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Physical Chemistry Laboratory CHEM 4643 Section 2

October 5, 2006

Lab Partner: Eric P

Intrinsic viscosities were calculated for native and denatured aqueous bovine serum

albumin at 25C. A stock solution of 0.0300 g/ml BSA was prepared and diluted into

15/25ml, 10/25ml, and 7/25ml samples using 0.010M KCl solvent. A portion of the stock

solution was denatured and diluted into 30/50ml, 20/30ml, and 15/30ml samples using

0.010M KCl adjusted to pH 3. Viscosities of the eight solutions and KCl solvent were

found using a Ostwald type viscometer which required calibration. Calibration was

carried out with the use of water. The individual viscosities and their concentrations were

used to graphically determine the intrinsic viscosity. The viscometer constant was

determined to be 1.562*10^-4 3.134*10^-7 cm2/sec2 and the viscosity of the KCl

solution was 8.698*10^-4 8.995*10^-7 kg/sec*m. The native stock and denatured

stock intrinsic viscosities were 2.720.55 kg/m*sec and 8.301.30 kg/m*sec respectively.

Introduction

The purpose of this experiment was to measure the intrinsic viscosities of native and

denatured bovine serum albumin (BSA). The viscosity of a fluid measures its resistance

to flow under an applied shear stress [4]. Generally in fluid flow there is a velocity

difference across the thickness of the fluid that ranges from zero at the point of contact

with a wall or tube to a maximum at the surface of the fluid [3]. It is this velocity

difference across the width of the fluid that induceses shear stresses that resist the motion

of the fluid and give it its characteristic thickness. Fluids with a higher viscosity are

difficult to pour whereas fluids with a low viscosity are easily pourable.

Serum albumin is the most abundant protein in blood and is used to maintain its pH. Two

forms exist; native, the more compact form and denatured, the elongated form. To

measure the viscosity of both the native and denatured forms of BSA we used an Ostwald

viscometer. The viscometer was operated isothermally in a water bath at 25C. Using

the efflux time, or the time that a controlled volume of fluid passes between the upper and

lower marks on the viscometer and the Poiseuille Equation 1, we were able to calculate

the viscosity of our solutions.

/ = Bt (1)

Where is the viscosity, is the fluid density, B is the viscometer constant, and t is the

efflux time. Rearranging equation (1) and utilizing tabulated values for the viscosity and

density of water at 25C the viscometer constant can be calculated.

B = (/)/ t (2)

A potassium chloride solution was used as the solvent for this lab and its viscosity was

calculated using equation 3.

o = Bt (3)

Specific viscosity is the fractional increase in viscosity due to a solute, in the case of this

lab BSA.

sp = (- o) / o (4)

Taking the limit as c goes to zero of the ratio of the specific viscosity and the

concentration is defined as the intrinsic viscosity. Intrinsic viscosity is a measure of a

solutes contribution to the solutions viscosity.

For the purposes of this lab the intrinsic viscosity is estimated graphically by plotting the

specific viscosity divided by the concentration of the respective solution versus the same

concentration. The best fit line to this plot is solved for the intercept or the intrinsic

viscosity. Intrinsic viscosity is sensitive to the shape of the molecules in solution and can

provide rough estimates for various physical attributes such as the effective spherical

radius.

= 10NaRe3/(3M) (6)

weight.

Alistofreagents,supplies,andadditionaldetailsareavailableinthelabhandout[1].

Experimental Methods

Severalreagentswereusedduringthissetofexperiments.Thefirstofwhichwas500ml

of0.010MKCl.Thissolutionwasusedasthesolventforallofthedilutionsinthelab

andwasalteredtodenaturetheBSAafterallofthenativeBSAsolutionswereprepared.

