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Webinar 4: Don‘t worry: use HIC and HILIC !

May, 29th 9-10 a.m. and May 30th 5-6 p.m. (CET)

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Summary part 3: Ion biochromatography

1. Capture step

2. Intermediate step

3. Polishing step

Where does it fit?

AC - SEC

AC – IEC - SEC

IEC – HIC - SEC

HIC – IEC - SEC

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Summary of principle in IEX

sample loading

separation according to their charge

elution

source: https://www.fh-giessen-friedberg.de/kmub/mitarbeiter/images/stories/Membrantechnik/membranchromatographie_vorlesung_hahn.pdf

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Now: HIC or HILIC?

HILIC basic principles in a rush

“HILIC [Hydrophilic Interaction] chromatography

provides retention of highly polar hydrophilic

analytes that are difficult to retain on RP

methods using simple combinations of mobile

phase and stationary phase”

Source: Mark Woodruff in LC GC the Column. The Ins and Outs of HILIC Chromatography Jan 24, 2011, Vol.7, issue 1

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Now: HIC or HILIC?

HILIC basic principles in a rush

“A highly polar stationary phase, often bare

silica, is used with largely a non-polar mobile

phase system (water acting as a very strong

solvent in HILIC)”

It is a very powerful method for LC-MS

Source: Mark Woodruff in LC GC the Column. The Ins and Outs of HILIC Chromatography

Jan 24, 2011, Vol.7, issue 1

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HILIC – separation

Source: machery-nagel www.mn-net.com

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HILIC – examples

Source: machery-nagel www.mn-net.com

HILIC is a key separation technology where other

techniques come to a limit !

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Separation of substances is based on

their varying strength of interaction

with hydrophobic groups attached to

an uncharged gel matrix

Hydrophobic groups on proteins are

sufficiently exposed to bind to the

hydrophobic groups on the matrix

Source: C.G. Huber, Biopolymer Chromatography,

Encyclopedia in Analytical Chemistry, 2000

HIC

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HIC - advantages

Large volume of sample

Samples with high ionic strengths

Good before gel-filtration (SEC), ion-

exchange (IEX) or affinity chromatography

Sample eluted with low salt

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HIC – influencing parameters

Hydrophobic ligand type

affects protein absorption

- interaction may not be

strictly hydrophobic

Choice of ligand is rather

empirical

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HIC – influencing parameters

Binding capacity of protein

to HIC surface increases with

alkyl chain length (A)

and increased degree of

substitution of immobilized

ligand (B)

Protein can bind multi-point

and is then difficult to elute!

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HIC – influencing parameters

The type of base matrix is also

important – widely used are cross-

linked agarose (like BioFox) and

synthetic copolymer materials

May be necessary to modify adsorption

and elution conditions – take notice in

protocols !

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HIC – influencing parameters

Protein binding can be seen

as “salting out”. More salt =

more protein bound

Bound proteins are desorbed

by washing the HIC column

with neutral, diluted buffer or

even water

Decrease of pH enhances

binding

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HIC uses hydrophobic sections of proteins; HILIC

is not suitable for complete proteins but for

peptides

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HIC and HILIC protocols

For highly charged compounds, at least a low salt

concentration (10mM) is needed to compensate for it

(ion supression) !

HIC protocols start with even higher salt

concentrations and use a decreasing salt gradient for

separation !

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HIC – where does it fit

AC - SEC

AC – IEC - SEC

IEC – HIC - SEC

HIC – IEC - SEC

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HILIC – examples

Source: machery-nagel www.mn-net.com

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HILIC – examples

Source: YMC Europe www.ymc-europe.com

Dry cheese

Dry cheese (melamine

spiked)

Standard 0.5µg/ml

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HILIC – examples

Source:EP Nesterenko et al. Anal.Chim.Acta 652 (2009) 3-21

Simultaneous

cation and anion

separation is a

HILIC/IEX

application

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HIC – examples

Source: Tosoh Bioscience

Optimized IgG

separation on a

commercial HIC phase

for protein separation

Comparison of different

ligands

1 RNAse

2 Lysozyme

3 a-Chymotrypsinogen

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Summary

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Biochromatography systems: fuchs@knauer.net

applications: naether@knauer.net

mailto:fuchs@knauer.netmailto:naether@knauer.net