Elution profiles

This section mainly focuses on the parameters that could lead to an evolution of the elution profile during a purification campaign. Several root causes are listed, which could impact the elution profile and modify the retention or disturb the peak shape. 

This should be taken into account to ensure the reproducibility of the chromatographic process and a temperature control with an online heat exchanger is preferred to get a reproducible process. 

A variation of the elution profile could potentially occur when the temperature is not controlled and when a fresh solvent is loaded in the eluent tank used to feed the system. The stored solvent could potentially be cooler or warmer, leading to a disturbance of the elution profile. 

The last (and probably most difficult) situation described is when a loss of purity 

Any resin, once packed, has a shelf life for a determined process. The number of cycles a packed chromatography column can be used has to be validated in order to guarantee specified yields and product quality. 

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In Figure 16 the elution profiles for samples from each group of seven plates are included together with the overall composite peak from the total charge. The calculations assumed a column efficiency of 5000 theoretical plates. The elution... 

Large cell volume can also have a very serious effect on solute resolution, but this can be examined theoretically. The situation is depicted in Figure 14. It is the elution profile of a peak eluted from a column 3 cm long, 3 mm I.D. packed with particles 3 pm in diameter. 

FIGURE 2.7 SEC elution profiles of dextran in clinical samples, serum ( ) and urine ( ). The first peak represent dextran and the second peak inulin (used as a reference for clearance). The content of carbohydrates was determined in collected fractions with the anthrone method. [Reproduced from Hagel ef of. (1993), with permission.]... 

Eigure 4.36 shows a comparison of elution profiles of dipalmitoylphospha-tidylcholine (DPPC) vesicles on a TSK-GEL G6000PW column and a Sephacryl S-1000 column (25). 

FIGURE 4.35 Elution profiles of adenovirus and vesicular scomacicus virus on TSK-GEL G6000PW and G50000PW. Column TSK-GEL G6000PW + G5000PW in series. Sample (A) adenovirus and (B) vesicular stomatitus virus. Elution 145 mA1 NaCI in 10 mM Na-HEPES buffer, pH 7.4. Flow rate ... 

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

Loss of resolution after a few steps Elution profiles are not reproducible... 

Sometimes elution profiles may also depend on the number of cycles. This can cause problems in the reproducibility of SEC separations. It should be mentioned that no regeneration procedure will give a completely new SEC support. 

Long-time reproducibility of elution profiles broad standard calibration with dextran T-500 transformation of a scb-type calibration function into nb/Icb-type via universal calibration dp of synthetic glucans in the presence of significant amounts of monomer mass and molar degree of polymerization of Triticale (hybride) starch... 

Establishing a calibration function with one single broad distributed sample is an alternative to traditional peak postion calibration of SEC systems with a set of narrow distributed standards. An obvious advantage of this technique is time for peak position calibration elution profiles for the set of standards need to be determined for broad standard calibration the elution profile of one sample needs to be determined only. Establishing a linear calibration function with a broad distributed standard includes startup information [M (true), Mn(true)] and an iterative (repeat.. . until) algorithm ... 

Preswelled Sephacryl S-1000 was prepared in a K26/100 column (88 X 2.6 cm). Equilibration with 0.005 M NaOH containing 0.002% NaN3 at a flow rate of 0.67 ml/min was achieved after 20 hr. Sample solutions were applied with a 5-ml injection loop. The mass and iodine-complexing potential of separated glucan components was determined off-line for each of the subsequently eluted 5-ml fractions. Based on the determined mass of carbohydrate for each of the fractions, elution profiles such as Fig. 16.1 were constructed. 

Reproducibility of separation for a Dextran T-500 sample was tested on a semipreparative Sephacryl system S-500/S-1000 (65 + 95x1.6 cm) over a period of 6 months. The elution profiles of Dextran T-500 could be superimposed with deviations in the elution axis of 3 ml ( 1 fraction), and deviations in carbohydrate content within 5% referring to the maximum value at V,e, = 213 ml (Fig. 16.8). 

