Zachary Vaz

Mentor: Dr. Susana Teixeira

8/17/2023

High-pressure sample environment and spectroscopic studies of biomolecular solutions

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RET project this summer (A tale of Two Talks)

HP Sample environment for BioSANS: equipment 

commissioning

Manual System

- cart-ready assembly

- backup for PMI

- For benchtop measurements 

(DWS, fluorescence,exbeamline incubations, etc)

LIPSS system

- McHugh cells: test orings on 

old cell; test new cell 

performance

PMI automated system

- testing/creating 

documentation

Optimizing HP-BioSANS experiment planning: a case 

study (BSA binding to polyphenols) 

Pre-incubating samples under pressure 

- pre-characterization

- testing more pressure steps, holding times, 

reversibility

Sample characterization using benchtop techniques

- Circular dichroism

- FTIR

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Background: Why do we need high-pressure SANS to study biomolecules?

High-pressure is ubiquitous: in-vivo, in laboratories and industrial environments

• SANS accesses sizes and concentrations in 

environments more relevant to in vivo or in 

industry

• SANS is non destructive: samples can be 

equilibrated at different pressures for 

various holding times (with HP-SAXS we are 

limited to 5min equilibration), different 

steps can be explored and pressurization 

rates can be explored

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LIPSS : Liquid Insertion Pressure System for SANS Studies

in-situ studies of P|T effects for Biomolecules - how it works

Teixeira et al. (2018). J. Neutron Res. 20(1), 13.

Teixeira S. (2019). Cur. Op. Colloid & Interf. Science 42, 99.

Critical parameters for Biomolecules:

Sample volume: 2 - 5 mL

Pressure: ambient to 3.5 kbar

Temperature: -20°C to +65°C

Temperature equilibration: 10 min to 2.5h

d-spacing: 20 - 6000 Å (0.001 - 0.3 

Å-1)

Concentration: > 5 mg/mL

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LIPSS : Liquid Insertion Pressure System for SANS Studies

Pressurizing 

Fluid Line + 

Valve

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LIPSS : Liquid Insertion Pressure System for SANS Studies

Separator

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LIPSS : Liquid Insertion Pressure System for SANS Studies

Sample Injection 

Valve and Block

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LIPSS : Liquid Insertion Pressure System for SANS Studies

McHugh Cell 

Containing Sample

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LIPSS : Liquid Insertion Pressure System for SANS Studies

A typical HP-SANS experiment - how it works

Aggregation of Monoclonal Antibodies: Role of the Fc and

Fab Fragments. Berger et al. (2022). J. Phys. Chem. B, 126

(24), 4431.

1. Collect data at ambient 

pressure and temperature

2. Pressurize to target 

pressure, keep T constant. 

Equilibrate to avoid 

heterogenous samples. Collect 

data.

3. Lower the temperature to 

-20C. Equilibrate and collect 

data. 

4. Warm up at constant P. Then 

depressurize. Collect data to 

monitor reversibility.

~2hr

2min

10-30min

~2hr

~2hr

10-30min+2hr

10min

10-30min

~2hr

Blue path (forced degradation studies):

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LIPSS : Liquid Insertion Pressure System for SANS Studies

in-situ studies of P|T effects for Biomolecules – what can be improved

• Reliability of operation: leaks are common, particularly at 

low temperatures. Continued pressurization while there 

are leaks can damage the separator.

• Assembly is difficult and time consuming. Sample 

changes require priming (vacuum to ensure injections fill 

the entire volume of the sample side of the assembly), 

this is a slow step (up to 45min)

• Lost time waiting for radioactive decay of one of the two 

McHugh cells available (3h)

• Temperature equilibration is very slow at subzero 

temperatures – Not addressed during RET project

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What’s new with HP-SANS?

NEW: “The LIPSS Cart” – New Platform and

Assembly Procedure

1. Plug and Play Valves

• Loaded Pressure cell can be placed

into sample chamber.

• Minimizes mistakes and risk of damage

to materials.

