HowTo

How to BiaCalculations

1. Calculations

This document contains a number of formulas used with surface plasmon resonance and
biomolecular interaction analysis. For the convenience of the reader, an Excel spreadsheet
(BiaCalculations.xlsm) is available on www.sprpages.nl that facilitates the use of these
formulas.

The abbreviations used in the formulas are:

Name Meaning
Da Dalton
RU response units
M Molar
Mr molecular mass in Da
R response in RU
Req equilibrium response in RU
Rmax maximal response in RU
-1 -1
ka association rate constant in M s
-1
kd dissociation rate constant in s
KD equilibrium constant in M
2 -1
D diffusion constant in m s

The units used in the formulas are:

Symbol Name SI units

-2
N Newton kg m s
-2
Pa Pascal Nm
J Joule Nm
 Liquid viscosity Poise = Pa s

The constants used in the formulas are:

Symbol Name SI units

23 -1
NA constant of Avogadro 6.0220 10 mol
-1 -1
R Gas constant 8.3144 J mol K
-23 -1
k constant of Boltzmann 1.3807 10 J K
-34
h constant of Planck 6.6262 10 J s
8 -1
c speed of light (vacuum) 2.9979 10 m s

Each worksheet is divided in two parts:

(yellow) input of data
(orange) output of calculations

Please note that in order to obtain useful results, the input values should be in the correct units
(as indicated per input cell).

Last update: February 29, 2016

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2. Conversions

2.1. Response to concentration conversion

The surface concentration Gamma (G), for a protein can be calculated by the following
formula (3). One Response Unit (RU) in the Biacore 2000/3000 machine corresponds to a
-6 -2
surface coverage of 10 g m for a typical protein. A 100 kDa protein generating a response
-8 -2
of 1000 RU, corresponds to a surface coverage of 10 moles m .

Formulas

Response  106
Gamma  -2
(mole.m ) (1)
Mr

response
surface density  (g m 2 ) (2)
1000000

surface density
binding sites  (mol m 2 ) (3)
molecular mass

response
concentration  (mol L1 ) (4)
100 * molecular mass

Input
Response 500 RU
Molecular mass 60000 Da

Calculated
-2
Binding sites 0.0083 pmol mm
-2
Binding sites 8.3E-09 mol m

-2
Surface density 0.5 ng mm
-2
Surface density 0.0005 gm

-1
Concentration 8.3E-05 mol L
-1
Concentration 5 mg ml

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2.2. Concentration calculation

-1
Calculate molar concentrations based on mg ml and molecular mass and calculate dilutions.

Stock concentration
-1
Protein mg ml Da M µM
A 2.7 150000 1.80E-05 18.00
B 4.66 150000 3.11E-05 31.07

Pre dilutions
stock volume end vol. conc
µM µl µl nM
A 18.00 10 720.0 250
B 31.07 10 310.7 1000

Use this table to calculate the concentration of small compounds when dissolving small
quantities in small volumes.

compound amount Mw amount conc volume conc

gram Da Mol M ml M

T101 0.005 248.77 2.01E-05 2.01E-05 0.5 4.02E-02

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2.3. Calculating immobilization

The table calculates the amount of ligand to immobilize to obtain a theoretical analyte
response. In general, a Rmax of 50 – 100 RU when the analyte is injected is sufficient (6).
Immobilization of too much ligand can lead to mass transfer limitation and non-Langmuirian
kinetics due to steric hindrance (4, 8).

Formula
Mrligand  Rmax
Rligand = (RU) (5)
Mranalyte  Valencyligand

Ligand
Molecular mass 60000 Da
Valency 1
Activity 100 %

Analyte
Molecular mass 2000 Da
Desired Rmax 100 RU

Calculations
Ligand immobilization 3000 RU
-2
Ligand sites 0.05000 pmol mm

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2.4. Calculations after immobilization

The table calculates some parameters after immobilization of the ligand and injection of the
analyte. The theoretical Rmax is calculated based on the molecular mass and amount of
immobilized ligand and the molecular mass of the analyte. The same is done for the ligand
sites and concentration. The amount of functional ligand (the ligand that is binding the analyte)
is calculated on the basis of the measured Rmax (2).