Figure 16.15 shows the resulting chromatograms for the three glucan fractions obtained by previous preparative separation on Sephacryl S-200/S-1000 (Fig. 16.14). From the normalized fraction chromatograms, the elution profile of the initial mixture has been reconstructed by mixing 50% fraction 1, 40% fraction 2, and 10% fraction 3. Compared to the chromatogram of the preparative Sephacryl S-200/S-1000 system, separation with the TSK/ Superose system yields improved resolution in the low dp (high V, ) domain.

These combined HDF and GPC separations require the use of detectors such as static light scattering or viscometers to help sort out the convoluted elution profiles seen in those type of experiments. It should also be remembered in these situations that the typical refractive index or ultraviolet detector responses may not be representative of the actual mass fraction of insolubles eluting from the column because of the significant light scattering that can occur with those large particles in the detector cell. 

Elution profile of a large macromolecule (excluded from pore.s) (V —V )... 

Fig. 6-6. Overload elution profiles of D,L-PA injected on a column (125 4 mm) packed with the L-PA imprinted stationary phase used in Fig. 6-5. Mobile phase MeCN TFA (0.01 %) FI O (2.5 %). The tendency for fronting and the increase in retention with sample load is attributed in part to saturation of the mobile phase modifier.

The authors found that the yield of 30-mer (a product with 5—6 linkages) was not much smaller than that of 10-mer or 12-mer. These facts indicate that the stability of the complex between the oligonucleotides and the complementary template is the most important factor in determining the extent of the condensation. The strong influences of template polymer (Poly C) are demonstrated in Fig. 9, in which the elution profile is shown of the polymerization products of (2 MeIp)6 in the presence of Poly C (B) and in their absence (A). 

Fig. 9. The elution profile of the polymerization products of (2 MeIplf, in the presence of Poly C (B) and in the absence of poly C (A) (Ref.

Under circumstances where two solutes are incompletely resolved and one of the pair is present at a much lower concentration than the other, the profile of the pair often resembles a normal peak with slight asymmetry. Consider the combined elution profile of the two peaks shown in figure 3. 

Fig. 11 Elution profile of the ws-material from microwave-heated spruce chips after SEC [218]. Detection by refractometry index (Rl) dotted line) and UV detection at 280 nm (full line). The arrows mark the elution volume of acetylated GGM fractions...

Figure 2. Gradient-elution profile (5) of 18 amino acids deriva-tized with NDA/CN . The peak numbers correspond to those listed in Table III. Each peak represents 20 pmol of the individual amino acids. The chromatographic conditions are described in the text.(Reprinted from reference 5. Copyright 1987 American Chemical Society.)...

Figure 1. Elution profile on HPAEC of apple MHR-S after treatment with RGase at 30°C and pH 5.0 for 24h [37],...

Figure 5. Selected HPLC elution profile of products obtained after incubation of 0.25% polygalacturonate with PGII, upper trace, and PGII H223A, lower trace, respectively, demonstrating the effect of the mutation on catalysis. G1 to G3 indicate the peaks of the corresponding oligogalacturonates. IS indicates the internal standard, glucuronate. The vertical axis shows the pulsed amperometric detector response while the horizontal axis shows the retention time.

Figure 1. Elution profile of ChSS on DEAE Sepharose Fast Flow. Eluent molarity (. galacturonic acid ( ) and neutral sugars ( ).

Figure 4. Elution profile on HPAEC of pea shoot pectin fractions obtained after Biogel P4 separation.

The main peaks on their HPAEC elution profiles were corresponded to the peaks of unsubstituted galactan oligomers and some peaks to galactan oligomers... 

Fig.5 Elution profile on HPAEC of fraction IPN 6 before (a) and after treatment with galactonase (b) and arabinofuranosidase (c).

Figure 6A-C. High-performance anion-exchange chromatography elution profile of isolated modified hairy regions without addition of enzymes (A), with Biopectinase OS (B) and with rhamnogalacturonase-containing culture filtrate from an A. awamori multicopy transformant (C). /xC ftCoulomb- Data taken from Ref. 6.

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