7 INSTRUMENTAL CHROMATOGRAPHY

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1 7 INSTRUMENTAL CHROMATOGRAPHY 7.1 Introduction There are two forms of chromatography, very widely used in analytical laboratories, which rely on electronic control of the process and detection of the species. Both are column-based: gas chromatography - where the mobile is a gas, and the separation is based on polarity attraction to the stationary and volatility attraction to the mobile high performance liquid chromatography where the mobile is a liquid, and separation is based on competition between the polarity of the two s 7.2 Gas Chromatography In gas chromatography (GC), components are carried through a heated column containing the stationary by an inert gas, which is the mobile. Mobile The mobile, often called the carrier gas, is most often nitrogen or helium. It has no chemical attraction for the components of the mixture being separated; it competes for the compounds by heat the more volatile (lower boiling) compounds will be attracted to the mobile. This method of attraction gives GLC its one important limitation: only volatile compounds can be analysed, since travel through the medium is in the gas. Stationary The stationary can be a solid (gas-solid chromatography, GSC) or more commonly, a liquid (gas-liquid chromatography, GLC). In the latter case, the liquid is high-boiling (> 400ºC) and is either coated on a inert powder material (a packed column), known as a support or on the inside walls of the column (a capillary column). The stationary competes for the analytes by polarity(glc) or adsorption (GSC). Stationary Polarity Compounds in sample Heat Mobile Instrument configuration The basic layout of a gas chromatography instrument is shown in Figure 7.1. Oven Carrier gas reserve Flow rate control Injection port Column Readout Detector FIGURE 7.1 Typical gas chromatograph

2 CARRIER GAS Carrier gases are supplied in compressed form in bottles, and the flow rate is then controlled by a tap mechanism on the instrument. Flow rates are typically 1-10 ml/minute. the faster the flow rate, the faster the components move through the column. INJECTION PORTS Sample sizes in GC are very small: typically 0.1 to 10 ul in solution. This is done manually or by a robotic controller with a calibrated microsyringe, through a rubber septum into a heated area at the head of the column. The septum provides a seal so that the sample doesn t vaporise back out the injection port! Manual injection is prone to errors because of the small volumes and likelihood of trapping air bubbles. Duplicate injection s are essential, and the use of internal standards even better 9see Applications) The injection port must be heated to immediately vaporise all of the sample, typically using temperatures of at least 50ºC above the boiling point of the least volatile component of the mixture. If the sample was not vaporised upon injection, the carrier gas would selectively evaporate the more volatile components, leaving the others behind. So when you are injecting a sample into a GC, be careful not to put your fingers on the metal port; otherwise you ll be rewarded with the smell of burning flesh!! COLUMNS In this subject, we will only deal with packed columns: these are not as efficient as capillary columns, but have other advantages. A typical packed column is made of stainless steel, and is 2-3 metres long. Its internal diameter is 3-5 mm. The column is coiled so that the oven it is placed into is as small as possible. Packed columns for GLC need the liquid stationary to be physically or chemically bonded to an inert support material. The solid support generally consists of small, uniform spherical particles with good strength and high surface area. The first, and still most widely used, supports are silicates prepared from diatomaceous earth, which consists of the skeletons of thousands of single-celled plants that inhabited ancient lakes and seas. Desirable properties for the liquid in a GLC column are: low volatility (b.p. at least 100ºC above maximum operating temperature) thermal stability chemical inertness suitable polarity characteristics to separate the mixture Table 7.1 lists some of the common stationary s. TABLE 7.1 Common stationary s for GLC Name * Max. Temp (ºC) Polarity Applications OV-1 350ºC non-polar hydrocarbons DNP 150ºC medium alkanones Carbowax 20M 250ºC polar essential oils, alkanols * the different companies making stationary s may have other names for equivalent materials DETECTORS The detector is responsible for measuring the gas stream as it emerges from the column, and responding rapidly to microgram amounts of the solutes. Other desirable properties of a detector are linear response, stability and uniform response to a wide variety of chemical species. Table 7.2 describes the two most common GC detectors. Sci Inst Analysis (Spectro/Chrom) 7.2

