bld 414 notes
TRANSCRIPT
BLD 414 Notes 1/9/12 8:54 PM
Osmolality
Osm/Liter
Osmole = species in solution (after disassociation)
Full Disassociation
1mol NaCl 1mol Na+ + 1mol Cl-
1 mole in 2 species in solution
1 mole NaCl = 2 Osm/L
1mol CaCl2 1mol Ca2+ + 2mol Cl-
1 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 SO42-
1 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 1 liter of 1M HCl
o HCl = 36.5g/mol
o SG = 1.029
o 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
o Use colon
True dilution: used more often
o Part solute : total volume
o 1mL serum and 9mL water
1/10 = true dilution
1mL in 10mL total volume
o Use slash
Volumes of Dilution
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 = ¼
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 Equivalent dilution factors
o n = tube number
o 1/x = DF
o 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+ + logOH-
o 14 = -logH+ - logOH-
-logH+ = pH
[H+] and [OH-] always expressed in M (mole/Liter)
o 14 = pH + pOH
ex) 3mM HCl, what is pH?
o Complete disassociation
o HCl H+ + OH-
o 3mM 3mM + 3mM
o 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
Weak Acids
Does not completely disassociate
Acetic acid
o HAC H+ + AC-
o 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
Low NPV
o High specificity needed
Low PPV
High NPV
o High 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: don’t 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 Y-
axis 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 1/9/12 8:54 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
Specimen arrival to lab until result output to LIS
Gathering specimen rate limiting step
Total 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 ½ 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 Beer’s 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 Beer’s 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 1/9/12 8:54 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: Beer’s 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 ½ 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
T2 found by running up enzyme concentration until non-
linear relationship of product vs. time, then find
inflection of slightly less (20%) enzyme concentration
Units of enzyme activity
o IU: micromole/liter/minute
o Product formed: monitored by spectrophotometry – color
change
o 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 Low pH: positive charged protein
o High pH: negative charged protein
o Anion (negative charged protein) pulled to anode
Rate of migration
o 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