Chromatography and

Capillary Electrophoresis

Master programme in forensic science

Analytical Methods for Forensic Sciences

© Analytical Pharmaceutical Chemistry, Uppsala university

Agenda

Chromatography I (1h) LC and GC

• Principle

• Method development

• Optimisation

• Applications

Capillary electrophoresis (1h)

• Principle

• CE method development

• Optimisation

• Applications

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SEPARATION (LC,GC)

Literature:

Chapter 19. Gas chromatography

Chapter 20. High-performance liquid chromatography

Learning outcomes

Chromatography

Know about basic chromatographic theory regarding retention, band broadening and

resolution.

Liquid Chromatography (LC)

Understand the principles of retention and selectivity for the chromatographic techniques

covered in the course (adsorption chromatography and chiral chromatography).

Be able to describe how retention and selectivity are regulated in liquid-adsorption

chromatography.

Understand the principles of gradient elution.

Know about the instrumentation used for LC.

Know about the most important detectors and their advantages and disadvantages (UV,

fluorescence, mass spectrometry).

Know the opportunities and limitations of the technique.

Gas Chromatography (GC)

Understand the principles of retention and selectivity in GC.

Understand the principles of, and differences between, the most common injection techniques.

Understand why derivatization is sometimes important in GC.

Be able to describe the instrumentation used in GC.

Know about advantages and disadvantages of the commonest detectors (FID, ECD, NPD,

MS).

Know the areas of application of the technique.

Liquid chromatography

applications within forensic science

• LC is the “work horse”(often together with

MS) for analysis that needs high selectivity

and sensitivity.

• Drugs, abused drugs, metabolites,

endogenous compounds in biological

matrices (e.g., blood) and in the environment

• Explosives

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Schematic diagram of an LC

c.f. Figur 20.1

77

The principle for a chromatographic

separation

The separation is based on the analytes affinity to the stationary phase

http://chemsite.lsrhs.net/Intro/images/Overheads_large/insect_chromatography.jpg

88

Adsorption chromatographyretention principle

The analyte and the mobile phase solvent competes

about a limited number of binding site on the stationary

phase:

Animation separation in LC

https://www.youtube.com/watch?v=MYSBOxbnuAw

Mobile phase component

Analyte

Adsorption chromatography

reversed phase

Injection volume: 1-50 mL (depending on loop

size)

Stationary phase: unpolar

C18 bonded (see Figure 20.5)

Mobile phase: polar water based buffer +

organic modifier (e.g., phosphate buffer+

methanol)

Detector: e.g., UV or MS

1010

Reversed phase chromatographyChange of retention and/or selectivity

• Amount of organic solvent in the mobile phase

Increased amount decreases the retention.

• Type of organic solvent (e.g., methanol, acetonitrile)

Different elution strenght, might also change the selectivity

• pH (for protolytes)

The uncharged form of the analyte is more retainedthen the charged form (hydrofobic column)

• Addition of an counter ion to the mobile phase (for charged compounds) The analyte is eluted as an ionpair

• Type of stationary phase

Another type of column might change the retention and the selectivity.

Adsorption chromatographyEluent strenght of solvents used in reversed

phase LC

(see Table 20.1)

Solvent e0 value on alumina

Acetonitrile 0.65

2-Propanol 0.82

Ethanol 0.88

Methanol 0.95

Water High

Increased

strenght

Buffer

• A buffer is used to maintain a constant pH in a

aqueous solution.

• A buffer has resisitance against pH-changes

when acid or base are added. A buffer contains

a weak acid and its corresponding base (or vice

versa).

e.g., acetic acid and acetate (HAc)+ NaAc

Phosporic acid and diphosphate H3PO4 och H2PO4-

• A good buffer has a pH in the range ± one pH

unit from the acid’s pKa and have a high ionic

strenght.

Optimisation of a separationRetentionfactor (k), selectivity(a) and resolution(Rs)

k

Alfa

Rs 2t1t

1R2Rs

ww

)tt(2R

o

oR

t

ttk

Rs≥1.5 =base line separation

Optimisation of a separation

Efficiency

Number of theoretical plates

(N)

The height of a theoretical

plate (H)

Optimisation of an LC separation

First choice: C18 column an a polar eluent

A) Adjust the mobile phase composition

B) try another type of column.