2. New separator (previous version was very

difficult to assemble without damaging the

o-rings  Note about Manual/Tutorial)

3. New McHugh cell

• 3 assemblies ready

• Cells activate. ~3hr downtime

4. New chiller baths to cool the Peltier

system

(very similar to commercial 

hydraulic cylinder seals)

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The LIPSS Cart – “They See Me Rolling...” 12
What’s old with HP-SANS

OLD

1. McHugh cells are ~ 30 years old. They are

starting to leak at lower pressures and are not

meeting the demands of lower temperature

operations (materials contract)

2. The automated PMI system is showing signs of

degradation (it is 4 years old). We measure

pressure losses even when it is not connected

to LIPSS.

Test new O-rings and sapphire window

performance.

Investigate the origin of the problem.

Characterize the inherent pressure drop of the

PMI to determine:

- if there is a ‘normal’ pressure drop that we

can assume is not caused by leaks

- if the system needs servicing

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My work with LIPSS

McHugh Cell O-ring Tests:

• Tested several different 

combinations of primary and 

back-up O-rings.

• Identified a no-leak candidate.

− Higher Durometer Primary 

o-ring

• Need more tests. PMI currently 

down.

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Separator O-ring Tests: My work with LIPSS

• Want to test if the separator is 

leaking

• Identify leaks using NanoDrop

UV-Vis Spectrometer

− Confirmed nanodrop can 

detect a 20 μL drop in a 

volume of 1mL

− Tests interrupted by 

torquing issue

Concentrated Blue dye 

in hex side of separator

Leak: 

Pressurizing 

Fluid Dilutes Dye

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Manual Pressurizing System

• Previously existed but was to

be replaced by automated

PMI system.

• Now want asa backup and

for benchtop measurements

(Diffuse Wave Spectroscopy,

Incubations, Fluorometry)

• Ordered necessary parts.

When assembled will go on

the LIPSS cart.

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Automated Pressurizing System – PMI 

• For HP SANs need to maintain

pressure for several hours.

• Previous behavior was stable.

System has aged...

• Pressure reading drifts over

time even when isolated (i.e.

not connected to LIPSS)

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Piston

V2

PMI – Under the Hood

Analog Gauge

Labview GUI

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Automated Pressurizing System – PMI 

What we want:

• Minimal drift at pressures up to

4kbar (58000 psi)

• This was achievable in the past

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Characterizing PMI Drift 

What we see now: • Pronounced drift followed up by

equilibration

• Put yourself in shoes of a user:

−Starting to pressurize...

Is there a leak or is the system

just drifting?

• Goal: Develop procedure for user to

determine if system is leaking

− ~30min ideal. These experiments are

long, need to optimize for time.

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Characterizing PMI Drift 

What we see now: Considered the effects of 3 different

pressurizing fluids

1. Regular DI Water

2. In-House Degassed Water

3. Commercially Degassed Water

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Characterizing PMI Drift 

What we see now: Collected data at 2 different pressure

thresholds and held to examine drift

Used fitting program developed by

Alan Ye to fit decaying exponential

curves to the drift.

Obtain a time constant τ for the

relaxation time.

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Characterizing PMI Drift Results 

Each run takes about 3-4 hours

These data are all with

commercially degassed water.

There may be a reuptake of air

as time goes on.

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Characterizing PMI Drift Results 

More data needed for in-house

degassed water and regular DI.

Most importantly need to

introduce a known leak, collect

data, and compare to natural

drift of system.

Hope to be able to quickly

distinguish between leak vs drift

using time constant.

Testing can continue when V1

is repaired/replaced.

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Development of a Novel Pre-Incubation Vessel

Would be ideal to be able to prepressurize a sample to cut down on

necessary beam time and use

complementary techniques (e.g. SAXS)

Preliminary data (shown later) indicates

this may be feasible.

Used pipettes and vacuum grease with

an empty separator tube to incubate

protein sample.

We incubated Bovine Serum Albumin

(BSA).

Vessel does not leak.

