Instrumental Exam 3 Ch 24 and Ch 25
attributes for good separation
0.5 < k < 20 resolution >/ 2 operating P <. 15 asymmetry factor 0.9 - 1.5
asymmetry factor
0.9 - 1.5
Under what conditions are split, splitless, and on-column injection used? I. Split injection is preferred for samples where the analyte of interest consitute > 0.1% of the sample 2. On column injection is used for samples that decompose above their BP 3. Splitless injection is preferred for introducing small volumes of sample onto the column 4. Split injection is preferred for samples with high boiling solutes in low boiling solvent 5. Splitless injection is preferred for trace analysis of analytes that are less than 0.01% of the sample
1, 2 , 5
What are the general steps in developing an isocratic separation for RP-HPLC with one organic solvent?
1. Choose column and solvent 2. Optimize by adjusting k by varying the organic solvent/water composition of mobile phase. 3. Check for low N 4. Adjust separation factor 5. Optimize column dimensions
types of detectors for GC
1. TCD = drop in TC results in change in V 2. FID = CH radicals, N2 is best 3. electron capture detector = insensitive to hydrocarbons, alcohols, ketones 4. alkali flame detector = N and P 5. flame photometric detector = optical emission from P, S< lead, tin 6. photoionization dector = UV 7. element specific plasma detectors (AES or MS)
advantages of FID
1. contaminated samples 2. 100 x more sensitive 3. linear response
disadvantages of FID
1. insensitive to carbonlyl, alcohols, halogens, amines, noncombustible gases
Why do smaller particles give better resolution in HPLC?
1. less distance the solute must diffuse (reduces C) 2. More uniform flow (reduces C) reduces H, inversely proportional to N/resolution
Why do OT columns provide geater resolution than packed column in gas chromatography?
1. longer column length (proportional to square root N = sq rt L) 2. shorter column diameter = decreasing plate height by decreasing the distance through which solute must diffuse 3. no A factor (path length) all have a uniform path
disadvantages of TCD
1. lower sensitivity (increase by increase filament I, decrease flow rate / detector T) 2. every substance by carrire gase
advantages of TCD
1. simple 2. 10^5 linear range 3. organic and inorganic 4. nondestructive
sample preparation techniques for GC
1. solid phase microextraction (SPME) 2. store bar sorptive extraction (SBSE) 3. purge and trap 4. thermal desportion
HPLC detectors
1. spectrophotometric = UV 2. fluorescence 3. evaporative light scattering = volatility 4. charged aerosol detector = nonvolatile 5. electrochemical detector = redox 6. refractive index detector
If the retention times are methane 0.5, octane 14.3, unknown 25.7 and none 18.5, find the retention index for the unknown.
100 [8 +(9-8) ((log(15.7)-log(14.3)) / (log(18.5) - log(14.3)) ] 836.27
store bar sorptive extraction (SBSE)
100x more sensitive --> trace analysis more sorbent
Which of the following solvent system has the highest elution strength for RP chromatography? a) 10% aceto / 90% water b) 30% aceto / 70% water c) 50 / 50% d) 70 / 30 3) 90 / 10
3) 90 / 10 most nonpolar
components of HPLC
6 autosampler pump sample injection valve high pressure chromatography colun detector column
cold trapping
A method of analyte condensation at the beginning of the column to produce a narrow injection band. column temp is 150 less than BP of ANALYTES
Why is superficial porous silca used for separating larger molecules, such as proteins?
Allow for faster diffusion/rapid mass transfer
bonded silica
C18 - base protection up to 11.5 polar embedded bulky (hihg temp acids) optically active to sep. enatiiomers
Gradient elution is used in HPLC. How is the gradient created for a reverse-phase HPLC experiment?
Decrease the polarity of the mobile phase over time by mixing with a less polar solvent.
Why do H2 and He allow more rapid linear flow rates in gas chromatography than N2 w/o loss of column efficiency?
