The Role and Importance of High Performance Liquid Chromatography

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.

Finally the overall goal of analytical chemistry is to achieve sufficient resolution of analytes (peaks) within the shortest possible time. By using long columns packed with small particles high performance of separations can be achieved. Use of very small particles requires very high pressure drop across the column is to be increased according to the following equation.

ΔP = φηLu/d2p Eq- (1)

Where:

ΔP = Pressure drop

φ = Flow Resistance Factor

L = Column Length

η = Viscosity of mobile phase

u = Linear Velocity of Mobile Phase and

dp = Particle Diameter.

Therefore higher pressures are required to run the columns with smaller particle size when compared to columns packed with higher particle size. Hence applicability of higher flow rates for separations using small particle packed columns is limited by the back pressure that the other components of the chromatographic system for example injector, column and values can withstand but at the same time lower flow rates result in degraded column efficiency because of increased band broadening by solute longitudinal diffusion.

Most conventional pumping systems have upper pressure limits of approximately 400 bar which would limit a column packed with a particle size of 1.5 µm and column length up to 3.3 cm. Fast separations can also be done by using such columns but the efficiency of the column will be lost due to such a short column and they are not suitable for the separation of complex samples.

In 1997, Mac Nair et al. first reported the use of ultra high pressures in liquid chromatography. Using pressures near 40 000 p.s.i, separations of combinatorial chemistry samples, pharmaceutical compounds and chiral compounds have been completed within less than 2 min, yielding more than 200 000 theoretical plates.

In LC high temperatures can be used to lessen the back pressure due to smaller particles. Increasing the temperature of mobile phase decreases its viscosity, allowing the use of higher flow rates at lower back pressure. Generally for fast separations, columns are operated at higher velocities than the optimum velocities in order to reduce time separation. These high velocities are demonstrated by the mass transfer characteristics of the column packing, typically leading to a loss of efficiency. However as temperature increases diffusivities

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