Analytical techniques and instrumentation Flashcards
It is a method of determining the chemical composition of sample (specimen) qualitatively and quantitatively
Analytic techniques
It pertains to instrument, system or device used for the measurement of an analyte in a solution.
Analytic techniques
Photometry
analytical techniques
Photoelectric colorimetry
Spectrophotometry
Atomic absorption spectrophotometry (AAS)
Flame emission spectrophotometry (FES)
Infrared spectrophotometry
Mass spectrometry (MS)
Electro chemistry
analytical techniques
Electrophoresis
Ion selective electrode (ISE)
Luminescence spectrometry
analytical techniques
Fluorometry
Chemiluminescence
Flourescence
most commonly used electromagnetic radiation in measuring different analyte in the sample.
visible light
the number of waves produce in a specific time (1 second).
frequency
Can see by the eye, made up of colors.
From 380nm to 780nm.
Visible light (narrow region)
The study of interaction between light and matter.
The study of electromagnetic radiation (light) emitted or absorbed by a chemical in a solution.
All molecules absorbed light.
Spectrometry
Chromatography
analytical techniques
GC-gas chromatography
HPLC- high pressure liquid chromatogaphy
Below 380 nm. Short wavelengths
Harmful to body due to extreme high energy which causes ionization.
Example gamma rays and X rays.
. UV light
Electromagnetic radiation (light) is a combination of electronic and magnetic vibration that travels in a wave-like manner (oscillation).
Electromagnetic radiation
the distance between 2 peaks of wave, measured in nanometer (nm)
wavelength
States that the intensity of transmitted light is inversely proportional to concentration of the solution while absorbed light is directly proportional concentration of the solution.
Beer’s law
Above 780 nm. Long wavelengths
Low frequency waves, not harmful. Non-ionizing
Examples radio waves, microwaves.
Infrared light
Transmitted light or Transmittance (T) can be calculated as follows:
T =I/Io
I= the intensity of light (transmitted light) after it passed through the sample.
Io= the intensity of light (incident light) striking the sample.
Transmitted light is inversely proportional to the concentration of solution.
Absorbed light is directly proportional to the concentration of solution
Beer’s law
Equation: A= abc
A- The absorbance of the solution ( no unit)
a- Molar absorptivity- a measure of how well the molecule absorbs the particular wavelength of radiation
b- path length -the distance travel by light through the solution ( in cm)
c- The concentration of the solution (mol/L)
Absorbance (Abs) is also called
optical density (OD).
The absorbance is directly proportional to the concentration of the solution and the diameter of the test tube or path length.
Beer-Lambert’s Law
States that the amount of absorbance by a color solution is directly proportional to the concentration of the solution and the length of a light path through the solution.
Beer- Lambert’s Law
The intensity of transmitted light decreases as the thickness or path length (diameter of test tube) increases through which the light travels.
Lambert’s law
REMEDIES for beer’s law
use of blank solutions
water blank
reagent blank
serum blank
use of allen correction
Transmitted light is inversely proportional to the path length (the diameter of the tube).
The wider the diameter of the test tube the lower the transmitted light and vice versa.
Lambert’s law
uses only visible light and measures only colored solution
PHOTOELECTRIC COLORIMETRY
White light passes through an appropriate filter which absorbs light of all wavelength except the wavelength denoted by the particular filter.
Light passes through the colored solution (unknown or standard) and this colored solution absorbs some of the light depending on the intensity of the color.
The transmitted light impinges on a photoelectric cell which converts the light energy to electrical energy and this is recorded on a galvanometer as either percent transmittance or absorbance (optical density).
PHOTOELECTRIC COLORIMETRY
A combination of spectrometer and photometer that measures the amount of light absorbed through light transmittance to determine concentration in the solution. It can measures concentration of solution in visible or UV lights.
Spectrophotometer
much like photoelectric colorimetry except the use of monochromator like diffraction grating or quartz prism to disperse white light into a systemic array of its component colors or wavelengths while colorimeter uses filters of specific wavelength.
