8/6/2019 High Performance Chromatography

1/13

HIGH PERFORMANCE

CHROMATOGRAPHY High pressure liquid

chromatography wasdeveloped in the mid-1970's and quickly

improved with thedevelopment of columnpacking materials andthe additionalconvenience of on-linedetectors. In the late1970's, new methodsincluding reverse phaseliquid chromatographyallowed for improvedseparation between

very similar compounds

8/6/2019 High Performance Chromatography

2/13

ABOUT HPLC :-What is HPLC ?

High performance liquid chromotography(HPLC) is a chemistry based is a tool for

quantifying and analzying mixtures of chemicalcompounds.

What is HPLC used for?

It's used to find the amount of a chemicalcompound within a mixture of other chemicals.

An example would be to find out how muchcaffeine there is in the cup of coffee (or tea, orcola) I have to get me moving in the morning

8/6/2019 High Performance Chromatography

3/13

What I must do to analyze the sample?Dissolve the sample in a solvent (like water or

alcohol), thus the term LIQUID chromatography.How do I measure the sample amount?A detector measures response changes between

the solvent itself, and the solvent & sample whenpassing through it. The electrical response isdigitized and sent to a data system. The graphicbelow shows basic system components

8/6/2019 High Performance Chromatography

4/13

Less volatile and larger samples can be used with

HPLC. It was discovered that better separation of the

components of the mixture occurs if the particles in

the stationary phase are very small. However, it was

also found that if very small particles were used in

the column, then the liquid passed very slowly

through the column. Therefore, a pump is used to

force the liquid through the column. This is notnecessary in GC but a shorter column can be used in

HPLC because the separation is more efficient.

Comparison of Chromatography HPLC

over Gas

8/6/2019 High Performance Chromatography

5/13

8/6/2019 High Performance Chromatography

6/13

8/6/2019 High Performance Chromatography

7/13

The Principles of High Pressure

(Performance) Chromatography

Chromatographyis the term used to describe a separationtechnique in which a mobile phase carrying a mixture is caused tomove in contact with a selectively absorbent stationary phase.Different components of the sample are carried forward at different

rates by the moving liquid phase, due to their differing interactionswith the stationary and mobile phases. There are a number ofdifferent kinds of chromatography, which differ in the mobile and thestationary phase used.

In HPLC: The Mobile Phase is a solvent. This solvent is pumpedunderhigh pressure through a column.

The Stationary Phase is a finely divided solid held inside thecolumn.

8/6/2019 High Performance Chromatography

8/13

8/6/2019 High Performance Chromatography

9/13

Pumps

Isocratic or Gradient

Analytical or Preparative

Vacuum Degassers

As Low as 200L internal volume Independent vacuum chambers

8/6/2019 High Performance Chromatography

10/13

Solvent Recyclers

Reduce solvent use up to 90%

Pays for itself in 60-90 days!

Compatible with all isocratic systems

Autosamplers

Variable Volume or Fixed Volume

120 position or 20 position autosamplers

8/6/2019 High Performance Chromatography

11/13

Column Heaters

Ambient to 150C

Single or multi-column

models available

Detectors

UV/VIS, PDA, Fluorescence,

RI, ECD, Conductivity,

IC Chemical Suppressor

8/6/2019 High Performance Chromatography

12/13

Fraction Collectors

Collect Fractions in 4 ways:

Time, Drop Count, Pumped Volume, Peaks

Post Column Reactors

Pickering Post-column

Derivitization Instrument

8/6/2019 High Performance Chromatography

13/13

Instrument Accessories and

Replacement Parts

Detector Lamps

Pump Replacement Parts

Agilent Instrument Replacement Parts

Gas Generators

Hydrogen Generators

Zero Air Generators

Nitrogen Generators