Formulas
Mranalyte  Rligand  Valencyligand
Rmaxanalyte  (RU) (6)
Mrligand

Responseligand
Ligandsites  (pmol.mm )
-2
(7)
Mrligand  Valencyligand

Rligand
Ligand concentration  -1
(mol.l ) (8)
100  Mrligand

Rmax Mrligand
Ligandfunctional    100 (%) (9)
Rligand Mranalyte

Ligand
Molecular mass 40000 Da
Immobilization 111 RU
Valency 1

Analyte
Molecular mass 92000 Da
Rmax 49 Da

Calculations
Rmax analyte 255 RU
2
Ligand sites 0.002775 pmol/mm
Ligand concentration 2.78E-05 mol/L
Ligand activity 19.2 %

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3. Flow and Flow cell

3.1. Flow rate

The table converts the flow rate (μl/min) in different units.

Formulas

 flow rate(ul/min) 
 
 60 
flowrate(l/s)  -1
(l.s ) (10)
10000

 flow rate(ul/min) 
 
 60 
flowrate(m3/s)  3
(m .s )
-1
(11)
10000000

-1
Flow rate f 25 μl.min

-1
Flow rate 4.17E-07 l.s
3 -1
Flow rate 4.17E-10 m s

3.2. Flow cell

The table calculates the volume of the flow cell and the refresh rate.

Formulas
flowcell volume = l  b  h 3
(m ) (12)

flow rate -1
refresh rate= (s ) (13)
flowcell volume

-1 3 -1
Flow rate f 3 µl min 5.00E-11 m s
Height h 0.05 mm 5.00E-05 m
Width w 0.3 mm 3.00E-04 m
Length l 0.8 mm 8.00E-04 m

3 3
Volume v 1.20E-02 mm 1.20E-11 m
-1 -1
Refresh rate 250 times per min 4.2 times per s

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3.3. Thickness of diffusion layer

The table calculates the thickness of the diffusion layer on basis of the diffusion coefficient
(see below), flow cell dimensions and flow rate (5).

Formula

d=3
D  h 2  w  l  (m) (14)
f

2 -1 2 -1
Diffusion coefficient D 5.00E-11 m s 5.00E-11 m s
-1 3 -1
Flow rate f 3 μl min 5.00E-11 m s
Height h 0.05 mm 5.00E-05 m
Width w 0.3 mm 3.00E-04 m
Length l 0.8 mm 8.00E-04 m

Thickness d 8.4 µm 8.43E-06 m

3.4. Time shift between flow cells

The table calculates the time delay between flow cells of the Biacore 1000, 2000 and 3000
machines.

Formula
0.3
ts = 60 (s) (15)
flow rate

-1
Flow rate f 10 μl min
-1
Flow cell volume v 0.3 μl min

Shift 1.8 s

3.5. Wall shear

The table calculates wall shear based on the flow rate and a constant.

Formula
q  jw  0.0121 -1
(ul.min ) (16)

-1
Flow rate q 25 μl min
Wall shear jw 2066 s-1
Constant f 0.0121

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4. Diffusion

4.1. Diffusion factor for globular proteins

The diffusion coefficient (D) for a globular protein can be approximated by the formula below
(3). The f/fo is the friction factor and can be set to 1.2 if unknown. For proteins with an
increasing degree of asymmetry (less globular), this value will become larger. The v/vo is the
viscosity of the solution relative to water of 20°C. For water, the value is 0.89.

Formula

1.1011  324.3  Mr
 1 
3
D (m .s )
2 -1
(17)
f v 
fo vo 

Molecular mass Mr 50000 Da

Friction factor f/fo 1.2
Solution viscosity v/vo 0.89
Factor 1 1.00E-11
Factor 2 324.3

2 -1
Diffusion factor D 8.27E-11 m s

4.2. Mass transport coefficient (km)

D2  f
km  3 (m.s 1 ) (18)
0.3  h2  w  l

2 -1
Diffusion factor D 6.94E-11 m s
flow rate f 5.00E-11 m/s
flow cell height h 5.00E-05 m
flow cell width w 3.00E-04 m
flow cell length l 8.00E-04 m
factor 1 0.3
factor 2 0.98

