The Role and Importance of High Performance Liquid Chromatography

Published: 2021-06-29 07:09:52
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1.1. Introduction:
As the demand for shorter analysis times continues to rise, emerging technologies are being developed to meet this demand. Many of these technologies involve speeding up chromatographic separations and include such techniques as ultra high pressure liquid chromatography (UHPLC), multidimensional chromatography (2D LC), and high temperature liquid chromatography (HTLC) among others. While all of these approaches are promising, HTLC may provide the greatest value due to the ease with which this technology can be adopted. Commercial column heaters are currently available which can provide precise temperature control up to 200oC as well as temperature gradient programming. There are also a growing number of commercial columns containing stationary phases that are stable at these high temperatures. With these products, traditional HPLC methods can often be easily converted to high temperature methods with little added method development time and with the benefit of dramatic reductions in separation times.
In order to take full advantage of the rapid separations provided by techniques such as HTLC, detectors with the capability of acquiring data at high rates are required. This is due to the fact that fast separations yield narrow chromatographic peaks and so more data points per time are necessary to achieve proper characterization and quantization. For such applications, time-of-flight mass spectrometry (TOFMS) has become the detection method of choice. Not only does TOFMS provide the requisite data rates, but accurate masses of analytes can also be measured which allows for identification based on a molecular formula. In addition, modern data acquisition systems are capable of providing improved dynamic range and so quantitative data can be readily obtained [1].
The key aspect of MALDI-TOF/ MS is to dilute and isolate macromolecules in a suitable matrix of highly laser light absorbing small organic molecules, such as a-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA) and 2,5dihydroxybenzoic acid (DHB), and then allowing it to dry on `a MALDI-target into a crystalline deposit throughout which the molecules of the analyte are dispersed. The excitation of the matrix by a high intensity laser pulse of short duration, the absorbed energy causing desorption (vaporization) and ionization of the analyte in a very dense MALDI plume. The ions are generated essentially at a point source in space and time then enter a vacuum where they are accelerated by a strong electric field in a 'flight tube' where they are then separated in time and finally hit the detector. An analyzer measures the time-of-flight (TOF) taken for particular ions to hit the detector. The flight time of an ion is related to its mass-to-charge ratio (m/z), thus, mass spectra can be generated from simple time measurement [2].
1.2. Background:
Over the last thirty years the name HPLC has been synonymous with high-speed liquid chromatography and during the last ten years we have experienced a dramatic increase in the speed of analysis. With a solid grounding in the chromatographic theories, column technology has been mainly responsible for the advances in this field. Recent development showed that columns packed with micropellicular or gigaporous stationary phases of the bi-disperse or the bimodal type facilitate rapid mass transfer between the mobile and stationary phases and thus can deliver high resolution separations in a very short time. This suggests that HPLC has the potential to be the prime analytical technique for on-line monitoring of biotechnological processes in real time. Further enhancement of the speed of separation comes from the use of elevated temperatures. The role of temperature in HPLC has largely been ignored and most commercial instruments are not equipped with appropriate temperature control.

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