Elution position

A SEC material should be hydrophilic if it is to be used for biological applications. One such material, introduced by PolyLC in 1990 (8), is silica with a covalently attached coating of poly(2-hydroxyethyl aspartamide) the trade name is PolyHYDROXYETHYL Aspartamide (PolyHEA). This material was evaluated for SEC of polypeptides by P.C. Andrews (University of Michigan) and worked well for the purpose (Fig. 8.1). Because formic acid is a good solvent for polypeptides, Dr. Andrews tried a mobile phase of 50 mM formic acid. The result was a dramatic shift to a lower fractionation range for both Vq and V, (Fig. 8.2) to the point that V, was defined by the elution position of water. 

FIGURE 8.6 Comparison of hexafluoro-2-propanol (HFIP) with formic acid as a denaturing agent in SEC. Eiution positions of neutral amino acids were similar with both agents. The elution positions of Lys and Asp shifted dramatically in C, as shown by the tie lines, but this was an effect of pH (see Fig. 8.7). The elution positions of a-MSH and formic acid are shown to demonstrate that the amino acids eluted within Vo and V,. Column Same as Fig. 8.1. Flow rate 1.0 ml/min. Mobile phase As noted. Detection Aiij = 0.1 AUFS. 

The continuous annular chromatograph can be described mathematically by a theoretical plate approach similar to the one developed by Martin and Synge [40] and exemplified by Said [41] for stationary columns [5]. The mathematical description results in algebraic expressions for the elution position of each solute relative to the feed point and for the bandwidth of the eluting zone as a function of the elution position or other system parameters. However, a series of simplifications have to be made in order to describe the CAC with the theoretical plate concept ... 

However, it is not only the pH of the eluting buffer that determines the relative elution position of the amino acids, but also the cation concentration of the buffer. Sodium citrate buffer solutions are commonly used and the positive sodium ions compete with the positively charged amino acids for the sul-phonic acid sites on the resin ... 

The temperature of the resin column must be carefully maintained to avoid changes in both the pH of the buffers and ionization of the amino acids. Although increasing temperature usually results in faster elution, the effect may be variable for different amino acids and the relative elution positions can be altered, making interpretation of results difficult. The temperature often chosen is 60 °C although lower temperatures are sometimes required to resolve two similar amino acids. Temperature programming, which entails an alteration in temperature at a specified time in the separation procedure, is widely used. 

Fig. 3.26. Typical elution HPLC profiles of MG residues extracted from (a) salmon spiked at 20 pg/kg LMG and MG each (b) residue-incurred salmon fillet (2.9 pg/kg). Analysis of the residue-incurred salmon was repeated using the LC-MS-MS system as shown in (c) the profile shows the monitoring of the m/z = 329.5 to m/z 313.3 fragmentation. The elution positions of MG, LMG and the internal standard brilliant green (BG) are indicated. Note BG is not detected in the m/z = 329.5 to m/z 313.3 trace (c) and its position is therefore depicted as an under broken arrow. Reprinted with permission from A. A. Bergwerft et al. [105],...

Wish, L., E. C. Freiling and R. Bunney Ion-Exchange as a Separation Method. VIII. Relative Elution Position of Lanthanide and Actinide Elements with Lactic Acid Eluant at 87° C. J. Amer. chem. Soc. 76, 3444 (1954). 

Fig. 4 Elution position of S-3-aminopropylcysteine (AP-C) in four different amino acid analysis systems. Purified 5-3-aminopropylcysteine was spiked into amino acid standards and subjected to amino acid analysis. Standards run on (A) an AminoQuant (OPA) system, (B) a Waters PICO-TAG (PITC) chemistry system, (C) a Varian Amino Tag (FMOC) chemistry system, Y = tyrosine and FMOC-Cl, (D) a Beckman System 6300 (nin-hydrin) amino acid analyzer. (From Ref. 90. Copyright 1994 Academic Press, Inc.)...