-1
km 1.0803E-05 ms

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4.3. Mass transport coefficient (kt)

kt  km  Mr  109 (RU.M 1.s 1 ) (19)

-1
km 1.08E-05 ms
Molecular weight Mr 66500 Da
Factor 1.00E+09

-1 -1
kt 7.18E+08 RU M s

4.4. Dynamic viscosity of water

 B 
 T C  
v  A * 10  
(Pa.s ) (20)

A 2.41E-05 Pa.s
B 247.8 K
C 140 K
T 298 K

v 8.93E-04 Pa.s

The dynamic viscosity of water is 8.90 × 10−4 Pa·s or 8.90 × 10−3 dyn·s/cm2 or 0.890 cP at
about 25 °C.
Water has a viscosity of 0.0091 poise at 25 °C, or 1 centipoise at 20 °C.
As a function of temperature T (K): (Pa·s) = A × 10B/(T−C)
where A=2.414 × 10−5 Pa·s ; B = 247.8 K ; and C = 140 K.[citation needed]

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5. Dilution
Use the dilution table to calculate minimal injection volumes and dilution steps.
Start with the flow rate, injection time and replicates.
Then transport the total volume value to the final volume (you can adjust the final volume).
Adjust the dilution step, highest desired analyte concentration and stock concentration.
In the dilution scheme, the dilutions are given. In addition, some parameters are calculated
from the input of the ka, kd and glycerol values. The column under Req (%) lists the Req value
in percentage of Rmax which can be reached when the injection time is long enough. The
values are green when the fall in the range of 10 – 90% Rmax.

Biacore
T100/200
2000/3000
Inject command KInject Inject Protein
Flow rate 30 30 ul/min Dilution step 2 x
Inject time 2 2 min Final volume 200 µl
Replicates 2 2 x Highest conc. 1000 nM
Extra volume 40 40 ul Stock concentration 8642 nM
Total volume 200 200 ul

Stock buffer mix Final solution KD Req Glycerol

Vial (nM) (µl) (µl) (µl) (nM) (µl) (x) (%) (%)
1 8642.00 46.29 353.7 400.0 1000.00 200.0 10.00 91 11.571
2 1000.00 200.0 200.0 400.0 500.00 200.0 5.00 83 5.786
3 500.00 200.0 200.0 400.0 250.00 200.0 2.50 71 2.893
4 250.00 200.0 200.0 400.0 125.00 200.0 1.25 56 1.446
5 125.00 200.0 200.0 400.0 62.50 200.0 0.63 38 0.723
6 62.50 200.0 200.0 400.0 31.25 200.0 0.31 24 0.362
7 31.25 200.0 200.0 400.0 15.63 200.0 0.16 14 0.181
8 15.63 200.0 200.0 400.0 7.81 200.0 0.08 7 0.090
9 7.81 200.0 200.0 400.0 3.91 200.0 0.04 4 0.045
10 3.91 200.0 200.0 400.0 1.95 400.0 0.02 2 0.023

6. Equilibrium

6.1. Theoretical Req

The table calculates the theoretical Req on basis of the association and dissociation constants
and the analyte concentration. Input of (calculated) values from the first analyte injection can
provide estimates of the range of concentrations needed for the future experiments. The Rmax
is included to visualize the actual data. The column with ‘x KD’ shows the relation between KD
and the analyte concentration.
The analyte concentrations come from the dilution page.

Formulas
ka  C
Req  Rmax (RU ) (21)
ka  C  kd

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Req
Req   100% (%) (22)
Rmax

C
xK D  (23)
KD

-1 -1
ka 1.00E+06 M s
-1
kd 1.00E-02 s

Rmax 100 RU
Rmax 95 %

C C KD Rmax Rmax
(M) (nM) (x) (RU) (%)
1.00E-06 1000.00 100.000 99 99
5.00E-07 500.00 50.000 98 98
2.50E-07 250.00 25.000 96 96
1.25E-07 125.00 12.500 93 93
6.25E-08 62.50 6.250 86 86
3.13E-08 31.25 3.125 76 76
1.56E-08 15.63 1.563 61 61
7.81E-09 7.81 0.781 44 44
3.91E-09 3.91 0.391 28 28
1.95E-09 1.95 0.195 16 16

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6.2. Time to equilibrium

The table calculates the time to reach equilibrium based on the association and dissociation
constants and the analyte concentration. The percentage of Req to reach is set to 95% of the
Rmax. This can be adjusted at the kinetics input.

