dynamic surface tension...

1

Dynamic Surface Tension Measurements

Emilia Bramanti

Italian National Research Council(CNR)

Institute for chemical and physical processes (IPCF)Laboratory of Instrumental Analytical Chemistry, Via G. Moruzzi 1, 56124- Pisa, ITALYTEL +39-050-3152292Fax +39-050-3152555

[email protected]

mailto:[email protected]

2

OutlineDESCRIPTION OF THE INSTRUMENTAL SET UPDynamic Surface Tension Detector (DSTD)TheoryOn line calibration of DSTDQuality of DSTD information

EXPLOITED APPLICATIONS FIA-DSTD study of polymer formulationsFIA-DSTD study of metal-surfactant interactionsLC-DSTD study of PEGs and proteins (globular proteins and caseins)FIA-pH gradient-DSTD analysis for a high-throughput screening of Protein Surface Activity FIA-DSTD study of the effect of denaturing agents on globular proteins

IN PROGRESS STUDIES•Development of a new drop detachment system (DDS) for DSTD•Study of PEG-protein interactions

• study of cement-polymer interaction by FIA-DSTD• study of nanoparticles by FIA-DSTD

3

Air Burst Capillary

Solenoid Valve

Air Supply

Drop Detachment System

Waste

HPLCPump

Sample

Injection Valve

Chromatographic Column

( Optional )

Dynamic Surface Tension Detector (DSTD) Schematic

Invention Disclosure: OTT # 1006 - 3198DL

Computer

Capillary Sensing

Tip

PressureSensor

P0

4

DSTD top view

5

DSTD front view

6

drop pressure is measured,

r(t)2γ (t)

P(t) = + C

In the DSTD system:

PC= the pressure offset (sensor position, viscosity losses)

= dynamic surface tensionr(t) = drop radius

2γ (t)

P

ΔP = 2 γ /r (Young-Laplace equation)

FlowPressure Sensor

Open to Atmosphere

Membrane

TheoryTheoryIn a static drop:In a static drop:

7

FlowPressure Sensor

Open to Atmosphere

Membrane

FlowPressure Sensor

Open to Atmosphere

Membrane

P(t) = 2 γ(t) / r(t) + PC

Diagram of pressure sensor and capillary tip during drop growthDiagram of pressure sensor and capillary tip during drop growthDisplacement of the sensor membrane during formation of a mobile phase droplet

Displacement of the sensor membrane during formation of a mobile phase droplet

Displacement of the sensor membrane during formation of a droplet containing surface-active analyte

Displacement of the sensor membrane during formation of a droplet containing surface-active analyte

8

0 10 20 30 40 50

0.26

0.28

0.30

0.32

0.34

0.36 Water1.5 mM SDS*

Pre

ssur

e, p

si

Drop Time, seconds

* Sodium Dodecyl Sulfate

Raw Sensor Data, P(t)

9

π(t)A = γ(t)M – γ(t)A = r(t) [ P(t)M – P(t)A ]2

a. Surface pressure of analyte solution, A :

Step 1: Removal of PC offset

Sensor Calibration

2

b. Surface pressure of standard solution, S :

π(t)S = γ(t)M – γ(t)S = r(t) [ P(t)M – P(t)S ]

π(t)Analyte

= γ(t)M

- γ (t)Analyte

Surface PressureSurface Pressure

10

Sensor CalibrationStep 2: Radius dependency, r(t), factored out

π(t)A [ P(t)M – P(t)A ]π(t) [ P(t) – P(t) ]=

S M S

Thus, surface pressure for analyte, A is then:

P(t)M – P(t)A

P(t)M – P(t)S π(t)A = π(t)S

Drop profile vectors

Raw data

Standard value

(acetic acid 5%) π(t)= 10.3

11

Calibration Dynamic Surface Pressure

Raw Sensor DataDrop Profiles, P(t)

