Chemistry Instrumentation SOLO 3

electrophoresis

• migration of charged solutes or particles in a liquid medium under the influence of an electric field

• separation technique used primarily for separating and identifying serum proteins in blood

Iontophoresis

migration of small ions

Zone electrophoresis

• used to separate macromolecules, such as proteins, in a porous support medium

• charged molecules migrate as zones

• migration takes place across a support medium

• sample is mixed with buffer solution

• separated protein zones are generated on the support media known as an electropherogram

• media stained with protein specific stain

• zones are quantified using a densitometer

ampholyte (zwitterion)

molecule that can become either negatively or positively charged based on the pH of the solution in which it resides

isoelectric point

the pH at which a molecule has a net charge of zero

The ability to separate proteins by electrophoresis is based on what?

the characteristics of amino acids

Direction of migration in electrophoresis

• an ampholyte will take a positive charge in a solution more acidic than its isoelectric point and migrates towards the cathode

• an ampholyte will take a zero net charge in a solution at which the pH equals the isoelectric point

• an ampholyte will take a negative charge in a solution more alkaline than its isoelectric point and migrates toward the anode

What are the five bands proteins are separated into during serum protein electrophoresis?

1. Albumin (fasstest)

2. Alpha 1 globulins

3. Alpha 2 globulins

4. Beta globulins

5. Gamma globulins (slowest)

migration inhibition factor phenomenon

at a pH of 8.6, the gamma globulins move toward the cathode, despite the fact they are negatively charged

The rate of migration in serum protein electrophoresis depends on:

• net electrical charge of the molecule (larger=faster)

• size and shape of the molecule

• electrical field strength

• properties of the supporting medium

• temperature of the operation

Basic components of an electrophoresis apparatus

• power supply

• two buffer chambers

• electrodes in buffer

• electrophoretic support media

• wick in buffer

• chamber lid

Power Supply of an Electrophoretic apparatus

• best to use one with a constant current as opposed to xonstant voltage to keep the migration rate relatively constant

• constant voltage power supplies cause a rise in current due to production of heat during electrophoresis

• rise in heat reduces resistance and increases the electromotive force due to:

  • Joule heat: flow of current through a medium that offeres resistance

  • heat causes thermal agitation of ions

  • water loss due to evaporation causes an increase in ion concentration

• temperature of gel must be controlled because heat also has the potential to dentaure proteins

Wick flow of an Electrophoretic apparatus

• caused by movement of buffer into the support medium

• moisture evaporation from the gel from heat generation during electrophoresis causes movement of the buffer into the gel

• gel absorption of buffer to replce the lost moisture affects the migration of sample molecules

• using a lid or cover during electrophoresis can prevent some of this evaporation

• methods that gernerate excessive heat utilize a cooling system during electrophoresis to prevent wick flow and other damage to sample solutes

Buffer of an Electrophoretic apparatus

• carries the applied current

• establishes the pH at which electrophoresis is performed

• determines the electrical charge on the solute

ionic strength

the sum of all charges present, negative or positive

Increasing ionic strength of the buffer results in:

• increases conductance of the support

• increases size of the ionic cloud surrounding a charged molecule

• yields slower migration rates

• yields sharper band separations

Support media of an Electrophoretic apparatus

• cellulose acetae (one of the first, used in isoelectric focusing)

• agarose gel (serum protein electrophoresis)

• polyacrylamide gel (used in PAGE, separates proteins using molecular sieving (m/z and size))

• automated systems

What is the importance of the ionic strength of the buffer used in gel electrophoresis?

a heat labile protien will become denatured if the ionic strength of the solution is too high

In electrophoresis, the best support medium to use because it is thermostable, transparent, and eliminates endosmosis due to the lack of charge is?

polyacrylamide gel

General operations in a conventional electrophoresis include:

• separation

• staining detection

• quantification

Stianing detection in electrophoresis

• serum proteins: amido black coomassie brilliant blue, ponceau S

• isoenzymes: formazan, nitrotetrazolium blue

• lipoprotein zones: fat red 7b, oil red o, sudan black b

• DNA fragments: ethidium brommide (fluorescent)

• CSF proteins: silver nitrate

A reliable and accurate quantification of electrophoresis requires:

• light of appropriate wavelength, may include visible and U.V. light

• linear response of the instrument

• transparent background in the strip being scanned

Usefule features of a densitometer include:

• ability to scan gels of appropriate length

• automatic gain, which chooses the peak of greatest intensity and sets it to full scale

• automatic background zeroing to select lowest point in the scan as a baseline

• variable wavelength control over the range of 400-700nm

• variable slits to allow adjustment of the beam size

• appropriate selectrion of cut points between peaks

• automatic indexing to move the scanner from one strip or sample channel to the next

• ability to measure ultraviolet fluoescence

Endosmosis

• preferential movement of water in one direction through an electrophoresis medium

• occurs due to selective binding of one type of charge on the surface of the medium

• minimal in AGE and PAGE because the surface charge of these gels is low

• much more significant in capillary electrophoresis

electrolysis

chemical decomposition produced by passing an electric current through a liquid or solution containing ions

Buffer technical considerations of electrophoresis

• highly susceptible to bacterial growth

• should be discarded after each use due to electrolysis

Common problems in electrophoresis

• discontinuties (dirty applicators)

• unequal migration of samples across the gel (dirty electrodes)

• distorted protein zones (bent applicators, air bubbles, over application, excessive drying)

• irregularities in sample application (excessively wet media)

Unusual bands in electrophoresis can be caused by?

• hemolyzed specimens (increase beta globulins)

• plasma (fibrinogen band)

• albumin (split zone caused by bis-albuminemia or more than one type of albumin)

Zone electrophoresis

• produces zones of prroteins

• migration depends on the ratio of charge to zise

• agarose, cellulose acetate, and polyacrylamide gel used

Isoelectric focusing

• separates amphoteric compounds sucha as proteins across a gell possessing a pH gradient

• based on separation of different molecules by their charge

• proteins migrate across a gel until they reach a zone that mathces the isoelectric point of the protien

• most commonly used for abnormal hemoglobin screens

immunofixation electrophoresis

detects specific ypes of immunoglobulins or kappa and lamba chains in serum or urine of patients with monoclonal gammopathies such as multiple myeloma or Waldenstrom’s macroglobulinemia

capillary electrophoresis

• uses the basic principles of electrophoresis carried out in a small-bore capillary tube which serves at the electrophoresis chamber

• tube is connected to a detector at its terminal end and to a high-voltage power supply via buffer reservoirs

Capillary electrophoresis buffers must:

• not interfere with the ability to detect the analyte of interest

• maintain the solubility of the analyte

• maintain the buffering capacity through the analysis

• produce the desired separation

Advantages of capillary electrophoresis

• produces efficient heat dissipation

• enhances separation efficiency

• reduces separation time in some cases to less than 1 minutes

two-dimensional electrophoresis

• 1st dimension: charge dependent isoelectric focusing

• 2nd dimension:molecular weight dependent electrophoresis

• used to study families of proteins, look for genetic or diseased based differences in proteins, and study the protein content of cells of various types

proteomics

the identification and quantification of proteins and their posttranslational modifications in a given system or systems

osmosis

process that constitutes movement of solvent across a membrane in response to differences in osmotic pressure across the two sides of the membrane

osmotic pressure

• pressure required to stop osmosis through a semipermeable membrane between a solution and pure solvent

• governs the movement of solvent across membranes that separate two solutions

osmole

1 gram molecular weight of a substance divided by the number of particles into which it dissociates

osmolality

expresses concentrations related to mass of solvent

osmolarity

expresses concentrations per volume of solution

major osmotic substances in normal plsama

• sodium

• chloride

• glucose

  • urea (BUN)

Osmolality formula

mOsm/kg= 2[Na(mmol/L)]+glucose[mg/dl]/18 + urea[mg/dl]/2.8

osmometry

• a technique that measures the concentration of dissolved solute particles in a solution that contribute to the osmotic pressure

• dependent on the number of particles in solution rather than size or charge

• expressed in units of mOsm/kg or /L

• includes osmolarity and osmolality

A solute dissolved in solvent causes the following changes in colligative properties:

• increased osmotic pressure

• lowered vapor pressure

• increases boiling point

• lowered freezing point

Freezing point osmometers

measurement of the freezing point of a solution by supercooling the specimen to -7C

vapor pressure osmometers

• measurement at the specimen’s dew point

• correlation shows more variation at freezing point than dew point so freezing point osmometers more commonly used

factors that affect osmo readings

• lipemic specimens (dont freeze well)

• prescence of lactic acid (increase freezing point)

