Chromatography Module

Error and Atomic Spectroscopy &

Chemistry Spectroscopy Chromatography Sensors
Review (Elements)

WEEK 6!
1. Liquid Chromatography Gas and Liquid
2. Instrumentation Basic Theory
Chromatography
3. Column selection and
issues

• Types of chromatography • Gas Chromatography

• Selecting Instruments • Liquid Chromatography
• Designing methoods

Week 4-6
 Chromatography:
Case Study 2
Week 6, Lecture 1

(A) About your case and situation

(B) GC and chocolate: authentication and PAH

(C) LC and chocolate: cool molecules in chocolate!

FoodStuff: A Commercial
Analytical Laboratory

A small business, 20 employees

• Serves the food and beverage industry:

o Food safety certification
o Nutritional labels
o Analyses specific to products

Great instruments and trained personnel

o Two HPLC systems

o One GC-MS system
Chromatography Instruments:
Specifics
Laboratory has an Agilent GC-MS:

An Agilent 7890 A GC with a Agilent 5975C Series

GC/MSD with a tandem axis detector. Also
equipped with an autosampler and FID. Oven can
run from room temp to 400 C.

Laboratory has an Agilent HPLC:

With solvent degasser, binary gradient

pumping, gradient mixer, injector capable of
10 μL injection (with autosampler), column
oven, fluorescence detector and data analysis
system
 A New Market: High
End Chocolatiers

• Projected to be a $4 billion
dollar market
• Increased spending on dark
chocolates driven by positive
health effects
• Global growth (India and
Valentine Chocolate Giving, 2007 China) even larger than in US

Sources: IRI FDMx Valentine Universe, 7 weeks ending 2/18/07 www.sbdcnet.org

 The Production of
Chocolate

By Sanjay Acharya, Creative Commons

1: Picking fruit with beans inside

2: Fermenting beans (sun)
3: Shelled and roasted
4: Nibs used for chocolate liquor
5: Conching and blending: milk and dark

Boutique chocolatiers by bars of conched chocolate to temper and

mold into high end truffles and candies

Some videos: http://www.thehersheycompany.com/about-hershey/our-story/making-our-chocolate.aspx

A good overall citation about chocolate: http://en.wikipedia.org/wiki/Chocolate
 GC-MS for Polycyclic
Aromatic Hydrocarbons

PAHs are a class of >10 molecules

Napthalene (see to the left)
Fluoranthene

Dietary exposures to PAH has now

been the subject of research

Grilling and roasting both can

Benzo [b]fluoranthene Benzo [a] pyrene increase PAH levels in food

Big challenge in GC analysis is the

high temperatures needed for these
big molecules, and column
contamination

Resources: 2GCChocolatePAH.pdf
Benzo [k]fluoranthene Dibenzo [a,e] pyrene
Resources: 15LCAgilentPAHOptional.pdf
 Authentication of
Chocolate Source
• Three types of cocoa beans:
Forastero, Criollo, and Trinitario
• Criollo is the best, but how can you be
sure you are always getting this?
• GC-MS ‘fingerprinting’ is an approach
that looks at all volatiles.
• Targeted: look for ‘signature’ peaks
that are only present in the real thing
(whiskey example)
• Fingerprint: look for patterns of peaks
that correspond to origin of interest

3GCWhiskey.pdf
http://online.wsj.com/article/SB10001424052702303292204577516833843957916.html#project%3DCOCOAQUIZ0712%26articleTabs%3Dinteractive
 Descriptors for ‘real’
whiskey
Fake Real Fake

n-propanol to
butanol ratio

5x: 3 methyl-1-
butanol to 2-
methyl-1-butanol

In the ‘real’ whiskey this ratio is 2!

3GCWhiskey.pdf
 Key Molecules in
Chocolate: Catechins
Flavan-3-ol monomers: may have health effects (+)

Over 900 HPLC analyses of these compounds

Wine, tea, grapes, chocolate

Concentrations diminish with fermentation and roasting

(+-) catechin

(+-) epi-catechin

5HPLCChocolate1Catechins
 Chocolate Molecules:
Xanthines

1 2

3 4

6HPLCChocolate2Xanthines
 Your Task: Design Methods
for GC and HPLC Analysis

• Case Study Option 1: Peer grading with

questions as described on handout (note: it will
not be identical but close)

• Case Study Option 2: A multiple choice and

short answer quiz format for equivalent points
but more directed towards the reading.

