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Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored.

The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites.

It is further demonstrated how information on competition or displacement effects can be obtained by these methods. Keywords: Affinity microcolumns, High-performance affinity chromatography, Biointeraction analysis, Zonal elution, Frontal affinity chromatography, Ultrafast affinity extraction 1.

Introduction Biomolecular interactions make up an important component of the many pathways and responses that are present in living systems. These interactions include the binding of substrates and co-factors to enzymes, antigens to antibodies, proteins to proteins, and sugars to lectins, as well as the binding of small molecules such as hormones and drugs with transport proteins and receptors [ 1 — 4 ]. These interactions can determine the eventual activity, distribution, excretion, metabolism, and effects of a solute or biomolecule in the body.

In addition, the binding of small molecules with proteins can determine the solubility of hydrophobic compounds and can be an important source of direct or indirect competition between different solutes with the same binding protein e. Various techniques can be employed for examining biomolecular interactions. These methods include X-ray crystallography, fluorescence spectroscopy, absorption spectroscopy, ultrafiltration, equilibrium dialysis, capillary electrophoresis, surface plasmon resonance SPR , and nuclear magnetic resonance spectroscopy NMR [ 5 , 6 , 10 , 13 — 27 ].

Two other, related methods that have seen increasing use in the study of biomolecular interactions are affinity chromatography and high-performance affinity chromatography HPAC, also known as high-performance liquid affinity chromatography or HPLAC [ 1 — 4 , 9 , 11 , 28 — 30 ]. These methods use a chromatographic column and support that contain an immobilized biologically related agent e. This immobilized agent can then be used to study the binding of injected compounds to the column or as a probe to examine the interaction of an injected compound with another binding agent in the mobile phase [ 2 , 3 ].

Various types of columns and formats can be used in these experiments [ 1 — 3 , 9 , 11 , 31 — 33 ]. However, one topic that has been of growing interest in the analysis of biomolecular interactions is the use of affinity microcolumns for such work i.

This review examines the developments and applications that have appeared in the use of affinity microcolumns as related to the characterization of biomolecular interactions. First, the basic principles behind affinity chromatography and HPAC are described, especially as related to the use of these methods in investigating biomolecular interactions.

1. Introduction

The general types of affinity microcolumns that have been reported for binding studies are next considered. These microcolumns range from open-tubular capillaries and packed capillaries to small columns based on monoliths or particulate supports [ 2 , 4 , 40 — 55 ].

The various approaches that have been used with affinity microcolumns for binding studies are then discussed. This discussion includes various formats based on zonal elution and frontal analysis [ 1 , 2 , 11 , 31 , 34 , 50 — 59 ].

In addition, techniques such as peak decay analysis, split-peak analysis, ultrafast affinity extraction, and band-broadening measurements are discussed [ 2 , 11 , 31 , 34 , 55 ]. The principles behind each method are described, along with the advantages and possible limitations of these methods. Results and Discussion 3. Yield of Organosolv Fractionation Experiments Ethanol organosolv pretreatment is known to be effective at separating cellulose from lignin as well as hemicellulose in multiple feedstocks, including woody biomass [ 31 ].

Results from the catalyzed and autocatalyzed organosolv fractionation experiments of larch sawdust are summarized in Table 2. The moisture content of the larch sawdust was No predrying was performed because high moisture content facilitates feedstock impregnation with pulping liquor and thus increases the delignification rate [ 32 ].

The pretreatment time was set to 0. The maximum yield of ethanol organosolv lignin EOL , The results also indicate that an increase of both temperature to K and catalyst loading 0.

The increase in lignin yield is due to more excessive ether bond cleavage at higher process severities [ 11 ]. Similarly, the increase in ASL is caused by further fragmentation of dissolved lignin polymers to more water-soluble species. The yield of solid substrate decreased with both increasing temperature and catalyst loading due to increased delignification as well as carbohydrate degradation.

The Klason lignin content of the solid substrate first decreased with increasing temperature and catalyst loading, reaching a minimum at K at 0. However, with increasing process severity, the rate of carbohydrate degradation surpassed that of delignification and the content of Klason lignin increased, to reach a maximum of Table 2: Process parameters, mass flows of the organosolv extraction experiments, and lignin content of the solid substrate.

A significantly higher yield was achieved in this study, However, Pan et al.

Analysis of biomolecular interactions using affinity microcolumns: A review

As the ethanol organosolv fractionations were performed at both low pH and high temperature, it is possible that pseudolignins were formed. They are hypothesized to form primarily from sugar degradation products, such as furfural and 5-hydroxymethylfurfural, via first rearrangement reactions followed by oxidation, polymerization, and condensation reactions to yield aromatic components [ 33 , 34 ]. These aromatic components can increase the Klason lignin content of the pulp phase and furthermore reduce enzymatic digestibility by unproductive binding with enzymes.