Thesecondwas100mlof0.0300gm/mlnativeBSAstocksolution.BSAwasinsolid

formandrequireddissolvingin80mloftheKClsolvent.Thestocksolutionwasstirred

untilnovisiblesolidBSAwaspresentandwasthenfilteredtoensurenosolidBSA

remained.Usingthenativestocksolutionthreedilutionswereprepared;15/25ml,

10/25ml,and7/25ml.A25mlsampleofthenativestockwasalsosetasideforviscosity

measurements.Next,40mloftheremainingnativeBSAstockwasdenaturedusing60ml

ofKClsolventwhichhadbecorrectedtopH3,anddropwiseadditionof1MHCl.

Oncethedenaturedstocksolutionwasprepared,threedilutionswereprepared;30/50ml,

20/30ml,and15/30ml.Theremainingvolumeofthedenaturedstocksolutionwasalso

retainedforviscositymeasurements.

Afterthesolutionsandtheirdilutionswerecreatedtheirviscositiesweremeasured.The

solutionsviscositiesweremeasuredbypipetting10mlofeachsolutionintothe

viscometerandmeasuringtheeffluxtimes.Theviscometerconstant,KClsolution,stock

BSAsolutions,andBSAdilutionsweredeterminedinthelistedorder.Allmeasurements

wereconductedisothermallyat25Cintheprovidedwaterbath.Asoutlinedabove,

literaturevaluesfortheviscosityanddensityofwaterarereadilyavailableandallowed

ustocalculatetheviscometerconstant.Severalrepetitionsofeachstepwerecompleted

toreduceourerror.Specialcarewastakentocleantheviscometerbetween

measurements.Afivestepwashingmethodthatinvolvedwater,DIwater,soap,and

acetonewaspreformedeachtimeanewsolutionwasintroducedintotheviscometer.

Adetaileddescriptionofthelabprocedures,equipment,andmeasurementdetailscanbe

foundinthelabhandout[1].

Results

Sample calculations for the various steps in this lab are hand written and attached at the

end.

Tabulated concentrations and efflux times for the native BSA and its dilutions are

presented in Table 1.

Native BSA solutions

c (g/mL) t1 (s) t2 (s) t3 (s)

Stock 0.02996 61 62.1 63

15/25 0.017976 59 59.1 59.2

10/25 0.011984 58.1 58 57.9

7/25 0.008389 57.2 57.3 57.1

Tabulated concentrations and efflux times for the denatured BSA and its dilutions are

presented in Table 2.

Denatured BSA solutions

c (g/mL) t1 (s) t2 (s) t3 (s)

Stock 0.011984 65.4 65.5 65.4

30/50 0.00719 60.9 60.8 60.6

20/50 0.004794 58.8 58.7 58.5

15/50 0.003595 57.9 58 57.7

The viscometer constant for each trial, the average viscometer constant, its standard

deviation, and efflux times are presented in Table 3. The constant was calculated using

Equation 2

Viscometer Calibration

B (cm^2/sec^2) w (gm/cm*sec) w (gm/cm^3) t (sec)

1.564E-04 8.949E-03 0.9971 57.4

1.564E-04 8.949E-03 0.9971 57.4

1.558E-04 8.949E-03 0.9971 57.6

1.562E-04 Average

3.134E-07 Standard Deviation

The KCl solvent viscosity as determined using Equation 3, efflux times, and the accepted

density of solution are presented below in Table 4.

KCl Solvent Viscosity

t

o (kg/m*sec) o (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) (sec)

8.709E-04 8.709E-03 1.562E-04 0.99751 55.9

8.693E-04 8.693E-03 1.562E-04 0.99751 55.8

8.693E-04 8.693E-03 1.562E-04 0.99751 55.8

8.698E-04 8.698E-03 Average

8.995E-07 8.995E-06 Standard Deviation

The native BSA solution viscosities as determined using Equation 3, efflux times, and the

accepted density of solution are presented in Table 5.