3 TABLE 7.2 Common GC detectors Name Mechanism of detection Features Thermal conductivity Flame ionisation Measures heat conduction of gas stream relative to carrier gas Gas stream passes through flame; combustible species produce ions which are measured Simple, cheap, responds to all species, relatively insensitive Requires separate hydrogen and air lines for flame, very sensitive to species that burn well (eg hydrocarbons), less sensitive to oxidised species (eg alkanoic acids), insensitive to water, CO 2 Improving Separation Efficiency The stationary polarity should be similar to that of the substances being separated. The only influence that the gaseous mobile in GC has on the components of the mixture is heat. To achieve the most efficient separation (good resolution of peaks in the shortest time possible), the most appropriate column in terms of polarity is chosen, and then the oven temperature is adjusted to improve the separation. CLASS EXERCISE 7.1 Complete the following sentences. The more volatile a compound is, the GREATER/LESS the time it will spend in the mobile, and the SHORTER/LONGER its retention time will be. The higher temperature in the oven, the GREATER/LESS the time all compounds will spend in the mobile, REDUCING/INCREASING retention times. CLASS EXERCISE 7.2 A mixture of alkanes pentane, hexane and heptane are to be analysed by GLC. (a) From the columns in Table 7.1, choose the most suitable. (b) Predict the retention order for the compounds. (c) The three chromatograms below are recorded at different temperatures. Comment on the differences and choose the optimum temperature. 60ºC 90ºC ºC Sci Inst Analysis (Spectro/Chrom) 7.3

4 Applications Trace analysis of organic analytes in a mixture is a difficult process, but gas chromatography enables many complex mixtures (containing chemically very similar components) to be determined accurately and rapidly. Both qualitative and quantitative work can be carried out on instruments which are generally robust, reliable and simple to operate. However, remember that only volatile compounds can be analysed. The methods of retention time comparison or spiking described in Chapter 6 are used for identification in GLC. Quantitative analysis is carried out by using a series of standards to prepare a calibration graph much in the same way as spectrophotometric analyses, but in this case, it is detector response as measured by peak area or height that is plotted on the vertical axis. However, as mentioned above, injection volumes are very hard to keep constant because of the small measures involved. An internal standard is used in a similar way to that in flame photometry. A constant volume of a compound chemically similar to the analytes, but not present in the sample, is added to all solutions. The analytical measure for the calibration graph becomes the ratio of the peak areas of the analyte to internal standard. 7.3 High Performance Liquid Chromatography GLC, until recently, has been the dominant form of analytical chromatography. It has, of course, the limitation of being only suitable for volatile analytes. Liquid chromatography where the mobile is liquid has been available since almost the very earliest days of the field, but has been plagued by poor separation efficiency: to achieve a reasonable separation generally took at least two hours! The long separation times were primarily the result of very small flow rates of mobile, caused by very large particle size in the column packing. Any attempts at pumping the mobile through reduced the efficiency of separation even further. However, recent developments in producing much small packing material have meant that the technique known as high performance liquid chromatography (HPLC) has become as important as GLC. Mobile The mobile is a liquid, frequently a mixture of pure solvents or containing a dissolved species to change the overall characteristics. It uses polarity attraction for the analytes to aid separation. Stationary Packed and capillary columns also exist in HPLC, though the former are far more common. As in GLC, the liquid stationary is coated physically or chemically onto the support material or the column walls. Like the mobile, the stationary attracts the analytes by polarity. Stationary Polarity Compounds in sample Polarity Mobile Instrument configuration A schematic diagram of a typical HPLC instrument is shown in Figure 7.2. Mobile reservoir Pump Injection port Guard column Main Column FIGURE 9.11 Typical HPLC instrument Readout Detector Sci Inst Analysis (Spectro/Chrom) 7.4

5 MOBILE PHASE STORAGE The mobile in HPLC is kept in reservoirs of L capacity. The reservoir outlets generally include a filter to remove suspended particles, and a degassing device, such as a sparger (in which the dissolved gases are carried out of solution by a stream of bubbles of an inert, insoluble gas). Nothing wrecks a HPLC system both the pump and the column - quicker than suspended solids, so it is absolutely essential to filter the mobile immediately before use. PUMPS A reliable pump was one of the things standing in the way of progress in liquid chromatography. The pressures needed to push the mobile through the packed column are times atmospheric pressure! INJECTION PORTS The simple injection ports in GC instruments are unsuitable in HPLC because of the high pressures encountered. You couldn t possibly force a syringe into a liquid being forced through at such high pressures. And even if you could, the flow would come spurting out the injection port. The most widely used method is known as a sampling loop, where the sample is placed into a non-pressurised chamber, and then a measured aliquot automatically transferred to the top of the column. The advantage of such a system is that the volume of sample is highly reproducible, since a large excess is placed into the sampling loop, and the mechanical system takes the microlitre amount of sample. This means the internal standards are not necessary in HPLC. COLUMNS HPLC columns are usually constructed from stainless steel tubing, because of the pressure requirements. Typically the main column will only be 30 cm in length. A guard column is placed in front of the main column: it is a miniature version (1 cm) of the main column, designed to catch any remaining solid material before it could damage the expensive main column. Stationary s were originally physically bonded to support material in the column, but they suffered the problem of slowly dissolving in the mobile. For this reason, most modern instruments use chemically-bonded packings, where covalent bonds are involved. Normal- HPLC stationary s are polar, while reversed- means non-polar. Ion exchange columns for anions and cations also exist. CLASS EXERCISE 7.3 Choose a column type to suit the following analyses. Chloride and nitrate in river water Sugars in fruit juice Caffeine (non-polar) in tea DETECTORS The most widely used detectors for HPLC are based on conventional laboratory instruments: UV/visible absorption refractive index conductivity Like GC detectors, they need to respond rapidly to microgram amounts of analyte, and must operate with very small volumes. The measurement cells must be flow-through to allow continuous measurement. Sci Inst Analysis (Spectro/Chrom) 7.5

