The calibration chromatogram

The column can be calibrated with a test mixture of compounds of accurately defined molecular mass, as a means of finding the elution volume, V, for a specihc molecular size. The sizes of test molecules must be selected to ensure that  

Monodisperse samples (a single type of molecule per peak) are best for calibration but polydisperse ones 

Molecules with a degree of polymerization n of between 14 and 1 are eluted between V) and Vq- The volume Vq — V) corresponds to the pore volume, Vp, of the 

The void volume Vq defined here corresponds to the elution volume of breakthrough time as presented in Section 2.3. However, in size-exclusion literature often as defined here, is termed Vq because it is the volume where the first peak can occur. 

8 ml it must be somewhere on the vertical branch at this volume but its log position is unknown. The black dots represent from left to right peak 2 with 

A chromatogram for styrene oligomers, with n= 1-14, may be taken as an example (Fig, 15,2). The peak on the far right may be derived from styrene. This is the smallest molecule (next to those of the mobile phase) and is eluted last. The void volume, Vq, is its relevant elution volume. The first small peak on the left is produced by excluded molecules, the relevant volume being the interstitial volume, Vz (liquid found between the individual particles of the stationary phase).  

Molecules with a degree of polymerization n of between 14 and 1 are eluted between Vz and Vo- The volume Vq-Vz corresponds to the pore volume, Vp, of the stationary phase (liquid in the pores). Vp is the only effective separation volume and should, therefore, be as large as possible. The peak capacity is a function of Vp. It is clear that peaks follow on more closely, the smaller is the difference in molecule size (mass). 

In an ideal case, a graph of log (molar mass) against elution volume is a straight line that characterizes the column (Fig. 15,3). There may also be some sort of curve in the calibration graph. 

In Fig. 15.14 a separation of milk proteins is presented. Draw a calibration curve which corresponds to this chromatogram. 

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From the calibration chromatogram, determine the response factor for each component as follows ... 

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. 

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. 

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. 

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... 

One possibility is that although averages for polystyrene standards require correction, those for PMMA would not According to symmetrical axial dispersion theory (5) the correction depends upon both the slope of the calibration curve (different for each polymer type) and the variance of the chromatogram of a truly monodisperse sample. Furthermore, the calibration curve to be utilized can be obtained from a broad standard as well as from monodisperse samples. The broad standard method may itself incorporate some axial dispersion correction depending upon how the standard was characterized. 

Mjj and My or [q] for the broad MWD standard are taken as known quantities. Fy(v) is the normalized chromatogram for the broad MWD standard obtained with a mass detector. D2 is the slope of the molecular weight calibration curve at the peak position of the chromatogram (the equation of the tangent is given by M(v) = Dj exp(-D2v). is the variance of the single-species chromatogram... 

Chromatograms of the ethanol-soluble fraction were obtained in THF on Toyo Soda Oligomer columns over a range of sample masses, as shown in Figure 5 The exclusion limit of these columns is 50,000 and Mp" values above 10,000 are inaccurate because the calibration curve is very steep in this region. Consequently, chromatograms were also obtained on the "Main Column" GPC with the results shown in Figure 6. 

SEC-FTIR yields the average polymer structure as a function of molecular mass, but no information on the distribution of the chemical composition within a certain size fraction. SEC-FTIR is mainly used to provide information on MW, MWD, CCD, and functional groups for different applications and different materials, including polyolefins and polyolefin copolymers [703-705]. Quantitative methods have been developed [704]. Torabi et al. [705] have described a procedure for quantitative evaporative FUR detection for the evaluation of polymer composition across the SEC chromatogram, involving a post-SEC treatment, internal calibration and PLS prediction applied to the second derivative of the absorbance spectrum. 

However, as soon as at the eluate-side H ions are replaced with an equivalent amount of Na or K ions, which elute, the then asymmetric cell acquires a potential that reflects the Donnan equilibrium potential on the basis of the ion mobilities concerned. Hence the potential change as a function of time represents the ionic chromatogram and the peaks concerned yield the alkali metal ion contents via calibration. 

Figure 5. A typical chromatogram. The solid curve is the part of the data that is used to characterize the molecular weight distribution. The dashed portions represent data that are not used. The solid straight line represents the baseline under the chromatographic peak. The vertical lines define the limits of the calibration function for the column set.

Obtain HPLC chromatograms of the calibration standards by injecting 20 jTL of each. There should be two peaks. The benzoate peak will be much smaller than the caffeine peak, and the detector attenuation will need to be changed after the caffeine has eluted. Record the peak size vs. concentration data. 

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