It is sometimes difficult to retain polar metabolites. For this

application one of these approaches might work:

• Ion pair chromatography

• straight phase mode (polar column-unpolar mobile phase)

• HILIC (HydrophILic Interaction Chromatography)

columns

a type of straight phase where AcN or alcohol is used as

the mobile phase.

1616

Gas chromatography

16Literature: Chapter 19

Principle: https://www.youtube.com/watch?v=iX25exzwKhI

Gas chromatography

applications (forensic science)

• Drug analysis (doping control, post mortem

analysis)

• Tear gas, war gas, explosives, volatiles

• Remaining after a fire

Ref: Chapter 190 “Forensic applications of GC” in Encyclopedia of Chromatography, Third

Edition 2009

17

181818

Schematic diagram (GC)

18

https://www.youtube.com/watch?v=iX25exzwKhI

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Gas chromatography (GC)

Injection volume: capillary GC 0.01-3 mL (often

split injection)

Stationary phase: Often a adsorbed liquid on a

solid support. E.g., methylsilicone (unpolar),

polyetylenglycole (PEG) (polar)

Mobile phase: gas e.g., H2,He,N2

Detector: e.g., FID or MS

19http://www.youtube.com/watch?v=q0pM-k0SvOQ

202020

Gas chromatography

How is the separation optimised in GC?

Temperature – the main variable. Increased temperature decrease the retention.

Type of stationary phase– might change the relative retention of the peak.

No interactions in the mobile phase (gas phase). Thus, the type of gas doesn’t influence the separation.

20

Gas chromatographythe effect of temperature on the retention

21

a) isothermal at 45°C

b) isothermal at 145°C

c) Temperature gradient

30-180°C

222222

Gas chromatography

isothermal vs. temperature gradient

22

Gas chromatography

optimization of the retention

• Temperature

Initial and final temperature, gradient (ramp rate)

• Type of column

• Derivatisation of the analytes

242424

Gas chromatography

derivatisation

• Increase the volatility and decrease the polarity of

the analyte.

• Improve the thermal stability of the analyte

• Increase sensitivity by incorporation of functional

groups that enhances the detector signal.

• Decrease peak tailing

24

Gas chromatography

hydrolysis

Conjugated drug metabolites are degraded by high

temperatures. The conjugation can be cleaved by

hydrolysis (enzymatically or chemically)

Advantage:

Gives a higher concentration of the parent drug.

Disadvantage:

Separate determination of parent drug and

metabolite is not possible after hydrolysis.

Liquid- vs gas chromatography

advantages with LC

• The analysis can be performed at room

temperature (decreases the risk of thermal

degradation).

• It is possible to analysis unvolatile analytes.

• The mobile phase can be used to optimise the

separation.

272727

Liquid- vs gas chromatography

advantages with GC

• high efficiency (N)

• Often faster separations

• Many different types of detectors with high selectivity and sensitivity are available.

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Interactive practises

HPLC

https://www.ncbionetwork.org/educational-

resources/elearning/high-performance-

liquid-chromotography-hplc

GC

https://www.ncbionetwork.org/educational-

resources/elearning/gas-chromatography-

gc

28

292929

Capillary electrophoresis (CE)

Literature:

Chapter 8 “Capillary electrophoresis, an

introduction” p 160-170 in Principles and Practice of

bioanalysis- Venn 1st ed.)

Learning outcomes (CE)

• Understand the principles of zone electrophoresis,

micellar electrokinetic chromatography (MEKC) and

chiral CE.

• Describe in general terms how electro-osmosis occurs.

• Explain the effects of electro-osmosis in various types

of separations.

• Know the most common detectors for CE.

• Be able to describe the advantages and limitations of

CE.

• Be able to decide which of the techniques are

appropriate in various types of applications.

