Chromatographic Separation Tools Advantages and Disadvantages

Chromatographic Separation Tools Advantages and Disadvantages

1. Advantages & Disadvantages Chromatographic Tools

2. Column Chromatography 01

3. 01 Column Chromatography Advantages: All different kinds of complex mixtures can be separated by column chromatography. Mobile phase is on a wide range. No limit for quantity as any amount of mixture can be separated by this technique. It is a robust method. The separated analytes can be reused. This process can be automated. It has a low separation power.

4. 01 Column Chromatography Disa dvantages: It is a time-consuming process for the separation of compounds. It is expensive as higher quantities of solvents are required. The automated process becomes complicated and therefore costly. It has a low separation power.

5. Ion-Exchange Chromatography 02

6. 02 Ion-Exchange Chromatography Advantages: Ion exchange chromatography is a very powerful separation technique that is used not only for preparative chromatography but also for analytical chromatography.  Conversely, its requirement for loading samples in buffers of low ionic strength makes ion exchange chromatography an excellent second purification step after  hydrophobic interaction chromatography (HIC) . Ion exchange chromatography, unlike some other chromatography methods, also permits high flow rates, which in some cases can be crucial to the recovery of active protein.

7. 02 Ion-Exchange Chromatography Disa dvantages: One of the main disadvantages of ion exchange chromatography is its buffer requirement: because binding to IEX resins is dependent on electrostatic interactions between proteins of interest and the stationary phase, IEX columns must be loaded in low-salt buffers. For some applications, this restriction may require a buffer exchange step prior to ion exchange chromatography. A limitation of weak ion exchangers is their pH dependence. When working outside of their optimal pH range, these resins rapidly lose capacity, and more importantly, resolution.

8. Gel-Permeation (Molecular Sieve) Chromatography 03

9. 03 Gel-Permeation Chromatography Advantages: Versatility Wide range of matrices commercially available Mild conditions of operation

10. 03 Gel-Permeation Chromatography Disa dvantages: Sample dilution Need for a low ratio of sample volume to column volume Considered as the most common, efficient, and fastest method that provides information about the polymer’s MW distribution.  Poorly reproducible in terms of molar mass analysis due to the difficulty to obtain a pure size-exclusion separation for various factors such as concentration effects, osmotic effects, side processes, secondary retention mechanisms, secondary exclusion, SEC band broadening, parasitic processes, and preferential interactions

11. Affinity Chromatography 04

12. 04 Affinity Chromatography Advantages: Involves many types of interactions between ligand and target such as steric effects, hydrogen bonding, ionic interactions, van der Waals forces, dipole-dipole interactions and even covalent bonds while other chromatographic techniques involve just one or a few of them. The combination of these multiple interactions leads to separation with high selectivity and retention in affinity chromatography.  Most specific and effective technique for protein purification 

13. 04 Affinity Chromatography Disa dvantages: Non-specific binding of irrelevant proteins during affinity purification and chemical modification of bioactive compounds of interest used as ligands. These drawbacks limit its extensive application. Selectivity of the ligand, recovery process, throughput, reproducibility, stability and economical criteria are some of the factors that influence the success of affinity chromatography process

14. Paper Chromatography 05

15. 05 Paper Chromatography Advantages: Q uick to perform and easy to master. With a correctly chosen mobile phase (chromatographic solvent), an analyst can rapidly determine the number of constituents of a mixture sample. Sometimes, paper chromatography even allows one to positively identify these constituents. Another advantage of this method is that it requires a relatively small sample and is very inexpensive - a big plus in today's cost-conscious world.

16. 05 Paper Chromatography Disa dvantages: Some mixtures are very difficult to separate by paper chromatography; and any species that is not coloured is difficult to observe on the chromatogram. Solely, an analytical method, not a preparative one. Because the sample size is so small, it is difficult to perform further analysis after the sample's contents have been chromatographically separated. Lastly, paper chromatography can only be used in qualitative analysis. It is not possible to extract meaningful information about the quantitative content of a mixture from a paper chromatogram.

17. Thin-Layer Chromatography 06

18. 06 Thin-Layer Chromatography Advantages: Low cost and the possibility of analyzing a large number of samples simultaneously. It is especially suitable for screening tests, in which pretreatment of the analytes can be avoided, even with dirty samples. The thin-layer format provides a better arrangement for high sample throughput, flexible detection strategies, and a greater tolerance of samples with a high-matrix burden. An easy method of  separation of the components. In this technique, fewer types of equipment are used. The separation is done in a very short time as the components elute rapidly.