Non-kinetically hindered analytes

0 1 20.24

0.28

0.32

Pres

sure

, psi

Drop Time,seconds

Mobile Phase1 mM SDSStandard

Surf

ace

Pres

sure

, dyn

/cm

0

5

10

15

Drop Time,seconds0 1 2

1 mM SDS

12

0

2

4

6

8

10

12

14

16

18

20

0.0 0.5 1.0 1.5 2.0 2.5Drop Time, seconds

Sur

face

Pre

ssur

e, D

yne/

cm

0.5 mM

3.0 mM

2.5 mM

2.0 mM

1.5 mM

1.0 mM

Water PMAX

I II III Regions

Dynamic Surface Pressure of Sodium Dodecyl Sulfate (SDS) Solutions

13

0

4

8

12

16

20

0 1 2 3 40

4

8

12

16

20

0 1 2 3 4SDS Concentration, mM

Sur

face

Pre

ssur

e, D

yne/

cmCalibration assessment

14

Sur

face

Pre

ssur

e, d

yn/c

m


0

5

10

15

250 ppmPEG 22.000

Dynamic Surface Pressure

CalibrationRaw Sensor DataDrop Profiles, P(t)

Kinetically hindered analytes

250 ppmPEG 22.000

Mobile Phase

Standard

Pre

ssur

e, p

si


0.24

0.28

0.32

15

Quality of DSTD informationQuality of DSTD information

Flow injection analysis (FIA)-DSTDFlow injection analysis (FIA)-DSTD Reproducible, fast, high-throughputReproducible, fast, high-throughput

Liquid chromatography (LC)-DSTDLiquid chromatography (LC)-DSTD Multidimensional informationMultidimensional information

0 1 2 3 4 51

23

4 5

10

15

20

Drop Tim

e, sec

sample injected

Surf

ace

Pres

sure

dyn/

cm

Elution Time, min0 1 2 3

0

5

10

15

Elution profile

Surf

ace

Pres

sure

, dyn

cm

-1

Elution time, minutes

16

Quality of DSTD informationQuality of DSTD information

Flow injection analysis (FIA)-DSTDFlow injection analysis (FIA)-DSTD Fast, high-throughputFast, high-throughput

Liquid chromatography (LC)-DSTDLiquid chromatography (LC)-DSTD Multidimensional informationMultidimensional information

0 1 2 3 4 51

23

4 5

10

15

20

Drop Tim

e, sec

sample injected

Surf

ace

Pres

sure

dyn/

cm

Elution Time, min0 1 2 3 4

0

5

10

15

Surf

ace

Pres

urre

, dyn

cm

-1 Kinetic Profile

Drop Time, sec

17

Outline

EXPLOITED APPLICATIONS (12 publications)FIA-DSTD study of polymer formulationsFIA-DSTD study of metal-surfactant interactionsFIA-DSTD study of the effect of denaturing agents on globular proteinsLC-DSTD for the study of PEGs and proteins (globular proteins and caseins)FIA-pH gradient-DSTD analysis for a high-throughput screening of Protein Surface Activity

DESCRIPTION OF THA INSTRUMENTAL SET UP DESCRIPTION OF THA INSTRUMENTAL SET UP Dynamic Surface Tension Detector (DSTD)Dynamic Surface Tension Detector (DSTD)TheoryTheoryOn line calibration of DSTDOn line calibration of DSTDQuality of DSTD informationQuality of DSTD information

YOUR SUGGESTIONS!

IN PROGRESS STUDIES•Development of a new drop detachment system (DDS) for DSTD•Study of PEG-protein interactions

• study of cement-polymer interaction by FIA-DSTD• study of nanoparticles by FIA-DSTD