Native BSA solutions: Stock

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)

9.503E-04 9.503E-03 1.562E-04 0.99751 61

9.675E-04 9.675E-03 1.562E-04 0.99751 62.1

9.815E-04 9.815E-03 1.562E-04 0.99751 63

9.664E-04 9.664E-03 Average

1.560E-05 1.560E-04 Standard Deviation

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)

9.192E-04 9.192E-03 1.562E-04 0.99751 59

9.207E-04 9.207E-03 1.562E-04 0.99751 59.1

9.223E-04 9.223E-03 1.562E-04 0.99751 59.2

9.207E-04 9.207E-03 Average

1.558E-06 1.558E-05 Standard Deviation

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)

9.051E-04 9.051E-03 1.562E-04 0.99751 58.1

9.036E-04 9.036E-03 1.562E-04 0.99751 58

9.020E-04 9.020E-03 1.562E-04 0.99751 57.9

9.036E-04 9.036E-03 Average

1.558E-06 1.558E-05 Standard Deviation

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)

8.911E-04 8.911E-03 1.562E-04 0.99751 57.2

8.927E-04 8.927E-03 1.562E-04 0.99751 57.3

8.896E-04 8.896E-03 1.562E-04 0.99751 57.1

8.911E-04 8.911E-03 Average

1.558E-06 1.558E-05 Standard Deviation

The denatured BSA solution viscosities as determined using Equation 3, efflux times, and

the accepted density of solution are presented in Table 6.

Denatured BSA solutions: Stock

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)

1.019E-03 1.019E-02 1.562E-04 0.99751 65.4

1.020E-03 1.020E-02 1.562E-04 0.99751 65.5

1.019E-03 1.019E-02 1.562E-04 0.99751 65.4

1.019E-03 1.019E-02 Average

8.995E-07 8.995E-06 Standard Deviation

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)

9.488E-04 9.488E-03 1.562E-04 0.99751 60.9

9.472E-04 9.472E-03 1.562E-04 0.99751 60.8

9.441E-04 9.441E-03 1.562E-04 0.99751 60.6

9.467E-04 9.467E-03 Average

2.380E-06 2.380E-05 Standard Deviation

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)

9.160E-04 9.160E-03 1.562E-04 0.99751 58.8

9.145E-04 9.145E-03 1.562E-04 0.99751 58.7

9.114E-04 9.114E-03 1.562E-04 0.99751 58.5

9.140E-04 9.140E-03 Average

2.380E-06 2.380E-05 Standard Deviation

(kg/m*sec) (gm/cm*sec) Bave (cm^2/sec^2) sln (gm/cm^3) t (sec)

9.020E-04 9.020E-03 1.562E-04 0.99751 57.9

9.036E-04 9.036E-03 1.562E-04 0.99751 58

8.989E-04 8.989E-03 1.562E-04 0.99751 57.7

9.015E-04 9.015E-03 Average

2.380E-06 2.380E-05 Standard Deviation

The specific viscosities and data used for the graphical determination of the intrinsic

viscosities for all BSA solutions are presented in Table 7.

Table 7: Specific Viscosities and Data used for Graphical Determination of Intrinsic Viscosity

Intrinsic Viscosity Plotting Data and Specific Viscosities of BSA Solutions

C sp/C

(gm/ml) (ml/gm) sp

Native BSA solutions: Stock 0.02996 3.706 1.110E-01

Native BSA solutions: 15/25 0.017976 3.255 5.851E-02

Native BSA solutions: 10/25 0.011984 3.238 3.881E-02

Native BSA solutions: 7/25 0.008389 2.918 2.448E-02

Denatured BSA solutions: 30/50 0.00719 12.288 8.836E-02

Denatured BSA solutions: 20/50 0.004794 10.586 5.075E-02

Denatured BSA solutions: 15/50 0.003595 10.130 3.642E-02

The graphical determination of intrinsic viscosity for native BSA is displayed in Figure 1.

A linear regression was also performed and is presented as Table 8.