Spectrophotometer
Light passes through a monochromator and is spread into a spectrum of colors. Slit blocks off all but a narrow band of the light. Sample absorbs some of this light and transmits the rest. The transmitted light strikes a detector which sends a signal to an amplifier. This small signal is amplified and presented as transmittance, absorbance or concentration units by a read-out devices like meters, digital displays, printed read-outs and recorders.
Spectrophotometer
used visible, UV and infrared light for measurement of analyte. It can analyzed both colored and non-colored solution.
Spectrophotometer
Light source
UV
visible light
Infrared light
UV
Hydrogen lamp
Deuterium lamp
Mercury arc lamp
Xenon lamp
Hollow cathode
Visible
Tungsten-iodine lamp
Mercury/sodium vapor lamp
Hollow cathode
Infrared
Merst glower
Globar (silicone carbide)
Basic components of spectrophotometer
Light source
Monochromator (with entrance and exit slits)
Sample cells (cuvettes)
Photodetector
Readout device
made up of parallel groves into aluminized surface of a flat piece of crown glass.
Wavelength is bent when it strikes the diffraction gratings.
Diffraction gratings
commonly used light source in visible and near infrared region
tungsten light bulb
light source used in UV region.
deuterium lamp
isolates the specific wavelength or band of light from the light source.
Monochromator
It has entrance and exit slits which regulates the degree of light the passes through.
Monochromator
Types of monochromator
Glass filter
Prism
Diffraction gratings
less expensive, but not precise. Made up of semi transparent silver film used in photoelectric colorimetry.
Glass filter
it disperse the white light into several spectrum. It is rotated to isolate desired
wavelength.
prism
the most commonly used because it isolates a linear and widely
distributed wavelength. It can operates in visible and UV light.
diffraction gratings
made up of glass, quartz or sodium chloride (wedge shape).
A narrow light focus on a prism is refracted as it enters the more dense glass.
Rotated to a specific wavelength.
prism
concentrate the white light towards the monochromator. Minimizes the entrance of stray light and scattered light into monochromator
entrance slit
concentrates the specific wavelength towards the sample cell. Allows a narrow of light beam to reach the cuvette or sample cell.
exit slit
is the most common cause of loss of linearity at high analyte concentration.
stray light
use to check the accuracy of wavelength setting in spectrophotometer.
holmium oxide
wavelength of
colors
RED
ORANGE
YELLOW
YELLOW GREEN
GREEN
BLUE GREEN
GREEN BLUE
BLUE
VIOLET
651-700
601-650
576-600
556-575
5506-555
496-505
476-495
431-475
350-430
The wavelength at which a substance has its maximum absorbance of light
Lambda max (λmax)
Must be free from scratches to prevent the bouncing of lights that passes through.
Sample cells-
It holds the colored solution or test sample.
Sample cells-
also known as cuvettes, made of glass round or square shape.
Sample cells-
converts light energy transmitted into electrical energy. The reading is directly proportional to the intensity of light.
Light/Photo detector-
Sample cells TYPES
glass cuvettes
quartz cuvettes
for UV light spectrophotometer.
quartz cuvettes
for visible light spectrophotometer. E.g. borosilicate, alumina silica.
glass cuvettes
displays the output of photodetector. It could be meter or digital display
Readout system-
Readout system types
galvanometer, ammeter, LED display
most commonly used detector due to its sensitivity and rapid response.
Photomultiplier tube (PMT)-
One measurement at a time at one specific wavelength.
Single beam spectrophotometer
Operates at 185 nm to 1000 nm wavelength
Double beam spectrophotometer
Operates att 325 nm to 1000nm
Single beam spectrophotometer
The light travels in a linear direction and the test solution and blank are read in the same.
Single beam spectrophotometer
Types of spectrophotometer
Single beam spectrophotometer
Double beam spectrophotometer
It has two beam of light split by monochromator. One beam is used for reference and the other for sample reading.