This phenomenon is most likely to have occurred in the high severity pulping runs, OS-F and OS-G, since significant amounts of carbohydrates could not be retained in the solid substrate phase. Furthermore, the lignin yield of the autocatalyzed fractionation experiment was low 6. Thus, with proper reaction optimization, higher removal efficiency is possible, but still inferior to the catalyzed organosolv experiments.

CSF analysis was used to be able to easily compare and correlate ethanol organosolv fractionation yields and physicochemical characteristics of the isolated larch lignins.

However, CSF was not used to analyze the processing conditions of the autocatalyzed experiment, as no acidic catalyst was used. Influence of Process Parameters on Characteristics of Larch Organosolv Lignin FT-IR analysis is a commonly used technique to analyze structural features of lignin, due to its speed and simplicity.

All spectra look similar, albeit small differences, indicating comparable chemical structures. The absorption bands of the lignin samples were assigned based on published studies [ 35 , 36 ], which are summarized in Table S1 in the Supplementary Materials. A relative decrease of all three peaks in the organosolv lignins compared to MWL indicates that structural degradation of the lignin structure occurred during the fractionation process.

High-performance size-exclusion chromatography was performed, to assess the impact of treatment severity on molecular mass distribution of the resulting organosolv lignin fraction.

Although average molecular mass estimation based on a polystyrene calibration is by no means able to produce accurate absolute masses, it is indeed a valid tool for comparative purposes. Results listed in Table 3 indicate that the differences between number average molecular weights Mn of organosolv lignins from low K, 1.

The average molecular weights and their variance between low and high severity fractionation experiments are significantly smaller compared to previously published data with similar fractionation conditions, with Mw ranging from 3. Table 3: Results from molecular weight analysis of larch organosolv samples. Proton NMR analysis is known to be a reliable tool to semiquantify the content of functional groups of acetylated lignin samples, specifically phenolic PhOH and aliphatic hydroxyl groups AlkOH as well as methoxyl groups MeO [ 38 , 39 ].

These results are in good accordance with the literature, where PhOH contents for organosolv lignins from 3. Table 4: Functional group content of the larch organosolv samples. On the contrary, the content of aliphatic hydroxyl groups is decreasing with increasing severity, from 4.

Thus, the same mechanisms leading to an increase of PhOH groups during the organosolv fractionation process led to a decrease of AlkOH groups. Additionally, during organosolv pulping, the whole side chain can be cleaved off, reducing the AlkOH content further.

The determined concentration range for aliphatic hydroxyl groups corresponds well to literature values, ranging from 3. As can be seen in Table 4 , the total amount of free hydroxyl groups does indeed not change significantly between all the acid-catalyzed fractionation experiments, just a slight increase from OS-C to OS-G can be observed. Both the MWL and the autocatalyzed organosolv lignin contain a significantly higher amount of total hydroxyl groups, 9.

Furthermore, the content of free carboxylic acid groups was low in all larch organosolv fractions, ranging from 0. The highest content was achieved in the autocatalyzed sample because of its comparably least severe method of extraction. However, no significant difference can be observed within the acid-catalyzed organosolv samples. Methoxyl group content ranges from 5. These observations are consistent with data published by Pan et al.

Additionally, the content of methoxyl groups is significantly lower in both MWL and the autocatalyzed organosolv lignin compared to the sulfuric acid catalyzed organosolv samples. This can be explained by nucleophilic addition of ethanol on the benzylic position during acid catalyst pulping and a general higher lignin extraction efficiency [ 45 ]. The high content of AlkOH in the autocatalyzed organosolv sample despite its comparably high content of PhOH can be explained due to the absence of an acidic catalyst as described above.

Of the eight different organosolv lignins investigated in this study, OS-G probably has the highest applicability as it contains the highest amount of phenolic hydroxyl groups.


A higher content of free hydroxyl groups correlates increased chemical reactivity, thus making OS-G an excellent candidate for use as an antioxidant or as a monomer for biopolymer production.

Table 5: Yields from thioacidolysis and GC-MS of the thioethylated guaiacyl G monomer derivatives mean of two independent analyses. To get a more complete view on the influence of pulping conditions on changes in the lignin structure, thioacidolysis of the larch organosolv samples was performed, and the results are summarized in Table 5.

