume. The experimental data are sufficiently accurate to show that the perturbed dimension determined with the Ptitsyn-Eizner relation does not give an adequate universal calibration.

The reproducibility of the photometric evaluation of spots on thin-layer chromatograms for the determination of the concentration of substances was investigated. The remission of some U.V.-absorbing test substances was measured at a wavelength of 254 nm. The deviation of the remission value on one plate, on plates of the same series and on plates of different series was determined. A calibration curve obtained for one series of measurements can be used for later measurements if calibration values at two different concentrations are determined again. When using plates that have not been specially selected, the relative standard deviation with which the concentration can then be determined from the remission value—at not too low concentrations—is on an average 10%. In the cases investigated, it was found that for low concentrations measurement of the remission value of the centre of the spot gives the most accurate results, while for medium concentrations aboutthe same accuracy is obtained when the remission values over the whole spot are averaged. The thickness and density of the silica gel layer primarily affect the ascension rate of the solvent front and consequently the spread of the spot in the direction of flow. Only a very slight influence of the thickness of the layer on the remission value could be detected, since several effects compensate each other.

The quantitative aspects of isotachophoretic analyses have been studied. The introduction of a calibration constant removes the necessity for constructing calibration curves for each ionic species. Calibration constants were determined for two electrolyte systems and the values obtained agreed with the theoretical values.

A study of a number of different pharmaceutical compounds has shown that with the calibration function A2 = m′c + n′ (A = peak area, c = concentration) linear calibration curves can be obtained with concentrations between 1 and 10 μg per spot. For concentrations in the range of 0.3 to 1.0 μg the function h = m″c + n″ (h = peak height) is preferred at higher amplification. This actually means that with the densitometric evaluation of directly spotted liquid samples concentrations in the range 0.03–5 mg/ml can be analysed. The time saved by direct sample spotting has been demonstrated with a typical example where nine analyses of a product with a 0.2 μg/ml concentration of active principle (2 μl/spot) can be carried out in one day, with a reproducibility of ±1.4% relative S.D. For the same samples, using classical elution procedures, 3–4 days are required and extraction of samples prior to spotting (with danger of decomposition) is necessary. The analysis of thyroacetic acid, which appears as an enzymatic breakdown product of thyronine and has a λmax. value of 235 nm, has been carried out by UV reflectance at concentrations of 50 ng/spot using a standard addition approach. This is a good example of the applicability of this method to very low concentrations and at relatively short λmax.. Instrumental modifications which facilitate the measuring process and data handling are discussed.

Porous glass of 370 A average pore diameter is useful for fractionating amylose and dextran of weight-average molecular weights ranging from 2 × 105 to 2 × 104 and at flow-rates of 2.4–2.9 ml/h,cm2 at room temperatures. Amylose and dextran in methyl sulfoxide-water (95:5) or 4 M guanidine hydrochloride as solvents exhibit the same calibration curve; hence, commercially available dextran fractions may serve to calibrate the column for use with amylose. Polystyrene fractions in tetrahydrofuran exhibit a different calibration curve. Thus the universal calibration concept does not apply to all three polymers in our column.

Basic considerations for a rational application of quantitative thin-layer chromatography Using the Lambert-Beer law one obtains for transmission measurements for quantitative thin-layer chromatography calibration curves according to the function y = ax, if oversaturation of the spots to be measured is avoided. Once the optimal conditions for the starting spot have been attained, they can be easily reproduced with an automatic spot applicator. Up to nine samples can be applied simultaneously in a very short time. The diameter of the starting spots can be chosen. The calibration curve according to the Lambert-Beer law and the automatic applicator permit national utilisation of quantitative thin-layer chromatography. The coefficient of variation for this method lies between 0.8 and 3.5%.

Five gas chromatographic methods have been developed whereby gas mixtures containing H2, A (or O2, N2, CO, CO2, CH4 and C2H0 can be analyzed on various lengths and combinations of three kinds of gas-solid adsorption columns. The adsorbents employed are molecular sieve 5A, silica gel and activated charcoal. Volume of gas mixture injected into the column is fixed at 0.5 ml, and therefore, volume percent of each component can be directly determined from its calibration curves. The calibration curves are constructed by plotting peak area versus percentage of the component in 0.5 ml sample of a series of standard mixtures with attenuation of the instrument as the parameter. When greater accuracy is desired, a normalization method is used. Preparation of gas samples for chromatography, including sampling and conditioning, are also presented and discussed in detail. All five methods are simple, rapid, and accurate. They have been satisfactorily applied to gas samples from coal gastification. Two of them are also applicable for on-stream analyses.