18

Formulation:Effects on Dynamic Surface Pressure

Brij ®-35

-

0

4

8

12

16

20

24

0 1 2 3 4 5Time, seconds

Sur

face

Pre

ssur

e, d

ynes

/cm

c

b

a

Additive Effects

a. 1mM SDS

b. 100 ppm

c. 1mM SDS +

100 ppm Brij ® 35

0

4

8

12

16

0 1 2 3 4 5Time, seconds

Sur

face

Pre

ssur

e, d

ynes

/cm c

b

a

b

a

a. 50 ppm PEG 1470

b. 100 ppm Brij ®-35

c. 50 ppm PEG 1470 +100 ppm Brij ®-35

Competitive Effects

Talanta 50 (1999) 1045–1056

19

Brij®35 polyoxyethylene 23 lauryl etherSDS C12H25-O-SO3NaPEG 1470 -O-(CH2CH2)n-

hydrophobic hydrophylic

Brij-SDSadditive effect Brij-PEG

competitive effect

Formulation:Effects on Dynamic Surface Pressure

Talanta 50 (1999) 1045–1056

20

Dodecyl Sulfate-Chromium (III) complexesSDS

[Cr(III)] = 0.50 mM

0.50 mM0.250.010.050.03

0.020.00water

FIA-DSTD study of metal-surfactant interactions

SDS

con

cent

ratio

nSD

S c

once

ntra

tion

Talanta 55 (2001) 551–560

21

0.400.200.10

0.05

0.030.01water

SDS

SDS-TBA

FIA-DSTD study of metal-surfactant interactions

DS-Cr (III) complexes Calibration

[SDS] = 0.50 mMC

r(III

) c

once

ntra

tion

SDS

DS-Cr(III)

•Ion pair formation (SDS/chromium(III) or SDS/TBA) increases the surface activity of 0.5 mM SDS of about 25-fold

•FIA-DSTD is a rapid analysis tool for the detection of multiply charged metal cations (Cr(III), Al, Co, Cu(II)…) in a cleaning bath

22

Size Exclusion Chromatography of surface active polymers with Dynamic Surface tension Detection

4

8

12

0.4

0.8

1.2

1.6

2.0

ba

Drop Time, seconds

, dyn

es/c

m S

urfa

ce P

ress

ure

Elution Time, minutes

PEG22.000 Da250ppm

PEG1470 Da250ppm


Dro

p Ti

me,

sec

onds

4 8 12 160.4

0.8

1.2

1.6

2.0

Anal. Chem. 2000, 72, 4372-4380

23

Polydispersity Determinations of Polymer Samples

Sample Concentration: 375 ppm

Mn = 2778 g/mol using PEG 1470, 4120, and 8500g/mol

Mw/Mn = 1.65 Mw/Mn = 1.58

2 4 6 8 100.4

0.8

1.2

1.6


Dro

p Ti

me,

sec

onds

2 4 6 8 100.4

0.8

1.2

1.6

Dro

p Ti

me,

sec

onds

2.02.0

00

Contour Plot

Polydispersity Determinations of Polymer Samples

Sample Concentration: 375 ppm

Mn = 2778 g/mol using PEG 1470, 4120, and 8500g/mol

Mw/Mn = 1.65 Mw/Mn = 1.58

2 4 6 8 100.4

0.8

1.2

1.6


Dro

p Ti

me,

sec

onds

2 4 6 8 100.4

0.8

1.2

1.6


Dro

p Ti

me,

sec

onds

2.02.0

00

A B

24

B - A

Mw/Mn = 1.65

Mw/Mn = 1.58

0 2 4 6 8 10 12

0

3

6

9

12

Difference of RI SignalsRI S

igna

l, ar

bitra

ry u

nits


Selectivity Advantage of Surface Pressure Signal

Refractive Index (RI) Signals

14708500 4120 14708500 4120

2.0

Difference of Surface Pressure Signals Enables Polydispersity

Deteminations!