Double beam spectrophotometer
The measurement of the concentration of the sample is determined by measuring the light absorb by an atom instead by a molecules. It uses hollow-cathode lamp as source of light. The atoms in the sample are vaporized (atomized) using a flame, the sample absorbs this light in proportion to its concentration and the intensity of the transmitted light for the particular element can be measured by photo detectors
Atomic absorption spectrophotometry (AAS)
Advantage is it eliminates the error which occurs due to fluctuations in the light output and the sensitivity of the detector.
Double beam spectrophotometer
FES Color of flame of metal ions:
Lithium
Sodium
Potassium
Calcium
Magnesium
Red
yellow
Lilac
brick-red/red orange
blue
designed to measure the vibrational spectrum of a sample by passing infrared radiation through it and recording the frequencies that have been absorbed and the extent to which they have been absorbed.
INFRARED SPECTROPHOTOMETRY
very sensitive technique which gives rapid results and avoids time-consuming chemical extractions
Atomic absorption spectrophotometry (AAS)
Occurs when a molecule absorbed light at a specific wavelength and reemits light at a longer wavelength (fluorescence). This fluorescence is detected and measured to determine the concentration of solution.
Fluorometry
The sample is atomized by flame, emitted light produced by excited atom is detected and measured to determine the concentration of solution. this emitted is proportional to the concentration of the analyte.
Flame emission spectrophotometry (FES)
the intensity of the fluorescent light emitted by a sample under constant input of light intensity is directly proportional to the concentration.
Fluorescence
determine the concentration of sample by measuring the light emitted by excited atom.
Flame emission spectrophotometry (FES)
It is more sensitive than spectrophotometry due direct measurement of emitted light.
But is sensitive to environment changes.
Fluorometry
determine the concentration of sample by measuring the light absorbed by ground state atom.
Atomic absorption spectrophotometry (AAS)
Fluorometer monochromators
Excitation monochromators
Emission monochromators
use to analyze the composition of calculi or stones
INFRARED SPECTROPHOTOMETRY
isolates light absorbed by molecules.
Excitation monochromators
absorbs visible light and transmits UV light Flourometer filter
Primary filter-
Fluorometer basic component (light source)
Mercury arc lamp- above 350 nm
Xenon arc lamp- 470 nm to 600 nm
Tungsten lamp- for visible region only
Turnable dye lasers- 360 to 650 nm
Detectors of fluorometer
Photo cell
Photo tube
Photomultiplier- widely used because of accuracy.
isolates light reemitted by molecules.
Emission monochromators
Made of glass or silica that is round or rectangular test tube.
The path length or diameter is 1 cm.
The surface should free from scratches and dust to prevent interferences.
Cuvettes
Absorbs UV light and transmits visible light.
Flourometer filter
Secondary filter
A chemical reaction (oxidation reaction) emits light which then detect and measure. The luminescence produced is directly proportional to the concentration of solution.
Chemiluminescence
These technique both measure concentration and size of the particulate.
Nephelometry and Turbidimetry
Light scattered (reflected) is measure at 90 º angle with respect to beam of light. The intensity of light scattered is directly proportional to the concentration of solution.
Used for the measurement of Ag-Ab complexes.
Nephelometry
Light transmitted (passing through the solution) is measured at 180 º angle with respect to beam of light. The transmitted light is inversely proportional to the concentration of solution. The more light is blocked the more concentrated the sample.
Turbidimetry
It does not require excitation of sample.
No monochromator is needed.
Enzyme is employed to maximize the efficiency.
Chemiluminescence
mostly in immunoassays (Ag-Ab reaction).
Chemiluminescence
used in determining the concentration of insoluble particulate suspended in a sample as a result of Antigen-Antibody interaction.
Nephelometry and Turbidimetry
Measures the activity of ions in a solution through detection of electrical potential . The concentration of an analyte in a sample is measured through its activity.