Give better resolution at higher flow rates Dm H2 > He > N2 which is inversely proportional to Cm. H is proportional to Cm because stationary phase is thin enough. This increase N
equations for H
H = (Cm + Cs) ux H= A + B/ux + C
types of carrier gas
He H Nitrogen
The criteria for an adequate separation for isocratic reverse-phase HPLC include which of the following? I. The retention factor for all peaks are in the range of 0.5 - 20. II. All peaks should be symmetric with an asymmetry factor B/A in the range of 0.9 - 15. III. For quantification, the minimum resolution between the two closest peaks should be 1.5, though 2.0 is best to compensate for small changes in separation conditions. IV. The pressure must be less than or equal to 15 MPa to prolong instrument life.
I, II, III, and IV
Which of the following statements are true for GC columns? I GC colum are capillaries composed of fused silica coated with a polyimide II Column inner diameter typically 0.10 - 0.53 and the columns are 15 cm to 100 cm in length, with 30 the most common III a wall-coated OT column has a liquid stationary phase coating the inside wall of the column IV a porous layer column is filled with a highly porous stationary phase V a packed column is a column filled witha stationary support coated in liquid stationary phase
I, III, V
Which of the following are characteristics of reverse phase chromatography? I. the stationary phase is polar II. the mobile phase is more polar the stationary phase III. less polar mobile phase has a lower eluent strengh IV more polar mobile phase has a higher eluent strength V. the stationary phase is nonpolar
II and V
The pressure required to push solvent through the column is dependent on several variables such as column length, particle diameter, etc. For column pressure, which relationship is INCORRECT?
Pressure is inversely proportional to column length.
Explain how to use a gradient for the first run to decide whether isocratic or gradient elution would be appropriate
Run a scouting gradient to investigate elution behavior. t/tg ratio will tell you < 0.25 iso >0.4 grad between depends, usually gradient
What is the main difference between SPME and purge trap method?
SPME removes portion of analyte purge and trap has the foal of 100% analyte removed from sample
T/F: Superficially porous particles allow fast mass transfer of macromolecules into thin porous layer.
T
True or False: The purpose of a guard column is to protect the analytical column from contamination.
True
efficiency of purge volume depends on
Vapor pressure of analyte solubility sample temp purge volume
Open Tubular column
a hollow capillary tube fused silica coated with polyimide
Which of the following statioanry phases would be appropriate to separate a mixture of hydrocarbons? a) diphenyl polysiloxane b) cyanopropyl polysiolxane c) polyethylene gycol d) boscyanopropyl polysiolxane
a) diphenyl polysiloxane
desired attributes for isocratic RP-HPLC
adequate resolution short run time rugged - not drastically effected
During method optimization, the relative retention is adjusted by altering all of the following, except:
adjusting sample injection volume.
Which of the following is/are CORRECT for quantitative or qualitative analysis with GC? a) selected ion monitoring identifies a peak by monitoring m/z characteristic for the compound b) internal standards quantify compounds by adding a known amount of compound to the sample and calculating the concentration from peak areas for the compound and internal standard, and the response factor for the two compounds c) spiking identifies a peak by a known compound to the unknown. if the peak area increase, the peak has been identified d) linear response for concentration and detector response is required for quantitative analysis
all of the above
splitless injection used for
analyte is <0.1
split injection used for
analyte is >0.1 high resolution work small volume analyzing neat or concentrated samples, introduce small volume inaccurate quant analysis b/c split ratio is not reproducible
gas-solid adsorption chromatography
analyte is adsorbed directly on solid particles of stationary phase
Which statement is NOT true for GC detectors? a) thermal conductivity detectors respond to the thermal conductivity difference between analyte and carrier gas b) flame ionization detectors are used only with inorganic compounds c) thermal conductivity detectors respond to all analytes d) flame ionization detectors generate signal by burning the analyte, generating electrons e) for thermal conductivity and flame ionization detectors, detector response is proportional to analyte concentration
b) flame ionization detectors are used only with inorganic compounds
For quantitative analysis with GC: a) the retention time reported by the instrument for a compound is proportional to the quant of that compound b) the area of the peak by the instrument for a compound is proportional to the quant of that compound c) the calculated response factor value for a compund is proportional to the quant e) the peak height is inversely proportional to quant e) the peak shape is proportional
b) the area of the peak by the instrument for a compound is proportional to the quantity of that compound
Which of the following is NOT true for HPLC sample injection? a) reproducibly load injector loop with < 0.5 or >3 loop volumes dependin on injection techniqye b) the injection loops must be removed and cleaned after each injection c) sample solvent strenth should be less than or equal to eluent solvent strength d) pass samples through a 0.5 um filter to prevent damage to the injection port e) use a blunt HPLC needle, not pointed, to remove particulate matter prior to injection
b) the injection loops must be removed and cleaned after each injection
retention based on what two characteristics
boiling point polarity of stationary phase
One reason why small particles improve the performance of HPLC column is: a) increase flow rate b) increase pressure of mobile phase c) reduce analytes time on the stationary phase d) provide more uniform flow through the column e) cause higher temperature as a result of increased frictional heating
d) provide more uniform flow through the column
After optimization of an isocreatic eltion w/ several solvents, the resolution of two peaks is 1.2 How might you improve the resolution without changing the solvents or the kind of stationary phase?
