BLD 414 Notes

1/9/2012 6:54:00 PM

Osmolality Osm/Liter Osmole = species in solution (after disassociation) Full Disassociation 1mol NaCl 1mol Na+ + 1mol Cl1 mole in 2 species in solution 1 mole NaCl = 2 Osm/L 1mol CaCl2 1mol Ca2+ + 2mol Cl1 mole in 3 species in solution 1 mole CaCl2 = 3 Osm/L 1 mole Glucose: does not disassociate o Therefore 1 mole = 1 Osm/L Partial Disassociation 1 mole MgSO4 disassociates 60% .4mol MgSO4 ---partial--- .6mol Mg2+ + .6mol SO421 mole MgSO4 = 1.6 Osm/L

Density

Mass/Volume Grams/cm3 cm3 = mL Usually for liquids H2O at 4 degrees Celsius to standardize o 1 gram per 1 cm3

Specific Gravity o For liquids only o Unitless o g/mL (implied) Make 1 liter of 2M MeOH (methanol)

o MeOH = 32 g/mol o SG = .87 o 2mol/L * 32g/mol = 64 g/L needed o .87g/mL = 64g/X X = 73mL QS to 1 liter Percent Purity Focus on liquids Done by weight Make o o o 1 liter of 1M HCl HCl = 36.5g/mol SG = 1.029 Bought 75% pure

o 1mol/L * 36.5g/mol = 36.5g/L o 1.029g/mL * .75 = .772g/mL o .772g/mL = 36.5g/X X = 47.3 mL QS to 1 liter

Dilution Ratio dilution: not common except in microbiology o Part solute : part solvent o 1mL serum : 9mL water 1mL to 9mL Use colon dilution: used more often Part solute : total volume 1mL serum and 9mL water 1/10 = true dilution 1mL in 10mL total volume o Use slash Volumes of Dilution o True o o

o Make 40mL of 1/5 dilution of serum in saline 1/5 = x/40 x = 8mL, add 32mL of water Dilution Factor (DF) o Final concentration = original concentration * DF o DF is the true dilution fraction o Want .5M HCl starting with 2M HCl .5 = 2*DF .5 = 2*(1/x) 1/x = .5/2 x=4 DF =

Independent Dilutions o Non-equivalent dilutions o Have 5M HCl and need 2M, .5M, & .2M Method 1: use this Add same amount of solute to each tube Figure out DF for each Calculate how much solvent to add to each Method 2 Add same amount of solvent to each Ex) add 4mL of water to each x/(4+x) x = volume of solute Serial Dilutions: common o o o o Equivalent dilution factors n = tube number 1/x = DF To calculate concentration of any given tube 1/x ^ n

pH Relative concentration of two species (H+ and OH-)

Kd = limited by the disassociation of water o H2O [H+] * [OH-] o Kd = [H+][OH-] = 10-14 H+ and OH- are always in 1:1 o -14 = logH+ + logOHo 14 = -logH+ - logOH -logH+ = pH [H+] and [OH-] always expressed in M (mole/Liter) o 14 = pH + pOH ex) 3mM HCl, what is pH? Complete disassociation HCl H+ + OH3mM 3mM + 3mM pH = -log[3*10-3] pH = 2.5 ex) pH = 8.5, what is [H+] o Take negative antilog o 10-8.5 = H+ o [H+] = 3.16*10-9 M o o o o