19. 06 Thin-Layer Chromatography Advantages: All components of UV light is achievable to visualize. The non-volatile compounds can be separated by this method. Microliter quantity of sample can also be separated in TLC The components of complex mixtures easily separate and recover.

20. 06 Thin-Layer Chromatography Disa dvantages: The results obtained from the experiment are difficult to reproduce. Applicable for soluble mixture components only. Qualitative analysis, not quantitative analysis. Not an automatic process. The humidity and temperature can affect the results as Thin layer chromatography works in an open system. Separation process takes place only to a certain length as plate length is limited.

21. Gas Chromatography 07

22. 07 Gas Chromatography Advantages: Due to its high efficiency, GC allows the separation of the components of complex mixtures in a reasonable time. Accurate quantitation (usually sharp reproducible peaks are obtained) Mature technique with many applications notes available for users. Multiple detectors with high sensitivity (ppb) are available, which can also be used in series with a mass spectrometer since MS is a non-destructive technique.

23. 07 Gas Chromatography Disa dvantages: Limited to thermally stable and volatile compounds. Most GC detectors are destructive, except for MS.

24. Hydrophobic Interaction Chromatography (HIC) 08

25. 08 Hydrophobic Interaction Chromatography Advantages: The perceived kinetic performance gains are due to the metabolites are more hydrophilic than the parent compound, especially if conjugation to the glucuronide has taken place. HILIC was highly effective for the analysis of purine, pyrimidine and low-molecular weight amide substances in a pharmaceutical setting, comparing bare silica and amino phases for this application. Entirely suitable technique for the analysis of seized narcotics which are both polar and basic. Using HILIC in the nano-bore scale for the separation of sympathomimetic drug substances.

26. 08 Hydrophobic Interaction Chromatography Advantages: Another advantage of the low viscosity HILIC eluent is the enhanced desolvation properties encountered with electrospray ionisation (and other aerosol based detectors). Up to 10 times greater signal was observed in HILIC mode versus reversed-phase. This affords greater sensitivity in comparison to more aqueous rich-mobile phases, enhancing ion transfer from solution into the gas phase.

27. 08 Hydrophobic Interaction Chromatography Disa dvantages: The main disadvantage in adopting HILIC is the reliance on acetonitrile during times of shortage of this solvent. HILIC is not as straight forward a technique as reversed-phase. There may be a perceived reluctance to adopt such a technique even when the advantages are distinct.

28. Fast Protein Liquid Chromatography 09

29. 09 Fast Protein Liquid Chromatography Advantages: Reproducible with excellent resolution. Very simple system programming. Inert construction against the very high salt concentrations and corrosive liquids hence columns have longer lifetime. Since lower pressures are used in FPLC than in HPLC, a wider range of column supports are possible. The wide flow range makes it suitable both for analytical and preparative chromatography.

30. 09 Fast Protein Liquid Chromatography Disa dvantages: Needs glass columns. Can not handle high pressure. Instrument does not support HPLC columns. Purifying thermo labile (heat sensitive) proteins is a tough task.

31. Pyrolysis Gas Chromatography 10

32. 10 Pyrolysis Gas Chromatography Advantages: Can examine materials and compounds that are not suitable for traditional GC-MS Able to study polymeric structures from pure systems to multiple block polymers Micrograms of samples are used for analysis Reduced sample preparation

33. 10 Pyrolysis Gas Chromatography Disa dvantages: On-homogenous samples might have variable results Does not detect most inorganic components Pyrolysis-GC-MS is a destructive technique

34. High Performance Liquid Chromatography (HPLC) 11

35. 11 High Performance Liquid Chromatography Advantages: The most important aspect of HPLC is the high separation capacity which enables the batch analysis of multiple components. Even if the sample consists of a mixture, HPLC will allows the target components to be separated, detected, and quantified. Also, under appropriate condition, it is possible to attain a high level of reproducibility with a coefficient of variation not exceeding 1%. Also, it has a high sensitivity while a low sample consumption. HPLC has one advantage over GC column that analysis is possible for any sample can be stably dissolved in the eluent and need not to be vaporized. With this reason, HPLC is used much more frequently in the field of biochemistry and pharmaceutical than the GC column.

36. 11 High Performance Liquid Chromatography Advantages: One of the main benefits of HPLC is its ability to elucidate the structure and determine the quantities of impurities in pharmaceutical formulations.3 HPLC is especially suitable for compounds that are not easily volatilised, thermally unstable and have high molecular weights. Therefore, it can quantify a drug in its pure and dosage form. 

37. 11 High Performance Liquid Chromatography Disa dvantages: It must be preceded by calibration tests which can increase costs.