2 4 6 8 100.4

0.8

1.2

1.6


Dro

p Ti

me,

sec

onds

0 12

B-AB-A

25

FIA-DSTD analysis of proteins: effect of denaturing agents onsurface pressure of proteins

Human serum albumin (HSA)Human serum albumin (HSA)

26


0 2 4 6 8

0

2

4

6

8

10

12

14

0 2 4 6 8

0

5

10

15

20

0 2 4 6 8-202468

10121416

0 2 4 6 8

0

2

4

6

8

10

urea

urea urea

GdmHCl

urea

GdmSCN

2σ

Denaturant, M Denaturant, M

Denaturant, M

Πm

ean,

dyn

cm-1

Πm

ean,

dyn

cm-1

Πm

ean,

dyn

cm-1

Cytochrome C

Πm

ean,

dyn

cm-1

Denaturant, M

GdmHCl

GdmHCl

GdmHCl

GdmSCNGdmSCN

GdmSCN

Ovalbumin

Myoglobin

Lysozyme

0 2 4 6 80

5

10

15

20

0 2 4 6 80

5

10

15

0 2 4 6 80

5

10

15

20

0 2 4 6 8

2

4

6

8

10

Πm

ean,

dyn

cm-1

Πm

ean,

dyn

cm-1

Πm

ean,

dyn

cm-1

β-Lactoglobulin

Πm

ean,

dyn

cm-1

Glyceraldehyde-3-P-Dehydrogenase

Human Hemoglobin

Denaturant, MDenaturant, M

Denaturant, MDenaturant, M

α-Lactalbumin

•Surface pressure increases as the denaturation proceeds •Denaturant effectiveness is GdmSCN>GdmHCl>urea

Analytical Biochemistry xxx (2006) xxx–xxx, in press.

27

0 2 4 6 81.001.011.021.031.041.051.061.07

UV

abs.

@ 2

92 n

m, a

.u.

-1

0

1

2

3

4

5

6

Lysozyme

Urea, mol dm-3

Πm

ean , dyn cm-1

0 2 4 6 8

1.7

1.8

1.9

2.0

2.1

2.2

2.3

β-Lactoglobulin

Urea, mol dm-3UV

abs.

ratio

281

nm/2

92 n

m

0

2

4

6

8

10

12

14

16

18

Πm

ean , dyn cm-1

UV and DSTD unfolding curves

•DSTD unfolding curves do not necessarily overlap UV unfolding curves because of the different properties monitored


Analytical Biochemistry xxx (2006) xxx–xxx, in press.

28

0.0 0.4 0.8 1.2 1.6 2.00

5

10

15

20

25

0.0 0.4 0.8 1.2 1.6

0

2

4

6

8

10

12

14

16Π

mea

n (pe

ak a

rea)

, dyn

min

cm

-1

GdmHCl

urea

GdmSCN

pbs

β-Lactoglobulin

protein, mg mL-1

GdmHCl

urea

GdmSCN

pbs

α-Lactalbumin

Πm

ean (

peak

are

a), d

yn m

in c

m-1

protein, mg mL-1

Calibration plot

•A significant enhancement of the surface pressure of proteins due to the denaturationprocess is observed. •The detection limit and sensitivity for protein determination by DSTD improves in the case of denatured proteins


29

05

1015

2025

12

34

56

78

Ovalbumin

Myoglobin

Cytochrome C

Drop Time, seconds

Surf

ace

Pres

sure

Elution Time, Minutes

Dynamic Surface Tension Detection of Denatured Globular Proteins separated by liquid chromatography (LC)

SEC-DSTD of proteins with post-column denaturation

ACS Symposium Series 893," A. M. Striegel Ed.; American Chemical Society: Washington, DC; BOOK CHAPTER N.16, 2004.

30Native proteins= NDNative proteins= ND

Denatured proteinsLODm 4 – 11 μg

Denatured proteinsLODm 4 – 11 μg

HIC-DSTD separation of a mixture of standard globular denatured proteins (3.0 M GdmSCN).

0 10 20 30 40

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

α-chyβ-CN

Myoα-lac

Elution time, minD

rop

Tim

e, s

ec

???

Dynamic Surface Tension Detection of Denatured Globular Proteins separated by liquid chromatography (LC)

Denaturing agentsDenaturing agents

31

0 10 20 30 40 50

0

5

10

15

1234

Surf

ace

Pres

sure

, dyn

/cm

Dro

p Ti

me,

sec

onds

β-caseinα-casein


HIC-DSTD separation of caseins in milk

0 10 20 30 40 500.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

α-CNβ-CN

Elution time, min

Dro

p Ti

me,

sec

J Chromatography A 1023 (2004) 79-91.