Capillary electroforesis

forensic applications

• DNA analysis

• Analysis of metal ions

(Cr3+, Pb2+, Hg2+, Ni2+) in water

• Proteins and peptides (e.g., CDT Carbohydrate-

Deficient Transferrin- alcohol marker)

• Drug analysis (e.g., alkaloids in urine)

• Explosives (portable system)

Reference. Pascali et al Electrophoresis 2012, 33, 117–126

Shimadzu MultiNA

3232

Capillary electrophoresis

instrumentation

Thermostated

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Capillary electrophoresis (CE)

Injection volume: in the nano litre range

Stationary phase: None (not a chromatographic

technique!)

Capillary: Fused silica or coated fused silica. Inner

diameter typically 50-75 µm (e.g., 50-80 cm in length)

Back ground electrolyte (BGE): water based buffer

Detector: e.g., UV or MS

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O-OH O- OH O- O- OH O- O- OH O- O- OH O-

O- OH O- O- OH O- O- OH O- O- OH O- O-

Capillary electrophoresis

electrophoresis

–Multiply charged ions have a stronger driving force

(Fi) than single charged→ higher velocity

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+-

-+ -

+ +Fi

Fi

2*Fi

Fi

Capillary electrophoresis

electrophoresis

–Smaller ions have a higher velocity than bigger.

35

+

+-

-+ -

Fi

Fi

Fi

Fi

Fd

Fd

Fd

Fd

iid vrF 6

radius v increase with

decreased radius

di FF

Capillary electrophoresis

= electrophoretic mobility + electro-osmosis (EOF)

+-

-

+

eofeffobs mmm

Mobility of ion EOF mobility

37

Capillary electrophoresis =

electrophoresis + electro-osmosis

eofeffobs mmm

Low pH --low EOF

+-

-+

o

o+ -

-+ --

o

o +

+

High pH-high EOF

3838

Flow profileCE

HPLC

CEHPLC

Tid

Abs

3939

Way to change the electro-osmosis

Variable Result

pH EOF increase with increased pH

Ionic strenght EOF decrese with increased ionic strenght

Covalent ”coating” Neutral coating decreases the EOF

Positive coating reverse the EOF

Electrical field EOF increase with increased E

(but…mEOF is constant)

40

Optimisation of a CE separation

• pH in the BGE

• type of buffer

• ionic strength of the buffer

• type and concentration of eventual micelle or chiral

selector

• voltage/capillary length …

• addition of an organic modifier (type and

concentration)

Creation of a robust CE method

• New capillary-with a clean cut (90◦) and removal of

polyimide coating in both ends.

• Conditioning of a new capillary as well as storage of

a capillary in use (acid/base wash)

• Conditioning of the capillary between the analysis

bakground elektrolyte (+ eventually acid/base wash,

and/or conditioning with voltage)

• Use of a BGE buffer with good buffer capacity

(change BGE regulary e.g., every tenth sample)

• If possible- use a high (9-10) or low (2-3) pH in the

BGE, avoid a pH near the pKa of the silanols (4-7)

• Particle free, degassed samples and BGEs41

Read chap 4+6 in Capillary Electrophoresis Methods for Pharmaceutical Analysis

Tip for

lab work

4242

Summary CE

• Able to separate charged analytes(CZE)

and if micelles (MEKC) is used, also uncharged

• Electro-osmosis both anions and catios can be

detected in the same analysis

Advantages

• More efficient peaks than in LC/GC

• Lower sample volumes than LC/GC

Disadvantages

• Needs higher concentration of the sample than LC

• Lower repeatability in injection volyme than LC

Injection medium

Note: The sample needs to be dissolved

(soluble) in the injection medium.

LC

A solvent with a lower elution strenght than the

mobile phase (e.g., a water based buffer)

GC

Volatile solvent

CE

The same conductivity as the BGE (the sample

is often dissolved in 1/10 dilluted BGE)

More reading

“Capillary Electrophoresis Methods for

Pharmaceutical Analysis” Satinder Ahuja and

M. Ilias Jimidar (Eds) e-book:

http://libris.kb.se/bib/13497300