Thioacidolysis is based on acid-catalyzed depolymerization of lignins with a high specificity to aryl ether bonds. A high content of releasable monomers therefore indicates more benign extraction conditions and consequently a less altered lignin structure [ 46 ].

Only guaiacyl G thioacidolysis monomers and vanillin, an oxidized guaiacyl derivate, were detected, neither p-hydroxyphenyl- H nor syringyl S monomers. This trend is in good accordance with the results of 1H NMR analysis, and the maximum content of releasable monomers correlates to the minimum of PhOH hydroxyl groups and vice versa.

Advanced Chemistry: Quantitative Analysis

The lower thioacidolysis yields of OS-A compared to OS-B could be explained due to the less-efficient lignin extraction in the presence of the same sulfuric acid loading. As expected, the content of releasable monomers is highest in the autocatalyzed lignin sample due to the relatively benign extraction conditions.

Correlation between Process Parameters and Antiradical Potential As parameter to assess the reactivity of isolated larch organosolv lignins, their respective antiradical potential ARP was chosen. As can be seen in Figure 3 , the pulping conditions do have a significant impact on the ARP of the resulting organosolv lignin fractions.

Larch sawdust extracted under the most benign conditions K, 1. With increasing process severity, the radical scavenging capability also rises, up to a maximum of This finding is in good accordance with previously published data on organosolv lignins of Miscanthus x giganteus [ 37 ], hybrid polar [ 28 ], and aspen, spruce, and birch wood [ 29 ].

ISBN 13: 9780321596949

Figure 3: Relationship between antiradical potential and content of phenolic hydroxyl groups; ARP is reported as mean , and the error bars represent the standard deviation.

The content of nonetherified phenolic hydroxyl groups is primarily responsible for the antioxidative potential of lignin [ 29 , 48 ].

Indeed, as can be seen in Figure 3 , the organosolv sample with the lowest content of PhOH exhibits the lowest antioxidative potential and vice versa Results further indicate that the contribution of methoxyl group to ARP is negligible.

However, more recent findings by Ponomarenko et al. This discrepancy can be explained by the large variety of lignins used in the aforementioned study, including grass, hardwood, and softwood lignins from different technical processes. The difference in average molecular weights between low and high severity pulping conditions is quite low, as discussed above, and significantly smaller compared to previously reported results [ 28 , 37 , 49 ].

Thus, the average molecular weights cannot be used as proper indicator for the potential antioxidative potential of larch organosolv lignins. The results by Pan et al. Their results for EC50 of hybrid poplar organosolv lignins range from 8.

This parity indicates that under similar pulping conditions, organosolv lignin from the European larch can achieve antioxidative potentials similar to that of hardwood lignins.

Conclusion In this study, for the first time, the relationship between physicochemical characteristics and pulping conditions of ethanol organosolv lignins from the European larch are investigated. This study should serve as the initial step for possible future applications of larch organosolv lignins as antioxidant or feedstock for biopolymer production. The harshest reaction conditions OS-G , K and 1.

Additionally, OS-G has the highest content of phenolic hydroxyl groups 4. The autocatalyzed organosolv lignin exhibited the highest aliphatic hydroxyl group 5. Both the high antiradical potential and content of free phenolic hydroxyl groups indicate that larch organosolv lignin indeed has potential for further valorization. Further investigations are planned, to properly assess potential future applicability of the material.

Data Availability The data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest The authors declare no conflicts of interest. Acknowledgments The authors would like to thank Prof. Anton Huber for his various suggestions and fruitful discussions, Prof. Bernd Werner for analyzing the lignin samples via NMR. Supplementary Materials The supplementary materials contain a detailed description of milled wood lignin isolation and purification, 31P NMR analysis, and a table containing a detailed assignment of the absorption bands from the IR analysis of the lignin samples.

Supplementary Materials References J.

Kleinert and K. Lora and W. Lesar, M. Humar, G. Hora et al. Nitsos, R. Stoklosa, A.

Karnaouri et al. Kleinert, and T.

Smit and W. Koumba-Yoya and T. Harris, U. Tschirner, A. Gillespie et al. Spigno, and J. Foulon, P. Soto et al. Stimm, A. Roloff, H. Weisgerber et al. View at Google Scholar J. Matras and L. Grabner, R. Wimmer, N.Similarly, both antiradical potential and phenolic hydroxyl group content exhibit a direct relationship to reaction temperature and catalyst loading. The results from the chemical characterization of larch sawdust are summarized in Table 1. Roloff, H. The moisture content of the larch sawdust was Furthermore, multiple studies have been published recently on possible applications for organosolv lignins.

View or edit your browsing history. Pan, D. Various techniques can be employed for examining biomolecular interactions.