HPLC

Caleb Gallops Intro

What is HPLC?

Heather's Project

• Determine the types of PAHs in bodies of water in New Orleans
• Water samples were collected at the Riverwalk and Lakefront
• Standard solution of 100ug/mLAnthracene, 1000ug/mLNaphthalene, 100ug/mLPhenanthrene, and 100ug/mLPyrene was prepared
• Data was compared to literature

• High-Performance Liquid Chromatography is a technique used to separate, identify, and quanitfy components in a mixture
• Uses pumps to pass a pressurized liquid solvent with the sample through a column containing adsorbent material
• Each sample component interacts differently with the adsorbent material, producing different flow rates and separations

Caleb Gallops Analyze

Caleb analyze

Uses

Mobile Phase

Mobile Phase:

• Common mobile phases are miscible combinations of water with organic solvents; most common are acetonitrile and methanol
• Some techniques use water-free mobile phases; can contain acids to assist in separation
• Can either be isocratic, constant, or gradient
• Choice ultimately depends on the nature of the column and sample

• Medical purposes such as detecting vitamin D levels in blood serum
• Legal purposes such as detecting performance enhancement drugs in urine
• Research such as separating components of biological or chemical samples
• Manufacturing pharmaceuticals

Pumps and Particle Size

• Pumps may vary in pressure capacity but their performance is measured on their ability to yield a consistent, reproducible flow rate
• Pressure can reach as high as 400atm
• The standard particle size is about 5um; anything smaller requires higher pressure which is more expensive

How does it Operate?

The Column

• Microliters of the sample are injected into the mobile phase and into the column
• Components of the sample move through the column at different speeds because of interactions with the stationary phase
• The time at which the analyte elutes is the retention time

• There are a variety of different columns to choose from
• The stationary phase can vary in particle size and nature of its surface chemistry
• Smaller size packing materials require higher operational pressure (backpressure) and improves resolution
• Particle may be hydrophobic or polar

photo credit Nasa / Goddard Space Flight Center / Reto Stöckli

Different Types

JP's Intro

JP Analyze

Schematic

• Partition Chromatography: Analytes partition between a liquid stationary phase and the eluent based on differences in their polarity
• Normal-Phase Chromatography: Separates analytes based on their affinity for a polar stationary suface
• Displacement Chromatography: Separates as molecules are displaced by others with affinities for the chromatography matrices
• Many more such as reversed-phase, size-exclusion, ion-exchange, bioaffinity, and aqueous norm-phase chromatography