CC1 Week5_LEC.pdf

Graph- wavelength Number of waves- frequency Visible region: most analytes measures/ falls Nominal- peak transmittance (light here...

Graph- wavelength Number of waves- frequency Visible region: most analytes measures/ falls Nominal- peak transmittance (light here passes thru sample) ○ Wavelength adjustments depend on the region of analytes. Even “slight error” (cause significant error in “absorbance readings”) Wavelength accuracy: “actual wavelength” passes thru (transmittance) thru “monochromator” Didymium/ Holmium oxide filter- No need to compute rather just representation to used to “calibrate/check…” & control their relationship (WAVELENGTH accuracy) NOTE: Neutral density filters & Wave frequency “inversely related” to dichromate solution- certifies wavelength & energy (ABSORBANCE Accuracy on Wavelength “directly proportional” to linearity or “Correct reading”) energy PRINCIPLES: ○ Darkly colored sample-high turbid/high concentration=”low transmitted/passedlight” ○ Light colored/ low concentration= “higher light transmittance” Colorimetry- color involved w/c “color” (effect on the sample reading) AKA: Photoelectric colorimetry -measure “light intensity” w/o consider wavelength -Primary used (spectro/filter photomet): Isolation “discreet Absorbance/Optical density portion of spectrum” Mathematically derived “%Transmittance” 2 TYPES of Measurement: It presented w/ “3 Formula (machine is the Spectro- narrow wavelength (either one compute): (abc, 2-log%T & -logT)” UV region, visible/IR) NOTE: Photometric- light intensity only Medtech determine in lab NOTE: Similarity of (Concentration of photoelectric/colorimetry: Sample (absorbed unknown-derived from light) “Cu/concentration of unknown formula”) ○ Au & As (computed by machine) ○ Cs (medtech) 1. Spectrophotometry-measure “light transmitted (light passes thru sample) Follows: (Beer’s law) Also “computed by machine”  a. Single spectro- simple (ONE measurement= ONE wavelength NOTE: Absorption maximum (must “known Blank/ clear solution: Light pass in advance”) thru “completely” or 100% Transmittance/T b. Double beam spectro- “splits” BLANKING Technique- Any reading must minochrom light in 2 components: do the this technique 1 beam (sample) Used to check the absorbance Other beam (reference See if “spectrophotometer is sol’n/ blank- needed to working properly” “corrects” (variation in light Ultracentrifuge- used if it is not source intensity) effective specially if its too turbid NOTE: Adsorption must recorded “directly” Turbidity causes: lipids/fats as electrical output of beam 2 TYPES of Double Beam: b.1 Double beam (In SPACE)-uses “2 Photodetectors” 1 photodetec (sample), Other (reference beam) b.2: Double beam (In TIME)- uses “1 Photodetector”(alternate pass to “chopper/rotating mirror”)  ○ Give “accurate absorbance”: light intensity must be “LINEAR/ 1 direction only” (light intensity increase= increase absorbance din) 2 TYPES of Light/Radiant source: 6 components of BOTH “Single/ Double beam” Light source→Display Ex: of “Single Beam Spectrophotometer” Alternatives (if no tungsten available) a. Continuum source- flexible but the change must still be “linear” Light/radiance source- provide b. Line source- “limited” type of light: “Polychromatic light”  -widely used in AAS/Atomic Absorption Spectro, molecular & flourescent… 3. Monochromator- bandpass (isolate the indiv wavelength) 2 TYPES: a. Filters-simple Disadvantage: bandpass has 2. Entrance slit- stray light (outside the “wind band” & “low band e w/c “not originate” transmittance” (polychromatic light source) Made by: semi-transparent Stray light-most common silver films (Magnesium cause of “loss of linearity” fluoride) w/c to prevent must have **Interference filters- produces “mono/1 “entrance slit” light” b. Prisms- narrow beam light= shorter wavelength refracted 4. Exit slit- more selection -can be “rotated” w/c better to use Required: bandpass “less than ⅕” compare to filter the natural bandpass Better if “narrow bandpass” c. Diffractions gratings- COMMON used -has “parallel grooves” Principle: wavelengths “bend” as 5. Cubet/ Absorption cell/Analytical cell/ they “pass” (sharp corner) sample cell Function: holds the solution d. Holographic “gratings”- more TYPES/KINDS: expensive a. Alumina (common used) b. Quartz/Plastic- for “visible & UV spectra” c. Borosilicate d. Soft glass  TYPES/ KINDS: NOTE: Cuvet w/ scratches (discarded) a. Photocell…- disadvantage: tempt Silica cuvette effective at a sensitive & requires “no external “wavelength” (>220 nm) voltage source” Alkaline sol’n (not left standing