Analytical Methods: Other

reflectance photometry / reflectometry

diffused light illuminates a reaction mixture in a carrier & reflected light is measured

inverse

relationship between intensity of light reflected and concentration of analyte in reflectance photometry

• bilirubinometer

• automated reagent strip reader

examples of application of reflectance photometry

flame emission photometry

measures light emitted by a single atom burned in a flame

blue (hottest)

flame color in flame emission photometry

• sodium → yellow

• potassium → violet

• lithium / rubidium → red

• magnesium / copper → blue

flame emission photometry

• sodium

• potassium

• lithium / rubidium

• magnesium / copper

sodium

flame emission photometry: yellow

potassium

flame emission photometry: violet

lithium / rubidium

flame emission photometry: red

magnesium / copper

flame emission photometry: blue

direct

in flame emission photometry, relationship of light intensity of atoms emitting energy & concentration

atomizers

covert ions to atoms

atomic absorption spectrophotometry (AAS)

measures light absorbed by atoms dissociated by heat

• nebulizer

• flame / graphite furnace

examples atomizer

atomic absorption spectrophotometry (AAS)

used in unexcited trace metals such as:

• calcium

• copper

• magnesium

• lead

• aluminum

• lithium

• zinc

flameless atomic absorption spectrophotometry (AAS)

uses electricity to break chemical bonds instead of flame

graphite cylinder

in atomic absorption spectrophotometry (AAS), it holds the sample → where electricity passes through to evaporate

fluorometry / molecular luminescence spectrophotometry

measure amount of light intensity emitted by a molecule after excitation by electromagnetic radiation

fluorometry / molecular luminescence spectrophotometry

requires a primary & secondary monochromator

fluorometry / molecular luminescence spectrophotometry

1000x more sensitive than spectrophotometer

fluorometry / molecular luminescence spectrophotometry

it causes quenching / diminishing effect

• pH

• temp

• chemicals

what are the causes of quenching / diminishing effect

chemiluminescence

rely on principle of oxidation-reduction

chemiluminescence

emission of light is created from a chemical or electrochemical reaction

chemiluminescence

requires no excitation radiation, no monochromator

source lamp

not needed in a chemiluminescent immunoassay analyzer ⭐️

oxidation

chemiluminescence reactions w/ luminol, acridinium esters, dioxetane, ruthenium

chemiluminescence

more sensitive than fluorometry

enzyme (bioluminescence)

enhances chemiluminescence

firefly

example of bioluminescence

turbidimetry

measures light blocked by a particle in a solution

turbidimetry

used for measuring abundant large particles

turbidimetry

like spectro, detector is behind the cuvette

• concentration

• size

turbidimetry is dependent on

nephelometry (na-fall → scatter)

determines amount of scattered light

direct

in nephelometry, what is the relationship between detector response & concentration

nephelometry

for measuring Ag-Ab complexes

nephelometry

detector is set at an angle

• wavelength

• particle size

nephelometry depends on

albumin

most negatively charged, abundant, and fastest protein

immunoglobulins

in electrophoresis, it’s the protein farthest from the anode

• acid: H+

• alkali: OH-

acid vs alkali charge

ampholyte

molecule, such as protein — net charge can be either positive or negative

isoelectric point (pI)

pH where protein has no net charge

electrophoresis

migration of charged particles from cathode to anode

8.6

in electrophoresis, at what pH do proteins migrate & divide → albumin, a1 globulin, a2 globulin, beta globulin, gamma globulin

iontophoresis

migration of small ions

zone electrophoresis

migration of charged macromolecules (protein)

capillary electrophoresis

migrate towards cathode

capillary electrophoresis

separation is performed in narrow-bore fused silica capillaries

capillary electrophoresis

molecules → separated by electro osmotic flow (EOF)

positive charge

in capillary electrophoresis, what charge moves faster (bc walls are (-) charged)

isoelectric focusing

molecules migrate through a pH gradient

isoelectric focusing

ideal for separating proteins of identical size but w/ different net charges

high resolution protein electrophoresis

separate proteins into as many as 12 zones

densitometry

measures absorbance of stain / density in electrophoresis

densitometry

scan & quantitate electrophoretic pattern

direct

relationship between UV, stain, and concentration

agarose

best supporting medium for electrophoresis bc of neutral pH

polyacrylamide gel

medium used to separate protein to 20 fractions

chromatography

separation of soluble components in a solution

agarose

most common medium used in blood culture

• mobile phase (gas or liquid)

• stationary phase (solid or liquid)

basic components of chromatography

Rf (retention factor value)

distance leading edge of component moves / total distance of solvent

paper chromatography (Whattman paper)

chromatography used for sugar & amino acids

thin layer chromatography (silica gel, alumina, cellulose, or cross-linked dextran)

chromatography used for drug screening

gas chromatography

elution order of volatiles usually based upon the boiling point

gas chromatography mass spectrometry

gold standard for drug testing / drug of abuse

15 days

drug test shall be challenge w/in ___ using GCMS

discard

spx for drug test not challenged for 15 days →

high performance liquid chromatography

most widely used chromatography technique

pressure

HPLC uses ___ for faster separation

HPLC

uses: Hgb, rapid HbA1C, Hb related dse

reverse HPLC

HPLC wherein mobile phase is more polar than stationary phase

MS/MS (Tandem mass spectrometry)

detect 20 inborn errors of metabolism

vacuum

needed in MS/MA to prevent collision between fragments

UP-NIH

NBS ref lab

Matrix-Assisted Laser Desorption Ionization Time of Flight

MALDITOF meaning

Matrix-Assisted Laser Desorption Ionization Time of Flight

used in peptides & protein analysis

Matrix-Assisted Laser Desorption Ionization Time of Flight

time required for an ion w/ a given mass to reach the detector is a nonlinear function of the mass, w/ larger ions requiring more time than smaller ions

volumetric (titrimetric)

unknown sample + known sample w/ indicator

CHloride test

uses sCHales & sCHales

osmometry

measurement of osmolality (serum, plasma, urine)

osmolality

number of osmoles of solute per kg of solvent

osmolality

measure of solute in a solution ⭐️

osmometry

measuring changes in colliquative properties of soln.

• osmotic pressure

• boiling point

• freezing point

• vapor pressure

colliguative properties of soln.

• glucose

• urea nitrogen

• sodium

osmotic particles

FaVor ≠ Osm

B = Osm

relationship of colliquative properties to osmolality

NaCl

osmometer reference soln.

freezing point depression osmometry

most commonly used mtd for measuring the changes in colligative properties in a soln.

ethanol

• freezing point osmometer sensitive

• vapor pressure osmometer not

• freezing point = decrease by -1.86 deg C

• vapor pressure = decrease by 0.3 mmHg or Torr

• boiling point = raised by 0.52 deg C

each osmole

• freezing point =

• vapor pressure =

• boiling point =

osmolal gap

difference between measured osmolality & calculated osmolality

osmotically active substances (E.g., ethanol)

osmolal gap indirectly indicates presence of __

lipid and protein ⭐️

not contributor to osmolality

electrochemistry techniques

measurement of current or voltage generated by activity of a specific ion

ion selective electrode (ISE)

contains selective membrane → responds primarily to a specific ion

ion selective electrode (ISE)

electrode placed in a soln. → potential (voltage) develops across the membrane

• selective membrane

• internal reference electrode

• internal reference soln.

• external reference electrode

• voltameter / pH meter

components of ISE ⭐️