Monolithic disk

Branovic K, Lattner G, Barut M, Strancar A, Josic D, and Buchacher A. Very fast analysis of impurities in immunoglobulin concentrates using conjoint liquid chromatography on short monolithic disks. J. Immunol. Methods 2002 271 47-58. 

The concept of monolithic columns in the shape of disks was first introduced at the beginning of the 1990s by Tennikova, Belenkii, and Svec. They prepared hydro-phobic methacrylate-based monolithic disks with a diameter of 20 mm and a thickness of 1 mm which demonstrated a very efficient separation of a protein mixture. After this initial research, monolithic disks of different diameters and thicknesses were prepared and used for various chromatographic separations. The smallest monolith had a diameter of only 1.8 mm, while the largest had a diameter of 50 mm. The thickness was between 0.3 and 7 mm. In this way, the monolith volume differed by several orders of magnitude. The monolithic disks contained different chemical moieties which were used for separations in ion exchange, hydrophobic, and affinity interactions, as well as reversed phase modes. 

Fig. 3 Flow characteristics of the monolithic disks (A) flow-independent resolution and (B) flow-independent dynamic binding capacity. (A) Conditions—buffer A 20 mM Tris-HCl buffer, pH 7.4 buffer B 1 M NaCl in buffer A, pH 7.4 flow rate 10 mL/min detection UV at 280 nm gradient 0-70% buffer B in 30 sec sample (1) myoglobin (0.5 mg/mL), (2) conalbumin (1.5 mg/mL), (3) soybean trypsin inhibitor (2 mg/ mL) injection volume 20 pL. (B) Conditions—binding buffer 20 mM Tris-HCl buffer, pH 7.4 flow rate 2, 4, and 8 mL/min detection UV at 280 nm sample human serum albumin dissolved in buffer A (1 mg/mL).

Because of their small thickness, one might speculate that the monolithic disks can only separate a few substances during a chromatographic run. However, by precise gradient adjustments, more than 10 substances can be separated in a few minutes. In Fig. 4, a separation of 14 oligonucleotides was performed in 3 min at room temperature —a feat hardly achievable with a conventional particulate column. 

Monolithic structures enable two additional interesting features. Because of their defined dimension, several monolithic disks can be stacked together forming a single chromatographic column. Such a column has much better performance in comparison to a group of several columns... 

The features clearly indicate that the monolithic disks enable efficient separations and purification of different types of molecules. This is demonstrated in a number of examples where these columns were used in the separation of complex biological mixtures. 

Monolithic disks have been successfully used in the separation of different types of molecules. Most of the applications, however, deal with the separation of proteins. Initially, the studies were conducted with test solutions to prove the power of the separation media, but many applications with real samples have also been... 

There are several reports about the application of the disk monolithic columns for the separation and purification of plasma proteins. " Purification and monitoring of clotting Factor IX (FIX) from human plasma were performed using the ion-exchange monolithic disks. In addition, separation of vitronectin from FIX was possible. A similar system was used for the separation of a complex between clotting Factor VIII and von Willebrand factor (FVIII-vWF). Another application was the monitoring of oti-antitrypsin production. Separation of impurities such as human serum albumin and transferrin was achieved in a few minutes. 

Monolithic disks seem to be very efficient columns for the separation of polynucleotides. They enabled not only the separation of pDNA from RNA, but also the separation of pDNA isoforms supercoiled, open circular, and linear forms.Besides a separation time of only a few minutes, the most outstanding characteristic of the monolithic disks is their high dynamic binding capacity for pDNA, which is in the range of 10 mg/niL of support. 

In addition, the monolithic disks seem to be very attractive for the purification of even larger molecules. 

More information about the applications of the monolithic disk columns can be found in several... 

Methacrylate-based monolithic disks are very suitable for the immobilization of various ligands because they contain epoxy groups which form very stable covalent bonds with the amino or sulfhydryl groups of the ligand. Recently, an extensive description of different immobilization procedures was published. 

Different high molecular mass affinity ligands were successfully immobilized to monolithic disks.Monolithic disks with immobilized heparin and collagen were used for the purification of membrane proteins and annexins. The heparin unit was also used in the quality control of the preparation of the plasma proteins Antithrombin 111 and clotting Factor IX. Purification of IgG was successfully performed by immobilizing Protein... 

Enzymes immobilized to monolithic disks were not applied only as biosensors, but also as bioreactors. 

The first report, in 1991, describes the immobilization of carbonic anhydrase. Interestingly, an increase in enzyme activity with the increase of flow rate through the bioreactor was observed. Recently, the immobilization of trypsin was reported. Contrary to the previous work, increased flow rate diminished the extent of protein degradation. In contrast to the previously mentioned experiments, where the immobilized enzyme was used for substrate degradation, the synthesis of polyriboadenylate from ADP was studied by polynucleotide phosphorylase immobilized on a monolithic disk. 

An obvious approach for scaling-up the monolithic disks is by increasing their diameter. Because of the mechanical properties, however, this approach seems not to be the most appropriate one. The problem of increasing the... 

Tennikova, T.B. Freitag, R. An introduction to monolithic disks as stationary phases for high performance biochromatography (review). J. High Resol. Chromatogr. 2000, 23, 27-38. 

Porous carbons can be prepared with a variety of macroscopic shapes (e.g., granules, powder, fibers, cloths, pellets, monoliths, disks). 

---