in Convert energy via “internal cuvet for “prolonged periods”) electron transfer” Path of “Cuvet length”: 1 cm BUT NOT Require (EXTERNAL “smaller path” (used in automated Voltage) systems) Increase sensitivity: 10 cm Increase absorbance: factor of 10 6. Photodetector- “detects & converts” Endpoint: Sample must “convert into energy” NOTE: If light pass thru anode it will converted to energy b. Phototube- contains (cathode & anode) REQUIRES (EXTERNAL Voltage)  7. Meter/ Read out device- “displays” the “result of readings” Color absorbed (seen in “Naked eye”)- sample’s color/ end color Complementary color (machine reads)- if you run in “machine” c. PMT- COMMON used, Most “sensitive” Advantage: detect “very low level light” (even if highly concentrated, possible to convert pa rin) Measures: Visible & UV light Disadvantage: limited to “low power radiation” Ex: serum- add diff. Reagent then end color NOTE: PMT should “never” exposed to is “violet”: ROOM Light -Wavelength (350-430 nm) then run in machine w/c recognized as “Yellow-blue” d. Photodiode- Not as sensitive BUT “Excellent Linearity” Useful: Simultaneous multichannel detector  Requires: Indirect Internal Standard (Lithium/ cesium) Application: measures “Excited ions” (Electrolytes: sodium, potassium, fluoride, calcium & magnesium) Flickering light= changes in the fuel reading NOTES: Higher molecular/ ions type= higher concentration… Turbid- “Blue region” w/c more turbid= bluer Absorbance check: use “Glass filters” Determination of “linearity”: Used “Optical filters” 3. AAS- measured light absorbed by “atoms” Principle: Element is “not excited/exit” BUT “dissociated” from its chem bond AAS Consists of the ff:(Atomizer, chopper-modulate/controls, anthanum-reduce interferences due to chem bond created by “phosphate” ) 2. Flame emission photometry -excitation of electrons from “lower to higher” by “burning it in the flame” Volumetric- used specifically for “Chloride & Calcium test” Nephelometry- measures “antigen-antibody” (Serology) Turbidimetry- measurement of abundant Scattered light (must be measured) “Large particles (Proteins) & bacterial Antigen-antibody complexes have suspensions” ○ diameter: 250-1500 nm Principle: Highly turbid, it will ○ Wavelength: 320-650 nm “blocked” to reduce the light W/c these diameter & wavelength ○ Blocked/ reduced light (must “produced” (forward “rayleigh-debye type”) be measured) PMT is the detector Amount of turbidimetry depends: Concentration & size Electrophoresis- separation of “charged particles” based on “electric field” Acid/ basis (determines the net charge of protein) Anion/ negative charge- only moves with this neg charge towards “anode/positive charge” NOTE: Using the “Buffer barbital/veronal” it gives 8.6 pH (requirements of the protein to be negative charge) Isoelectric focusing- adjust the pH to migrate Capillary electro- uses “Electrical & Osmosis”  Positively charge- emerge early & EOF and ion movements are in “same directions” Negatively charged- specimen move “towards capillary outlet” BUT “slower rate” Separate “soluble components” w/c migration/separation depends on “physical/chemical characteristics” 2 FORMS: b. Column-use for separation of “steroids, barbitu, blood, alcohol & lipids” Elution order “based” (boiling point) a. Planar- perform separation on “flat surface” i. Paper chromo- uses “Whatman paper” for (fractionation of sugar & If tandem sila amino acid) Endpoint: Fingerprint patterns are created= ii. TLC- gives you presence of metabolites “semiquantitative/value” for drug screening test Drug extraction: pH “dependent”  1 monochromator- absorb wavelength 2nd monochromator- prevent “entry of incident light” Disadvantage: Affected by “quenching” (loss of fluorescent/radiation) Involatile- cannot be converts by GC-MS Chemiluminescence- NOT involved (Energy) rather only (Chem/ electrochemical reactions) Principle: Chem reaction produces “light” then produce “emission” (must be measured) Fluorometry- using “radiation” (excites the molecules) to produce “light” Osmolality depends on: Colligative Determines: difference in voltage… properties Uses/Application: pH & pCO2 test Osmolality “Increases” Ion selective electrode uses the “potentiometry” -Not specific (depends on “voltage differences”) NOTE: membrane used: Sodium- glass membrane electrode Potassium- liquid membrane + valinomycin Chloride- Silver membrane Among the 4 colligative properties, electrode “freezing point” (one being used) Endpoint is detected by “amperometry” Interferences (must not have/not seen in the sample dapat because it will contaminate)