Proteins in milk:βLactoglobulin NO surface activityα-lactalbumin NO surface activityκ-casein \ modest surface activityα- and β-caseins HIGH surface activity

32

0 10 20 30 40 500

5

10

15

20

α-CN

β-CN

Πm

ean,

dyne

s/cm

Elution Time, min

Cow’s milk

0 10 20 30 40 5005

1015202530

β-CN

Πm

ean,

dyne

s/cm

Elution Time, min

Goat’s milk

HIC-DSTD separation of caseins in raw milk (1:10 dilution)

Minutes0 10 20 30 40 50

mA

U

0

200

400

600

800

7.42

36.8

5

41.0

8

38.3

5

Minutes

0 10 20 30 40 50

mA

U

0

200

400

600

800 7.23

23.9

8

36.5

3

39.6

241

.30

DSTD signal DSTD signal

UV @280nm UV @280nm

33

Waste

PumpSample

Injection Valve

B. Capillary Sensing

Tip and Drop

C. Air Burst Capillary

E. Solenoid Valve

A. Pressure Sensor

Po

D. Air Supply

Computer

(Pneumatic Detachment)

Drop Collection Vessel

Mixing Coil

60 μL/min pH meter30 μL/min

30 μL/min

pH Gradient System

High-Throughput Screening of Protein Surface Activity via Flow Injection Analysis-pH Gradient-Dynamic Surface Tension DetectionHigh-Throughput Screening of Protein Surface Activity via Flow Injection Analysis-pH Gradient-Dynamic Surface Tension Detection

Analytical Chemistry 77 (2005) 250-258

34

High-Throughput Screening of Protein Surface Activity via Flow Injection Analysis-pH Gradient-Dynamic Surface Tension DetectionHigh-Throughput Screening of Protein Surface Activity via Flow Injection Analysis-pH Gradient-Dynamic Surface Tension Detection

0 2 4 6 8 10 122468

1012

pH

Time (min)

10 pH units in 12 min!10 pH units in 12 min!

Analytical Chemistry 77 (2005) 250-258

35

DSTD in the on line process controlE.g.: Production/purification of recombinant proteins (vaccines,...)

Process flow

on line sampling

PumpDSTD

Reagent reservoir (e.g. denaturant, acid buffer…)

0 5 10 15 20 25 30012345

Surf

ace

pres

sure

Time, minutes

Mixing T

E.g.: β-LG but not Lysis surface active in 5.0 M urea

DSTD signal is more specific than UV signal. Thus, if the surface tension properties of the denatured analytes and possible interferents in a process are know, the process can be monitored from a qualitative and quantitative point of view.

DSTD signal is more specific than UV signal. Thus, if the surface tension properties of the denatured analytes and possible interferents in a process are know, the process can be monitored from a qualitative and quantitative point of view.

Pump

36

Outline

EXPLOITED APPLICATIONS (12 publications)FIA-DSTD study of polymer formulationsFIA-DSTD study of metal-surfactant interactionsFIA-DSTD study of the effect of denaturing agents on globular proteinsLC-DSTD for the study of PEGs and proteins (globular proteins and caseins)FIA-pH gradient-DSTD analysis for a high-throughput screening of Protein Surface Activity

DESCRIPTION OF THA INSTRUMENTAL SET UP DESCRIPTION OF THA INSTRUMENTAL SET UP Dynamic Surface Tension Detector (DSTD)Dynamic Surface Tension Detector (DSTD)TheoryTheoryOn line calibration of DSTDOn line calibration of DSTDQuality of DSTD informationQuality of DSTD information

YOUR SUGGESTIONS!