Three methods are described which allow the quantitative analysis of hydrogen in gaseous mixtures, when helium is the carrier gas. These methods are respectively based on the simultaneous calibration with air, the simultaneous calibration with hydrogen and the determination of the correction factor curve. All the methods can be applied to other carrier gases and samples.

Five nucleo derivatives have been chosen for quantitative in situ determination by reflectance spectroscopy after two-dimensional separation from a complex mixture by thin-layer chromatography on cellulose. The use of an internal standard to compensate for fluctuations in the chromatographic procedure resulted in an improvement in the reproducibility of data by about 3–4% relative standard deviation. Average accuracies obtained with the method in the analysis of an artificial mixture ranged between 5.1 and 4.0%. The use of a linear calibration curve of substance to standard ratio plotted versus square root of concentration proved most advantageous. The use of one set of calibration curves for the analysis of mixtures on various days gave an average error of about 16% but was tremendously timesaving. The method was successfully applied to the analysis of nucleo derivatives in an extract of cartilage red bone marrow.

Hydroxylesterification and esterification reactions were made on α,ω-dicarboxypolybutadiene polymers and the products characterized by both analytical and preparative gel permeation chromatography. The choice of a calibration curve to study transformation of polymers is discussed, as well as the reasons for adopting the Moore calibration curve relating size of polymers to elution volume. An automatic Fortran program was used to calculate number-average and weight-average sizes of polymers from the chromatograms. Problems associated with changes in the distribution of a polymer during a reaction are examined. To relate sizes of polymers in solution to molecular weight, measurements were made by vapor phase osmometry, light scattering and viscosity. The constancy of the refractive index increments with polymer size has been verified.

The mass detector has been usef for the analysis of a wide range of materials embracing low boiling and high boiling liquids. Some low boiling materials were quantitatively detected without calibration by operating the detector below room temperature. The quantitative analyses of a wide variety of mixtures of normal and high boiling liquids were carried out and satisfactory results obtained without the necessity for calibration. The response of the detector toward gases has been studied.

Microquantitative determination of aliphatic amines with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole Fluorometric determination of microanalytical quantities of aliphatic amines with 7-chloro-4-nitrobenzo-2-oxa-1-3-diazole is described. Reaction conditions in a one-phase system, clean-up methods and column or thin-layer chromatographic separation procedures are investigated in order to know the potential of the reagent. For quantitative investigation, separation on polyamide sheets with subsequent in situ measurement by means of a Zeiss chromatogram spectrophotometer is particularly suitable. A linear calibration curve is obtained for quantities between 15 and 150 ng per spot. The high stability of the reaction products and the efficient reproducibility of the procedure justify recommendation of the reagent for the microanalytical determination of amines.

Analysis of N-nitroso compounds. II. Separation and quantitative determination of N-nitroso-N-alkylureas and N-nitroso-N-alkylurethans by high-speed high-pressure liquid chromatography . Physical data, elementary analysis data and IR, UV, NMR and mass spectra have been determined for N-nitroso-N-alkylureas and N-nitroso-N-alkylurethans. The fragmentation patterns in the mass spectrometer induced by electron impact are discussed. A fast separation is described of model mixtures of alkyl homologous N-nitroso-N-alkylureas and N-nitroso-N-alkylurethans by reversed-phase liquid—liquid distribution in a liquid chromatograph. Detection is carried out by UV absorption at 254 nm. Calibration curves for the quantitative determination of separated N-nitroso-N-alkylureas are given. The lowest detection limit for N-nitroso-N-methylurea was 10 ng.

