Exam 3 Flashcards
Which statement is true of an isosbestic point?
It is a point on an absorption spectra where two species have the same molar absorptivity.
what describes flow injection analysis best?
continuous flow
What characteristics describe sequential injection analysis?
uses a six way valve, interrupted flow, can monitor industrial processes continuously
what characteristics do flow injection analysis and sequential injection analysis share?
reacts sample with a reagent in the carrier.
How does ELISA detect the analytes of interest?
An enzyme, attached to an antibody bound molecule of analyte, catalyzes multiple cycles of a reaction that generates a fluorescent product.
list the steps of how ELISA is used
- an antibody specific for the analyte is bound to a polymer support
- sample containing analyte is incubated with the polymer-bound antibody
- the surface is washed to remove the unbound sample
- the antibody-analyte complex is incubated with an enzyme like antibody specific for a different region of the analyte
- the surface is washed to remove unbound enzyme linked antibody
- a nonfluorescent reactant is introduced
- the enzyme converts the nonfluorescent reactant into a fluorescent product
Which of the sources is commonly used as a continuum source in ultraviolet (UV) spectroscopy?
deuterium lamp
Which of the sources is commonly used as a continuum source in visible spectroscopy?
tungsten lamp
Which of the sources is commonly used as a continuum source in infrared (IR) spectroscopy?
globar
reference cuvet
houses the reagent blank
mirror
passes the beam to the detector
sample cuvet
holds the sample with a defined pathlength
amplifier
increases the detector signal for ease of display and quantitation
beam chopper
mirror that rotates to direct light to either the sample or the reference cuvet
light source
provides electromagnetic radiation in the UV, visible, or IR region of the spectrum
detector
typically a photomultiplier tube that generates an electrical current proportional to the photon flux
scanning monochromator
wavelength selector that passes a narrow band of wavelengths to the sample by scanning through a user defined range of selected wavelengths
Which change will increase the dispersion (Δ𝜙/Δ𝜆) of a grating?
decreased groove spacing
reflection grating
it diffracts radiation into different wavelengths and disperses it at different angles
collimating mirror
it splits radiation into parallel rays
exit slit
it is where monochromatic radiation leave the monochromator
focusing mirror
it refocuses the collimated radiation
focal plane
it is what the refocused radiation of different wavelengths is focused onto
entrance slit
it is where polychromatic radiation from the source enters the monochromator
Which characteristics (i-iii) are advantages of a double-beam spectrophotometer over a single-beam
spectrophotometer?
(i) No exchange of the sample cell and reference cell is needed.
(ii) More accurate absorbance readings above values of 2 AU (absorbance units).
(iii) More accurate absorbance readings over time.
i and iii
Which statements (i-iii) accurately describe gradient elution used in high-performance liquid
chromatography (HPLC)?
(i) The column temperature is gradually increased during the chromatographic run.
(ii) The solvent composition is gradually changed during the chromatographic run.
(iii) The method is typically used when the sample has many components to separate.
ii and iii
Which statements (i-iii) accurately describe the process of fusion used for inorganic sample preparation?
(i) The process uses concentrated mineral acids (like HNO3) to dissolve inorganic samples.
(ii) The process occurs at temperatures ranging from 300 to 1200 °C.
(iii) The process is usually conducted by heating samples in a Teflon vessel.
only ii
Which statements (i-iii) accurately describe bonded stationary phases used for HPLC column packings?
(i) Polar bonded phases are used for normal-phase HLPC.
(ii) Cyano bonded phases (where R = (CH2)3C≡N) are used for both normal- and reverse-phase HPLC.
(iii) The solid support used for bonded stationary phases is usually porous silica particles.
all
Which statements (i-iii) accurately describe the different types of stationary phases used to separate
analytes by gas chromatography (GC)?
(i) The stationary phases in used GC columns are liquids or solids.
(ii) Liquid stationary phases physically adsorb analyte molecules onto their surface.
(iii) Analyte molecules partition into solid stationary phases by an equilibrium process.
only i
Which characteristics (i-iii) are TRUE for Enzyme-linked Immunosorbent assay (ELISA)?
