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
A
- 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
