Chem 321 Lecture 23 - Liquid Chromatography 11/19/13

Transcription

1 Chem 321 Lecture 23 - Liquid Chromatography 11/19/13 Student Learning Objectives High Performance Liquid Chromatography With the advent of relatively inexpensive and reliable pumps, the development of a wide range of detectors and very efficient columns, and the ability to analyze nonvolatile materials, high performance liquid chromatography (HPLC) is now as widely used as gas chromatography. Although the principles of chromatographic separations apply to liquid chromatography, the components of a basic HPLC system differ in significant ways from those in a gas chromatograph. In HPLC both the mobile phase and the stationary phase are liquids. This means that they both interact with the sample components, and in fact compete for these components. In our HPLC experiment, the stationary phase consists of a nonpolar, 18- carbon alkane (C 18 ) covalently bonded to small (5 μm diam.) particles of silica. This is known as a reversed-phase column since the stationary phase is less polar than the mobile phase (just the reverse of the liquid chromatography columns used in organic chemistry which have a polar stationary phase like alumina or silica gel). This is also a bonded stationary phase (one that is covalently bonded to the support particles). This stabilizes the stationary phase to a greater extent than when the liquid is simply mechanically coated onto the support. Bonded GC columns are also available. Silica is chemically attacked by basic solutions so these columns are used with liquids having a ph below 8. The small support particles are generally packed into a metal column (a typical size is 4.6 mm inside diameter x 25 cm in length). We are using cartridge columns in our system. These are very short columns (3.5 cm) sealed at each end with a metal frit. Two cartridges are piggy-backed to serve as the stationary phase. Check for Understanding What is the advantage of using two cartridges in succession? When the separation efficiency of these cartridge columns starts to degrade, they are simply discarded because they cost only a fraction of the cost ($100-$300) for a typical 30-cm analytical column. In order to protect the more expensive columns, a guard column is generally used. This is a very short column placed before the analytical column and consists of the same type of stationary phase as the main column (except the support particles are usually larger). This column serves to trap any sample components that might otherwise enter the main column and adhere. As the guard

2 page 2 column gets contaminated, it is replaced at a very modest cost, or perhaps the packing at the inlet end is refreshed. The small size of the packing particles in a HPLC column requires that the liquid mobile phase be pumped through the column using very high pressures (1000's of psi). This requires a pump capable of delivering a very smooth flow of the mobile phase at these high pressures. This component contributes about half the HPLC hardware costs. Flow rates are typically close to 1 ml/min. The mobile phase (and samples) used for HPLC are filtered through micron-size filters and degassed. Removal of dissolved air minimizes the formation of bubbles that can interfere with a flow-through spectrophotometer cell such as we are using in the detector, or the bubbles can create voids in the column that degrade column efficiency. HPLC-grade reagents are available for use in the mobile phase. In our experiment, the sample components are separated using a 55/45 (v/v) methanol/water mobile phase throughout the elution. This is known as an isocratic elution. A very powerful way to enhance the efficiency of separation is to carry out a gradient elution. In this case, the mobile phase composition changes during the elution. You will use a step-wise gradient elution for the separation of cobalt ion in the ion-exchange chromatography experiment. Since the mobile phase is competing with the stationary phase for the sample components, the composition is changed so that the mobile phase becomes more like the stationary phase during the elution. Check for Understanding For a reversed-phase column, how should a methanol/water mobile phase composition be changed for a gradient elution? Variations in the column temperature during elution (temperature programming) is the corresponding approach used in gas chromatography to improve the efficiency of separation.

3 page 3 Injection of the liquid sample onto the HPLC column usually occurs through a valve similar to the one diagramed in Figure W. H. Freeman and Company Figure 16.1 Diagram of a typical injection valve for HPLC A key feature to this injection port is the sample loop. This is a small section of metal capillary tubing that is filled to capacity during the loading process and then flushed into the column when the sample is injected. Thus, a known and very reproducible volume of sample gets injected. Notice that in the load position (Fig. 16.1a), the plumbing connects the syringe port to the sample loop so that the loop can be flushed and filled with the sample. At this point, the mobile phase flow bypasses the loop. Several (3-4) syringe volumes are generally applied to ensure complete filling of the loop. When the valve is moved to the inject position (Fig. 16.1b) the mobile phase flow is re-directed to flush the contents of the loop into the column. Sample loop sizes generally vary from 10s to 100s of microliters. Note that the very small section of tubing between the syringe port and the waste port in the inject mode (Fig. 16.1b) contains the last sample applied to the injection valve. This can be flushed out by rinsing while in the inject position or by using multiple syringe volumes when in the load position.

4 page 4 Detection of the eluted materials is done continuously with a variety of detectors in HPLC. We are using a variable wavelength spectrophotometer cell in our system. This flow-through cell is diagramed in Figure Although the flow-through cell volume is generally only a few microliters this design results in a pathlength of cm. A deuterium lamp serves as the source of the 275-nm radiation that is used to detect the caffeine in our experiment. This detector is also nondestructive, so the separated components could be collected for use if desired W. H. Freeman and Company Figure 16.2 Diagram of a flow-through cell for HPLC Check for Understanding What criterion will you use to identify the caffeine peak in the chromatogram of your unknown? Since you are able to inject the same volume of sample for each HPLC separation, a simple comparison of component peak areas for a standard and sample

5 page 5 can be done to get the concentration in the unknown. For any component A, where C unk and C std are the concentrations of A in the unknown and standard, respectively (same concentration units). However, rather than compare your unknown with a single standard, several caffeine standards will be analyzed and plot of peak area versus standard concentration will be prepared. The caffeine concentration in the sample will be determined from the equation for the linear least-squares line for the calibration curve and the caffeine peak area for the unknown. In our experiment, each standard will be run once and the unknown sample will be run several times. Calculate the concentration of caffeine in the unknown for each run and average the results. Exercises for Liquid Chromatography