7. High-performance Liquid Chromatography (HPLC)
CHEM 231 Lab
Technique Primer
7. High-performance Liquid Chromatography (HPLC) While there are many analytical instruments found in the typical chemical laboratory, perhaps the most common and most flexible is the HPLC. Based on the principle of chromatographic separation, HPLCs are used to determine the composition of complex mixtures (such as plant extracts), to establish the provenance of artifacts (such as evidence at a crime scene), to gauge the extent of a reaction, or to verify the purity of a product.
20 uL loop
pump solvent
column
The typical HPLC consists of four key components (Figure 1). Solvent is pumped at high pressure through a column packed with derivatized silica gel. Upstream of the column is an injector to allow for introduction of a sample. Downstream of the column is a detector (typically one that measures UV-Vis absorbance).
The sample is introduced into the HPLC system by way of an injector. When the injector is turned to the “load” position (counterclockwise), a syringe is used to fill up (or load) a sample loop of a precise volume. When the injector is then turned to the “inject” position, the loop is placed in line with the column (Figure 3).
injection port
solvent
pump
injector
column detector
Figure 1. Basic components of the HPLC system
pump solvent
column
As individual components exit the column, they create an absorbance signal in the detector, resulting in a peak on the HPLC chromatogram (Figure 2). The y-axis of the chromatogram is a measure of the intensity of absorbance (in units of 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 about 45 mAU and a retention time of about 3.9 min, whereas compound 5 has a peak absorbance of about 12 mAU and a retention time of about 7.6 min.
waste
20 uL loop
injection port
waste
Figure 3. HPLC Injector plumbing in the “load” (top) and “inject” (bottom) positions
Figure 2. An HPLC chromatogram (or “trace”) of a multicomponent mixture
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 more strongly than polar ones. Thus, non-polar compounds tend to have longer retention times.
Technique Primer
7. High-performance Liquid Chromatography (HPLC) While there are many analytical instruments found in the typical chemical laboratory, perhaps the most common and most flexible is the HPLC. Based on the principle of chromatographic separation, HPLCs are used to determine the composition of complex mixtures (such as plant extracts), to establish the provenance of artifacts (such as evidence at a crime scene), to gauge the extent of a reaction, or to verify the purity of a product.
20 uL loop
pump solvent
column
The typical HPLC consists of four key components (Figure 1). Solvent is pumped at high pressure through a column packed with derivatized silica gel. Upstream of the column is an injector to allow for introduction of a sample. Downstream of the column is a detector (typically one that measures UV-Vis absorbance).
The sample is introduced into the HPLC system by way of an injector. When the injector is turned to the “load” position (counterclockwise), a syringe is used to fill up (or load) a sample loop of a precise volume. When the injector is then turned to the “inject” position, the loop is placed in line with the column (Figure 3).
injection port
solvent
pump
injector
column detector
Figure 1. Basic components of the HPLC system
pump solvent
column
As individual components exit the column, they create an absorbance signal in the detector, resulting in a peak on the HPLC chromatogram (Figure 2). The y-axis of the chromatogram is a measure of the intensity of absorbance (in units of 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 about 45 mAU and a retention time of about 3.9 min, whereas compound 5 has a peak absorbance of about 12 mAU and a retention time of about 7.6 min.
waste
20 uL loop
injection port
waste
Figure 3. HPLC Injector plumbing in the “load” (top) and “inject” (bottom) positions
Figure 2. An HPLC chromatogram (or “trace”) of a multicomponent mixture
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 more strongly than polar ones. Thus, non-polar compounds tend to have longer retention times.