Weak Acids Does not completely disassociate Acetic acid o HAC H+ + ACo Some HAC remains o Kd = ([H+][AC-])/HAC Want [H+] o [H+] and [AC-] are equal (what if not?) o Kd = x2/HAC x Use quadratic equation Get rid of x in denominator if small enough pK = -log[Kd] If pK > 4, disregard x in denominator Ex) Have .5M HAC, what is the pH? o pK = -logKd = 4.76 o Kd = x2/HAC o 10-4.76 = Kd = 1.74*10-5

o 1.74*10-5 = x2/.5 o x = 2.95*10-3 o pH = -log[2.95*10-3] pH = 2.5 Henderson-Heiselbach HAC H+ + AC Kd = ([H+][AC-])/HAC Kd/[H+] = [AC-]/HAC o logKd logH+ = log(AC-/HAC) o logH+ = -logKd + log(AC-/HAC) o pH = pK + log(AC-/HAC) Buffers Solution contain weak acid resists change in pH in presence of added base Buffering Capacity (BC) o From [base] = 0 to end of flat line region o How much base to add to use all available protons Protons from free H+ and from non-disassociated acid o When acid molarity doubles, need 2x more base to reach BC o Middle of flat line: AC- = HAC and pH = pK pK = constant ex) Prepare a 1 liter buffer solution of .1M acetate with pH = 5.2 and pK = 4.76 o pH = pK + log(AC-/HAC) o 5.2 = 4.76 + log(AC-/HAC) o .44 = log(AC-/HAC) o 2.75 = AC-/HAC maintain this unit-less ratio o BC = .1M = [AC-] + [HAC] o [AC-] = .1 [HAC] plug into ratio o 2.75 = (.1 HAC)/HAC [HAC] = .027M o AC = .1 - .027 [AC-] = .073M o Combine [HAC] and [AC-] and QS to 1 liter

Quality Assurance Predictive Value Theory Relevance of testing: epidemiology Diagnostic Sensitivity (%) o Performance of lab test on a known diseased population o Correct lab result: true positive (TP) o Incorrect lab result: false negative (FN) o TP/(TP+FN) Diagnostic Specificity (%) o Performance of lab test on a known non-diseased population o Correct lab result: true negative (TN) o Incorrect lab result: false positive (FP) o TN/(TN+FP) Predictive Value (PV) o Index of degree of confidence associated with a positive or negative result Needed because of the effect of prevalence due to lab performance done in a general population o Positive PV TP/(TP+FP) o Negative PV TN/(TN+FN) o Diagnostic Efficiency (TN+TP)/(TN+TP+FN+FP) Diagnostic Indicators to Make Decisions o High sensitivity needed High PPV o High o High Low NPV specificity needed Low PPV High NPV sensitivity and high specificity needed High PPV High NPV

Normal Reference Range Normal RR = 95% confidence interval on Gaussian curve o Measure of variance: Z-test o %CV = SD/mean * 100% Misrepresentation of normal distribution results in compromising diagnostic sensitivity and specificity o Normal RR too wide Increased specificity Increased false negative Decreased sensitivity Decreased false positive o Normal RR too narrow Increased sensitivity Increased false positive Decreased specificity Decreased false negative Use exclusion criteria to identify normal RR o Disease, drugs, etc. Use partitioning factors to make subclasses of population o Age, gender, diet, etc. o Priori: separate data using known information o Posteriori: analyze collected data then partition o Z-test Requirements o Ensure accuracy and precision, avoid transference o 95% CI of normal population in parametric (Gaussian) distribution Chi Square Goodness of Fit Test

Pass = parametric Pass: median is +/- 3% of mean Can transform non-G to G if Chi2 test has 95% CI o Reference data array n = total frequency Upper limit: (n+1)*.975 Lower limit: (n+1)*.025 Receive Operated Characteristic Curves (ROC Curves)

Cutoffs: dont use 95% confidence interval (x) = 1-specificity, (y) = sensitivity Want to find inflection point o Part of curve that has most change o Highest sensitivity o Lowest false positive o Best diagnostic performance

