Chromatogram, calibration

Last but not least, modern chromatography data systems are equipped with a powerful report generator. An integrated Excel-compatible spreadsheet, for example, makes it easy to analyze data and to present results in the way the consumer wants to see them. Report workbooks can include result tables, chromatograms, calibration plots, spectra, audit trails, and even custom equations and charts. Every cell, table, and chart updates instantly if any of the source data changes. Thus, the operator never has to worry about consistency. 

Fig. 38 Companson of manual dipping (A) with mechanized dipping (B) on the basis of scans and calibration curves [114] — 1 = cM-diethylstilbestrol, 2 = traw-diethylstilbestrol, 3 = ethinylestradiol Scanning curve 2 ng of each substance per chromatogram zone = 313 nm, /n > 390 nm Dipping solution water — sulfuric acid — methanol (85 + 15 + 1)...

FIGURE 2.4 Calibration curve of dextran on Sephacryi S-300 SF. Calibration curves were calculated from one chromatogram of a broad MWD reference sample using data for the molecular mass distribution as obtained by a calibrated gel filtration column ( , upper curve) and on-line MALLS ( ). The calibration curve was found useful for estimating the size of globular proteins. [Reproduced from Hagel et al. (1993), with permission.]... 

Water-soluble polymers obtained through a radical polymerization [e.g., poly(acrylic acid) PAA] often contain sodium sulfate Na2S04 as a decomposition product of the initiator. The peak of Na2S04 is eluted before the dimer. In the interpretation of the chromatogram, a typical GPC program has to be truncated before the Na2S04 peak, or at a Mpaa value of about 200. The calibration curve in this region can be flattened by an additive small pore column as well, but the principle problem remains unsolved. 

A commercially available cationic standard that can be used for the calibration of CATSEC columns is poly(2-vinyl pyridine), or PVP. Cationic PVP can be characterized easily on CATSEC columns over a broad range of molecular weight. DRI chromatograms of two cationic PVP standards using a bank of CATSEC columns (100-, 300-, 1000-, and 4000-A pore size) and a mobile phase of 0.05 N NaNOi/0.1% TFA are shown in Fig. 20.10. 

Procedure. Extract a series of aqueous aluminium solutions containing 5-25 mg aluminium in 5 mL, using the procedure described above under Sample. Calibrate the apparatus by injecting 0.30 L of each extract into the column and recording the peak area on the chromatogram. Plot a graph of peak area against concentration. 

Artifact removal and/or linearization. A common form of artifact removal is baseline correction of a spectrum or chromatogram. Common linearizations are the conversion of spectral transmittance into spectral absorbance and the multiplicative scatter correction for diffuse reflectance spectra. We must be very careful when attempting to remove artifacts. If we do not remove them correctly, we can actually introduce other artifacts that are worse than the ones we are trying to remove. But, for every artifact that we can correctly remove from the data, we make available additional degrees-of-freedom that the model can use to fit the relationship between the concentrations and the absorbances. This translates into greater precision and robustness of the calibration. Thus, if we can do it properly, it is always better to remove an artifact than to rely on the calibration to fit it. Similar reasoning applies to data linearization. 

It is appropriate at this juncture to illustrate the power of chemiluminescence in an analytical assay by comparing the limits of sensitivity of the fluorescence-based and the chemllumlnescence-based detection for analytes in a biological matrix. The quantitation of norepinephrine and dopamine in urine samples will serve as an illustrative example. Dopamine, norepinephrine, and 3,4-dihydroxybenzy-lamine (an internal standard) were derivatized with NDA/CN, and chemiluminescence was used to monitor the chromatography and determine a calibration curve (Figure 15). The limits of detection were determined to be less than 1 fmol injected. A typical chromatogram is shown in Figure 16. 

Up to this point, all chromatographic information is in terms of elution volume. If a calibration file is available to the program, the chromatogram may be converted to log hydrodynamic volume. The chromatogram in terms of log hydrodynamic volume may be written to a file (with extension HYD) for later use. 

The emission of the indicator is reduced in places where there are substance zones that absorb at 2 = 254 nm present in the chromatogram. This produces dark zones (Fig 4A), whose intensity (or rather lack of it) is dependent on the amount of substance appUed. If the plate background is set to 100% emission the phosphorescence is reduced appropriately in the region of the substance zones. When the chromatogram is scanned peaks are produced, whose position with respect to the start can be used to calculate Rf values and whose area or height can be used to construct calibration curves as a function of the amoimt applied (Fig. 25). 

Similarly, estimation of chemical composition of soluble polymer was also dependent on selectivity of the UV detector. Polymerized acrylonitrile has no significant UV absorbance at 230 and 254 nm. Thus, UV chromatograms were used to estimate amounts of polymerized methylacrylate and styrene In each resin system. The refractometer detector was sensitive to polymerized methylacrylate and styrene, as well as to polymerized acrylonitrile. It was therefore necessary to calculate comonomer contribution to refractometer peak areas In order to estimate concentration of polymerized acrylonitrile. This was done by obtaining a refractometer calibration for all three homopolymers. Quantity of polymerized comonomers measured by UV were then converted to equivalent refractometer peak areas. Peak areas due to polymerized acrylonitrile were then calculated by difference, and used to calculate amount of polymerized acrylonitrile. 

Size exclusion chromatography (SEC) separates molecules of a polymer sample on the basis of hydrodynamic volume. When the chromatograph is equipped only with a concentration-sensitive detector, i.e. conventional SEC, a molecular weight distribution (MWD) can be obtained from the chromatogram only through use of a calibration function relating molecular weight and elution volume V (2). 

The calibration technique used in conventional SEC does not always give the correct MWD, however. The molecular size of a dissolved polymer depends on its molecular weight, chemical composition, molecular structure, and experimental parameters such as solvent, temperature, and pressure ( ). If the polymer sample and calibration standards differ in chemical composition, the two materials probably will feature unequal molecular size/weight relationships. Such differences also will persist between branched and linear polymers of identical chemical composition. Consequently, assumption of the same molecular weight/V relation for dissimilar calibrant and sample leads to transformation of the sample chromatogram to an apparent MWD. 

Compare molecular size/weight characteristics of branched and linear species eluting at V in each chromatogram. Under the universal calibration formalism branched and linear components have the same hydrodynamic volume at V ... 

Once a value for M[ is assumed, this leaves one unknown b which can be determined from the SEC chromatogram and the measured whole polymer intrinsic viscosity in the following manner. First, one estimates a value for b, and calculates M CV) and [n](V) across the chromatogram using the universal calibration curve and equation (3). Then the whole polymer intrinsic viscosity is obtained from... 

Note that the weight and Z average molecular weights so obtained are only approximate and less than the true values. This is a consequence of the universal calibration method giving the instantaneous number average molecular weight across the chromatogram. 

In analysis of homopolymers the critical interpretation problems are calibration of retention time for molecular weight and allowance for the imperfect re >lution of the GPC. In copolymer analysis these interpretation problems remain but are ven added dimensions by the simultaneous presence of molecular weight distribution, copolymer composition distribution and monomer sequence length distribution. Since, the GPC usu y separates on the basis of "molecular size" in solution and not on the basB of any one of these particular properties, this means that at any retention time there can be distributions of all three. The usual GPC chromatogram then represents a r onse to the concentration of some avera of e h of these properties at each retention time. 

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