instru Flashcards
chromatography is derived from greek word
CHROMA
GRAPHEIN
greek word chroma means
COLOR
greek word graphein means
TO WRITE
Chromatography was first developed by
RUSSIAN BOTANIST MIKHAIL TSWETT
he produced a colorful separation of plant pigments through a column of calcium carbonate
RUSSIAN BOTANIST MIKHAIL TSWETT
developed into an invaluable laboratory tool for separation and identification of compounds
CHROMATOGRAPHY
are compared to known standards
SEPARATED COMPOUNDS
- this is to show the mechanism of specimen compounds as they travel to a medium and how they interact together with mobile and stationary phases.
- use to an unknown substance measurement
CHROMATOGRAPHY
smallest unit of matter
ATOM
consist of atom or things that cannot be broken down
ELEMENT
combined or union of elements or two or more atoms
COMPOUND
can either be liquid or gas
MOBILE PHASE
- firm or not moving
- a place wherein compounds are to be separated
STATIONARY PHASE
- molecule attraction
- the compound is adsorbed to a solid support such as silica or alumina
ADSORPTION
separates compounds based on their partition between the liquid mobile phase and liquid stationary phase coated on a solid support
PARTITION
uses column packing material that has charge-bearing functional groups attached to a polymer matrix
ION-EXCHANGE CHROMATOGRAPHY
used immobilized biochemical ligands as the stationary phase to separate a few solutes from other untreated solutes
AFFINITY CHROMATOGRAPHY
- separates molecules according to different sizes
-As the solute travel through, small molecules enter the pores whereas the larger ones, cannot and will elute first from the column
SIZE EXCLUSION
molecule separation or removal from another (eluent)
ELUTION
-designed for purification
COLUMN
the stationary phase is a thin film of liquid that is fixed onto small granular particles packed into the column
PACKED
can be made narrower than packed columns because their stationary liquid phase is actually coated as a very thin film directly onto the column’s inner wall
CAPILLARY
- the time it takes for a compound to elute
- this value is characteristic of a compound and is related to the strength of its interaction with the stationary and mobile phase
-term to determine a compounds identity
RETENTION TIME (Tr)
a phenomenon based on the electronegativity difference between two atoms
POLARITY
phenomenon of nonpolar (hydrophobic molecules) to not dissolving water (e.g oil and waxes)
HYDROPHOBICITY
useful in volatile compounds or compounds that can be easily converted to volatile form
GAS CHROMATOGRAPHY
retention of the compound in GC is determined by its __
- VAPOR PRESSURE
- VOLATILITY
retention of the compound in GC is determined by its vapor pressure and volatility depending on the interaction on the __
STATIONARY PHASE
act as mobile phase
GAS CYLINDER
should be high purity and flow must be tightly controlled to ensure optimum column efficiency and reproducibility
CARRIER GAS
samples are injected using an
AUTOMATED SYRINGE PIPET SYSTEM
- the injection port
- is heated very high temperatures
PLASTIC SEPTUM
must be packed
COLUMN
change indicator
DETECTOR
- for data and acquisition
- must be automated to provide more precise and efficient separation
COMPUTER
is necessary a reaction to make it volatile
DERIVATIZATION
GC Applications
ORGANIC MOLECULES AND DRUGS
2 types of SP in GC
- SOLID ABSORBENT
- COATED LIQUIDS
-uses low temperature for separation to achieve better separation of thermolabile compounds
LIQUID CHROMATOGRAPHY
- it is simple to recover a sample
- sample can be processed further or reanalyzed
- the mobile phase can be removed
-suitable alternative of GC with some restrictions
- similar in many aspect of GC and therefore instrumentation is similar
LIQUID CHROMATOGRAPHY
manual or automatic, achieved using a syringe and depositing the sample loop
- uses microprocessor control autosampler
SAMPLE INJECTOR
- must provide precise and accurate rate flow
- must have internal volume that does not react with solvent
MECHANICAL PUMP
Mechanical pump must provide precise and accurate rate flow at ____
6000PSI pressure
detects based on differential measurement and physical property measurement because the sample and mobile phase are often very similar
DETECTOR
uses small, rigid supports and special mechanical pumps producing high pressure to pass the mobile phase through the column
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
- columns are used many times without regeneration
- the resolution achieved is superior to other forms of liquid chromatography
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
- reproducibility can be greatly improved
-widely used clinical application is the separation and quantitation of various hemoglobins associated with specific disease