(i) Low sensitivity – due to having to wash away unbound antibodies and target antigens
(ii) Signal Amplification – due to the linked enzyme causing the reaction to occur multiple times
(iii) High Selectivity – due to antibodies having bio-recognition
ii and iii
Which characteristics (i-iii) are TRUE for Flow Injection Analysis (FIA)?
(i) high sample throughput
(ii) high measurement precision
(iii) easy automation
all
Which statements (i-iii) are TRUE about the guard column used in HPLC?
(i) It is located at the exit of the column packing.
(ii) It prevents any particles from clogging the main column.
(iii) It prolongs the lifetime of the main column.
ii and iii
Which statements (i-iii) are TRUE about normal- and reverse-phase HPLC?
(i) Normal-phase HPLC commonly employs silica for the stationary phase.
(ii) Reverse-phase HPLC commonly employs hydrocarbon chains in conjunction with silica for the
stationary phase.
(iii) Reverse-phase HPLC is more commonly used compared to normal-phase HPLC.
all
Which statements (i-iii) are TRUE about the reciprocating piston pumps used in HPLC?
(i) Reciprocating piston pumps create a constant flow of solvent.
(ii) Pulse dampeners are used to dampen the pulse-like flow that reciprocating piston pumps create.
(iii) Two ball check-valves open and close alternately during each cycle of piston motion.
ii and iii
Briefly explain why isosbestic points are sometimes observed for overlaid absorbance spectra.
Overlaid spectra derived from a mixture of 2 species can display a point where every spectrum intersects (the isosbestic point). This occurs when the 2 species interconvert via an equilibrium
reaction and they share a common wavelength where their absorptivity coefficients (ελ) are the
same.
Briefly explain why retention factors (k) for chromatographic peaks should be between 2 to 10.
Analytes with retention factors less than 2 do not have enough affinity for the stationary phase to adequately separate from other early eluting components. Analytes with retention factors. greater than 10 have too much affinity for the stationary phase, take too long to exit the column and suffer poor resolution (and low detector response) due to excessive peak broadening.
Briefly explain why temperature programming is used in gas chromatography.
Temperature programing is used in GC when the sample has large number of components that span a wide range of boiling points. Increasing the column temperature effectively separates low. BP components early in the run AND high BP components late in the run. Isothermal GC effectively separates components only in one BP range; which reflects the general elution problem.
Briefly explain why HPLC is considered ‘high performance’
The ‘high performance’ of HPLC is related to the small (2 – 50 µm dia) column packing particles used as the stationary phase support. This smaller packing size leads to greater N (plates) and better separation efficiency. However, smaller packing also requires greater liquid pressure to generate adequate flow through the column, so ‘high pressure’ is associated with the ‘high
performance’.
Briefly explain why an electron capture detector is useful for gas chromatography
The electron capture detector provides a highly selective and sensitive signal for compounds that contain electrophilic functional groups (e.g., -Cl, -Br, -NO2). The selectivity reduces matrix interference for complex samples (e.g., environmental or biological) that contain many different compounds. The sensitivity allows trace levels to be detected.
Which characteristics (i-iii) are advantages of a double-beam spectrophotometer over a single-beam
spectrophotometer?
(i) No exchange of the sample cell and reference cell is needed.
(ii) More accurate absorbance readings above values of 2 AU (absorbance units).
(iii) More accurate absorbance readings over time.
. (i) and (iii)
Which statements (i-iii) accurately describe gradient elution used in high-performance liquid
chromatography (HPLC)?
(i) The column temperature is gradually increased during the chromatographic run.
(ii) The solvent composition is gradually changed during the chromatographic run.
(iii) The method is typically used when the sample has many components to separate.
(ii) and (iii)
Which statements (i-iii) accurately describe the process of fusion used for inorganic sample preparation?
(i) The process uses concentrated mineral acids (like HNO3) to dissolve inorganic samples.
(ii) The process occurs at temperatures ranging from 300 to 1200 °C.
(iii) The process is usually conducted by heating samples in a Teflon vessel.
Only (ii)
Which statements (i-iii) accurately describe bonded stationary phases used for HPLC column packings?
(i) Polar bonded phases are used for normal-phase HLPC.
(ii) Cyano bonded phases (where R = (CH2)3C≡N) are used for both normal- and reverse-phase HPLC.
