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
Disadvantages:
 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
Disadvantages:
• 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
Disadvantages:
 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
Disadvantages:
• 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:
• Quick 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
Disadvantages:
• 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
Disadvantages:
 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
Disadvantages:
 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
Disadvantages:
• 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
Disadvantages:
• 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
Disadvantages:
• 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
Disadvantages:
• It must be preceded by calibration tests which can increase costs.