Instrumentation Flashcards
1
Q
STANDARD CURVE
A
- Method of converting quantity measured (e.g., absorbance) to that desired (e.g., concentration)
- Measure signal from samples with known concentration
- In simplest form, graph results; can compute equation for line as well
2
Q
Standard Curve Diagram
A
3
Q
STANDARD CURVE 2
A
- Standards - concentration known from some independent means (e.g., weight of substance in known V)
- Some methods show “matrix effect” - result varies with sample makeup
- Calibrators - similar to patient samples, conc. related to standards
4
Q
TERMS
A
- In all methods, limited range of results where same equation applies, often termed reportable (linear) range
- Detection Limit - lowest value distinguishable from zero
- Functional Sensitivity - lowest value with acceptable reproducibility
- Analytical Sensitivity - sometimes used as synonym for detection limit, more properly, slope of line for determination of concentration
- Analytical Specificity - ability to measure substance of interest but not chemically similar compounds
5
Q
PHOTOMETRY
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- Use of light absorbance to determine amount of substance present
- Principles similar for related techniques such as nephelometry, fluorometry, flame emission, and atomic absorption
6
Q
MEASUREMENT
A
- Relates intensity (I) of light passing through sample to that when no sample is present (I0)
- Ratio (I/I0) termed Transmittance (T) often multiplied by 100 (%T)
- Absorbance - amount of light absorbed, A = 2 - log (%T)
- Although absorbance can go to infinity, there is a practical limit in distinguishing small change in I
- Older photometers reliable from 10-90% T (0.05-1.0 A)
- Newer instruments may work to A of 2.5
7
Q
BEER-LAMBERT LAW
A
A = a * b * c, where:
A - absorbance at wavelength
a - absorptivity constant of compound of interest (L•mol-1•cm-1)
b - path of light through solution
c - concentration
8
Q
BEER-LAMBERT LAW 2
A
Simple form of Beer’s law works for single compounds in solution.
In biological fluids, many substances are present, producing a more complex version of the equation:
9
Q
BEER-LAMBERT LAW 3
A
- If product (a * c) for one compound >> that for all others, then can use simpler form (e.g., Hgb, bilirubin)
- Can perform chemical reaction that produces compound with large (a * c)
- Problematic if more than one compound absorbs at wavelength
- If reagent and/or serum absorb, measure A before adding key reagent and reset to 0 (“blank”)
- If A of interferent varies, measure A at points equidistant on either side, subtract average from A; termed “Allen correction” (e.g., OD450)
10
Q
BEER-LAMBERT LAW 4
A
- If only two compounds absorb (e.g., that of interest and Hgb), can solve by measuring A at two wavelengths
- Select one wavelength where both have same absorbance, ax (isobestic point), second where interferent has ax and compound of interest has a = 0
11
Q
BEER-LAMBERT LAW Image
A
12
Q
SPECTROPHOTOMETRY
A
Several elements contribute to performance:
- Light source
- Monochromator
- Specimen container
- Detector/Recorder
13
Q
SPECTROPHOTOMETER LIGHT SOURCE
A
- Usually use continuous spectrum source (quartz halogen, tungsten)
- May use discrete spectrum source (mercury, deuterium vapor, xenon)
- Can also use high intensity single wavelength source (laser)
14
Q
MONOCHROMATOR
A
- If continuous spectrum, need to select wavelength of interest
- Usually use prism, grating, or interference filters
- Want narrowest range of wavelengths possible to pass through, measured as width of peak T at half height (band width, half band-pass)
15
Q
DETECTOR
A
- Photomultiplier tube - multiple stages (dynodes) that amplify # electrons released by light
- Diode array - strip of semiconductor cells that release electrons in response to light of specific wavelengths; allow measurement at multiple wavelengths simultaneously
16
Q
NEPHELOMETRY
A
- “Cloud” meter - measure of dispersion of light by particles in solution
- Key feature: similar to photometry, but detector at angle to light source
- Turbidimetry - measured decrease in light (A) by particles, use photometer
17
Q
NEPHELOMETRY 2
A
- Major application is for detection of antigen-antibody complexes
- Can also be used for amylase, lipase (clearance of particles of starch, triglycerides)
- Major interference - lipemia (causes light scattering)
18
Q
FLUOROMETRY
A
- Fluorescent compounds - when excited by light, emit light of lower energy (longer ) after delay; difference in Stokes shift
- Requires second monochromator, detector at angle to light source
- Limited to compounds that exhibit fluorescence
19
Q
FLUOROMETRY 2
A
- Few endogenous compounds show fluorescence (cortisol, quinidine)
- Can attach fluorescein or other fluorescent labels
- Inner filter - absorbs exciting light; quenching - absorbs emitted light
20
Q
VARIATIONS
A
Time resolved - use fluorescent compounds with long emission time (europium) to distinguish from interfering fluorescent substances
Fluorescence polarization inhibition (FPI) - can be used to distinguish small, large fluorescent compounds
21
Q
FPI
A
- Small compound - rotates before emitting light, little polarized
- Large compound - does not rotate, most fluorescence polarized
- If label antigen, can distinguish free (small), large (bound) forms, simplifying measurement
22
Q
FLAME PHOTOMETRY
A
- Heat from flame excites group I ions (Na+, K+, Li+)
- Emit light of characteristic wavelength ; amount related to concentration
- Signal also dependent on flame temperature, concentration of other group I ions
23
Q
