CHEM 231 Lab Technique Primer

7. High-performance Liquid Chromatography (HPLC)

While there are many analytical instruments found The sample is introduced into the HPLC system by in the typical chemical laboratory, perhaps the most way of an injector. When the injector is turned to common and most flexible is the HPLC. Based on the the “load” position (counterclockwise), a syringe is principle of chromatographic separation, HPLCs are used to fill up (or load) a sample loop of a precise used to determine the composition of complex volume. When the injector is then turned to the mixtures (such as plant extracts), to establish the “inject” position, the loop is placed in line with the provenance of artifacts (such as evidence at a crime column (Figure 3). scene), to gauge the extent of a reaction, or to verify 20 uL loop the purity of a product. The typical HPLC consists of four key components (Figure 1). Solvent is pumped at high pressure pump through a column packed with derivatized silica gel. solvent Upstream of the column is an injector to allow for column introduction of a sample. Downstream of the column is a detector (typically one that measures UV-Vis absorbance).

injection port

injector column pump solvent detector

Figure 1. Basic components of the HPLC system waste 20 uL loop As individual components exit the column, they create an absorbance signal in the detector, resulting in a peak on the HPLC chromatogram pump

(Figure 2). The y-axis of the chromatogram is a solvent measure of the intensity of absorbance (in units of column mAU, or milli-Absorbance Units). The x-axis is in units of time (typically minutes), and is used to determine the retention time (tR) for each peak. For example, compound 2 has a peak absorbance of injection about 45 mAU and a retention time of about 3.9 port min, whereas compound 5 has a peak absorbance of about 12 mAU and a retention time of about 7.6 min. waste Figure 3. HPLC Injector plumbing in the “load” (top) and “inject” (bottom) positions When making up a sample for the HPLC, a good target concentration is about 1 mg/mL. Usually, acetonitrile is a good solvent to use for making up samples. If your HPLC trace shows more intense absorbances than 1500 mAU, then the sample is too concentrated & should be diluted. Keep in mind that HPLC is performed in “reverse phase” mode, so that the stationary phase retains non-polar compounds Figure 2. An HPLC chromatogram (or “trace”) of a multi- more strongly than polar ones. Thus, non-polar component mixture compounds tend to have longer retention times.