Quality Control Observed error < allowable error Observed error = accuracy error + precision error o Accuracy: difference between true value and mean (x bar) o Precision: 2 standard deviations out from mean Allowable error o Consensus: government, many labs Analytic error: use mean as true value 2 SD/mean * 100% o Medical Allowable Error: insurance $, professional societies, few labs Weighted average 2 segments/mean * 100% o Use lower error of the two when publishing o Expressed as %, [], or SD from mean Internal and external QC Establish temporary QC values o T-test o Mean +/- allowable error = floating mean o Mean = (temporary) Target o Once n = 200, final target mean established Change in mean is small as n increases o Outdate: due to time with specimen degrading, etc. Concentration of QC Material o One for normal and for one for diseased populations Monitoring Controls o Usually once per day

o More frequent with drift: analytic system change, needs recalibration Detect QC Problems o Using Westgard Rules on Levy-Jennings graph: internal 2 graphs: normal and abnormal, ran simultaneously 12s 13s 22s R4s 41s 10x o External QC: Proficiency Testing Law Certifying agencies Blind specimen, use values determined by allowable error (MAE or consensus) 3 strikes in a row is bad

Method Evaluation Compare observed to allowable error Verify analytic machine technical specifications Analytic Range: Quantitative Test o Standard curve and working range o Want working range as wide as possible May need dilution to achieve this o Upper Limit: Calculate R value Best: R = 1 Look for point of inflection on graph of R values on Yaxis and # of points on X-axis, then use that many data points o Lower Limit Analytic Zone near origin where value is always 0 Functional: sig figs Regression line with 95% CI around it n = same for each x column

Analytic Specificity: Accuracy o Interfering substances o Percent Recovery Make one tube the control and find the [base mean] Measure the [observed] of the sample tubes Report the % shy or over: is it within allowable error? o Paired-Difference: want maximum physiologic concentrations Make one tube the control and find the [base mean] Make a 95% CI (2SD on each side of x) around the [base mean] Measure the [observed] of the sample tubes with different possible interfering substances If [observed] is within the 95% CI around [x], then the substance is not interfering Find point of interference Monitored response on Y-axis [interfering substance] on X-axis Make regression line using the same n for each x Make 95% CI around regression line Point of no overlap

Error o Observed compared to allowable Observed: accuracy and precision/random o MDL: concentrations of substances to diagnose Within run: 2SD / mean = %CV Between run: change everything about assay except specimen Random/Precision Error (RE) Between run between day 2 SD of the observed mean Systematic/Accuracy Error (SE) Compare using reference method vs. candidate Plot reference = x, candidate = y Draw regression: y = mx + b Ideal: y = x Constant error: same slope, different y-int

Proportional error: different slope o Can be both (usually is) Total Error = RE + SE Take TE / [MDL] * 100%

BLD 414 Notes

1/9/2012 6:54:00 PM

LIS: Laboratory Information Systems HIS: Hospital information system o Allows for communication via LAN In-house request o Admission: gather patient info, wristband = barcode o Panel: test most pertinent for situation/condition Pre-prepared/standardized CPOE: Computerized Physician Order Entry o Accession number: _ _ _ _ _ _ _ _ / _ _ (identification) o Compile/sort based on priority Routine: at leisure, 24 hours; time and location Timed: prior to timed test, location; certain analytes at precise times STAT: critical conditions; goes straight to lab without sorting o Compile to batch list List of info per patient, printed after positive identification o Collect specimen, take to lab, sort/ID Conveyor belt with forks o Order any tests necessary via LIS Upload: transfer info from instrument to mainframe Download: transfer info from mainframe to instrument o Data verification Auto: computer verifies normal RR results Also does delta check for intraindividual variation Manual: if failed delta check o Output data Auxiliary features: new/future o Patient and specimen auditing o QC: Westgard rules o Balance department work load