FIGURE 9.3 Schematic diagram of a SI separation system using a 6-port 2-position valve to isolate the separation column. The 6-port valve is shown in the column elution position. 

Based on chemical shifts and peak multiplicities, the on-flow HPLC-NMR characterisation of the majority of the components in the mixture of 27 tripeptides was achieved and demonstrated that this approach is likely to be an effective method for compound mixtures. The elution positions of all of the alanyl-containing peptides were determined, with the exception of A-M-M-NH2, which may have co-eluted with another peptide or may have been synthesised in a much smaller quantity. The only other tripeptides for which assignments have not been obtained are the MY2-NH2 isomers and two of the three M2Y-NH2 isomers. These eluted towards the end of the gradient run and are not as well resolved under these HPLC conditions. Additionally, with changes in the relative chemical shifts of the solvent signals, the intensities of the non-TV-terminal a-CH protons and the methionyl [3-methylene signals from these peptides may have been reduced by the effects of the solvent suppression irradiation of the water and acetonitrile resonances, respectively. With further optimisation of the elution conditions, it is possible that all 27 analytes could have been resolved and characterised. 

As long as units are consistent throughout, the measurements can be recorded as time or volume. The measurement V0 is the time taken for a non-retained component to travel from the injection port to the detector. The elution positions of the retained components, Vj and V2, respectively and the width of the first peak at half height, W1/2 are used to calculate the number of theoretical plates, N and the separation factor, a. 

Fig. 9. Fractional extraction, %p, of Nb, Ta, Pa, and Zr/Hf vs. HC1 molarity in the system TiOA-HCl/HF. The bold bars encompass the upper and lower limits of %p deduced from the Db elution positions. The bar for the extraction of Db from 12 M HC1/0.02 M HF is not included in the figure for clarity. The figure suggests that the element with the unusual behavior is Ta. Reproduced from [43] with the permission of Oldenbourg Verlag.

The data for 0.05 M a-HiB show that the elution position and the width are quite reproducible. As the elution obviously takes more time with 0.0125 M a-HiB, see Figure 14, 0.05 M a-HiB was finally selected for the dubnium experiment. 

Figure 1.4 Separation of substrates and products of an adenosine kinase reaction on ion-paired reversed-phase HPLC. The separation was carried out on a prepacked Ctg (/xBondapak) column with a mobile phase of 65 mAf potassium phosphate (pH 3.7) containing 1 mAf tetrabutylammonium phosphate and 5% methanol. The column was eluted isocratically, and the detection was at 254 nm. Four relative elution positions (elution times) arc shown.

Figure 2.3 A representative HPLC chromatogram showing the separation of compounds A and B. The time of injection is taken as zero time, and the elution position is shown as a function of time after injection. The amount of each compound in the original sample is given by the peak height or area, as represented on the tracing by the letters B and A.

Figure 9.76 Chromatograms of the digest of PA-sialyllactose by urine. PA-sialyllac-tose (0.167 mM) was incubated with dialyzed urine in 0.1 M sodium acetate buffer (pH 5.0) at 37°C for 2 hours. (A) Substrate + urine heated at 100°C for 10 minutes (control). (B ) Substrate + urine. The arrows show the elution position of PA-lactose. (From Omichi and Ikenaka, 1982.)...

Hyl was assigned by the elution position of the PTH-amino acid [between Val and DPTIJ (13)] as shown in Fig. 2 this assignment was confirmed by sequencing a Hyl standard (not shown). Hyp77 is foimd within a Xaa-Lys-Gly sequence, consistent with other known hydroxylysine modification sites. 

Figure 4.22. Separation of PTH-Amino Acids. PTH-amino acids can be rapidly separated by high-pressure liquid chromatography (HPLC). In this HPLC profile, a mixture of PTH-amino acids is clearly resolved into its components. An unknovm amino acid can be identified by its elution position relative to the known ones.

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