Calibration curve columns

A detailed examination of the correlation between Vj and M is discussed in references on analytical chemistry such as Ref. 6. We shall only outline the problem, with particular emphasis on those aspects which overlap other topics in this book. To consider the origin of the calibration curve, we begin by picturing a narrow band of polymer solution being introduced at the top of a solvent-filled column. The volume of this solvent can be subdivided into two categories the stagnant solvent in the pores (subscript i for internal) and the interstitial liquid in the voids (subscript v) between the packing particles ... 

An interesting outgrowth of these considerations is the idea that In r versus K or Vj should describe a universal calibration curve in a particular column for random coil polymers. This conclusion is justified by examining Eq. (9.55), in which the product [i ]M is seen to be proportional to (rg ), with r = a(rg 0 ) - This suggests that In rg in the theoretical calibration curve can be replaced by ln[r ]M. The product [r ]M is called the hydrodynamic volume, and Fig. 9.17 shows that the calibration curves for a variety of polymer types merge into a single curve when the product [r ]M, rather than M alone, is used as the basis for the cafibration. 

The preliminary precipitation of proteins from milk is realized through the addition of solutions of acetic acid (1,7 mol/1) and sodium acetate (lmol/1) at t = 40-45°C before chromatographic isolation of OxTC. The precipitated proteins are separated by filtration. OxTC is detenuined in filtrate after its isolation on chromatographic column. Contents of OxTC was determined on calibration curve which is linear within concentration range 0,01-1,0 p.g/ml. 

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.]... 

FIGURE 4.2 Polyethylene oxide, dextran, and protein calibration curves for TSK-GEL SW Columns. Column TSK-GEL SW, two 7.S mm x 60 cm columns in series. Sample , proteins Q, polyethylene oxides O, dextrans. Elution dextrans and polyethylene oxides distilled water proteins 0.3 A1 NaCI in 0.1 M phosphate buffer, ph 7. Flow rate 1.0 ml/min. Detection UV at 220 nm and Rl. 

TSK-GEL PW type columns are commonly used for the separation of synthetic water-soluble polymers because they exhibit a much larger separation range, better linearity of calibration curves, and much lower adsorption effects than TSK-GEL SW columns (10). While TSK-GEL SW columns are suitable for separating monodisperse biopolymers, such as proteins, TSK-GEL PW columns are recommended for separating polydisperse compounds, such as polysaccharides and synthetic polymers. 

The range of pore sizes in which TSK-GEL PW and TSK-GEL PWxi columns are available permits a wide spectrum of water-soluble substances to be analyzed. Calibration curves for polyethylene glycols chromatographed on... 

Por analytical purposes, TSK-GEL PWxl columns are preferred. Por preparative work, or for other cases in which large amounts of sample must be used, TSK-GEL PW columns are recommended because of their larger loading capacity. To select the proper TSK-GEL PW type column for a particular sample, consult the separation ranges listed in Table 4.6 or the calibration curves in Pig. 4.11. 

H type resins are available in different pore sizes. Examples of calibration curves for polystyrene standards are shown in Figs. 4.38 and 4.39. Other series of H type columns have similar calibration curves. Exclusion limits are listed in Tables 4.12-4.16. 

FIGURE 4.43 Calibration curves for globular proteins on toyopearl resins. Column 22 mm X 30 cm. Sample Protein standards. Elution 0.06 A1 phosphate buffer, pH 7, in 0.06 A1 KCI. Legend elution volume V column volume. 

Linear type columns are especially designed to have wider linear molecular weight ranges. These linear-type columns are highly recommended for correcting nonlinear sections of molecular weight calibration curves (Table 6.2). 

Figure 6.3 shows a comparison of elution patterns of standard polystyrene between a linear-type column and a standard-type column. Because of the high linearity of its calibration curve, the linear series has improved the efficiency of oligomer domain separation. 

FIGURE 6.1 Calibration curves of Shodex GPC KF-BOO series. Column Shodex GPC KF-800 series 8 mm i.d. X 300 mm. Eluent THF. Sample Polystyrene standards. 

FIGURE 6.40 Calibration curves of Shodex PROTEiN KW-800 series. Column Shodex PROTEiN KW-802.S, KW-803, KW-804, 8.0 mm i.d. X 300 mm. Eiuent Pullulan, PEG PEO Purified water, Protein SO mM Sodium phosphate buffer + 0.3 M NaCI (pH 7.0). Flow rate 1.0 mL/min. Detector Pullulan. PEG PEO Shodex Rl Protein Shodex UV (220 nm). Column temp. Ambient. 

FIGURE 7.13 Preparative separation of various proteins on Fractogel EMD BioSEC (S). The length of the column was 1000 mm and the inner diameter 100 mm. The flow rate was 6.2 ml/min with 20 sodium phosphate buffer (pH 7.2) containing 0.3 M NaCI as the eluent. The injected standard proteins can be used to create a calibration curve. 

Another important parameter for column selection is the proper choice of sorbent porosity. The pore size of the sorbent determines the fractionation range of the column. The best way of doing this is by looking at the calibration curves of the columns, which are normally documented by the column vendor (cf. Fig. 9.3 for PSS SDV column calibration curves and PSS SDV fractionation ranges) (7). 

Figure 9.4 illustrates a simple way of selecting the best column for SEC work based on the calibration curves of two sorbents with different pore sizes (4). 

The linear column (PSS SDV 5 /mm linear) has a wider molar mass fractionation range while keeping the analysis time roughly the same. Therefore the slope of the calibration curve is much steeper and the resolution will be poorer in this case. The second column with a single pore size (PSS SDV 5 /mm 1000 A) separates only below 50,000 Da, but does this very efficiently in the same time. 

All packing materials produced at PSS are tested for all relevant properties. This includes physical tests (e.g., pressure stability, temperature stability, permeability, particle size distribution, porosity) as well as chromatographic tests using packed columns (plate count, resolution, peak symmetry, calibration curves). PSS uses inverse SEC methodology (26,27) to determine chromatographic-active sorbent properties such as surface area, pore volume, average pore size, and pore size distribution. Table 9.10 shows details on inverse SEC tests on PSS SDV sorbent as an example. Pig. 9.10 shows the dependence... 

For proteins, the most useful columns are those with pores of 100-500 A, as seen in Fig. 10.2, because most proteins elute on the linear portions of the calibration curves. Figure 10.5 illustrates an analysis of a protein mixture on SynChropak GPC100. Small peptides can be analyzed on the 50-A SynChro-pak GPC Peptide column with appropriate mobile-phase modifications. Many peptides have poor solubility in mobile phases standardly used for protein analysis, as discussed later in this chapter. 

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