FLAME PHOTOMETRY 2
A
- Use large amount of group I ion not measured (either K+ or Li+) as “internal standard” (e.g., ratio of Na+ /Li+ or K+/Li+)
- Also serves as “radiation buffer”, negating effect of change in Na+ and K+ on each other’s measurement
24
Q
ATOMIC ABSORPTION
A
- Elements absorb light emitted by same element in cathode tube
- Decrease in light intensity (A) while passing through vaporized element directly related to concentration
- Flame or furnace used to free element from chemical bonds
25
CHROMATOGRAPHY
* Separation of compounds based on difference in solubility between stationary, mobile phases
* Multiple applications based on differing characteristics of phases
* Versatile, generally slower than most photometric techniques
26
CHROMATOGRAPHY 2
* Sample pretreatment usually needed to remove substances with high affinity for stationary phase
* Inadequate removal leads to accumulation of substance on stationary phase, altering its ability to function in separation process
27
CHROMOTOGRAPHY PHASES
* Most chromatography separates on basis of difference in polarity
* “Standard” chromatography - mobile phase non-polar, stationary polar
* “Reversed” phase - mobile phase polar, stationary non-polar
* Elution - removal of compound(s) from solid to mobile phase
28
CHROMOTOGRAPHY RESOLUTION
* Ability to separate closely related compounds
* Dependent on surface area of stationary phase, difference in polarity between two phases
* Increased by increasing length of column, decreasing particle size, altering mobile phase
* Other factors do not change resolution: temperature, rate of flow of mobile phase, use of pressure (which changes rate of flow)
* These may alter time for separation, and are thus useful in shortening procedure if resolution still adequate
29
CHROMOTOGRAPHY FORMS
* Most chromatography uses columns; coated plates (thin layer) also used
* Stationary phase usually solid, but may be liquid (in gas chromatography)
* Mobile phase usually liquid, may be gas in gas chromatography
30
ION EXCHANGE
* Stationary phase is charged: anion exchange, positive charge, cation exchange, negative charge
* Oppositely charged compounds attach to stationary phase
* Use of increasing ionic strength mobile phase allows elution
31
AFFINITY
* Stationary phase binds specific compounds only
* Examples: Staph. Protein A for IgG, boronate for glycated proteins such as hemoglobin A1c
* After other compounds leave column, can elute by changing pH
32
MOLECULAR SIEVE
* Stationary phase has “pores” that trap small compounds
* Large molecules pass between beads
* Examples: Sephadex
33
GAS CHROMATOGRAPHY
* Separates compounds based on volatility
* Stationary phase high boiling point liquid or solid, mobile phase inert gas
* Can make many compounds volatile by producing derivatives
34
IDENTIFICATION
* In TLC, distance migrated relative to solvent (Rf) used
* In column methods, time to elute from column (retention time) used
* Usually compared to a known compound added to each sample (internal standard)
35
DETECTION
* For most, photometric methods most widely used
* In GC, can use flame ionization (organic), electron capture (halogen)
* Quantification usually by peak area, compared to standards
36
ELECTROPHORESIS
* Separates compounds based on relative charge density
* Components include electricity source, buffer (where separation occurs), inert support
* Constant voltage used; potential affects rate of migration
37
ELECTROPHERESIS SEPARATION
* Proteins have varying charge
* pI, isoelectric point - no net charge
* If pH \> pI, charge negative
* If pH \< pI, charge positive
* Buffer adjusts pH; ionic strength determines size of ion cloud, rate of migration, band sharpness
38
ELECTROPHERESIS SUPPORT MEDIA
* Polyacrylamide, starch - separate on charge, size
* Agarose, cellulose acetate - have negative charge on membrane
* Solvent molecules (H3O+) attracted to membrane, cause physical current; causes paradoxical migration of some molecules (electroendosmosis)
39
ELECTROPHERESIS QUANTIFICATION
Use densitometer, similar to spectrophotometer, after staining
Proteins - Coomassie blue, Ponceau red-S usually used
DNA - ethidium bromide used
40
CAPILLARY ELECTROPHORESIS
* Use of capillary tube allows small volumes, continuous sampling, automation
* Proteins pass by detector, generate “peaks”; no physical gel to view
* Can be combined with mass spectrometer to identify proteins (e.g. Hgb A1c)
41
MASS SPECTROMETRY
* Involves ionization of compounds in two major formats
* Weak ionization used for mixtures of compounds
* Strong ionization fragments compounds in characteristic ways
* Ions separated based on mass/charge (m/z) ratio
42
MASS SPECTROMETRY 2
* Strong ionization used to identify compounds separated by other means (e.g., GC/MS)
* Weak ionization used to separate mixtures of compounds (e.g., steroids) after extraction of sample
* May be combined (tandem MS)
43
RADIOACTIVITY
Isotopes emit 3 types of radioactivity
* alpha particles - He nucleus, low energy, short penetrance
* beta particles - electron or positron; moderate energy
* gamma rays - high energy photons
44
RADIOACTIVITY
Radioactive decay random; related to specific activity constant, gamma , for each isotope: (equation)
Time for activity to fall to 50% of baseline (half-life) usually used to describe isotopes, = 0.693/gamma
45
RADIOACTIVITY MEASUREMENT
* Beta particles measured by scintillation
* Crystal scintillation - NaI
* Liquid - 2,5 phenyl oxazole (PPO)
* gamma radiation - gas filled tubes (e.g., Geiger counter), radiation ionizes gas molecules, current measured