IN PROGRESS STUDIES•Development of a new drop detachment system (DDS) for DSTD•Study of PEG-protein interactions•Study of cement-polymer interaction by FIA-DSTD•Study of nanoparticles by FIA-DSTD

37

Development of a new drop detachment system for DSTD. Preliminarydata

Advantages of this DDS:•No critical alignment •low cost ($30 against about $1000)•it can be miniaturized•In perspective it can be adapted to the study of liquid/solid interactions

water

Rotatingmotor

Capillary tip

38


39

water

500 μL loop; 30 μ L/min flow rate; Meantdrop=2.25 sec

Acetic acid 5%

SDS* 5 mM


Pre

ssur

e, p

siP

ress

ure,

psi

Pre

ssur

e, p

siP

ress

ure,

psi

Pre

ssur

e, p

siP

ress

ure,

psi

* Sodium Dodecyl Sulfate

40

0.0 0.5 1.0 1.5 2.00

5

10

15

20

SDS 5 mM

SDS 2 mM

S

urfa

ce P

ress

ure,

dyn

cm

-1

Drop time, sec

Mean tdrop=1.64 sec

0 1 2 3 4 5 6 7 8 9-2

0

2

4

6

8

PEG 35 kDa 100 ppm

SDS 2 mM

PEG 10 kDa 100 ppm

water

Sur

face

Pre

ssur

e, d

yn c

m-1

Drop time, sec

Mean tdrop=8 sec

0.0 0.5 1.0 1.5 2.0 2.5-5

0

5

10

15

20

BSA 1000 ppm, pbs pH 2.5

water

SDS 5 mM

PEG 20 kDa 235 ppm

Sur

face

Pre

ssur

e, d

yn c

m-1

Drop time, sec

Mean tdrop=2.26 sec

Although the cohesive force mask the information on kinetic, this detachment system could be fine for obtaining raw information in the process analysis, answering two questions:i) Are there surface active compounds?ii) What is their concentration?

41

020

4060

80100

20004000

60008000

10000

23

4

5

6

7

PEG MW, Da

Surfa

ce P

ress

ure,

dyn

/cm

PEG, ppm

020

4060

80100

20004000

60008000

1000001234567

PEG MW, Da

Surfa

ce P

ress

ure

dyn/

cm

PEG, ppm

DSTD study of interaction between β-lactoglobulin and polyethylenglycols (PEGs)

β-lactoglobulin 1000 ppmphosphate buffer pH 3.0

β-lactoglobulin 1000 ppmphosphate buffer pH 7.2

PEG concentration range = 0.0 – 100.0 ppmPEG MW range = 1000 – 10.000 Da

42Operating conditions: flow rate 50 ul/min; 4 sec drops; 200 ul loop; Mobile phase: MilliQ water

FIA-DSTD experiments on cement suspension, HSP111 a commercial co-polymer used as additive to cements, and their mixture.

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

0

2

4

6

8

10

12

Cement suspension 10 mg/ml

HSP111 30 ppm

Cement/HSP111 mixture

Time, minutes

Surf

a ce

Pres

sur e

, dy n

cm

-1

43

FIA-DSTD analysis of 10 nm nanoparticles covered by anionic surfactant AOT (sodium bis(2-ethylhexyl)sulfosuccinate)

0 100 200 300 400

20

40

60

80

Πm

ean, d

yn c

m-1

ppm injected

Calibration curve

0 10 20 30 40 50 60 70 80 90 100-10

0

10

20

30

40

50

60

70

80

Elution time

200 ppm

100 ppm

400 ppm

20 ppm

Surf

ace

Pres

sure

, dyn

cm

-1

200 ppm injected

Drop time Elution time

Operating conditions: Flowrate= 60 uL min; mobile phase= MilliQ; 4 sec drops(50 point/sec)

44

AKNOWLEDGEMENTSProf. Robert E. Prof. Robert E. SynovecSynovec, Department , Department of Chemistry, University of of Chemistry, University of Washington, SeattleWashington, Seattle

Massimo Massimo OnorOnor, Roberto , Roberto SpinielloSpiniello, Daniel , Daniel ToncelliToncelli, , CNR, Italian National Research Council, PisaCNR, Italian National Research Council, Pisa

CPAC, Center for Process Analytical Chemistry, SeattleCPAC, Center for Process Analytical Chemistry, Seattle

45

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