Ultra- centrifugation (Gradient centrifugation), Viscometry, UV-VIS

absorption spectroscopy, Mass spectrophotometery

Mitesh Shrestha

Sedimentation

• Sedimentation describes the motion of molecules in solutions or particles in suspensions in response to an external force such as gravity, centrifugal force or electric force.

Sedimentation Theory

solvent

solvent

Ff: the Frictional Force

= f * v

FC: The Centrifugal Force

=M0* 2* r

FB: the Buoyant Force (Archimedes)

=Mw* 2* r

Centrifugal force = buoyant force + frictional force

1. The Centrifugal Force

• Mo is the particle weight, or molecular weight

• (omega)= angular velocity (radians/sec)

• r is the radius of rotation

This equation says that the larger the molecule, or the faster the centrifugation, or the longer the

axis of rotation, the greater the centrifugal force and the rate of sedimentation.

Fc = M * w * r 0

2

The Centrifugal Force

A more common expression is the relative centrifugal force (RCF):

r = Radial distance of particle from axis of rotation

rpm = Revolutions per minute

RCF reports centrifugal force relative to earth’s gravitational force; commonly refer to as “number times g.” A sample rotating at 20, 000 rpm with r = 7 cm will experience RCF=

33,000 x g.

2

1000**18.11

rpmrRCF

2.The Buoyant Force

• The buoyant force opposes the centrifugal force. • where Mw is the mass of the solvent displaced by the

particle. The net force= (Fc-FB) will determine whether a

particle floats or sediments

• Particles with higher density will experience smaller buoyant force, and thus, sediment faster.

FB = Mw * w * r 2

The Frictional force

Frictional force (resistance of a molecule to movement)

• v = velocity relative to the centrifuge tube, • f = frictional coefficient.

Ff = f V

• The frictional coefficient depends upon:

1. the size

2.shape of the molecule,

3.the viscosity of the gradient material.

• The frictional coefficient f of a compact particle is smaller than that of an extended particle of the same mass.

Centrifuge

• Centrifuge is a piece of equipment, generally driven

by a motor, that puts an object in rotation around a

fixed axis, applying a force perpendicular to the axis.

The centrifuge works using the sedimentation

principle

• Theory: The amount of acceleration to be applied

to the sample, rather than specifying a rotational

speed such as revolutions per minute.

Centrifuge rotors

Fixed-angle

axis of rotation

At rest

Swinging-bucket

g

Spinning g

Geometry of rotors

b c

rmax

rav

rmin

rmax

rav

rmin

Sedimentation path length

axis of rotation

a

rmax rav rmin

k’-factor of rotors

• The k’-factor is a measure of the time taken for a particle to sediment through a sucrose gradient

• The most efficient rotors which operate at a high RCF and have a low sedimentation path length therefore have the lowest k’-factors

• The centrifugation times (t) and k’-factors for two different rotors (1 and 2) are related by:

2

211

k

tkt

Calculation of RCF and Q

2

100018.11

QrxRCF

r

RCFQ 299

RCF = Relative Centrifugal Force (g-force) Q = rpm; r = radius in cm

gd

vlp

18

)(2

Velocity of sedimentation of a particle

v = velocity of sedimentation d = diameter of particle

p = density of particle l = density of liquid

= viscosity of liquid g = centrifugal force

Differential centrifugation

• Density of liquid is uniform

• Density of liquid << Density of particles

• Viscosity of the liquid is low

• Consequence:

Rate of particle sedimentation depends mainly on its size and the applied g-force.

Differential centrifugation of a tissue homogenate (I)

1000g/10 min

Decant supernatant

3000g/10 min etc.

Differential centrifugation of a tissue homogenate (II)

1. Homogenate – 1000g for 10 min

2. Supernatant from 1 – 3000g for 10 min

3. Supernatant from 2 – 15,000g for 15 min

4. Supernatant from 3 – 100,000g for 45 min

• Pellet 1 – nuclear

• Pellet 2 – “heavy” mitochondrial

• Pellet 3 – “light” mitochondrial

• Pellet 4 – microsomal

Differential centrifugation (IV)

• Poor resolution and recovery because of:

• Particle size heterogeneity

• Particles starting out at rmin have furthest to travel but initially experience lowest RCF

• Smaller particles close to rmax have only a short distance to travel and experience the highest RCF

Differential centrifugation (VI)

• Rate of sedimentation can be modulated by particle density

• Nuclei have an unusually rapid sedimentation rate because of their size AND high density

• Golgi tubules do not sediment at 3000g, in spite of their size: they have an unusually low sedimentation rate because of their very low density: (p - l) becomes rate limiting.

Density Barrier Discontinuous Continuous

Density gradient centrifugation

How does a gradient separate different particles?

