2. Principal Chemical and Analytical Methods Used in Reverse Engineering
2.4 Chromatography
2.4.4 High-Performance Liquid Chromatography (HPLC)
column flow. The major part of the split sample is vented to the atmosphere using a needle valve to vary the split ratio.
Some samples contain trace quantities to be analyzed and thus require the whole sample to go onto the column to ensure adequate sensitivity. The splitless mode was developed to allow this feature. This splitless mode is ideal for low concentrations of components.
2.4.3.7 Sample Handling Techniques
Different types of samples are analyzed by gas chromatography. Depending on sample characteristics, the handling of the sample plays an important role in gas chromatography.
Some of the sample handling techniques are:
• Gas sampling
• Pyrolysis
• Headspace analysis 2.4.3.7.1 Gas Sampling Gas sampling requires:
• Constant temperature (high enough to prevent any condensation of the compo- nents in the mixture)
• Constant pressure
• Gas-tight seals on sample transfer lines and the sampling device
• Fixed amount of sample injected into the GC 2.4.3.7.2 Pyrolysis
Pyrolysis is a technique applied mainly to solid samples, usually polymers. The pyroly- sis system involves the thermal decomposition of the sample—the degradation products enter the gas chromatograph where they are separated and identified.
There are two types of pyrolyzer in general use:
• Filament type: this system is capable of operating in the range of 500 to 1000°C.
• Curie point: in this case, the sample is coated onto the probe.
2.4.3.7.3 Headspace Analysis
In headspace analysis, a sample of the vapor produced by a liquid or solid material is obtained and introduced into the gas chromatograph. The sample is placed in a sealed con- tainer and thermostated for a period sufficient to establish equilibrium in the headspace above the sample. When the headspace sample is injected, a chromatogram is obtained with peaks corresponding to the components.
specific interactions between sample molecules with the stationary and mobile phases. These interactions are not present in the mobile phase of gas chromatography. There are varieties of stationary phases for HPLC, which allows a greater variety of these selective interactions and more possibilities for separation. Sample recovery in HPLC is easy. Recovery is usually quantitative, and separated sample components are readily isolated from the mobile-phase solvent. A typical instrumentation diagram is shown in Figure 2.20.
The general instrumentation for HPLC incorporates the following components:
• There is a solvent reservoir for the mobile phase.
• The mobile phase must be delivered to the column by some type of pump. To obtain separations either based on short analysis time or under optimum pres- sure, a wide range of pressure and flows is desirable. The pumping system must be pulse-free or have a pulse damper to avoid generating baseline instability in the detector.
• Sampling valves or loops are used to inject the sample into the flowing mobile phase at the head of the separation column. The sample should be dissolved in a portion of the mobile phase to eliminate an unnecessary solvent peak.
• Ahead of the separation column there may be a guard column or an in-line filter to prevent contamination of the main column by small particulates.
• To measure column inlet pressure, a pressure gauge is inserted in front of the separation column.
• The separation column contains the packing needed to accomplish the desired HPLC separation. These may be silicas for adsorption chromatography, bonded phases for liquid-liquid chromatography, ion-exchange functional groups bonded to the stationary support for ion-exchange chromatography, gels of specific poros- ity for exclusion chromatography, or some other unique packing for a particular separation method.
• A detector with some type of data handling device completes the basic instrumen tation.
Waste Detector Signal to processor Sample
injection Pump to produce high
pressure
Solvent reservoir
Processing unit and display
HPLC tube
FIGURE 2.20
Typical instrumental diagram of HPLC.
2.4.4.1 Mobile Phase
The mobile phase is to be delivered to the column at different flow rates and pressures.
The pump, its seals, and all other connections are made of materials that are chemically resistant to the mobile phase, as HPLC needs to handle a wide variety of organic and inorganic solvents. A degasser is used to remove dissolved air and other gases from the solvent. Another desirable feature of the solvent delivery system is its capability to gener- ate a solvent gradient.