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Small Angle Scattering studies of BSA/Polyphenols

Epigallocatechin 

Galate

(EGCG)

Catechin

EGCG: principal green tea polyphenol

(≈270 mg/L)

Potential food preservatives

Therapeutic effect limited by poor

stability under physiologic conditions

Health benefits: Ohishi et al. (2017). Molecules 26(2): 453, 656.

Adapted from Scepankova et al. (2018). Plant Foods for Human Nutrition 73: 253.

Untreated leaves HPPE, 3kbar 10min Heat Reflux extraction

SEM 

images of 

green tea 

leaves

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BSA: a model protein in food

 Main carrier protein in blood plasma

 Dietary protein found in beef and cow milk.

Used as a food additive (emulsifying

properties, enhanced after HPP processing)

 P > 100 MPa: secondary structure affected

 P > 300 MPa: protein unfolds under certain

conditions (pH, salt, temperature)

Physiological blood 

concentration: 40 mg/mL

BSA image adapted from: 

Tang et al. (2017). J. Agric. Food Chem. 65, 656. 

SAXS
DOI: 10.1021/acs.jafc.6b04630

J. Agric. Food Chem. 2017, 65, 656−665

Adapted from: 

Tang et al. (2017). J. Agric. Food Chem. 65, 656. 

Stronger binding 28
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HP-SANS scattering profile

100 mM MES pH 6.5, 50 mM KCl

20 MPa/min ramp

20 min equilibration

Prior HP-SANS and HP-SAXS studies of complexes
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Intrinsic HP- Fluorescence

• Cathecin/BSA complex is not very sensitive to

pressure. Minor shifts in fluorescence, reversible

(local chemical environment of the Tryptophans)

• EGCG/BSA complex partially unfolds under

pressure. Effect is irreversible.

Prior Spectroscopic data

HP-Fluorescence data collected at the University of Delaware
CD Deconvolution using Dichroweb

[Miles et al. (2021). Protein Science: https://doi.org/10.1002/pro.4153 ] and 

Reference Data #7 [Sreerama et al. (2000). Analytical Biochem.287,252-260]

Prior Spectroscopic data – Far UV Circular dichroism

S. Teixeira

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Circular Dichroism Far UV Data (D2O solutions, [Protein]=2.5 mg/mL)

100 mM MES pD 6.5, 50 mM KCl

CD Deconvolution using Dichroweb

[Miles et al. (2021). Protein Science: https://doi.org/10.1002/pro.4153 ] and 

Reference Data #7 [Sreerama et al. (2000). Analytical Biochem.287,252-260]

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FTIR Data (D2O solutions, [Protein]=10 mg/mL) – Collected at 18 angles (1 angle shown here)

10 mg/mL solutions

100 mM MES pH 6.5

50 mM KCl

Ongoing analysis

Collaboration with Curtis Meuse 

(IBBR)

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BSA has

- 2 Trp residues

- 20 Tyr, 27 Phe

- 7 intrachain disulfide bonds with one

free thiol group at residue 34

• Can see from Near UV difference in

tertiary structure for different

incubation procedures

Circular Dichroism Far UV Data (D2O solutions, [Protein]=2.5 mg/mL) 34
What Will I take Back to the Classroom?

1. The techniques you are introduced to in science

courses are used routinely in contemporary

work.

• e.g. UV-Vis. Used an elementary technique to tackle a

problem and develop a solution.

2. Recognition that scientific progress is not linear.

• There is a great deal of trial and error, creative

exploration, and dead-ends.

3. Newfound patience for students who are

learning things for the first time.

• Interactions with everyone at the NCNR are helpful,

positive, and extremely encouraging.

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Acknowledgements

I would like to extend a heartfelt and gracious thank you to...

• Yamali Hernandez for her coordination and placing me in the RET

• High Pressure Group

• Paul Butler, Juscelino Leao, Alan Ye, Cedric Gagnon for their

unwavering support and collaboration

• The one, The only, THE INCOMPARABLE...

My Mentor: Susana Teixeira

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