HPLC has matured over time and has seen several innovations to meet the expectations of chromatographers engaged in academic research and industry.
Some of theexpectations from scientists are:
- Higher separation efficiencies in short analysis times
- Low column back pressures
- Reduction of solvent consumption
- Lower levels of quantification and detection
- Separations covering wide temperature ranges
- Availability of wider pH analysis range
- Greater robustness of the columns and with longer useful lifespans
- Larger samples load capacities of columns
Emerging trends
Advances in the Stationary Phases:
Stationary phase characteristics are a major deciding factor in selection of the column for a particular application.Silica based columns are primarily used for performing major majority of separations. Present developments in stationary phases have multiplied the scope of applications several fold. New innovative stationary phases have evolved around monolith packings, core-shell technology andzirconia based packing materials.
UPLC or ultra-pressure liquid chromatography:
The efficiency level of column increases remarkably with reduction in the size of stationary phase particles. It was in the year 2003 when sub- 2μm particle were introduced which resulted in fast separations. Now, you can easily reduce the length and diameter of the column for offering faster separation with increased sensitivity. Small size particles pose problems of increased column back pressure butthese can be overcome through advances in instrument design. UPLC permits higher separation efficiencies under high pressure ranges and in short analysis times.
Microbore and Nanobore HighPerformance Liquid Chromatography:
Limited sample size availability has always posed a challenge to the chromatographer. Nanobore columns provide viable solutions in such situations.Femtomole level detection withhigher resolutionimproves quantification and identification. The IDs of these nanocolumns are as small as 75 μm, and the flow rate is as low as 300nL per minute.This leads to appreciable saving in cost of expensive mobile phases as well as saving of valuable time
Microbore HPLC columns have id’s around 1mm and flow rates range from 50- 75 µL/minute .The potential of such columns has been exploited in mass spectrometric detection systems. These columns are mainly used for analyzing peptides in different biological matrices.
Hyphenated LC technique:
LC-FTIR, LC-MS, and LC-AAS are examples of some hyphenated techniques which have contributed to the separation and identification of hitherto unknown detection of difficult separations. LC-MS is the most popular technique used for separation range and quantification of complex substances mainly in bio analytical and pesticide residue separations.
Fast Protein Liquid Chromatography:
FPLC is used in purification of proteinsin biological matrices. It has the preparative scale potential for milligram to even kg scale separations. Isolation and separation are based on charge separation, taking place between protein and resins. Buffer compositionvariation helps in elution rate changes.
Affinity chromatography:
Affinity chromatography shows promise in separating biochemical molecules. Some examples are protein purification and nucleic acids in biological fluids like in blood. This is mainly based on the interactions, taking place between antibody and antigen, substrate and enzyme or ligand and receptor molecules.
Chiral chromatography:
Chiral Chromatography has been useful in separation of optically active components of complex mixtures.
The scope of HPLC innovations is limitless and hundreds of new applications are reported worldwide in reputed journals based on these emerging technologies.
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