2.4.4.2 Pumps
Mobile phase flow through the column at high pressure should be continuous and pulse- free. The pumps for analytical applications should be able to deliver up to 20 mL/min of elu- ent at pressures up to 300 to 400 atm. The following pumps are suitable for solvent delivery:
1. Single-stroke piston pumps with constant eluent flow
2. Reciprocating piston pumps and diaphragm pumps with a pulsating flow and constant stroke frequency
3. Reciprocating piston pumps with variable stroke frequency 4. Gas-driven displacement pumps
The pumps are designed for either constant pressure or constant flow operation.
2.4.4.2.1 Single-Stroke Piston Pumps
In these pumps the piston is driven at a slow, constant rate, and the mobile phase is deliv- ered continuously. The advantages of these types of pumps are the absence of valves and the delivery of a constant, pulse-free flow of solvent. The disadvantage is the necessity of more frequent interruptions to refill at the higher flow rates. Pumps of this type are rela- tively expensive.
2.4.4.2.2 Reciprocating Piston Pumps and Diaphragm Pumps
These pumps deliver a continuous but pulsating flow. Diaphragm pumps are recom- mended for chromatographic purposes because the parts that are contacted by the elu- ent can be readily made from inert materials such as stainless steel. In such pumps the piston movement is transmitted by means of hydraulic fluid to the diaphragm and then to the eluent. Since the piston seals contact only hydraulic fluid, the sealing problems are reduced, and the reliability is increased. The disadvantage of this type of pump is in the dependence of the delivered amount on back pressure caused by the dead volume and the valves. Their output decreases with increasing pressure.
2.4.4.2.3 Reciprocating Piston Pumps with Variable Stroke Frequency
These pumps are particularly well suited for installation in a gradient system.
2.4.4.2.4 Gas-Driven Displacement Pumps
Gas-driven pumps, in their simplest form, consist of a plastic vessel within a pressurized chamber that is connected to a gas cylinder. In an even simpler method, pressure from a gas cylinder is used to drive the eluent contained in a long tube. These pumps were used at the beginning of the development of HPLC.
2.4.4.3 Sample Introduction
Introduction of sample is either by syringe injection through the septum of an injection port into the eluent stream or by a sample loop from which it is swept into the system by the eluent. The sample should reach the column without any appreciable mixing with the eluent. Furthermore, the pressure and flow equilibria should not be disturbed during sample introduction.
2.4.4.4 The Column
Columns are generally constructed with heavy-wall, glass-lined metal tubing to withstand high pressure up to 680 atm and the chemical action of the mobile phase. The interior of the tubing must be smooth with a uniform bore diameter. Straight columns are preferred and are operated in the vertical position.
The properties of the most important tubing materials may be described as follows:
• Glass: smooth inner wall, transparent, inert, usable up to about 70 atm
• Stainless steel: relatively corrosion resistant, can be passivated, no pressure limitations
• Tantalum: smooth inner wall, largely inert, hard, difficult to shape
• Copper: easy to work, subject to corrosion
Column end fittings and connectors must be designed with zero void volume. Most col- umn lengths range from 10 to 30 cm.
The method employed for column packing depends on the particle size of the station- ary phase. Many HPLC separations are done on columns with an internal diameter of 4 to 5 mm. Such columns provide a good compromise between efficiency, sample capacity, and the amount of packing and solvent required. Although a decrease in response is associated with an increase in column diameter, there are benefits of using the wider- diameter radial compression columns. A decrease in the overall operating pressure allows decreasing analysis time by increasing solvent flow. Decreasing the internal diam- eter of the column by a factor of two increases the signal of a sample component by a factor of four the square of the change in diameter. This is the case with the narrow-bore columns. Narrow-bore columns also reduce solvent consumption and as a result save the solvent cost.