Automation Many accomplishments Computing Relationships

o Integrated: all parts from single vendor; seamless system; limited to what vendor can offer non-modifiable; expensive o Interfaced: multiple vendors; compatibility issues software; flexible modifiable; cheaper; more common Total Lab Auto o No humans involved o Processing/pre-analytical (similar to conveyor belt) o Analytical, post analytical (storage) Partially Auto Labs o Most common Instruments o Analytical Batch analyzer Dedicated system: for one task Multichannel analyzer Group of tests chosen by need Continuous flow uses this Random access Chosen from very large menu of tests Upload and download o Configuration Discrete Each test separated into cuvettes in a chain Tray or wheel Continuous flow Buffer flows continuously Bubble-specimen-bubble Multichannel series and parallel Series: non-destructive no addition of reagent; direct detection Continuous reagent: specimen introduced to reagent

Terms/concepts o Dwell time Time per test Want to verify salesmen claims

 Varies per analyte o Throughput Test in one hour Relates to dwell time Varies per analyte Varies per pipetting rate Wash cycle: more = lower throughput Tests/specimen: more = higher throughput o Turn Around Time Lab TAT Total Specimen arrival to lab until result output to LIS Gathering specimen rate limiting step TAT Test ordered by doctor until result output

o STAT

Highest priority/life-threatening Not all tests offered 1 hour Acquiring specimen rate limiting step

Basic Measurement Express data o Qualitative o Semi-quantitative: relative scale, visual, correlate to values o Quantitative: most data, instruments, standard curve, units Signal = response o Change in monitored value related to change in []: scale o Light-based = lumens o Electronic based = flowing electrons o Physical: partial pressure, osmometry, etc. o Change in signal Amplitude: magnitude Rate of generation Per unit or kinetic Frequency Duration

o Detection Direct: limited Indirect: convert signal to different unit; processor Measurement Scale o Standard curve Interpolation: have signal, find [] Direct (positive) or Indirect (negative) Linear or nonlinear Transform nonlinear to linear by taking log Want to go through origin: blanking o Made from standards Primary: government, NBS, 99% pure, expensive, bought by manufacturers Secondary: used by lab after [] established by manufacturers Factors Effecting Relationship o Drift: stability of standard curve; redo curve when drift seen Change in response over time Upward drift: signal increase Downward drift: signal drop Monitor with QC o Noise Property of electronic analog systems More apparent at high signal Less apparent at low signal Signal : Noise ratio High signal for low detection is good Low noise: good

Big signal: high slope on standard curve o Artifactual output Point error Analytic Systems Review Spectrophotometry 2/10/12 Electromagnetic radiation

o Wavelength: 1 complete cycle; nanometers o E = hc/wavelength More energy = smaller wavelength o Pertinent regions (increasing in wavelength) Gamma: ionize X-ray UV: far = 10-180; near: 180-380 Visible: purple = 380; red = 750 Infrared: generate heat Radiowaves: RFID o Intensity of Light: dim or bright; lumens Bright = higher amplitude Power = ability to do work o Interference Constructive: in phase, positive Same wavelength Both maximums hit together Increase amplitude: brighter Destructive: 180 deg out of phase, negative Max of one hits min of other Negate each other: no light o Diffraction Light bends around edge More bending = less intensity Laser: monochromatic = single wavelength Banding (constructive and destructive) Blaze: closest degree band to original light Highest intensity Shorter wavelength: larger degree of banding Sunset: red glow at start, purple at end o Refraction: light bends moving between states of matter More bend moving from gas to solid More density change = more bend Longer wavelength = bent less Shorter wavelength = bent more o Polarized light

Natural light is omnidirectional Dichroic crystal: layers of glass in single direction Absorb all but one plane of light Omniplanar in uniplanar out Absorption Spectrophotometry: Part 1 o Each molecule has unique absorption o Light hits electron and is absorbed o Electrons close to nucleus has shorter wavelength Higher energy o If photon hitting electron is same wavelength: light absorbed Electron thrown into high energy state o Each peak: electron math at given wavelength o Seen wavelength = no absorbed o White light: total spectrum o Colorimetry Visual Serial dilution of high purity standard used to compare unknown Duboscq Depends on volume (depth) of test tube and concentration of color Pre-light bulb Eyepiece: try to adjust to produce 2 identical (standard & unknown) colors by changing volume (depth) Cs * hs = Cu * hu; find Cu o Beer/Lambert -log(transmittance) = absorbance transmittance: out/in need to linearize data by taking the log used filter absorbance is unit less Molar absorptivity Abs = epsilon * b * concentration Epsilon = liters/mole*cm