(iii) The solid support used for bonded stationary phases is usually porous silica particles.
all
Which statements (i-iii) accurately describe the different types of stationary phases used to separate
analytes by gas chromatography (GC)?
(i) The stationary phases in used GC columns are liquids or solids.
(ii) Liquid stationary phases physically adsorb analyte molecules onto their surface.
(iii) Analyte molecules partition into solid stationary phases by an equilibrium process.
Only (i)
Which characteristics (i-iii) are TRUE for Enzyme-linked Immunosorbent assay (ELISA)?
(i) Low sensitivity – due to having to wash away unbound antibodies and target antigens
(ii) Signal Amplification – due to the linked enzyme causing the reaction to occur multiple times
(iii) High Selectivity – due to antibodies having bio-recognition
(ii) and (iii)
Which characteristics (i-iii) are TRUE for Flow Injection Analysis (FIA)?
(i) high sample throughput
(ii) high measurement precision
(iii) easy automation
all
Which statements (i-iii) are TRUE about the guard column used in HPLC?
(i) It is located at the exit of the column packing.
(ii) It prevents any particles from clogging the main column.
(iii) It prolongs the lifetime of the main column.
(ii) and (iii)
Which statements (i-iii) are TRUE about normal- and reverse-phase HPLC?
(i) Normal-phase HPLC commonly employs silica for the stationary phase.
(ii) Reverse-phase HPLC commonly employs hydrocarbon chains in conjunction with silica for the
stationary phase.
(iii) Reverse-phase HPLC is more commonly used compared to normal-phase HPLC.
all
Which statements (i-iii) are TRUE about the reciprocating piston pumps used in HPLC?
(i) Reciprocating piston pumps create a constant flow of solvent.
(ii) Pulse dampeners are used to dampen the pulse-like flow that reciprocating piston pumps create.
(iii) Two ball check-valves open and close alternately during each cycle of piston motion.
(ii) and (iii)
Which statement(s) BEST describe
the types of errors compensated by
a double-beam spectrophotometer,
compared to a single-beam?
(i) cell position differences
(ii) drift/changes in source
intensity
(iii) drift/changes in detector
sensitivity
all
Which statements BEST describe the
performance of a filter versus a monochrometer
for wavelength selection in spectrophometry?
(i) A filter is cheaper and more rugged than a
monochrometer.
(ii) A filter has better wavelength resolution than
a monochrometer.
(iii) A filter allows more convenient wavelength
adjustment.
(i) only
which statements are true?
i. PDA is more sensitive
ii. PMT’s are used for very strong signals
iii. PDA can convert one photon to one electron
only iii
Luciferase is a bioluminescent protein extracted from a firefly’s abdomen that is used in
various biochemical applications. What role would luciferase fulfill in an ELISA assay?
I) Analyte to be measured by the assay
II) Acts as a non-specific secondary antibody
III) Enzyme bound to the secondary antibody
III only
A Flow Injection Analysis does what?
i) Measures the absorbance of a colored dye product from the
reaction of a continuously flowing reagent and an added sample
ii) Can hold and perform up to 14 different analyte tests on a single
sample
iii) Utilizes a Peristaltic Pump to push the analyte and sample
through the flexible tubing and then the mixing coil, until eventually
the absorbance is measured, and the solution is pushed out to a
waste beaker
i and iii
Which statement(s) BEST describe(s) the following definitions
for the retention times tM, tR , t’R
* i. tm is the time between injection and the detection of the
analyte.
* ii. tr is the adjusted retention time that represents the time
the analyte molecules spend in the stationary phase.
* iii. t’r is time between injection and when a component that
is unretained comes out of the column.
none of the above
Which of the following statement are true regarding peak resolution?
i. Increasing the theoretical plate number increases peak resolution.
ii. Peak resolution should be above 1.5 but below 15.
iii. Either the width or half the width of the peak can be used to calculate peak
resolution.
iv. The area of the overlapping region between peaks increases as peak resolution
increases.
ii and iii
Which of the following statements are true?
i. In Gas-Solid Adsorption Chromatography, higher
temperatures can be used since liquid is absent.
ii. The column is used to seal the injection port and it can be
used as an interface for injecting the sample.
iii. The carrier gas serves as the mobile phase gas in gas
chromatography
(i) and (iii)