Least dense

Most dense

Applied Centrifugation • Parameters you need to know:

1. Type of rotor:

• fixed angle, swinging bucket, vertical

2. Type of centrifuge:

• Low speed , high Speed, ultracentrifuge

3. Type of centrifugation

• Differential, preparative, or analytical

– Also, the Speed and duration of centrifugation

1. Types of Rotors

swinging bucket rotors: * Longer distance of travel may allow better separation* Excellent for gradient centrifugation * Easier to withdraw supernatant without disturbing pellet

.

fixed-angle rotors:* Sedimenting particles have only short distance to travel before pelleting* Excellent for fractionation purposes* The most widely used rotor type.

.

Other types include vertical rotors and continuous-flow rotors

2. Type of Centrifuge 2-1.Low-speed centrifuges • Also called: microfuge, Clinical, Table top or

bench top centrifuges

• Max speed ~ 20,000 rpm

• Operate at room temperature

• Fixed angle or swinging bucket can be used

• Commonly used for rapid separation of coarse particles

– E.g. RBC from blood, DNA from proteins, etc. – The sample is centrifuged until the particles are

tightly packed into pellet at the bottom of the tube. Liquid portion, supernatant, is decanted.

2-2. High-speed Centrifuges

Preparative centrifuges. • Max speed ~ 80,000 rpm

• Often refrigerated, and requires vacuum to operate

• Fixed angle or swinging bucket can be used

• Generally used to separate macromolecules (proteins or nucleic acids) during purification or preparative work.

• Can be used to estimate sedimentation coefficient and MW, – No optical read-out

2-3. Ultracentrifuge The most advanced form of

centrifuges: (specialized and expensive)

• Used to precisely determine sedimentation coefficient and MW of molecules, Molecular shape, Protein-protein interactions

• Uses very high speed and/or RCF • Uses small sample size • Uses relatively pure sample • Built in optical system to analyze

movements of molecules during centrifugation

Analytical Ultracentrifuge

Ultracentrifuge

• The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high as 1,000,000 g (9,800 km/s²).

• There are two kinds of ultracentrifuges, the preparative and the analytical ultracentrifuge.

• Both classes of instruments are used in molecular biology, biochemistry and polymer science.

3. Types of Centrifugation

There are basically three modes of

centrifugation

3-1.Differential or pelleting

– Cellular fractionation and/or separating coarse

suspension

– removal of precipitates

– crude purification step

3-2. Preparative or Density gradient

centrifugation:

– Separation of complex mixtures

– Finer fractionation of cellular components

– Purification of proteins, nucleic acids, plasmids

– Characterization of molecular interactions

3-3. Analytical

– determining hydrodynamic or thermodynamic

properties of biomolecules in the absence of solid

supports (vs. electrophoresis, chromatography)

–Relative MW

– Molecular shape

–Aggregation behavior

– Protein-protein interactions

Analytical Ultracentrifugation – Applications

• determine sample purity

• characterize assembly and disassembly mechanisms of biomolecular complexes

• determine subunit stoichiometries

• detect and characterize macromolecular conformational changes

• measure equilibrium constants and thermodynamic parameters for self- and hetero-associating systems characterize the solution-state behavior of macromolecules under various conditions

• Viscosity is a property that represents the internal resistance of a fluid to motion.

• The force a flowing fluid exerts on a body in the flow direction is called the drag force, and the magnitude of this force depends, in part, on viscosity.

Viscosity

Viscosity Measurement

• A viscometer (also called viscosimeter) is an instrument used to measure the viscosity of a fluid.

• For liquids with viscosities which vary with flow conditions, an instrument called a rheometer is used.

• Viscometers only measure under one flow condition.

U-tube viscometers

• Glass capillary viscometers or Ostwald viscometers.

Falling sphere viscometers

Bubble viscometer

Ultraviolet–visible spectroscopy

• Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region.

• Uses light in the visible and adjacent (near-UV and near-infrared [NIR]) ranges.

Ultraviolet–visible spectroscopy

• Molecules containing π-electrons or non-bonding electrons (n-electrons) can absorb the energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals.

• The more easily excited the electrons, the longer the wavelength of light it can absorb.

• Based on the fact of four type of transition- π-π*,n-π*,σ-σ*,n-σ*.

• The energy required for various transitions obey the following order σ-σ*>n-σ*>π-π*>n-π*.

Ultraviolet–visible spectroscopy

• The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length.

• Thus, for a fixed path length, UV/Vis spectroscopy can be used to determine the concentration of the absorber in a solution.

• It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration curve.

