HIGH PERFORMANCE LIQUID CHROMATOGRAPH

The High Performance Liquid Chromatograph (HPLC) is used for the analysis and identification of compounds that are either non-volatile or degrade under the heat of a gas chromatograph’s injection port. This makes the HPLC directly applicable for the Pharmaceutical Industry in the analysis of drugs and biologics. Whereas, the gas chromatograph is only applicable to the analysis of organic compounds that have a molecular weight of about 350 or less, HPLC is applicable to much larger organic compounds and, to some extent, doesn't have a moleuclar weight limit. As long as the components can be dissolved or suspended in liquid and passed through a liquid chromatographic column, they can be separated, detected and counted.

As it’s name implies, the mobile phase is liquid. The stationary phase consists of porous solid beads. Depending on the type of components that are to be separated and the composition of the stationary phase, the mobile phase can be anything from water to hexane. Typically, two miscable solvents (e.g., water and methanol or hexane and dichloromethane) are used as a gradient to gradually change the polarity of the mobile phase and elute each component according to it’s polarity.

The HPLC system consists of solvent reservoirs, the solvent injection valve, the pumping system, the chromatographic column, the detector and the computer system. No single component is more important than the next. From the solvent reservoirs to the computing device, each component is significant and plays an important role in producing a precise and accurate answer.

SOLVENT RESERVOIR

Although, easy to be over looked, the solvent reservoirs are an important part of the chromatographic delivery system. The solvents should be delivered to the pumping system, air free and contaminant free. Dissolved air in the system makes it difficult for the pumping system to deliver solvent in a consistent manner. And contaminants in the solvents cause mechanical problems in the pumping system and shorten the life of chromatographic columns.

The solvents used in HPLC are not simply off the shelf solvents. The solvents used for HPLC are typically glass distilled, passed through submicron filters and tested for lot-to-lot consistency. It is not unusual for a Chromatographer to make sure the solvents used in developing a method or performaing a series of important analysis are from the same lot. Minor, undetectable differences in solvent can make significant differences in the anaysis of complex mixtures.

SAMPLE INJECTOR VALVE

Introducing a sample onto the chromatographic column is not a simple task. The solvent going onto the chromatographic column can be under thousands of pounds of pressure. Attempting to inject a sample consistently and repeatedly from a syringe can be challenging. The sample injector valve consists of a set of valves and a sample loop that allow for the introduction of a liquid sample onto the head of the chromatographic column consistently, repeatedly and easily. The valves allow for sample introduction at ambient pressures. And the sample loop provides for reproducibility in sample size. The most commonly used sample loop is 10 microliters in size. That is, the sample loop holds 10 microliters. If more than 10 microliters is introduced, the excess goes out the sample loop to waste. So, with a 10 microliter sample loop, 10 microliters or less can be introduced onto the chromatographic column. The benefit is that 10 microliters can be accurately and reproducibly inserted onto the chromatographic column. The disadvantage is that if more than 10 microliters has to be injected, the sample loop must be replaced with a larger sample loop.

PUMPING SYSTEM

The pumping system is responsible for delivering a consistent flow to the chromatographic column. It is also responsible for accurately mixing solvents in a consistent, repeatable manner to produce the requested solvent gradient. The range of flow rates for analytical pumps is 1 to 10 ml/min and for preparation (prep) to semi/prep applications is up to 100 ml/min. The analytical pumps can typically pump at pressures up to 6000 psi.

There are two general types of pumping systems: isocratic and gradient. Isocratic systems deliver a single solvent while gradient pumping systems deliver multiple solvents. Typically, gradients systems deliver two solvents (although it's not uncommon to have an HPLC system that can deliver four solvents). The solvent mixture is varied over time. One might start the chromatographic analysis with 100% solvent A and 0% solvent B. The pumping systems is then programmed to vary the solvent mixture so that, at the end of the chromatographic analysis, the solvent mixture is 0% solvent A and 100% solvent B.

HPLC COLUMN

There are four major types of chromatographic columns: normal phase, reverse phase, ion-exchange of size exclusion. Normal phase liquid chromatography uses columns packed with polar stationary phases combined with nonpolar mobile phases. Less polar solutes move the fastest throught the column followed by solutes of increasing polarity. Normal phase is used for compounds that are soluable in organic solvents. The most common solvent combination for normal phase liquid chromatography is hexane and dichloromethane. Reverse phase is used for compounds that are soluable in water. The typical solvent combinations used for reverse phase are water/methanol, water/acetonitrile and water/tetrahydrofuran. Ion exchange is used for ionic compounds and size exclusion is generally used for large molecules and separates compounds according to size.

DETECTOR

The majority of chromatographers use either an Ultra Violet detector or a Refractive Index detector. Fluorescence detectors are also a popular choice. The choice of detector is based on the physical characteristics of the components to be detected. Compounds with good UV absorbance such as benzene and it's aromatic relations are ideal for a UV detector. Most UV detectors are capable of varying the wavelength. This allows for adjusting the wavelength to get maximum sensitivity. Some systems also include the ability to scan the UV spectrum while the components are eluting from the column. This provides maximum flexibility in UV detection.

COMPUTING SYSTEM

The computing device is involved in every aspect of the chromatographic analysis. It is used to control the system during the analysis, as well as pre and post computational aspects of the chromatographic analysis. Computer systems are used as input devices, used to control the pumping system, receive the response from the detector and provide any qualitative or quantitative calculations required. They are also used for establishing instrument calibration. These systems can take the form of a simple pen plotter, to an electronic integrator to a fully equipped computer system. The instrument can be connected to it's own individual computing device or connected to a much larger Laboratory Information Management System (LIMS).

HIGH PERFORMANCE LIQUID CHROMATOGRAPH

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