b = 1cm; abs = epsilon * concentration want biggest slope of abs vs. conc large signal/noise ratio epsilon at specific wavelength maximum Absorptivity Used when molecular weight unknown a = liters/gram * cm o Quantitative Analysis Direct: compounds that naturally absorb Chromogenic reaction: absorbing substance made through chemical reaction Derivitization: substance binds to analyte, separate free and bound o Determining the [unknown] Standard curve: graph y = mx + b m = epsilon = molar absorptivity b = zero x = concentration: solve for this y = absorbance, measure this Direct: no graph Concentration = absorbance/epsilon Conc = mole/liter Assume 1cm test tube Ratio: common for large medically allowable error Assays with drift Cu = (Absu/Abss) * Cs Limited by upper limit of linearity Big allowable error: 2 point curve o Origin and one other point Small MAE: multipoint curve

o Interference Reagent blank Proteins Find absorbance in absence of analyte Specimen blank

Calcium Find absorbance before adding color reagent Chemical separation 2/15/12: Alan Correction No discrete peak of interferent Abs(a) = Abs(i) [(Abs2 + Abs1)/2] Abs(a) at wavelength max Bichromic/Simultaneous Analysis When interfering peak in same region as analyte

Total absorbance proportion by interferent calculated by equations on handout Absorption Photometry Instrumentation Light source o Intensity of emissions from sources vary of range of useful wavelengths: non-constant emission Wavelength selectors o Optical filter: glass/sandwich o Interference filter: one-way mirrors Mono-chromatic

Diffraction grating: can dial in desired wavelength o Long wavelengths bend less, and vice-versa o Linear separation of wavelengths o Transmission: many edges; increase light intensity Less pivot: more red More pivot: more purple o Reflection Reflection at different angles gives different wavelength Not monochromatic, range instead Like a CD in the sun Refraction: pass between states of matter o Greater change in density: greater bend o Select wavelength by pivot Red: little pivot Purple: more pivot

o Nonlinear distribution of wavelengths Long wavelengths close together: reds Short wavelengths far apart: purple Bandpass o Measure of monochromicity o Bandwidth: wavelength range with zero light intensity o Bandpass: wavelengths associated with max intensity, easy to measure o Narrow bandpass is better (for specificity) o Narrow bandpass gives steeper slope in standard curve of Absorbance vs. Concentration Narrow bandpass: good for lower limit of detection (sensitivity) Higher signal to noise ratio o Wavelength selectors Prism: non-linear = more separated wavelengths better bandpass Grating: linear; best; slit width dependent o Cuvet Material and diameter Absorbing UV is bad Detectors o Want to extend upper limit of linearity, limit noise o Photo Electric Cell Selenium: shift to conductor when light hits Create current: more light = more Amps Not good for dim light for detection Slow to react o Phototube/Vacuum Photo Diode Fast Linear response o PMT Multiplier: 1 e- hits, 6 3- released o Silicone Photo Diode Standardizing o Zeroing