Formulas
 ln(1  )
t  (s) (24)
k a C  k d

-1 -1
ka 1.00E+06 M s
-1
kd 1.00E-02 s

Rmax 100 RU
Rmax 95 %

C Time Time Time

(M) (s) (min) (hour)
1.00E-06 3 0 0.0
5.00E-07 6 0 0.0
2.50E-07 12 0 0.0
1.25E-07 22 0 0.0
6.25E-08 41 1 0.0
3.13E-08 73 1 0.0
1.56E-08 117 2 0.0
7.81E-09 168 3 0.0
3.91E-09 215 4 0.1
1.95E-09 251 4 0.1

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6.3. Complex half-life

The table calculates the time to dissociate to half the starting value. The complex half-life
depends only on the dissociation constant (1). As a rule of thumb, the dissociation curve
should decrease at least 5% before analysis can be attempted (7).

Formula
ln2
t 12 
-1
(s ) (25)
kd

kd t1/2 t1/2 t1/2

(s-1) (s) (min) (hour)
1.00E-01 7 0.1 0.0
1.00E-02 69 1.2 0.0
1.00E-03 693 11.6 0.2
1.00E-04 6931 115.5 1.9
1.00E-05 69315 1155.2 19.3
1.00E-06 693147 11552.5 192.5

6.4. Time to 5% dissociation

Formula
ln(100  95)
t1/ 2  (s 1 ) (26)
kd

kd t t t
(s-1) (s) (min) (hour)
1.00E-01 1 0.0 0.0
1.00E-02 5 0.1 0.0
1.00E-03 51 0.9 0.0
1.00E-04 513 8.5 0.1
1.00E-05 5129 85.5 1.4
1.00E-06 51290 854.8 14.2

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6.5. Time to 95% dissociation

Formula
ln(100  5)
t1/ 2  (s 1 ) (27)
kd

kd Time Time
(s-1) (min) (hour)
1.00E-01 0.5 0.0
1.00E-02 5.0 0.1
1.00E-03 49.9 0.8
1.00E-04 499.3 8.3
1.00E-05 4992.9 83.2
1.00E-06 49928.9 832.1

7. Kinetic plot
Create a kinetic plot in Excel by adding the values in the table.

8. Report points
Reorder the report points from a Biacore 2000/3000 report point table.

9. Kinetic converter
Convert the Excel notation to the ‘power off’ notation (sort of).

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10. Simulation
Simulate the association and dissociation in Excel.

11. References
1. BIACORE AB; Kinetic and affinity analysis using BIA - Level 1. 1997.
2. BIACORE AB; BIACORE Technology Handbook; 1998.
3. BIACORE AB; BiaSimulation. 1998.
4. Edwards, P. R. and Leatherbarrow, R. J.; Determination of association rate constants by an optical
biosensor using initial rate analysis. Anal.Biochem. (246): 1-6; 1997.
5. Glaser, R. W.; Antigen-antibody binding and mass transport by convection and diffusion to a surface: a
two-dimensional computer model of binding and dissociation kinetics. Anal.Biochem. (213): 152-161;
1993.
6. Karlsson, R., Michaelson, A, and Mattson, L; Kinetic analysis of monoclonal antibody-antigen
interactions with a new biosensor based analytical system. J.Immunol.Methods 229-240; 1991.
7. Katsamba, P. S. et al; Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple
Biacore users. Anal.Biochem. (352): 208-221; 2006.
8. O'Shannessy, D. J. and Winzor, D. J.; Interpretation of deviations from pseudo-first-order kinetic behavior
in the characterization of ligand binding by biosensor technology. Anal.Biochem. (236): 275-283; 1996.

12. Disclaimer

This document is compiled from the publications mentioned in the reference list and my own
research. The document is provided as is in a good scientific manner to share knowledge.
There is no warranty that the content is accurate or up to date.

You may use this document for your own research purposes. You may copy the document as
long as you keep it intact. It is not allowed to use this document or parts of it for commercial
purposes.

For more information go to the SPR Pages at www.sprpages.nl

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