Yes, you may do both if you really want to for extra credit
 Chromatography:
Liquid Chromatography
Week 6, Lecture 2

(A) LC is different than GC in important ways

(B) LC has many more ‘modes’ or operation than GC

(C) Nature of the partition distinguishes LC types

 Liquid versus Gas 
Chromatography

• Only 23% of 
substances can be 
separated by gas 
chromatography
• Using liquids as the 
mobile phase vastly 
expands 
chromatography  Longer columns
applications
 Different Types of 
Chromatography
Purple is the  Purple is the 
mobile phase mobile phase

Red is  Red is 
analyte analyte

Stationary 
Phase

• Solute adsorbs on the surface of the stationary phase – Adsorption 
• Solute dissolves in the liquid bonded to the surface of a column – Partition 
• Ions interact with oppositely charged groups on stationary phase – Ion Exchange
• Specific binding groups on stationary phase attach molecule – Affinity
• Solute avoids the small pores in a porous support – Molecular Exclusion
 Separation in Liquid 
Chromatography: Words

1. Normal phase chromatography
• POLAR stationary phase and a NONPOLAR mobile phase
• Polar bonded‐phase silicas are common – use for weakly polar compounds

2. Reversed phase chromatography
• Non‐polar stationary phase and a POLAR mobile phase
• Bonded‐phase silicas are also common – can pick up subtle differences in 
• Subtle differences in organic structure and composition
3. Ion‐exchange chromatography
• Weakly ionic stationary phase and a aqueous mobile phase w/electrolytes
• Bonded‐phase silicas are also common: ionic analytes
4. Size exclusion chromatography or Gel Permeation Chromatography
• Non‐interacting stationary phase 
• Size distribution of pores in stationary phase is essential
• Proteins, polymers and larger molecular weight substances
 Molecular Size and Polarity 
Matter to LC Method

Our focus will be on 
these examples
 Chromatography:
LC Instrument Overview
Week 6, Lecture 3

(A) The block diagram of a liquid chromatography

system

(B) LC Solvents: finicky and expensive

(C) Pressures in an LC system

 Understanding the 
Magic Box
Liquid Chromatography is Versatile: From 
Food to Biotechnology

Inject 
Sample
Here

• Diagram the instrument (major components)
• List options for each major component – know details!
• Select options based on measurement need
 Components in an LC 
Instrument

Sample injection

Mobile phase management 
and the pumps

The column and separation 
process

The detector
 Liquid Chromatography

Solvents

Vacuum degasser

Binary pump

Autosampler

Column

Detection system
Sample Injection: The 
Loop
 HPLC Solvents: Finicky 
and Expensive

Acetonitrile LC grade (<99.9%) $115/500ml

Acetonitrile reagent (<95%) $70/500ml

• Mobile phase choice – essential for method development (next lecture)
• Solvents must be high purity
• Generally degassed – bubbles bad and do not want oxygen
• In normal phase cannot have any water in sample (e.g. purged)
• Often use many solvent types, which change over a separation (gradient 
elution) – typically binary pumps
High Pressures

83 Bar or 1200 psig
 HPLC – Up to 1200 bar (!)

http://www.lcresources.com/resources/getstart/2b01.htm

Standard HPLC: 5 micron beads, p = 50 – 350 bar

Ultra‐HPLC: Smaller beads (d< 3 microns)  p>400 bar or 6000 psig
 Chromatography:
LC Columns Physical Parameters
Week 6, Lecture 4

(A) Columns generally have packed beads

(B) Particle size: small is GOOD for resolution

(C) Particle size: small is BAD for pressure demands

• High-performance Liquid Chromatography

OR

• High-pressure Liquid Chromatography

 Zooming into the column

 Must withstand high pressures
 Be non‐reactive
 With fittings that don’t leak
 Microstructure of the Column

•Small bead sizes decrease plate height 2 reasons
More uniform flow, less of term A
Distance for diffusion to stationary phase is less
•A rough rule of thumb 
N ~ 3000 L(cm)/diameter(um)

www.pall.com
http://www.sorbtech.com/integrated/images/MN_CHROM/Nucleo20.jpg
 Optimizing Flow
Rate
Goal: we want a small plate height, or efficient separation

Broadening
Broadening
dominated by
dominated by
mobile phase
mass transfer
diffusion
Particle Size is Key 
Parameter in LC
 Cost of Small 
Particles: Pressure

If you go from 4 micron beads in a 4.6 mm ID column to 
1.7 micron beads in a 2.1 mm ID column what are the 
advantages?
• More efficient (e.g. faster) 
• Higher resolution, lower plate heights
How much more pressure do you need to make 
the smaller bead sizes work?

New pressure ~ old pressure *(4 )^2* 2.1/(1.7^2)*4.6 

Or 2.5 times more pressure!
 Understanding the column: 
physical structure

Silica particles are made by agglomerating smaller 
condensed siloxane polymers

They are like rigid sponges 
with internal surface areas 
>> larger than external 
surface areas.

PACKING PORE SIZE is different from BEAD DIAMETER
 Emerging Trends:
Monoliths and Poroshells

Merck KGaA

In packed columns, space is wasted (e.g. voids between spheres)

Poroshell surfaces are ideal for large, slowly diffusing analytes (e.g. proteins)