To prolong the life of analytical columns, guard columns are often inserted ahead of the analytical column where they act as both chemical and physical filters. Guard columns are relatively short, usually 5 cm, and contain a stationary phase similar to that in the analyti- cal column. They protect the analytical column from particulate contamination that may arise from the contaminated mobile phase. A guard column extends the lifetime of the expensive separation column.
2.4.4.5 Detectors
The composition of the column effluent is continuously monitored by a detector. The two most frequently used detectors in LC are the UV and differential refractometers. UV detectors are the most sensitive for samples having relatively high absorption coefficients at appropriate wavelengths. Differential refractometers are very sensitive to temperature and pressure fluctuations. The following criteria are used for the characterization and
description of detectors: The noise level governs the lowest detection limit. A chromato- graphic peak can only be recognized as such if its height is at least twice that of the highest noise peak. A drift in the baseline is undesirable.
In considering the sensitivity, distinction must be made between the absolute and the relative sensitivity of a detector. The sensitivity is one of the most important characteristics of the detector.
2.4.4.5.1 Ultraviolet (UV) Detectors
With low susceptibility to temperature and flow rate fluctuations, UV detectors are widely used. Most of the instruments operate with a single wavelength of 253.7 nm. In some instruments a band at 280 nm is also used. The UV cells should have an optical path length of 5 to 10 mm and very small volume. The instruments may be single or double beam. The disadvantage of UV detectors is in their specificity. Only molecules that absorb in the UV region near the wavelength of the detector can be monitored. The sensitivity of UV detectors depends strongly on the molar absorption coefficients of the sample components. Because of its sensitivity and selectivity, a UV detector is applicable to gradient elution only if the eluent has no UV absorption in the region of the wave- length used.
2.4.4.5.2 Differential Refractometer
A differential refractometer measures the bulk refractive index of a sample-eluent sys- tem. In order to obtain adequate sample response, the refractive index of the mobile phase must be compensated by means of a differential technique. Any substance with a refractive index that differs sufficiently from that of the eluent can be detected. However, as this type of detector detects every change in eluent composition, it cannot be used for gradient elution unless solvents are chosen with identical refractive indices. Another disadvantage is in the strong temperature dependence of the refractive index. To attain adequate sensitivity, the temperature of the eluent and measuring cells must be held constant at ±0.001°C.
Other detectors for HPLC are as follows:
• Micro-adsorption detector
• Transport detector (FID)
• Fluorescence detector
• Electrochemical detector
• Conductivity detector
• Capacity detector
• Radioactivity detector
• Mass detector
• Infrared detector 2.4.4.6 HPLC Separation
Most of the LC separations are based on adsorption effects. Such separations are governed by the interaction of adsorbent, solute, and eluent. A linear adsorption isotherm is essen- tial for reproducible chromatographic work.
2.4.4.6.1 Effect of Water on Separation
The oxide adsorbents such as alumina and silica gel are good drying agents for nonpolar organic solvents. The absorbed water exerts a considerable effect on the chromatographic properties. The addition of small amounts of water or other polar moderators to an adsor- bent or eluent reduces the retention volumes to the extent that nonpolar compounds are no longer retained.
2.4.4.6.2 Effect of Eluent on Separation
The choice of the proper solvent frequently affects the success of a separation more than the selection of the stationary phase. Depending on the properties of the eluent, on a given adsorbent a sample may be excessively retained or not at all, or its retention time may fall into the desired level. The eluent should not interact irreversibly with either the sample or adsorbent. In some critical cases, eluent viscosity can be another criteria for eluent selec- tion, because the smaller the viscosity, the lower is the required pressure drop to achieve a given flow velocity.
2.4.4.6.3 Effect of Sample Structure
The molecular structure of the sample determines the elution order to a greater extent compared to the properties of the solid stationary phase and the eluent. Knowledge of the composition of a sample and the structure of its components simplifies the choice of sys- tem and enables predictions to be made about the elution order.