Small angle scattering study of NIST 

SRM1549a: 

Bovine Whole Milk Powder

Lisa Hong (U. Maryland Baltimore County)

Summer Undergraduate Research Fellowship

at the NIST Center for Neutron Research

Image from https://www.pinterest.com/explore/funny-cows/
Image from: http://www.borrisoleigh.ie/2009-11-18/

To contribute towards:

• a better assignment of the casein micelle 

scattering profile

a better understanding of

structure and applications

Goals

?

?

Typical Small Angle Scattering of Milk

Adapted from Ingham et. al (2016). Soft Matter. 12: 6937-6953
Milk has been part of the human diet for ∼8000 years. 

What’s new?

We don’t fully understand it yet

• None of the current models explains all experimental results for the particles 

that stabilize milk : CASEIN MICELLES

• NIST SRM 1549a was the chosen sample for this project: 

• certified mass fraction values 

• contributions from this project’s findings

https://phys.org/news/2009-08-years-central-europe.html Image from: https://alfafoods.co.nz/wp-content/uploads/2016/01/milk1.jpg
What are casein micelles?

Sub-micron particle with various sizes 

depending on many factors

~ 200 nm

Nanocluster model

MW 2x108 Da 

(10 000 peptide chains) 

~ 800 CPN

Calcium Phosphate Nanoclusters 

(CPN)

Calcium 

Phosphate 

Core

β-casein

cross-linker

α-caseins

shell

Core Radius

≈2.4 nm CPN: MW 61 kDa, ≈ 8 nm diameter Casein phosphopeptide

shell thickness ~ 1.6 nm

~82% water Image from Dalgleish. (2011). Soft Matter (7): 2265-2272.

Electron microscope Image

Peter Hristov, et. al. (2016). Milk Proteins - From Structure to Biological Properties and Health Aspects

Adapted from: C. Holt (2016). International Dairy Journal. 60: 2-13
Function/Applications of Casein Micelles

• Milk is supersaturated in calcium 

phosphate. Caseins stabilize it.

Biomineralization

Inhibit amyloid fibrils 

• Biocompatible, biodegradable

Drug & Nutrient Carriers

• Micelle size influences processing and 

texture of dairy products

Dairy quality & performance

Adapted from Holt et. al. (2012). Journal of Evolutionary Biology. 25: 1253–1263 Bouchoux et. al. (2015). Soft Matter. 389-399
Research Strategy

Stabilize Simplify

• Buffer mimics 

milk

• Added natural 

stabilizers

• Temperature 

control

Simulated 

Milk Ultra 

Filtrate

Use SANS w/ 

contrast variation:

CPN CM

Fractionate the 

Casein Micelles

Skim the milk

Ultracentrifugation

90000 g

Adapted from: http://www.creative-biostructure.com/custom-sizeexclusion-chromatography-service-259.htm
Simulation Why neutrons?

Fuzzy Sphere R=150 nm (CM)

Monodisperse 

Polydisperse

Simulation

CPN Nanoclusters R= 4 nm

Monodisperse Polydisperse

Monodisperse 

Polydisperse

?

?

Monodisperse Polydisperse
Why neutrons?

H2O 70% D2O 98% D2O
Small Angle Neutron Scattering (SANS)

SANS is the least intrusive, most informative technique

• Covers broad range of angles

• Does not require cooling, additives, etc. (less artifacts from sample preparation)

UV-Visible Signal easily saturated

Used instead:

- UV-vis of diluted fractions

- Dynamic Light Scattering 

BT5 (desmeared) &

NGB30 data

"Reduction and Analysis of SANS and 

USANS Data using Igor Pro", Kline, S. R. J 

Appl. Cryst. 39(6), 895 (2006).

RH from DLS (nm):

Skim – 361 ± 72

Pool 1 – 242 ± 57 

Pool 2 – 87 ± 7

Pool 3 – 82 ± 10

Pool 4 – 83 ± 21

Pool 5– 79 ± 11
SANS profiles in D2O buffers have better signal-to-noise but also match out the CPN contributions.

The change in size distributions is however still clear. 

Faster flow, 2ml fractions, more fractions pooled 

together. Run at 4°C

Less column saturation, 0.5 ml fractions, less 

volume pooled together. Run at 10°C

Fractionation helps – Optimization of SEC runs

1st COLUMN 2nd COLUMN

NGB30 data NGB30 data

Guinier Rg (nm)

Skim 276 ± 46

Pool 1 220 ± 10 

Pool 2 190 ± 10 

Pool 3 213 ± 11 

Pool 4 193 ± 9
Fractionation helps – Contrast Variation 

RH (DLS)

147 ±14 nm

RH

79 ±12 nm

Pool 1

Pool 2

Pool 3

Pool 4

Skim Milk
Conclusions

• SEC can reduce the natural polydispersity 

of milk for improved resolution

• Mid-q feature of the spectrum is matched 

out in D2O buffers (CPN matched out). 

• USANS-SANS provides information on the 

different components but sample 

monodispersity can only be kept for 1-2 

days. 

80-322 nm

Micelle Rg in SRM

Nanoclusters

in the Micelle

*VSANS please!*
Acknowledgments

• S. Teixeira (SURF Mentor, U. Delaware/NIST)

• T. Hoopes (IBBR), T. Cleveland, & M. Shremshock (NIST)

• L. Wood (MML Material Measurements Laboratory)

• B. Brooks (RET program)

• NCNR SURF team: J. Dura & J. Borchers

DISCLAIMER

Certain commercial equipment, instruments, or materials were identified to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute

of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

Image from: https://www.snorgtees.com/milk-i-am-your-father

Image credit: Susana Teixeira (NIST)
Peter Hristov et. al (2016). Measurement of Casein Micelle Size in 

Raw Dairy Cattle Milk by Dynamic Light Scattering, Milk Proteins -

From Structure to Biological Properties and Health Aspects

Holt et. al (2012). Darwinian transformation of a ‘scarcely 

nutritious fluid’ into milk. Journal of Evolutionary Biology

•Size Exclusion Chromatography: Separation 

by Size

•Akta Purifier System

•Hiprep 26/60 Sephacryl S-500 Column

(Matrix: cross-linked copolymer of allyl 

dextran and N,N’-methylene bisacrylamide)