On the basis of the Townsend equation for the gas amplified current and of the Dutton—Powell equation for the enhancement of the primary ionization coefficient in gas mixtures due to the Penning effect, a mathematical model of the chromatographic helium detector is proposed. Because both calculations of the relative current increase for E/p = 4, 5 and 6 V/em·torr and actual measurements lead to linear calibration graphs for the sample calibrations at the parts per million level, a simplified and linearized relationship was developed, although further experimental confirmation is needed. The suggestion is made that the derived equation will make possible measurements of the 0 number of metastable states produced per electron per centimetre of drift in an electric field, and the cross-sections for the Penning effect. The rate constants for Penning ionization by triplet metastable atoms of helium for hydrogen sulphide and ethylene were evaluated but the results are of a relative character. The shortcomings of the model are considered: the lack of explanation of negative peaks, the role of specially added admixtures to the carrier gas, and no account of recombination phenomena are the most important. A brief scheme of necessary investigations is outlined.

A description is given of a simple gas chromatographic method for assay of plasma levels of carbamazepine. This uses OV-1 as stationary phase and avoids high column temperatures. The calibration curve is linear and the results are reproducible. Plasma levels in patients under treatment with carbamazepine alone ranged from 1.1 μg/ml to 22.4 μg/ml.

Limits of error in the direct photometry of paper chromatograms Verification of the errors in measurement made in the direct photometric evaluation of paper chromatograms (modified wedge-shaped strips) shows that only a part of the calibration curve, depending on the cations being determined and on the reagent, is suitable. Under optimal conditions the standard deviation for repeat determinations may be expected to lie between 5 and 10%, while the comparative 3tandard deviation may be expected to be greater than 10%. The accuracy of the measurements can be increased by using internal standards.

The quantitative analysis of a number of N-methyl carbamate insecticides by thin-layer chromatography and in situ fluorometry of their dansyl derivatives is investigated. Statistical analysis indicates a relative standard deviation of 4.5% for concentrations ranging from 5 to 500 ng per spot of carbamate. Calibration curves (peak area versus concentration) are linear up to 300–400 ng per spot and generally exhibit an increase in slope with time, due to an increase in fluorescence of the spots. Analyses are performed on spiked water samples. Mesurol and Landrin are simultaneously extracted and reacted with dansyl chloride, after which the resulting dansyl-phenol derivatives are analysed quantitatively following separation on Silica Gel G—silver oxide layers with benzene—acetone (98:2). Baygon, Mesurol and Landrin are simultaneously extracted from water samples and determined as total N-methyl carbamate by measuring the fluorescence of the dansyl-methylamine derivatives. Recoveries from the samples extended from 90–106% at concentrations in the low p.p.b. range* range. The method offers improved specificity over existing techniques because two derivatives are formed with each carbamate, both of which can yield quantitative results.

A simple gas chromatographic method is described for the determination of dichloro-, trichloro-, and tetrachlorobenzene impurities in 1,3,5-trichlorobenzene samples. The sample is dissolved in carbon disulfide, and an aliquot is chromatographed on a 6-ft. long 10% Bentone 34 column. The peak heights and peak areas are measured and related to concentration by means of calibration curves. 1,3,5-Trichlorobenzene samples were found to contain small amounts of m-dichlorobenzene and 1,2,4-trichlorobenzene. From these data and thin-layer chromatographic analyses of crude 1,3,5-trichloro-2,4,6-trinitrobenzene, an explanation is proposed for the origin of the 1,3-dinitro-2,4,5,6-tetrachlorobenzene impurity found in 1,3,5-triamino-2,4,6- trinitrobenzene samples. It is concluded that 1,3-dinitro-2,4,5,6-tetrachlorobenzene is produced during the nitration of 1,3,5-trichlorobenzene.

