5) Lab instrumentation Flashcards
spectrophotometer
instruments used to measure the “optical density” of a solution.
Interaction of ———– in the form of photons with matter provides the principal means of measuring analytes in biological fluids.
electromagnetic radiation (EMR)
EMR exists both as ———– and as streams of ——–
Maxwell’s waves
photons
Length of the electronic vector at max peak height
amplitude, A
Time in seconds required for the passage of successive maxima or minima through a fixed point in space
period (P)
Number of oscillations of the waveform in a second
frequency, v
Linear distance between any two equivalent points on successive waveforms.
Wavelength, λ
visible light
380-750 nm
white light
polychromatic light
all colors
3 primary colors
red, blue, green
photometry measures…
reflected light
refractometry measures…
bent light
nephelometry measures…
scattered light
spectrophotometry measures…
transmitted and absorbed light
spectrophotometer is set to read —— with the “blank”, negating any absorbance occurring due to solvent
100% T
transmittance =
P/P0
A =
A = abc = -log(T)
concentration vs —– is nonlinear
concentration vs —– is linear
%T
ABS
States that for parallel monochromatic radiation that passes through an absorber material of constant concentration, the radian power decreases logarithmically as the light path increases arithmetically.
Lambert’s law
States that for monochromatic radiation, absorbance is directly proportional to the light path, b, through the medium and the concentration, c, of the absorbing species.
Beer’s law
define variables in Beer’s law
A = absorbance
a = absorptivity (L g-1 cm-1)
b = cell length (1cm)
c = concentration (g/L)
there is a deviation from the line when A approaches…
2
spec methodologies generally account for …..
7% lipemia
combines measuring scattered and forward light
nephelometry
turbidimetry
5 components of spectrophotometer
- A stable source of radiant energy
- A device that isolates a specific region of the electromagnetic spectrum
- A sample holder
- A photo detector
- A read- out device
2 types of radiant energy source
- continuum (Tungsten)
- line (mercury or sodium vapor)
wavelength selector
Monochromators
Wavelength in nanometers at peak light transmittance
nominal wavelength
Range of wavelengths at a point halfway between the baseline and the peak.
effective bandwidth
Total range of wavelengths transmitted
bandpass
different types of monochromators
Absorption filters
Interference filters
Prisms
Grating monochromators
Monochromator Slits
Photovoltaic or Barrier Layer Cell
Vacuum Phototubes
Silicon Diode Transducers
Multichannel Photon Transducers
Photodiode Arrays
Charge-Transfer Devices
types of photodetectors
commonly used when radiant power is very low, which is characteristic of very low analyte concentrations.
Output signal amplified up to approximately one million times.
Photomultiplier Tubes (PMT)
sources of spec error
lipemia
hemolysis
icterus
Temperature
pH
Standards and Standardization
Preparation of Solutions/Reagents
Clean cuvettes
Wavelength selection
Presence of Stray Light
Incorrect Blank Used
Clerical Errors
——- is performed using glass filters or solutions that have known absorbance values for a specific wavelength.
Assessment of photometric accuracy
Optical instrument used for visual identification of atomic emission lines
spectroscope
User compares the observed color of the unknown sample to a standard or a series of colored standards of known concentrations
colorimeter
Consists of a light source, monochromatic filter and photoelectric transducer, signal processor, and readout.
photometer
An instrument that provides information about the intensity of radiation as a function of wavelength or frequency
spectrometer
2 types of double beam specs
Double beam in space
Double beam in time
A filter photometer that measures the quantity of light reflected by a liquid sample that has been dispensed onto a nonpolished surface.
reflectometer
a urine dipstick is a type of…
reflectometer
2 types of reflectance
Specular reflectance—Occurs on a polished surface (e.g., a mirror)
Diffuse reflectance—Occurs on nonpolished surfaces
Used for quantitative analysis of metals such as arsenic, lead, copper, mercury, zinc, and aluminum.
atomic absorption spectroscopy (AAS)
AAS measures the amount of EMR absorbed at a specific wavelength by elements ….
in their ground state (Go) with a zero-net charge
components of AAS different from spec
hollow cathode lamp
flame serving as cuvette
measures atoms
Based on an energy exchange process that occurs when valence shell electrons absorb EMR, become excited, and return to an energy level lower than their original level
fluorometry
high specificity and sensitivity
fluorometry
fluorescence vs phosphorescence
single excited state
triplet excited state
Conventional design of ——— places the detector at a 90° angle to the polychromatic light source.
fluorometers
Light is produced from a chemical or electrochemical reaction and not from electromagnetic energy stimulation of electrons, resulting in emission of photons.
chemiluminescence
Rayleigh theory
Mie theory
Rayleigh-Debye theory
nephelometry
Used to identify unknown compounds, determine concentrations of known substances, and study the molecular structure and chemical composition of organic and inorganic material
mass spectroscopy
mass spec steps
- Atomization
- Conversion of a substantial fraction of the atoms to a stream of ions
- Separation of the ions on the basis of their mass-to-charge ratio (m/z)
- Counting the number of ions of each type or measuring current produced when the ions formed from the samples strike a transducer.
Electrospray ionization (ESI)
Matrix-assisted laser desorption ionization (MALDI)
Surface-enhanced laser desorption ionization (SELDI)
types of mass spec
