About
High Performance Liquid Chromatography
Agilent 1100 Series HPLC
Thermo Separation Products HPLC
Dionex DX-120 Ion Chromatograph
Spare Pumps, Injectors, and Detectors
HPLC Applications
About 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 staionary 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, and 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.
More sophistocated HPLC's 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 contral 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 phase allows one to apply HPLC to an enormous variety of complex mixtures from blood serum to hot sauce.
Our Agilent 1100 HPLC system is the most recent addition to our Chormatography resources. This system is fully automated using Agilent's Chemstation software. The pump is capable of mixing four different solvents to produce the mobile phase. We have an autosampler capable of automating the injection of up to 100 samples. The column is thermostatted, and we currently have two detectors attached to this system: a multiwavelength detector capable of monitoring 5 wavelengths at a time, and a refractive index detector which can detect components that don't absorb light in the UV or visible regions (like sugars).
Determination of the amount of capsaicin and dihydrocapsaicin in a hot sauce.
Thermo Separation Products HPLC
This TSP HPLC has components that are similar in function to the Agilent listed above. It is a fully automated system with an autosampler, but the detector is capable of monitoring one wavelength at a time.
Dionex DX-120 Ion Chromatograph
The Dionex Ion Chromatograph (IC) is designed to separate ions rather than neutral molecules. The separation also involves a 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
We have plenty of additional HPLC components. Undergraduates use these components to piece together their own HPLC system 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: how to seal a compression fitting, priming a pump, column installation, troubleshooting, electrical connections for the detector, etc.
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
1 Waters 401 Refractive Index Detector
Testing a HPLC system they just constructedHPLC is one of the most widely applied analytical separation techniques. When you start to look at some of the web sites 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.
Pharmaceutical
Tablet dissolution of pharmaceutical dosages
Shelf-life determinations of pharmaceutical products
Identification of counterfeit drug products
Pharmaceutical quality control
Environmental
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
Forensics
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
Clinical
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
HPLC Resource Sites
The
Analytical Chemistry Springboard
http://www.anachem.umu.se/cgi-bin/jumpstation.exe?Chromatography
The
HPLC Portal
http://www.hplcweb.com/
Separations
NOW
http://www.separationsnow.com
Topics
in Chromatography
http://www.forumsci.co.il/HPLC/topics.html
Several Vendors of HPLC Equipment
Waters
http://www.waters.com/
Agilent
http://www.chem.agilent.com/Scripts/PCol.asp?lPage=181
Dionex
http://www1.dionex.com/en-us/index.html
Several Vendors of HPLC Columns and Accessories
Phenomenex
http://www.phenomenex.com/
Alltech
http://www.alltechweb.com/US/Home.asp
Supelco
http://www.sigmaaldrich.com/Brands/Supelco_Home.html
Restek
http://www.restekcorp.com/