Assumptions

42

UV Spectroscopy

II. Instrumentation and Spectra A. Instrumentation

1. The construction of a traditional UV-VIS spectrometer is very similar to an IR, as similar functions – sample handling, irradiation, detection and output are required

2. Here is a simple schematic that covers most modern UV spectrometers:

sam

ple

re

fere

nc

e d

ete

cto

r

I0

I0 I0

I

log(I0/I) = A

200 700 l, nm

monochromator/

beam splitter optics

UV-VIS sources

43

UV Spectroscopy

II. Instrumentation and Spectra A. Instrumentation

3. Two sources are required to scan the entire UV-VIS band: • Deuterium lamp – covers the UV – 200-330 • Tungsten lamp – covers 330-700

4. As with the dispersive IR, the lamps illuminate the entire band of UV or

visible light; the monochromator (grating or prism) gradually changes the small bands of radiation sent to the beam splitter

5. The beam splitter sends a separate band to a cell containing the sample solution and a reference solution

6. The detector measures the difference between the transmitted light through the sample (I) vs. the incident light (I0) and sends this information to the recorder

44

UV Spectroscopy

II. Instrumentation and Spectra A. Instrumentation

7. As with dispersive IR, time is required to cover the entire UV-VIS band due to the mechanism of changing wavelengths

8. A recent improvement is the diode-array spectrophotometer - here a prism (dispersion device) breaks apart the full spectrum transmitted through the sample

9. Each individual band of UV is detected by a individual diodes on a silicon wafer simultaneously – the obvious limitation is the size of the diode, so some loss of resolution over traditional instruments is observed

sam

ple

Polychromator

– entrance slit and dispersion device

UV-VIS sources

Diode array

Components of instrumentation:

• Sources: Agron, Xenon, Deuteriun, or Tungsten lamps

• Sample Containers: Quartz, Borosilicate, Plastic

• Monochromators: Quarts prisms and all gratings

• Detectors: Pohotomultipliers

Biological chromophores 1. The peptide bonds and amino acids in proteins

• The p electrons of the peptide group are delocalized over the carbon, nitrogen, and oxygen atoms. The n-p* transition is typically observed at 210-220 nm, while the main p-p* transition occurs at ~190 nm.

• Aromatic side chains contribute to absorption at l> 230 nm

2. Purine and pyrimidine bases in nucleic acids and

their derivatives

3. Highly conjugated double bond systems

Effects of solvents

• Blue shift (n- p*) (Hypsocromic shift) – Increasing polarity of solvent better solvation of

electron pairs (n level has lower E) – peak shifts to the blue (more energetic) – 30 nm (hydrogen bond energy)

• Red shift (n- p* and p –p*) (Bathochromic shift) – Increasing polarity of solvent, then increase the attractive

polarization forces between solvent and absorber, thus decreases the energy of the unexcited and excited states with the later greater

– peaks shift to the red – 5 nm

Spectral nomenclature of shifts

APPLICATION OF ULTRAVIOLET/VISIBLE ABSORPTION SPECTROMETRY

Absorption measurements based upon

ultraviolet and visible radiation find widespread

application for the identification and

determination of myriad inorganic and organic

species. Molecular ultraviolet/visible absorption

methods are perhaps the most widely used of all

quantitative analysis techniques in chemical and

clinical laboratories throughout the world.

51

UV Spectroscopy

II. Instrumentation and Spectra D. Practical application of UV spectroscopy

1. UV was the first organic spectral method, however, it is rarely used as a

primary method for structure determination

2. It is most useful in combination with NMR and IR data to elucidate unique electronic features that may be ambiguous in those methods

3. It can be used to assay (via lmax and molar absorptivity) the proper irradiation wavelengths for photochemical experiments, or the design of UV resistant paints and coatings

4. The most ubiquitous use of UV is as a detection device for HPLC; since UV is utilized for solution phase samples vs. a reference solvent this is easily incorporated into LC design

Mass spectrometry (MS)

• Mass spectrometry (MS) is an analytical technique that ionizes chemical species and sorts the ions based on their mass to charge ratio.

• In simpler terms, a mass spectrum measures the masses within a sample. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.

• A mass spectrum is a plot of the ion signal as a function of the mass-to-charge ratio.

• These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules, such as peptides and other chemical compounds.

Mass spectrometry (MS)

• In a typical MS procedure, a sample, which may be solid, liquid, or gas, is ionized, for example by bombarding it with electrons.

• This may cause some of the sample's molecules to break into charged fragments.

• These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same amount of deflection.

• The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier.

• Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio.

• The atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.

How does a mass spectrometer work?

Ion source: makes ions

Mass analyzer: separates ions

Mass spectrum: presents

information

Sample

What does a mass spectrometer do?

1. It measures mass better than any other technique.

2. It can give information about chemical structures.

What are mass measurements good for?

To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers

• Pharmaceutical analysis Bioavailability studies Drug metabolism studies, pharmacokinetics Characterization of potential drugs Drug degradation product analysis Screening of drug candidates Identifying drug targets

• Biomolecule characterization Proteins and peptides Oligonucleotides

• Environmental analysis Pesticides on foods Soil and groundwater contamination

• Forensic analysis/clinical

Applications of Mass Spectrometry