Fluorigenic labelling techniques have been used for several years in conjunction with thin-layer chromatography, and are now being used also to good advantage in high-speed liquid chromatography. In this work, reagents such as 4-chloro-7-nitrobenzo-2,1,3-oxadiazole, dansyl chloride and dansyl-hydrazine were used for the fluorigenic labelling of carbamates, ureas, organophosphorous compounds, aliphatic amines, aldehydes, ketones, biphenyls and some compounds of pharmaceutical interest. The labelling reactions and the nature of derivatives are discussed, together with the column liquid chromatography properties of these derivatives in both liquid—liquid and solid—liquid modes. Detection limits obtained for most compounds with a new fluorimetric detection device range between 1 and 10 ng per 4-μl injection volume. The method is suitable for quantitative analysis, with a reproducibility of ca. 3% relative standard deviation. Linear calibration plots for peak area or peak height versus concentration were observed up to 600 ng per injection. The method has been applied successfully to the environmental sample analysis of pesticides in water, soil and vegetables without the necessity for a preliminary clean-up. The method is comparable in sensitivity and accuracy to gas chromatography. The advantages of this method can be summarized as follows: improved detection properties, such as better sensitivity, flow-insensitive detection mode and higher specificity; lowering of polarity of the compounds, hence greater ease of separation; suitable for automation and routine analysis; the range of possible applications is limited only by the available labelling reagents and separation procedures.

A rapid gas chromatographic method has been developed for determining the composition of the organic phase of the continuous TNT process. The method provides analyses for isomers of mono-, di-, and trinitrotoluenens in samples from nitration phases and for residual dinitrotoluenes and asymmetrical trinitrotoluenes from the TNT purification process and in the final product. The column used for separation was 10% UC-W98 silicone rubber on 80–100 Diatoport-S. Detector response factors were determined and used to calculate relative percent components to eliminate time-consuming calibration and achieve better reproducibility.

A typical pellet-type alkali flame detector was monitored photometrically. Its predominant (??? exclusive) mode of response is negative, i.e. alkali emission decreases when gas chromatographic effluents are burned in the flame. As a detector for organic halides, the optical emission of the alkali flame appears to simulate some characteristics of its more often used electrical conductivity response. For instance, it is possible to distinguish between chlorine, bromine and iodine, and the (negative) response for chlorides is proportional to the amount of chlorine entering the flame. Calibration curves for selected chlorinated hydrocarbons approach linearity on a logarithmic scale within two orders of magnitude, with minimum detectable amounts between 3–5 ng. Carbon compounds give signals approximately fifty times weaker than monochlorinated compounds. All characteristics considered, the pellet-type alkali source seems to differ considerably in its spectral behavior from other types of sources which have been described in the literature, especially those using a resistance-heated wire coated with sodium sulfate.

The pulse chromatograph technique was applied to the measurement of adsorption isotherms in systems that involve large adsorbed amounts and some “irreversible” adsorption. The assumptions necessary for successful measurement were verified. A method that permits direct calibration on strongly sorbing sorbent was developed. The adsorption isotherms of aliphatic amines on cobalt and alumina were measured over a wide temperature range and the isosteric heats of adsorption were calculated.

Some applications of a viscosimetric detector in gel permeation chromatography In this paper a detector which permits direct measurement of the viscosity of the eluate, leading to a more quantitative use of gel permeation chromatography, is described. If the elution volume and the concentration of polymer (determined by refractometry) are known, it is possible, with the use of a gel permeation chromatography universal calibration curve, to determine the molecular weight of each fraction, thus giving rapid, complete and accurate characterization of an unknown polymer. This technique has been applied to: (1) the study of linear polymers and the relation between viscosity and molecular weight; (2) the characterization of branched polymers and the determination of the degree of branching as a function of molecular weight; (3) the study of copolymers of different composition. Here the composition of each fraction was determined with the aid of a third detector, allowing evaluation of polydispersity in weight as well as in composition of the copolymers.

A new method for the automatic recording of the elution curve in ion exchange chromatography is proposed. It is shown that measurement of the difference in the electrical conductances at the inlet and outlet of the column provides the recording of the ordinary elution curve. The same measurement of the difference across a dead space at the column outlet makes it possible to record the full-wave rectified derivative curve of the elution curve. The derivative method gave a calibration curve of good linearity in an experiment using cupric ion eluted with ethylenediamine hydrochloride.

The ideal detector for quantitative analysis by gas chromatography would need neither calibration nor the application of integration procedures to a differential chromatogram. This paper describes the experimental realization of a detector possessing these two desirable characteristics. The “Brunel” mass integral detector yields an integral chromatogram in which the absolute masses of the sample components are directly recorded as step-heights.

