HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
At the beginning of this chapter the original experiments involving separation of plant pigments were described. These are early examples of LC. The analytes were dissolved in a liquid and then poured through a bed of silica. The liquid is the mobile phase and the silica is the stationary phase. This process is relatively slow because it depends upon gravity to get the mobile phase through the stationary phase. Vast improvements have been made in LC since the first experiments. Stationary phases have become much more efficient in separating components of an analyte and they are much more sensitive. As a result, the process can be sped up by having the mobile phase pushed through the stationary phase using pumps. This makes the experiment go much faster while keeping the high resolving power of the technique. This type of chromatography is called high performance liquid chromatography or HPLC. Some people refer to this technique as high pressure liquid chromatography, but this is technically not correct. In HPLC, packed columns are routinely used and the stationary phase can be similar to those in packed column GC or can be very different. In fact, one of the most popular HPLC stationary phases used in forensic science is a C18 hydrocarbon (octadecane). This material has the approximate consistency of candle wax. Mobile phases can be either a single liquid or a solution containing two or more miscible liquids.
HPLC has some significant advantages over GC. In GC, for example, the sta tionary phase is always more polar than the mobile phase (nothing can be less polar than an inert gas such as nitrogen or helium). In HPLC, stationary phases and mobile phases can be designed so that the stationary phase is less polar than the mobile phase. Octadecane is an example of a very nonpolar stationary phase. In such cases, the chromatography is referred to as being reverse phase. This can be a great help in separating a mixture of nonpolar substances that would not separate well using a polar stationary phase. Another advantage of HPLC over GC is that the composition of the mobile phase can be altered during the run. This is called gradient chromatography. At times it is desirable to start with a relatively nonpolar mobile phase and then gradually increase its polarity by adding more and more of a polar solvent. This can be easily accomplished using two or more solvents and two or more pumps. A computer controls the amounts of each solvent, thus changing the polarity of the mobile phase on the fly. Gradient chromatography is used when the analyte contains components of varying polarity. Its use can help separate simi lar low weight substances while keeping the experiment to reasonable time. It is somewhat analogous to temperature programming in GC. When the mobile phase stays constant during an HPLC run, it is called isocratic chromatography. Because liquids are used as the mobile phase, HPLC is commonly run at room temperature to remove the danger of pressure build-ups that can occur when liquids get near their boiling point.
FIGURE 1 A pyrogram of a polyester fiber. Each peak represents a product of the pyrolysis. The extreme heat of the pyrolysis in the absence of oxygen causes decomposition of the polyester polymers into smaller fragments. These, in turn may further decompose or react chemically with other fragments, creating new substances that may react or degrade further. This results in a large number of substances and a large amount of data for comparison of known and unknown substances.