• volatile substances such as ethanol (decrease freezing point_

Chromatography

a group of separation techniques that sepaate analytes by differential distribution between a stationay phase and a mobile phase

stationary phase in chromatography

a solid or liquid phase that interacts with components of the mobile pahse

mobile phase in chromatography

a gas ro liquid that flows in a chromatographic system and carries the sample past the stationary phase

As mobile phase flows past the stationary phase, the solutes may:

• reside only on the stationary phase (no separation)

• reside only in the mobile phase (migration with the mobile phase)

• distribute between the two phases (differential migration)

Basis of separation

• those solutes with high affinity for the stationary phase reside in the stationary phase and migrate slower

• those solutes with low affinity for the stationary phase reside mostly in the mobile phase and migrate faster

• strongly bound solutes subsequently are displaced from the stationary pahse by changing the physical or chemical nature of the mobie phase

planar chromatography

• separation technique in which the stationary phase is on a thin support such as paper or solid surface

• used in identification of drugs in urine, analysis of amino acid and analysis of lipids in amniotic fluid

• migration of a solute is expressed by its retention facotr value, the ratio of solute migration to solvent front migration

• includes paper and thin layer chromatography

paper chromatography

• the stationary phase consists of a layer of water or a polar solvent coated onto the fibers of a sheet of paper

• separation takes place between the nonpolar mobile phase and the polar stationary phase

thin layer chromatography

• the stationary phase consists of a thin layer of particles of a material such as silica gel that is uniformly spread on a glass plate, plastic sheet, or aluminum sheet

• when the thin layer consists of particles of a small diameter, the technique is known as high-performance thin layer chromatogrpahy (HPTLC)

Retention factor formula

distance from application point to solute center/ distance from application point to mobile pahse front

What phase of chromatography carries the sample?

mobile phase

Column chromatography

• separation technique in which the stationary phase is packed into a tube or is coated on the inner surface of the tube

• includes gas and liquid chromatography

gas chromatography

• gas mobile pahse or carrier gas is used to carry a mixture of volatile solutes through a column containing the stationary phase

• carrier gas is normally nitrogen, argon or helium

• to be analyzed, a compound must be volatile or be made volatile

liquid chromatography

• separation is based on distribution of solutes between a liquid mobile phase and a stationary phase

• ligh performance liquid chromatography (HPLC) uses small diameter particles

Detector in column chromatography

• a device that responds to the prescence of analyte in the mobile phase, the magnitude of which is used to identify and quantify analytes

• flame ionization detector most used in GC

• produces an electronic signal that is plotted as a funcion of time, distance or volume

• results in a chromatogram

chromatogram

a plot of detector resopnse used in column chromatography to the prescence of analyte in the mobile phase as a function of time or mobie phase volume

retention time

time interval between specimen injection and solute reaching the detector

column chromatography Resolution (Rs)

• measure of chromatographic separation

• requires that two peaks have different elution times for the peak centers and sifficiently narrow bandwidths to eliminate or minimize overlap

• incomplete separation occurs when the calculated value is <0.8

• baseline separation is obtained when the value is >1.25

Improved resolution is achieved by enhancing column efficiency by:

• using smaller particles

• changing flow rate

• using a longer column

• minimizing dead volume (volume of tubing, connectors, etc)

Enhanced resolution is achieved by changing the selectivity of separation by altering factors such as:

• the composition of the mobile phase

• stationary phase

• temperature

which component of a gas chromatograph introduces an aliquot of sample to be analyzed into the column?

injector

Chromatographic separations are classified by the chemical or physical mechanism used to separate the solute and include:

• ion exchange

• partition

• adsorption

• size exclusion

• affinity mechanisms

ion exhange chromatography

• based on an exchange of ions between a charged stationary surface and ions of the opposite charge in mobile phase

• cation exchange particles and anion exchane particles

• displacement of ions bound to the stationary phase depends on competition by other ions in the mobile phase

• retention is decreased either by higher ionic strength, adjustment of pH to decrease the charge of analytes, or adjustment of pH to decrease the charge of the stationary phase

cation-exchange particles

contain negatively charged groups in the stationary phase that bind positively charged components in the mobile phase

anion-exchange particles

containe positively charged groups in the stationary phase that bind negatively charged components in the mobile phase

partition chromatography

• differential distribution of solutes between two immiscible liquids is the basis for separation by partitiona chromatography