o Use occlude: maintain intensity over wavelength range o Going from dim to bright: apparent absorption is negative Errors in Spectroscopy Incident light is not monochromatic: wind bandpass o Lets many wavelengths through: low sensitivity o Solution: decrease bandpass by decreasing slit width This also decreases light intensity to a limited point Spectral stray light o Outside of absorbed wavelengths Stray Light Instrument o Stray light is constant as concentration of analyte changes o Inappropriate light reaching detector: cuvette or leak Above ideal behavior o Insoluble particles: precipitate does not absorb Fluorescence Molecule in high energy electronic state If light wavelength hits an electron of same wavelength o Transfers energy o Moves electron to different energy orbital Wants to return to ground state Fluorescence o Rapid return to ground state o Longer wavelength emitted than wavelength absorbed o High intensity light Phosphorescence o Slower return to ground state o Low intensity light Internal Conversion o Infrared: heat release o Step-wise return to ground state o No light emitted Photochemical reaction o Unstable o Ion pair created Fluorescent Spectroscopy

o Use absorbed wavelength that hits electron: excitation/primary wavelength o Secondary wavelength emitted Monitored response is intensity o F = IC F is secondary wavelength emitted intensity I is primary wavelength absorbed intensity C is concentration, solve for b is assumed 1cm for Beers law is molar absorptivity is efficiency constant

Direct correlation of intensity of light Fluorescent Spectrophotometer (fluorometer) o Want brightest light source Requires less molecules in cuvette to cause emission Highly sensitive o Selecting for I: primary wavelength o Primary filter: excitation wavelength o Secondary filter: emission wavelength o Sources Mercury: non-continuous intensity Xenon: flicker; better Compensated fluorometer: 2 detectors Calculate mean of I: primary wavelength intensity Produces constant F intensity Quantitative Fluorescent Assay o Want to find concentration o Lower limit of detection and upper limit of linearity o RFU vs concentration Relative fluorescent u_ Direct Assay o Rare o Interfering substances in the way o Hit cuvette with primary, observe secondary Fluorogenic Assay

o Reaction makes fluorescent compound o A+R=F o A-F bound Fluorescent polarization o Common o Basis Di_crystal Absorb light in specific plane of field o o Application: 2/22/12 Competitive-binding assay Bind of antibody increases molecular weight, and slows random Brownian motion Increases chance of emission in same plane as primary wavelength = only those detected Antigen = variable, want to determine Ag-F: created HMW, monitored More Ag, less Ag-F (vice-versa) Time-Delayed

o Extend duration of fluorescence with Europium (Eu) chelates o Phosphorescing Light Scattering Photometry Basic theory o Particulate must be greater than .2 micron to use light scatter o Small particle: back-scatter o Intermediate particle: side-scatter o Large particle: forward-scatter Turbidity o Only non-scattered light detected o Compare light in to light detected More particulates is less light detected o Follows Beers Law o Clinical application for CFU counter and PT times Nephelometer o Detects scattered light

o Do not use wavelength of light that can be absorbed by particulate o Minimize blank reading to improve signal:noise Non-divergent light source Angle of detection Use angle that gives max intensity o Clinical application for immunoassay and flow cytometer PIN detector Tiny barrier cell model with a circuit Hydrodynamic focusing: physical property

BLD 414 Notes

1/9/2012 6:54:00 PM

2/24/12: Reflectance Photometry Quantitate intensity of reflected light Types o Specular Glare: changes reflection = bad Not subject to change in intensity o Diffuse From matte surface Instrumentation: reflectometer o Blank pure porcelain as pure white: max intensity o First blank with black AlO3 to zero light out Reflectance: Beers Law o R value = Rout/Rmax o Linear relationship of density (Dr) vs. concentration Luminescence Assays: monitored response is generation of light (h) o Chemiluminescence A + Reag Product + h More h = more A Not all reactions produce light Correct with H2O2 A + R P + H2O2 Acridinium ester + H2O2 h o Bioluminescence Enzyme catalyzed Fireflies A + enzyme P + h Enzyme: lucerferase Depends on ATP and reduced luciferin = Analyte: ATP ATP + reduced luciferin (enzyme) ADP + oxidized luciferin + h Derivitization o Immunoassay: ELISA o Cannot use area under curve Instrumentation: luminometer