6 Improving Separation Efficiency As with all forms of chromatography, the stationary polarity should match that of the analytes. Therefore, column choice in HPLC is relatively simple. However, choice of mobile is not so easy. Its polarity cannot be so different that the compounds will not dissolve at all, and therefore never move. However, if it is too similar, then the stationary will lose the battle to attract the analytes, and no separation will occur. CLASS EXERCISE 7.4 The chromatograms below were recorded using a non-polar column and the mobile shown. Suggest how each separation could be made more efficient by changing the mobile. Use the table of dielectric constants in Chapter 6 to help you select a different mobile. Stick to pure solvents. (a) ethanol (b) trichloromethane Applications Not bound by the limitation of analyte volatility, HPLC is essentially capable of separating and detecting all classes of compounds at trace levels: chlorinated hydrocarbon pesticides, vitamins, sugars, amino acids, ions (cations or anions), the list of possibilities is endless. It finds substantial usage in the pharmaceutical industry where drugs of various chemical nature are qualitatively and quantitatively analysed. Qualitative and quantitative analysis is the same as for GC, except that internal standards are not necessary. Table 7.3 compares the basic aspects of both forms of instrumental chromatography. TABLE 7.3 Comparison of instrumental chromatography methods Characteristic GC HPLC Classification Column, elution Column, elution Mobile Inert gas Liquid Stationary Separation mechanism Introduction of sample Liquid coated on a powder or on the walls of the column; can be a powdered solid also Compounds are attracted between polarity of stationary and the heat of the mobile By syringe injection Chemically bonded to silica particles Polarity attraction to both s By syringe injection Detection By electronic measurement By electronic measurement Type of analytes Volatile compounds only All analytes possible Sci Inst Analysis (Spectro/Chrom) 7.6

7 CLASS EXERCISE 7.5 Determine which technique GC or HPLC would be better for the following analyses. Identification of volatile components of lavender oil Water content in ethanol Sugars in fruit juice Aspirin in tablets What You Need To Be Able To Do for each technique studied: - draw a schematic diagram of the components of the instrument - briefly describe aspects of instrument components - describe some common applications - describe how separations can be improved Terms And Definitions Match the term with the definition. 1. carrier gas 2. packed column 3. reversed- 4. normal- A. polar HPLC column B. GC or HPLC column filled with fine particles coated with stationary C. mobile in GC D. non-polar HPLC column Review Questions 1. Explain why gas chromatography is limited to volatile components. 2. Why would increasing the temperature of a column oven speed up the elution of all components? 3. Why do compounds of similar polarity elute from a GLC column on the basis of their boiling points? 4. A hydrocarbon mixture is suspected to consist of the alkanes (b.p. in ºC): heptane (98), octane (126), 2,3,4-trimethylpentane (113), 3-ethylhexane (119) and 2,2,3,3-tetramethylbutane (106). Gas chromatographic analysis shows five peaks as expected. (i) Predict the order of elution. (ii) Describe how you would confirm that this prediction is correct. (iii) What type of stationary would be used in this analysis? Give an example. (iv) Choose a internal standard for this analysis. (v) The retention times are relatively long. What could be done to improve them? What problem could occur? 5. What GC column would suitable for the separation of the components of lavender oil? 6. What advantages does the high pressure pumping of mobile give to HPLC? 7. A mixture of pharmaceuticals is analysed by HPLC, using a reversed- column and propanone as mobile. The separation does not work, since all compounds elute very quickly in one peak. What could be done to improve the separation? Sci Inst Analysis (Spectro/Chrom) 7.7

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