Ion selective electrode (ISE)
ISE components
Ion selective electrode-
Internal reference electrode-
Milli-voltmeter
Analytical technique that is used for separation of molecules from mixture. It is accomplish by applying electric current on the molecules which separates them based on difference of net charges.
Electrophoresis
found inside the ISE made up of silver wire coated with AgCl suspended in KCl this solution contains the same ion to be analysed.
Internal reference electrode
selects the ion of interest and excluding the other
Ion selective electrode-
connects the ISE and the reference electrode. detects the electrical potential.
Milli-voltmeter
It is not a diagnostic procedure and employed only for specimen preparation.
It uses gel as medium to facilitate the separation.
Electrophoresis
In clinical chemistry is mostly used for the separation of complex molecules like protein and lipid.
Electrophoresis
use of solid support medium and is routinely done in the laboratory
the material (serum) to be electrophoresed is applied as a small spot or narrow band and separation occurs into discreet spots or bands
ZONAL ELECTROPHORESIS
example of support media
paper, cellulose acetate, agarose gel, polyacrylamide gel
use in assessing protein abnormalities particularly dysgammaglobulinemia
IMMUNOELECTROPHORESIS(IEP)
dye such as bromphenol blue and Ponceau S is used to stain the various fractions so that they can be quantitated by densitometry
ZONAL ELECTROPHORESIS
process by which serum proteins are first separated by electrophoresis and then allowed to react by diffusion against polyvalent or monospecific antiserum
IMMUNOELECTROPHORESIS(IEP)
important in the fractionation of serum and CSF proteins, lipoproteins, hemoglobin and isoenzymes
ZONAL ELECTROPHORESIS
Widely used in clinical laboratory for separation , identification and quantification of:Sugar, amino acids, proteins, lipids, drugs and its metabolites, hormones, enzymes, vitamins, DNA in biological fluid
Chromatography
Is a process of separating complex mixture (sample) on the basis on solubility (polarity) or ionic charges of molecules.
Chromatography
Basic components of Chromatography
stationary phase
mobile phase
Composed of layer of solid or liquid absorbed on a solid support or column.
Stationary phase
Composed of liquid or gas mixed with sample.
Mobile phase
Uses pressure pump to force the solvent and the sample into a separation column. For fast separation.
High performance liquid chromatography (HPLC)
Separation is based on difference in polarities.
High performance liquid chromatography (HPLC)
Most commonly used technique in clinical laboratory for separation of drugs, hormones and drug metabolites due to less toxic and less flammable.
High performance liquid chromatography (HPLC)
Components are separated as vapors
Gas chromatography (gas-solid/gas-liquid)
gas or volatile (sample mixed with gas and then vaporized). what phase
mobile phase
Measures mass at the molecular level.
Mass spectrometry (MS)
Separation is based on mass - charge ratio (m/z).
Mass spectrometry (MS)
column coated. what phase
stationary phase
Coupled with GC or HPLC for maximum sensitivity and specificity
Mass spectrometry (MS)
gold standard for the detecting the drug metabolites in the blood. Confirmatory test for drugs of abuse.
Gas chromatography mass spectrophotometry (GC/MS)-
use to measure the rate of sedimentation of different classed of proteins with a gravitational field of about 250,000 grams.
ULTRACENTRIFUGATION
Used in
Toxicology
Vitamin D determination
Testosterone determination
Liquid chromatography mass spectrophotometry (LC/MS)-
In ultracentrifugation
lipoproteins can be studied by using solvent with higher density so that lipoproteins float groups found are :
HDL, LDL and VLDL
rate of sedimentation is expressed in ________ (s) which is equal to 10-13 absolute units (cm per second per dyne per gram)
Svedberg unit (s)
also used to clear lipemic serum
ULTRACENTRIFUGATION
Example of Volumetric (titrimetric)
Schales and schales method (chloride test)
EDTA titration method (Calcium test)
Unknown sample is made to react with known solution in the presence of indicator. volume of reagent, as a solution is added, is measured in a buret and the process of adding a reagent from a buret is called TITRATION.
Volumetric (titrimetric)
