sose 2006 alexander semmler: hplc 1 alexander semmler hplc high pressure liquid chromatography high...

SoSe 2006 Alexander Semmler: HPLC 1

Alexander Semmler

HPLC

High pressure liquid chromatography

High performance -‘‘-


Basic Principle

Sample Injection

Separation

Detection

Utilisation by integration and calculation

Injector

Column

Detector

EDU


Device


Step 1

Sample Injection


Sample injection

Sample is inserted into the device

Syringe with or without needle

Sample is mixed with eluent solution

Mixture is inserted into column


Injector


Injection (II)

I N J E C T O R Separation

Elu-ent

Sample

PUMP

syringe

bottle with eluent solution


Step 2

Separation


Separation

mobile phase: eluent-solution, liquid

stationary phase: column, solid

separation by different polarities

packed columns


Separation (II)

Pre-column

C O L U M N Det Waste

to save the column


Step 3

Detection


Detection

one substance group after the other flows through the detector

detection by intensity of different detectors

sending data to data utilisation


Possible detectors

refractive index – RIUV/VISIRconductivitymass spectroscopy – MS (coupled)


Detection (II)

DetectorSolution

separated components

DU

Waste


Step 4

Data Utilisation


Data Utilisation

PC/integrator – printer/plotter

intensity against time graph

calculation of retention time & integration of the peaks

qualitative information: retention time (Rf-value)

quantitative information: peak size (area)


Data Utilisation (II)

Detector Computer

Result-database

Printer

Calibration-database


Chromatogramm

Chromatogramm

0

20

40

60

80

100

0 5 10 15

time t [min]

sig

nal

baseline peaks


Answering Questions

For further information please contact your analytical chemistry teacher


Thank you for listening

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sose 2006 alexander semmler: hplc 1 alexander semmler hplc high pressure liquid chromatography high...

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