Native BSA Linear Regression

Coefficients Standard Error Lower 95% Upper 95%

Intercept 2.7171 0.1283 2.1648 3.2692

X Variable 1 32.924 6.7760 3.7687 62.078

The graphical determination of intrinsic viscosity for denatured BSA is displayed in

Figure 2. A linear regression was also performed and is presented as Table 9.

Denatured BSA Linear Regression

Coefficients Standard Error Lower 95% Upper 95%

Intercept 8.3046 0.3015 7.0069 9.6022

X Variable 1 512.76 39.666 342.08 683.42

The intrinsic viscosities for the native and denatured BSA and the effective spherical

radius are presented in Table 10.

(ml/gm) Re (nm)

Native 2.7171 3.06

Denatured 8.3046 4.44

Discussion

The purpose of this lab was to determine the intrinsic viscosities of native and denature

bovine serum albumin. Values obtained in lab, error estimates and literature values [2]

can be found in Table 11.

Lab Results Literature2

Students Accepted

(ml/gm) 95% CI (ml/gm) 95% CI (ml/gm)

Native 2.72 0.55 3.4 0.6 3.7

Denatured 8.30 1.30 11 4 11

Our results are similar to those of the students found in [2], but differ slightly from the

accepted values. This can be demonstrated because the error associated with each value

causes their ranges to overlap indicating no difference between means. This is best

demonstrated in graphical form. Figure 3 displays our values, the students values, and

the accepted values. Overall we showed that the native BSA has a lower viscosity than

the denatured BSA.

In addition to the intrinsic viscosities the effective spherical radius was also calculated.

We showed that the effective spherical radius for the denatured BSA (4.44nm) was larger

than the native BSA (3.06nm). This agrees well with the results of the viscosity tests

because the denatured solution had a higher viscosity due to its larger molecule size.

The calibration of the viscometer with water resulted in a constant of 1.562*10^-4

3.134*10^-7 cm2/sec2.Thisvalueiscrucialtotherestofthelabandthusseveral

repetitionsoftheeffluxtimeswereconducted.Theerrorassociatedwiththeconstantis

threeordersofmagnitudesmallerwhichisgood.

TheviscosityoftheKCLwasalsocrucialtothesuccessofthelabresults.Theaverage

valuewas8.698*10^-4 8.995*10^-7 kg/sec*m. Thisvalueiscrucialtotherestofthe

labandthusseveralrepetitionsoftheeffluxtimeswereconducted.Theerrorassociated

withtheconstantisthreeordersofmagnitudesmallerwhichisgood.

Some of the differences between our data and the accepted values and those of other

students could stem from a number of sources. This lab is very sensitive to temperature,

timing, and concentration. The use of the water bath allowed us to control the

environment in which we ran the viscosity tests. However, the water temperature did

change due to convective losses to the room and conductive losses to the beakers and

viscometer we place in the bath. If the bath were insulated there may be better

temperature control, however this would make it difficult to observe the viscometer.

Secondly, the measurement of the efflux times is done by hand. This introduces a lot of

human error. Finally, the change in concentration of the stock solution due to filtering

may also affect the end results. When filtering the BSA solution it was observed that it

foams easily and that not all of the BSA dissolves into solution. There are no doubt

losses in this step of the procedure.

Finally it should be noted that during the course of the two week lab we encountered

troubles. We had several human errors that ultimately left us with unusable data. As a

result, I spoke with Dr. Siders and was supplied with data to conduct the analysis. The

report and data analysis were conducted as if the data was my own to avoid confusing

wording and nomenclature. In the future we need to pay more attention to unit

conversions and what glassware is supplied with the lab.

References

Viscosity of Aqueous Bovine Serum Albumin, 2006

Chemistry Experiment Using Molecular-Level Models in the

Interpretation of Macroscopic Data Obtained from Simple Measurements.

Journal of Chemical Education 1993, 70(8), 685-689.

Thermodynamics, & Kinetics, Benjamin Cummings, 2005.

Bike, 1999

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