- it can measure the whole blood in 5 minutes
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
5 SEPARATION TECHNIQUES COMMONLY USED IN LC
PASAI
PARTITION
ADSORPTION
SIZE
AFFINITY
ION-EXCHANGE
- based on the fragmentation and ionization of molecules using a suitable source of energy
- the resulting fragment masses and their relative abundance yield characteristics mass spectrum of the parent molecule
- used technique for drug of abuse testing
MASS SPECTROMETRY
STEPS OF MASS SPECTROMETRY
- CONVERSION OF THE PARENT MOLECULE
- SEPARATION OF IONS MASS/CHARGE RATIO
- COUNTING OF NUMBER IONS
COMPONENTS OF MASS SPECTROMETRY
- ION MASS
- MASS ANALYZER
- ION DETECTOR
maintained at high temperature, has vacuum to provide ionizing vaporized sample molecules
ION SOURCE
ION SOURCE (3)
- ELECTRON - IMPACT IONAZATION
- CHEMICAL IONIZATION
- FAST ATOM BOMBARDMENT
ion source has both positive or negative gaseous accelerated into
MASS ANALYZER
usually an electron multiplier or ion photon conversion detector
ION DETECTOR
MASS SPECTROMETRY TECHINIQUE
VIADD
VAPORIZATION
IONIZATION
ACCELERATION
DEFLECTION
DETECTION
Proved to be such a gold standard because of great sensitivity and its reliability
- combined GC and MS
GAS CHROMATOGRAPHY - MASS SPECTROMETRY
- provides superior specificity and sensitivity compared to direct injection methods
-used to develop highly accurate and reproducible assays
- viable replacements for many immunoassays
LIQUID - CHROMATOGRAPHY - MASS SPECTROMETRY (LC - MS)`
deals with the analysis of molecular interactions with IR light of the electromagnetic spectrum
IR SPECTROSCOPY
used for the identification and quantitation of volatile substances in body fluids such as blood and urine
- like ethanol, methanol, acetone, isopropanol,ethylene glycol
GC - MS
- drugs commonly quantitated by
- used for the convenient screening of a wide range of toxic compounds such as chloro-phenols in water and soil ….
GC- MS
done in using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic
THIN- LAYER CHROMATOGRAPHY
used primarily by North American laboratories for drug testing in animal sports to provide a basic, acidic, and neutral drug.
THIN- LAYER CHROMATOGRAPHY
is passed through the absorber (constant concentration) a logarithmic decrease is seen in the radiant power of path length (Lambert);
Monochromatic EMR/radiation
spectrophotometer basic components:
- Light source
2.Wavelength selector - Sample holder
- Detector system
- Data readout
is accomplished by measuring the amount of light absorbed by that solution after appropriate treatment.
spectrophotometry
• take most several forms, them most recognizable being radiant heat and light energy
• The lighter absorbed, the higher the concentration of the material under study.
SPECTROPHOTOMETER
allows the application of several different approaches that provide a full analysis of the materials being studied.
SPECTROPHOTOMETER
developed the absorption principle
Bouguer
develop fundamental principles of Absorption spectroscopy;
Lambert and Beer
The main objective is To emphasize the interactions of light and matter.
ABSORPTION SPECTROSCOPY / AAS
• Also known as the Absorption spectroscopy
BEER-LAMBERT’S LAW
• The basic principle of spectrophotometry involves measuring the amount of light absorbed by a solution and relating that absorption to the solutions’ concentration.
• The difference between the amount of incident light and transmitted light can be expressed mathematically and is referred to as the absorbance of the solution.
TRUE
• measures the amount of light that the substance absorbs at a specified wavelength.
• a measure of the quantity of light absorbed by a sample
• first ascertain the wavelength of maximum absorbance for the compound of interest.
• Each chemical has a region where light is absorbed and the other regions where little or no light is absorbed.
ABSORBANCE
is directly proportional to path length and the concentration of the solution.
Light absorption
change indicator (of the variables in its environment such as pressure, temperature, electrical charge, electromagnetic radiation, nuclear radiation, particulates or molecules).
Detectors
(current producer)
- devices that convert information in nonelectrical domains and the converse.
It includes photo iodides, photomultipliers and those that produce current *sensors-chemical species monitoring (electrodes).
Transducer
Each chemical has a region where light is absorbed and the other regions where little or no light is absorbed.
TRUE
When a light is passed through a colored solution, lights of certain wavelengths are selectively absorbed by the solution, which gives a plot of the absorption spectrum of the solution. The wavelength of light at which maximum absorption occurs is called an absorption maximum (max) of that solution.