• one of the immiscible liquids serves as the stationary phase

• as the mobile phase carries the solute through the column, separation occurs

• classified as gas-liquid chromatography or liquid-liquid chromatography

normal phase liquid-liquid chromatography

stationary phase is polar and the mobile phase is relatively nonpolar

reversed phase liquid-liquid chromatography

the stationary phase is nonpolar and the mobile phase is relatively polar

adsorption chromatography

• based on differential adsorption of solutes on the surface of the stationary phase

• hydrogen bonding and hydrophobic interactions mediate separation

• retention depends on the surface area of the stationary phase and the affinity of the solutes for the stationary phase

• In GC, used to separate low molecular weight compounds and compounds that are normally gases at room temperature

size exclusion chromatography

• gel-filtration chromatography

• separates solutes on the basis of their molecular size in solution

• a variety of materials are used as stationary phase for size exclusion

  • beads have pores that allow small molecules to enter and to be retained to a greater extent than large molecules that are excluded from pores

Affinity chromatography

• one component of a highly-specific molecular interaction pairs is immobilized in a stationary phase and is used to capture molecules form the mobile phase

• interaction pairs are strong, highly specific, and may include enzyme inhibitor, hormone receptor, antigen-antibody, or aptamer-ligand

What is the method used for unknown identification of analytes in column chromatography?

comparison of retention time

mass spectrometry

• works by ionizing a target molecule into ions of a specific mass-charge ratio

• separates and measures those ions

• produces a mass spectrum

mass spectrum

the relative abundance of each ion is plotted as a function of its mass-charge ratio

mass chromatogram

abundance pltted at a function of time

mass analysis

the process by which ions are identified according to m/z ratios

resolution (MS)

• the mass of a given compound divided by the width of the corresponding peak

• “full width at half max”

mass accuracy

how accurate the instrument is by comparing measured mass assignment for an ion to the expected mass

sensitivity

what concentrations of an analyte can be detected

ionization sources

• electron ionization

• electrospray ionization (ESI)

• Atmospheric Pressure Chemical Ionization (APCI)

• Matrix Assisted Laser Desorption Ionization (MALDI)

Electrospray ionization

• applicable to a wide range of analytes

• minimal fragmentation of molecular ion

• high molecular weights can be analyzed

• high sensitivity

• ionization suppression (competition for the ions to leave the droplet)

• mobile phase composition can affect sensitivity

Atmospheric Pressure Chemical Ionization (APCI)

• low polarity molecules can be ionized

• wide dynamic range

• mass sensitive detection dependency

• higher buffer concentration allowed

• analytes must be thermally stable

• analytes must be volatile

MALDI

• applicable to a wide range of analytes, especially for large biomolecules

• sample in solid state

• more sophisticated workflows

• fast analysis

• high background for low m/z ratio region from matrix effects

• limited matrix selection for different compound types

Mass analyzer types

• quadrupole

• ion trap

• tandem mass spectrometry (MS/MS)

• high resolution MS

• time of flight

• orbitrap

Quadrupole

• four parallel rods in which the opposite pair of rods are connected to the same DC and RF voltage

• rapidly changing polarity and magnitude of voltage applied to filter by m/z

• full scan or selected ion monitoring

Ion trap

• trap and store ions over time for greater sensitivity

• electric field is adjusted to selectively eject ions based on m/z

Scanning modes

• full scan- detects all ions

• selected ion monitoring- detects ions of one selected m/z

• product ion scans- select ions of one m/z and fragments ions, all ion fragments pass through

• selected reaction monitoring- select ions of one m/z, fragments ions, fragments of only one m/z allowed to pass through

Tandem mass spectrometry

• important technique in clinical and analytical labs

• quantitative analysis, compound identification, structural characterization

high resolution MS

• can measure large number of analytse simultaneously in complex matrices

• calculaate mass to 0.001-0.0001 Da

time of flight

• flight time- time it takes to traverse the flight tube

• commonly used with continuous flow or pulsed ion sources

• high mass accuracy and resolution

MS detectors

• series of dynodes with increasing potential

• signal amplifies to the order of 1 mil or greater

  • includes discrete, continuous and microchannel

faraday cup

ion abundance is so high that it would saturate the output of an electron multiplier

clinical application of MS

• small molecule analysis (toxocology, Vit D, Testosterone, immunosuppressants)

• proteomics (shotgun approach)

• pathogen identification

• point of care