decrease flow rate increase column length decrease particle size
improve resolution, without changing optimized solvent by
decrease flow rate increase column length decrease particle size
temperature programming result
decrease tr sharpen peak
A mixture of 14 compounds was subjected to RP gradient separation going from 5% to 100% acetonitrile w/ a gradient time of 60 mn. The sample was injected at t = dwell time. All peaks were eluted between 22 and 41 min. Is the mixture more suitable for isocratic or gradient? if the next run is a gradient, select the starting and ending % acetonitrile and the gradient time
delta t = 19 tg = 60 19/60 = 0.3 either depending 40-70% / 80% for 40-60 minutes
which is hotter, detector or column?
detector gaseous analytes
choice of carrier gas depends on
detector desired efficiency desired speed
Which comparison of open tubular columns to packed columns is INCORRECT? a) OT have higher resolution than packed b) OT have increased linear velocity or a longer column or both c) PC have decreased sensitivity d) PC have longer analysis times e) PC have a lower sample capacity
e) PC have a lower sample capacity
Which of the following is not true for split injectoin? a) the sample is completely vaporized and mixed in the mixing chamber by a combination of higher temperature and brisk carrier flow b) at the split point, a small fraction of vapor enters the column, but most passes through the waste vent c) the proportion of sample that does not reach he column is the split ratio d) split ratios typically range 50:1 - 600:1 e) after injection, the waste vent is closed and flow rate increased
e) after injection, the waste vent is closed and flow rate increased
Which is not a sample prep technique for use w/ GC? a) purge and trap b) solid phase micoextraction c) thermal desportion d) stir bar sorptive extraction e) matrix matching
e) matrix matching
HPLC operation requires special solvent, HPLC grade. Additionally, HPLC grade solvent require pretreatment before use. Which of the following is NOT a characteristic of HPLC grade solvent or pretreatment required before operation? a) prevent degradation of costly columns with impurities b) must be sparged prior to use if the instrument is not equipped with a degrasser c) minimizes background contamination signals in the detector d) must be filtered to remove particulate matter e) must be stored in special cabinetry to prevent solvent degradation
e) must be stored in special cabinetry to prevent solvent degradation
Which of the following is not a detector used with HPLC? a) electrochemical b) ultraviolet c) refractive index d) evaporative light-scattering e) thermal conductivity
e) thermal conductivity
Polychlorinated biphenyls (PCBs) are major environmental pollutants. Which of the following detectors would be most suitable for GC analysis of PCBs?
electron capture detector (ECD)
sample preparation process
extraction preconcentrate remove/mask interfering derivatizing
packed columns
filled with fine particles of solid support coated with nonvolatile liquid silica is silanized to prevent H bonding to polar used for prep separations that -require lot of SP -OR separation of gases that are poorly retained
how does the column maintain sufficient vapor pressure? why?
high temperature for analytes to be eluted in a reasonable time
When you try separating an unknown mixture by normal phase HPLC with 50% hexane/ 50% methyl t-butyl the peaks are too close together. Should you use a higher or lower hexane in the next run?
higher - more nonpolar, slower
Why are silica stationary phases generally limited to operating in the pH range 2-8?
hydrolyzed by acid/base
effect of sample solvent on band shape symmetry
if sample has a greater mobile phase strength, doubled peaks & altered tr
pressure programming used to
incerase flow of mobile phase decrease tr
effects of smaller particles
increase N shorter run time lower detection limit higher pressure
temperature programming used to
increase analyte VP decrease retention time
What is the advantage of increasing temperature in GC?
