High Performance Liquid Chromatography ( HPLC )

History The origins of Liquid Chromatography began in the early 1900’s with the work of the Russian botanist, Mikhail S. Tswett. His famous studies focused on separating compounds (leaf pigments), which were extracted from plants using a solvent.

This was followed by pure solvent. Two specific materials that he found useful were powdered chalk (calcium carbonate) and alumina. He poured his sample (solvent extract of homogenized plant leaves) into the column. This was followed by pure solvent. As the “sample” passed down through the column by gravity, different colored “bands” could be seen separated, because some moved faster than others. Fig.1

Definition Tswett named his process: Chromatography from the Greek words “chroma”, meaning “color”, and “graphy”, meaning “writing” (literally “color writing”) to describe his “colorful” experiment

This was followed by pure solvent. Two specific materials that he found useful were powdered chalk (calcium carbonate) and alumina. He poured his sample (solvent extract of homogenized plant leaves) into the column. This was followed by pure solvent. As the “sample” passed down through the column by gravity, different colored “bands” could be seen separated, because some moved faster than others. Fig.1

Stationary phase Mobile phase The immobile phase involved in the chromatographic process. The stationary phase in liquid chromatography can be a solid, a bonded or coated phase on a solid support, or a wall coated phase. The stationary phase used often characterizes the LC mode. Mobile phase The solvent that moves the solute through the column.

Chromatography Theory

Distribution of analytes between phases A mobile A stationary K=[A]s / [A]m K: partition coefficient

tR: retention time k'A = t R - tM / tM K': capacity factor α: selectivity factor α = k 'B / k 'A

The Theoretical Plate Model of Chromatography HETP: Height Equivalent to a Theoretical Plate HETP = L / N

The Rate Theory of Chromatography HETP = A + B / u + C u Van Deemter equation

A - Eddy diffusion The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths. Fig.2

B - Longitudinal diffusion The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. Fig.3

C - Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. Fig.4

Resolution

The selectivity factor, α, can also be manipulated to improve separations. When α is close to unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable time. In these cases, k' is optimised first, and then α is increased by one of the following procedures: Changing mobile phase composition Changing column temperature Changing composition of stationary phase Using special chemical effects (such as incorporating a species which complexes with one of the solutes into the stationary phase

Types of Chromatography Method Stationary Phase Mobil Phase Liqiud Chromatography Liqiud Liquid Chromatography (LLC) Liqiud Liquid Liqiud Soild Chromatography (LSC) Solid Gas chromatography Gas Liquid Chromatography (GLC) Gas Gas Solid Chromatography (GSC) Soild

Types of Liquid Chromatography (LC) Liquid chromatography can be performed in three primary approaches: 1. Thin Layer Chromatography (TLC) 2. Paper Chromatography 3. Column Chromatography

1. Thin Layer Chromatography (TLC) The sample is “spotted” onto, and then flowed through a thin layer of chromatographic particles fixed onto the surface of glass plates

Fig.5

2. Paper Chromatography The sample is “spotted” onto paper to which solvent is added to create flow.

Fig.6

3. Column Chromatography The sample passes through a column or a device containing appropriate particles.

Fig.7

What are the parts of an HPLC instruments?

Fig.8

HPLC System Diagram Fig.9

1. Solvent : The HPLC uses a liquid to push the sample. We call this liquid the “Mobile Phase”—or sometimes, we just call it the “solvent.” It sits in a bottle right next to the HPLC, so we have plenty of it to use.

Isocratic and Gradient Operation There are 2 basic types of chromatographic mobile phase operating modes which can be used in HPLC.

Fig.10

Fig.11

2. Pump : We need a pump to pump the solvent through the HPLC. We use very high pressures in HPLC. We pump some mobile phase solvents at 1900 psi.

3. Sample injection loop Fig.11

4. Columns: The goal of the column is to contain the chromatographic packing material (stationary phase), which will be used to effect the separation.

Fig.12

Separation Performance Fig.13

5. Detectors: The instrument component in the HPLC system that senses the separated compounds eluting from the column, and provides the electronic signal to the Computer Data Station or Recorder to create the chromatogram.

1. UV (Ultraviolet) Lamp FlowCell Grating/Lens - Wave length FlowCell PhotoDiode - Differential Light Output

2. RI (Refractive Index ) Universal analyte detector Solvent must remain the same throughout the separation VERY temperature sensitive Somewhat difficult to stabilize baseline

3. FD (Fluorescence ) Excitation wavelength generates fluorescence emission at higher wavelength Analytes must have fluoropore group Can react analyte with fluoropore reagent Very sensitive and selective More difficult methods transfer Results vary dependent upon separation conditions

4. MS ( Mass Spec ) Mass to charge ratio (m/z) Allows specific compound ID Several types of ionization techniques electrospray, atmospheric pressure chemical ionization, electron impact

Separation Mechanism Adsorption chromatography Partition Chromatography Size Exclusion Chromatography ( Jel permeation chromatography) Ion exchange chromatography

For separation of compounds with low polarity 1. Adsorption Chromatography Stationary phase: Silica gel Alumina Calcium carbonate For separation of compounds with low polarity

2. Partition Chromatography Stationary phase: bonded phase ODS or C18 C8 -NH2 -CN

Partition Chromatography Mechanisms Normal Phase For separation of compounds with different functional groups 2. Revered Phase For separation of homologue compounds

Fig.14

Revered Phase Fig.15

Normal Phase Fig.16

3. Size Exclusion Chromatography An HPLC technique where the different sizes of the compounds are used to create the separation. There is no chemical attraction involved. Molecules, such as proteins, DNA and peptides, can also be separated by size.

4. Ion exchange chromatography The stationary phase surface displays ionic functional groups that interact with analyte ions of opposite charge. Cation exchange chromatography: retains positively charged cations because the stationary phase displays a negatively charged functional group such as a phosphoric acid. Anion exchange chromatography: retains negatively charged anions using positively charged functional group such as a quaternary ammonium cation

Fig.17

Analysis using HPLC Qualitative analysis: peak identification, sample identification. 2. Quantitative analysis: peak integration, area normalization, external standards, internal standards.

Qualitative analysis: Fig.18

Quantitative analysis: Fig.19

External standards Fig.20