The potentialities of organic adsorbates for a simpler and more rapid gas-solid chromatographic method for determining surface area have been explored. It has been observed that the logarithm of the surface area of adsorbents varies linearly with the slope of the universal straight line obtained by plotting retention times versus the number of carbon atoms of a selected group of n -paraffins. Using this as a calibration plot, the surface areas of sixteen solids of different chemical compositions have been determined. The advantages of this method over existing gas chromatographic methods and the methods of B runauer et al. have been discussed. A strong influence of the nature of carrier gas on the position of the slope-surface area plot has been observed.

A new chromatographic method for the direct determination of C9-C14n-paraffins in kerosene fractions of an intermediate base has been developed. The analysis is carried out by using an open tubular column. The contents of n-paraffins are calculated by an absolute calibration method, i.e., by comparing the n-paraffin peak heights in the sample and reference standard chromatograms. A synthetic mixture of n-paraffins dissolved in a molecular-sieve deparaffinized kerosene distillate of a corresponding base was used as reference standard. A coefficient of variation of 1–2 % and a relative error of 2–3% indicate the reliability of the method. The proposed method is advantageous compared with known methods for individual n-paraffin determination in kerosene fractions because of the precision and accuracy of the results as well as its relative simplicity and speed. With similar advantages, the new method may replace the usual adsorption methods for the determination of the total content of n-paraffins in kerosene fractions.

The separation and quantitation of traces of carbon monoxide from air have been performed using molecular sieve columns and helium ionization detectors. Calibration curves obtained by the exponential dilution method are linear down to about 50 parts per billion. A comparison of the curves obtained using helium or air as diluting gases is made.

Multicomponent mixtures of acetone, methanol, propanol and butanol were analysed using calibration mixtures, constant area factors and a linear relationship. The latter is simple to apply and, compared with the former methods, gives satisfactory accuracy.

A simple pyrolytic gas chromatographic method, capable of determining 1–40 micrograms of polystyrene in a plastic-bonded RDX explosive, is described. The polystyrene is converted to the monomer by controlled pyrolysis. The styrene monomer is determined by gas chromatography and related to polymer concentration by means of a calibration curve. Synthetic mixtures and explosive samples were successfully analyzed by the method, and the method has been applied to the study of composition variations in small PBX granules.

A pyrolytic gas chromatographic method for determining small amounts of Eastman 910 polymer in certain plastic-bonded explosives is described. The Eastman 910 polymer is separated from the plastic-bonded explosives by solution in nitromethane-chloroform, and an aliquot of the solution is converted to the monomer, methyl 2-cyanoacrylate, by controlled pyrolysis for gas chromatographic analysis. The peak area of the monomer is related to polymer concentration by means of a calibration curve. It is shown how the method may be used to study the penetration of the adhesive into glued pellets of the explosive.

The Possibility of quantitative analysis of products separated by thin-layer chromatography without eluting the products The possibility and precision of quantitative analysis of a series of homologous substances separated by thin-layer chromatography are determined by measuring the areas of the spots. The ideal conditions are determined and a calibration curve is constructed. The results of application of the method to substances belonging to the series studied are given.

Carbon disulphide obtained from the acid decomposition of pure and commercial dithiocarbamates and bis-dithiocarbamates was entrained in a stream of helium and condensed in a trap immersed in liquid air. The entire condensate was then analysed by removing the trap from the liquid air and connecting it directly to an appliance for introducing gaswous substances into a gas chromatographic column. The experimental procedures for preparing the calibration curves and for the determination of the compounds in question are reported, and advantages of the proposed method are described. One of these advantages is that, besides eliminating any risk of interference which may be encountered in other methods, this technique enables amounts down to a few γ of carbon disulphide to be determined. With the aid of the method described it is therefore possible to determine quantitatively trace amounts of dithiocarbamates, or of any compounds which can liberate carbon disulphide.

A technique for the quantitation of flat-bed chromatograms or electropherograms involving the minimum exposure time necessary for a spot just not to appear on a photograph has been developed. For calibration purposes the minimum exposure time is plotted against the amount of substance spotted. The amount of a substance present in an unknown spot is determined from the minimum exposure time. The procedure is inexpensive, simple and particularly suitable for occasional quantitations.