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Course Separation Science


Alniek van Zeeland

on 15 August 2012

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Transcript of Course Separation Science

Course Separation Science Table of Contents: Brief history
Chromatographic systems
Chromatographic columns
Pretreatment of samples
Questions Brief history: Chromatographic systems: Chromatography High Pressure Liquid Chrom. - HPLC
Ultra Pressure Liquid Chrom. - UPLC
Ion Chromatography - IC
Gel Permeation Chrom. - GPC (SEC)
Gas Chromatography - GC Chromatographic columns: Detectors: Pretreatment of samples: Questions: Reversed Phase
Normal Phase
Ion Exchange Refractive Index - RI
Ultra Violet-Visible - UV-VIS
Photo Array Detector - PAD
Electrochemical Detection - ECD
Mass Spectrometry - MS Dilution of samples
The use of Internal Standard - I.S.
Protein precipitation
Filtration Mikhail Tsvet - (1872–1919)
Russian botanist who invented adsorption chromatography He used liquid-adsorption column chromatography with calcium carbonate as adsorbent and petrol - ether/ethanol mixtures as eluent to separate chlorophylls and carotenoids from plant material. http://www.waters.com/selectivitychart The principle of HPLC Methods available:
Organic acids
Amino acids Resolution is the ability to separate two components from each other. The resolution can be influenced by:
solvent strength (mobile face)
diameter of particle in column (stationary face)
and the column length Smaller particles gives:
higher resolution
faster runtimes (10 to 15 minutes)
less eluent consumption (to 80% reduction)
but requires a system which can handle higher pressures (up to 1200 BAR) Gel permeation chromatography (GPC):

Suitable for:
The detection of polymers (e.g. lignin, protein)
Chain length determination of polymers Gas Chromatography (GC):

Suitable for:
volatile components or detection of gas
the analyte must be thermo stable

More information:
Hans Beijleveld
Dep. Environmental Technology Refractive Index (RI):

based on the difference of light refraction
between a reference cell and the measurement cell.

Universal response
Low sensitivity to dirt and air bubbles in the cells
The ability to cover the entire refractive index range
from 1.000 to 1.750 RI

Sensitive for temperature fluctuations
Low sensitivity
Only suitable for isocratic chromatography Photo Array Detector (PAD):

Generates a 3D signal of a UV spectrum (e.g. 200-350 nm)
and the retention time.

Identity of the components are based on RT and UV information
Gives more component (purety) information

A bigger scope of UV, gives a declinement of the sensitivety Mass Spectrometry (MS):

Based on the difference of molecular masses.

Identity of the components are based on RT and on mass differences
This generates extra resolution
Fragmentation information is possible (Iontrap)
Accurate mass information is possible (Orbitrap, FT-ICR)

Relative expansive system (250-800 K€)
Not suitable for buffers Ultra Violet-Visible (UV-VIS):

Based on light absorption of the components caused by
non-covalent π-interactions.

High sensitivity
Suitable for gradient chromatography

Not all components show UV-absorption

Derivatisation can be used to obtain a better separation on a reversed phase column and to get a higher capacity for light absorption for UV. John Martin "left" and Richard Synge "right" - (1941) They modernized the chromatographic technique by using silica and earned the Nobel prize for their work in 1952.
Round 1970, the first HPLC was introduced.
Up till now this technique is modernized and frequently used in research area’s. Identification of “unknown” components is obtained by:
separation of components
and then by comparing the retention time of this component
with the retention time (RT) of a standard.
A calibration curve is used to determine the amount of the component of interest.

HPLC can handle pressures up to 3500 PSI (approximately 250 BAR) and uses a cycle time between 45 and 60 minutes. The principle of U-HPLC Ion Chromatography (IC): Normal Phase: polar stationary phase
Pure silica

Reversed Phase: non-polar stationary phase
Carbon 4 (C-4)
C-18 Ion Exchange Chromatography: Waters Shodex (kation exchange)
Dionex CarboPak PA1 (anion exchange) Why need samples to be diluted?

to prevent tailing
to prevent overload of the detector
to prevent shortage of derivatisation reagent Pipetting: Internal Standard (I.S.):

Corrects for injection errors
Corrects for system errors
Localizes derivatization problems (e.g. amino acids)
Control for correct sample preparation Why filtration?

Filtration and/or centrifugation of the samples
prevents blocking of the injection needle, loop and (pre-)column. Why protein precipitation?

Prevents flocculation of protein in the LC-system.

by using an organic solvent such as
acetonitrile or methanol (70%)
or by using a strong acid (1M)

This treatment disconnects the hydrogen bond of the protein and it will precipitate out of the solvent. IC is a form of HPLC, but modified for ion exchange purposes.

Suitable for:
an-organic anions and cations (like salts)
amino acids
and organic acids Electrochemical Detector (ECD):

Based on potential differences between the working electrode and the reference electrode.
Mostly used by ion chromatography.
Suitable for the detection of sugars, amino acids, organic acids and salts (ions). Question 1:

What can you do to increase the resolution of your HPLC method, to obtain a better separation for your components? Question 2:

Which component has "most likely" the highest UV absorption, and why?



Acetic acid

or Phthalic acid Question 3:

Assume that you have a calibration curve with an upper limit of 0.5 mM and a lower limit of 0.01 mM. What will happen if you have a sample with a concentration of approximately 0.05 mM and you dilute this ten times?

What will be the concentration in your injection vial and are you still able to accurately determine the concentration? Question 4:

What can happen if the concentration of your samples is too high? Question 5:

Why do you use an internal standard? Question 6:

What is the order of separation of the following components on a reversed phase column?
Which component will come first off the column and which one comes last? Vanillin 5-HMF 2-Methoxyphenol Furfural Question 7:

When and why do you use a derivatization? Question 8:

How can you precipitate your protein (e.g. enzymes, yeasts, or microbes) out of your samples? Question 9:

Why is it not wise to use strong acid for protein precipitation before using the AA method? Question 10:

Why is it wise to filter your samples before injection? Question 11:

What kind of analytical technique will you pick for the analysis of polymers?

D. IC Question 12:

What kind of analytical technique will you pick for the analysis of volatile but stable components?

D. IC Question 13:

Which pipet is the best pick for pipetting 200 µL?

A. 2-20 µL pipet
B. 50-200 µL pipet
C. 200-1000 µL pipet
D. 1000-5000 µL pipet Question 14:

Which volume can you pipet most accurately with a 200-1000 µL pipet?

A. 200 µL
B. 400 µL
C. 800 µL
D. 1000 µL
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