The world leader in serving science

M. De Pra, G. Greco, N. Bartsch, E. George, M. M. Martin, F. SteinerThermo Fisher Scientific, Germering (Germany), Bremen (Germany), San Jose (USA)

Effects of Metal Contamination Caused by Iron-Free HPLC Systems on Peak Shape and Retention of Drugs with Chelating Properties

For research use only. Not for use in diagnostic procedures.

2

Schematic of fluidic components in a HPLC

Biocompatible HPLC Systems

• Biocompatible (U)HPLCs are used in the analysis of biomolecules

• Biomolecules are often analyzed with high salt concentrations in the mobile phase

• Conventional stainless steel HPLCs form rust

• In biocompatible UHPLC titanium is used instead of stainless steel

Is titanium always inert?

3

Titanium weakness

Stress Corrosion Cracking ofTi in pure MeOH

• Relevant problem in material science

• Not a problem for UHPLC systems

Any effect on chromatography?

5

Ti Leaching from a Biocompatible System

• ICP-MS analysis of effluents collected w/o column: 2 ppb Ti

• All Ti fluidic components contribute to Ti leaching

• The solvent frits are most critical (very large surface)

How do Ti ions affect chromatography?

H2O MeOHACN

A B

6

7 fluoroquinolones thiamphenicol

Prone to tailing No tailing

Investigating the Mechanism: The Experiments

• ISOCRATIC (H2O, MeOH, ACN, formic acid) pH 2.9

• Non-corrosive conditions (Any part of fluidic always in contact with water)

Amide polar embedded reversed phaseThermo Scientific™ Acclaim™ PA2 RSLC (2.2 µm, 2.1x150mm) column

New column

Column contaminated by titanium• High peak asymmetry• Permanent change • Ti is immobilized in the column

+

!

7

Retention Mechanism Model with Leaching Titanium

N

(CH2)3

(CH2)3O

(CH2)16O

CH3

SiO

OO

O

SiO

O

SiO

O

Ti4+

Ti4+

Ti4+

Ti4+

Ti4+

N

F

N

ON

O

O

OH NH+

Ti4+

Ti4+ Ti

4+

N

F

N

ON

O

O

O NH+

Ti4+

H3O+

2. Complex formation* of fluoroquinolones with immobilized titanium

1. Dissolved titanium binds to the stationary phase

*Uivarosi V. Molecules 2013 “Metal complexes of quinolone antibiotics and their application”

N

(CH2)3

(CH2)3O

(CH2)16O

Ti4+

CH3

SiO

OO

O

Ti4+

SiO

O

SiO

OTi

4+

Ti4+

8

• Reversed phase is the main retention mechanism

• Ti cations make the contaminated column less hydrophobic

However this does not explain the tailing

Influence of Organic Content on Retention

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

0 5 10 15 20 25

ln k

'

% organic

Ciprofloxacin

New column Contaminated column

Ti4+

Ti4+

Ti4+

Ti4+

9

Influence of Salt Content on Retention

1.2

1.4

1.6

1.8

2

2.2

2.4

2 4 6 8

ln k

'

ln 1/[Na]

Ciprofloxacin

New column Contaminated column

Ti4+

Ti4+

Ti4+

Ti4+

• Without salt - Contaminated column is much less retentiveWith salt - Column behavior becomes more similar

• Contribution of electrostatic repulsion to the mechanism (Retention increases with salt)

However this does not explain the tailing

Increasing Na2SO4

10

Influence of Organic Content on Peak Asymmetry

012345678

0 10 20 30

As (E

P)

% organic

Ciprofloxacin

N

F

N

ON

O

O

OH NH+

Ti4+

Ti4+ Ti

4+

N

F

N

ON

O

O

O NH+

Ti4+

H3O+

• Complex formation of fluoroquinolones with immobilized titanium

• complex stability is influenced by organic content

• Tailing is due to complex formation

organic 5-15%

organic 20%

Increased complex stability

Decreased fluoroquinolone acidity

11

• Leaching of titanium from biocompatible systems observed when anhydrous methanol/acetonitrile was used as eluent.

• Peak shape of some drugs is strongly affected.

• Titanium ions are immobilized on the stationary phase and reduce column hydrophobicity.

• Fluoroquinolones form complexes with the immobilized titanium, creating a secondary interaction.

• Conventional C18 columns are not affected by titanium.

Conclusions

For research use only. Not for use in diagnostic procedures.