increase analyte vapor pressure decrease retention time of late eluting as a result = good separation, all compounds elute -decrea retention time - sharpen peaks
ways to increase resolution in GC
increase column length (+N) decrease column diameter ( - H, +N) increase stationary phase thickness
What is the advantage of increasing pressure in GC?
increase flow of mobile phase decrease retention time saves (compared to temp programming)
List ways in which rate of resolution b/w two closely spaced peaks can be improved
increase rate of equilibrium of analyte between the mbolie and stationary phases change = decrease elutio strength change stationary phase of the column
effects of narrow columns
increase resolution decrease operating pressure, sample capacity
what happens as pH -> Pka
increase retention
effect of increasing stationary phase thickness
increase retention time increase sample capacity increase resolution of early eluting peaks (will increase k for k </5) (- H) reduce tailing (shield from silica)
As particle size decreases for HPLC, the required solvent pressure ____, the plate number ________, and the resolution _______.
increases increases increases
What is the difference between isocratic and gradient chromatography
isocratic - constant solvent composition gradient - continuous change of solvent composition in increasing mobile phase strength
elution with a constant composition of mobile phase is
isocratic elution
SCOT compared to WCOT
larger sample size
Reversed-phase chromatography uses a:
less polar stationary phase and more polar mobile phase.
When you try separating an unknown mixture by RP HPLC with 50% acetonitrile/ 50% water the peaks are too close together. Should you use a higher or lower acetonitrile in the next run?
lower so it comes out slower
What is the differnce between normal phase and reverse phase chromatography?
normal phase -polar SP (increase retention w/ increase polarity) - less polar solvent (eluent strength increase w/ polarity, higher P') RP -nonpolar SP (increase retention based on volatility for NP = Troutons rule) -more polar solvent (eluent strength increase w/ NP, lower P')
ethylene bridged silica
pH 8-12
normal phase chromatography uses a ____ stationary phase and a ____ mobile phase. The more _____ the mobile phase becomes, the stronger the eluent strength.
polar, nonpolar, polar
PLOT SP
porous polymers molecular sieves
Derivatization can be applied in HPLC in order to:
prepare compounds suitable for UV and fluorescence detection
what to do if overloading is occurring
reduce sample mass by 10 check if tr increase or peaks are narrower
sparging system
removes dissolved gases by sweeping them out of solution by fine bubbles or intet gas
solid phase microextraction (SPME)
removes portion of analyte extractions from liquid, air, sludge w/o solvent
purge and trap
removing VOLATILE analytes from liquids or solids --> thermal desportion CONCENTRATING analytes INTRODUCING into GC 100% analyte goal removed purge gas strips from sample matrix
Hydrophilic Interaction Chromatography (HILIC)
separate molecules that are TOO polar to be retained by RP stationary phase = strongly polar
stationary phase retention and selectivity depend on
silica pore size bonded phase
Why is high pressure needed in HPLC?
small particles give increased resistance to flow. High pressure is required to obtain a usable flow rate.
how to adjust separation factor
solvent composition column temperature type of organic solvent column type
solvent trapping
solvent contains 10^4 solvent : analyte column temp is 40 below SOLVENT BP trapped in narrow band = sharp peaks
Tailing, a distortion of the Gaussian bandshape, occurs when
some sites retain solute more strongly than other sites
Cold trapping and solvent trapping us used with ___ injection to give ___ peaks
splitless sharper
The term "column bleeding" refers to:
stationary phase decomposition.
gas-liquid partition chromatography
stationary phase is a nonvolatile liquid bonded to the inside of the column or to a fine solid support
why is OT column coated with polyimide
support protection from atmospheric moisture
The maximum volume of sample injected in HPLC is limited by:
the size of the loop.
on column
thermally unstable solutes and high boiling solvent best for quant analysis
dwell time (td)
time required for gradient to reach column (td) dwell volume/flow rate
dwell volume
volume between point at which solvents are mixed and at beginning of column
types of OT columns
wall coated (WCOT) - thick film of liquid stationary phase solid supported (SCOT) - solid particlescoated with liquid stationary phase porous later OT (PLOT) - stationary solid phase on inside wall