TRUE
- used to measure concentration by detecting absorption of electromagnetic radiation by atoms rather than by molecules.
ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)
ATOMIC ABSORPTION SPECTROSCOPY (AAS) COMPONENTS
• Hallow Cathode lamp- has anode in a gastight chamber./cylindrical
• Gases- Helium and Argon
• Filter Gas+ Voltage=ionized/ionization → collision of ionized ion and metal
• Metal→ knocks atoms off to be excited → upon excitation they return to ground state = LIGHT EMISSION
measured by Copper hollow cathode lamp
Cu
a conductor that is used to make contact with a nonmetallic part of a circuit. Becomes the new light source for the AAS
Electrode
filled argon excites an element to emit a spectrum
The bulb
is alternative of a cuvette
FLAME
is a long and narrow slit, to permit a longer path length for absorption of incident radiation to occur.
The burner
ATOMIC ABSORPTION SPECTROSCOPY (AAS) Principle:
• A. Light from the hollow lamp passes through the sample of ground-state atoms in the flame.
• B. The amount of light absorbed is proportional to the concentration.
• C. When a ground-state atom absorbs light energy, an excited atom is produced. The excited atom then returns to the ground state, emitting light of the same energy as it absorbed.
• D. The emitted energy forms the flame will go in all directions, and it will be a steady emission.
• Because the purpose of the instrument is to measure the amount of light absorbed, the light detector must be able to distinguish between the light beam emitted by the hollow cathode lamp and that emitted by excited atoms in the flame.
• To do this, the hollow-cathode light beam is modulated by inserting a mechanical rotating chopper between the light and the flame or by pulsing the electrical supply to the lamp.
• Because the light beam absorbed enters the sample in pulses, the transmitted light also will be in pulses. There will be less light in the transmitted pulses because part of it will be absorbed.
is used to isolate the desired emission line form the other lamp emission line. In addition, it serves to protect the photodetector from excessive light emanating from flame emissions (Bishop, 2000).
MONOCHROMATOR
• SODIUM, LITHIUM AND POTASSIUM MEASUREMENT
• MEASURES LIGHT EXCITED ATOMS
• SAMPLE+ ASPIRATED TO FLAME→ ENTRY ON ELECTRONIC STATE→ MONOCHROMATOR ISOLATION PHOTODETECTOR ELECTRIC SIGNAL MEASUREMENT
• ELECTRICAL SIGNAL PROPORTIONAL TO THE CONCENTRATION OF ATOMS
FEP
detects the amount of photons absorbed and then sends a signal to a galvanometer or a digital display.
Photometer
a combination of spectrometer and photometer, quantitatively measure light intensity using wavelengths.
Spectrophotometer
- is a method(spectrum and light)
Spectrophotometry
is a device that produces, typically disperses and measures light; contained by Spectrophotometer
Spectrometer
is a method of studying and measuring a specific spectrum
Spectrometry
Spectro- specere; specter meaning
to look; image (i.e spectrum)
• Scopy maning
-viewing or examination. (microscopy or endoscopy)
are based on the interaction of radiation with matter.
Spectroscopic methods
absorption of light at all frequencies is monitored simultaneously based on interference, and recorded as an ____
interferogram
- is subjected to Fourier transformation, which calculates the contributions of the constituent frequencies and plots the spectrum as intensity versus wavelength, or wavenumbers (cm-1), as is commonly used in infrared spectroscopy.
INTERFEROGRAM
One of the major advantages of Fourier transform infrared (FTIR) spectroscopy is that it can give detailed qualitative and quantitative chemical information without destroying the sample.
• In most cases, the samples used for FTIR spectroscopic investigations can be completely recovered and used for further analysis elsewhere.
TRUE
based on the interactions of reflected infrared light with the sample.
REFLECTANCE
- is the possibility to investigate the spatial distribution of chemical changes and sample heterogeneity, and to follow reaction fronts and reactions at certain sites, as well as precise sampling, such as when only measuring a certain area in a sample.
• highest achievable spatial resolution:
MICROSPECTROSCOPY
are capable of recording images at diffraction (he spreading of waves around obstacles) limited spatial resolution
Modern FPA detectors
refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region.
ULTRAVIOLET VISIBLE SPECTRUM
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis)
This technique is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.
ULTRAVIOLET VISIBLE SPECTRUM
is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules.
UV SPECTROSCOPY
- may be used as a detector for HPLC
- • The presence of an analyte gives a response assumed to be proportional to the concentration.
UV/Vis spectrophotometer
an analytical procedure for identifying elements
FLAME TEST
