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