Bookmark and Share

High Performance Liquid Chromatography


In general, chromatography is a term that describes techniques used to separate components of mixtures. In liquid chromatography, a liquid is used to carry a mixture across a bed of material. Because the liquid moves, it is called the mobile phase. The bed of material, on the other hand, is called the stationary phase because (you guessed it) it doesn't move. As the mobile phase carries the mixture across the stationary phase, some of the components of the mixture "stick" to the stationary phase more than others. Therefore, the components travel at different rates across the stationary phase, and exit the stationary phase at different times. The components of the mixture have been separated!

In high performance liquid chromatography (HPLC) the liquid mobile phase is forced through the stationary phase using pressure. A simple HPLC would include a solvent reservoir to hold the liquid mobile phase, a pump to pressurize the liquid mobile phase, an injector to allow injection of a small volume of the sample mixture under high pressure, a column containing the bed of stationary phase, a detector to detect the presence of components as they exit the column, and some means to record the detector signal.

HPLC Schematic

More sophisticated HPLCs may involve more than one pump to generate mobile phases mixtures, robotic arms capable of injecting perhaps 100 samples unattended (autosamplers), small ovens to control column temperature, mass spectral detectors capable of identifying components as they exit the column, and complete computer control for automation.

Since its introduction in the late 1960's, HPLC has become one of the most widely used separation techniques! The ability to choose among a wide array of stationary phases allows one to apply HPLC to an enormous variety of complex mixtures from blood serum to hot sauce.


Agilent 1100 HPLCs

Agilent 1100Our two Agilent 1100 HPLC systems are the most recent additions to our chromatography resources. These systems are fully automated using Agilent's Chemstation software. The pumps are capable of mixing four different solvents to produce the mobile phase. The autosamplers are capable of automating the injection of up to 100 samples. One system is equipped with a multiwavelength detector capable of monitoring 5 wavelengths at a time, and the other has both a variable wavelength detector and a refractive index detector which can detect components that don't absorb light in the UV or visible regions (like sugars).

Below: determination of the amount of capsaicin and dihydrocapsaicin in a hot sauce.

Hot Sauce Determination Graph

Dionex DX-120 Ion Chromatograph

The Dionex Ion Chromatograph (IC) is designed to separate ions rather than neutral molecules. The separation also involves both mobile and stationary phases, but the stationary phase carries an electrical charge to attract ions to various degrees. The detector in this system monitors the conductivity of the mobile phase. When an ion exits the column, the conductivity changes. This system is fully automated, and includes an autosampler. The autosamplers allow us to perform repetitive injections overnight, and retrieve data for analysis without wasting precious class time.


Spare Pumps, Injectors, and Detectors

Students with their own HPLC systemWe have plenty of additional HPLC components. Undergraduates use these components to piece together their own HPLC systems for laboratory experiments or research projects that don't require multiple pumps and automation.

By constructing an HPLC system, students learn practical aspects of HPLC such as: sealing a compression fitting, priming a pump, installing a column, troubleshooting, creating electrical connections for the detector, and others.

(At left: two students testing a system they constructed.)

Spare HPLC Parts
2 Beckman 110 B Pumps
3 Waters 6000A Pumps
1 Rheodyne 7725 Manual Injector
1 Altex Manual Injector
1 Waters Manual Injector
1 SSI 500 Variable Wavelength Detector
1 Dionex UV/Vis Detector
1 Beckman 164 Variable Wavelength Detector
1 Shodex R-71 Refractive Index Detector

Applications of HPLC

HPLC is one of the most widely-applied analytical separation techniques. When you start to look at some of the websites of some of the vendors that sell HPLC columns, you start to get a glimpse of just how widespread these applications are. If you can think of a complex mixture, there is probably an HPLC method that has been developed to tackle the separation. I have broken out a few application areas below, and listed a few specific separations under each of these. Keep in mind that this is a miniscule sampling.

Tablet dissolution of pharmaceutical dosages
Shelf-life determinations of pharmaceutical products
Identification of counterfeit drug products
Pharmaceutical quality control

Phenols in Drinking Water
Identification of diphenhydramine in sediment samples
Biomonitoring of PAH pollution in high-altitude mountain lakes through the analysis of fish bile
Estrogens in coastal waters - The sewage source
Toxicity of tetracyclines and tetracycline degradation products to environmentally-relevant bacteria
Assessment of TNT toxicity in sediment

A mobile HPLC apparatus at dance parties - on-site identification and quantification of the drug Ecstasy
Identification of anabolic steroids in serum, urine, sweat, and hair
Forensic analysis of textile dyes
Determination of cocaine and metabolites in meconium
Simultaneous quantification of psychotherapeutic drugs in human plasma

Quantification of DEET in Human Urine
Analysis of antibiotics
Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis
Detection of endogenous neuropeptides in brain extracellular fluids

Food and Flavor
Ensuring soft drink consistency and quality
Analysis of vicinal diketones in beer
Sugar analysis in fruit juices
Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits
Trace analysis of military high explosives in agricultural crops
Stability of aspartame in the presence of glucose and vanillin