o Has photo multiplier tube o Monitor generation of light 2/27/12: Enzyme Kinetics Correlate enzyme activity to concentration of substrate Michaelis-Menton o Hold enzyme concentration constant o Vary substrate concentration Low substrate concentration: 1st order Intermediate substrate concentration: pseudo 1st order High substrate concentration: zero order Reaches saturation point Vmax o If enzyme concentration changes, rate changes proportionally o Use zero order, high substrate concentration Gives linear relationship o Km = substrate concentration at Vmax Affinity of substrate to enzyme Low Km = high affinity; takes less substrate to saturate enzyme Determining enzyme concentration o Kinetic assay Zero order Linear relationship: ideal Product formed per unit time Initial lag phase: substrate starts occupying enzyme active site Intermediate zero order/linear range phase: only use this Final bend over phase: substrate depletion o Continuous monitor: lab instrument o Fixed period: more practical than continuous Use 2 point slope: assumes constant reaction rate T1 after lag: lower limit

Units o o o

T2 found by running up enzyme concentration until non-linear relationship of product vs. time, then find inflection of slightly less (20%) enzyme concentration of enzyme activity IU: micromole/liter/minute Product formed: monitored by spectrophotometry color change EXAMPLE 1mL serum: specimen Plus 4mL substrate containing buffer DF Dilution factor: 1/5 correlate to enzyme activity T1 = 30sec, Abs = .1 T2 = 2min 30sec, Abs = .4 Change in absorbance: .3 in 2 minutes .15/min Beers Law: .15/molar absorptivity (epsilon: 7500) Gives mole/liter Convert to micromole Multiply by 5 due to DF Answer: 100 IU

3/2/12: Osmometry Units o Osmolarity: osmoles/liter Depends on temperature o Osmolality: osmoles/Kg solution Temperature independent o Osmoles: number of species in solution after disassociation Colligative Properties o Osmolarity increase, freezing point decrease Add salt on icy roads o Osmolarity increase, vapor pressure decrease Add salt to boiling water Vapor Pressure Osmometry o Air-tight chamber o Components o Low osmolality: high vapor pressure, high humidity

o Underestimate osmolality when sample is organic Freezing Point Depression Osmometry o Freezing point of a solution decreases 1.86 deg C for each osmole/Kg solution o Components Ethylene glycol absorbs heat of test tube sample Cool sample below freezing point but still liquid Rapid freeze seeded by agitation with iron rod Crystallization starts at seed then expands out Heat of fusion warms solution to freezing point

Measure Osmolality o COP: automated method Quantitation o Series of standards with CPU 3/12/12: Electrophoresis Basis o Isoelectric point: neutral charge protein; no migration o Quaternary protein: positive charge o Tertiary protein: negative charge o o o Rate o Low pH: positive charged protein High pH: negative charged protein Anion (negative charged protein) pulled to anode of migration Charge on molecule: more absolute charge = faster Charge to mass ratio Lower: slower Larger: faster o Friction

Viscosity Weight: heavier moves slower Physical barriers: restricting system/sieving/pores Endo-osmosis o At pH 8.6 all proteins have negative charge o Barbital is positively charged at pH 8.6 o Protein migrates in opposite of expected direction due to Barbital mobile-phase/friction

Heat: not good o Associated with current = Amps (in constant voltage system) o Increase heat, increase conductance o Increase conductance, increase migration rate o May denature proteins o More heat more evaporation = decreased resistance, high salt concentration, increased Amps, increased heat o Wick effect o Minimize heat Constant current source: decrease voltage, decease rate of migration Refrigerate Cut down evaporation: glass sandwich Electrophoretic Apparatus o Horizontal or vertical o Tube: capillary 3/14/12: Support Media o Paper: positive charge Non-restricting: huge pores (cellulose channel) May interact with proteins High endo-osmosis Trialing edge: bad o Cellulose Acetate: neutral charge Non-restricting, charge to mass ratio takes effect Low endo-osmosis No trailing edge