9(18)-10 Flashcards
MS could provide _______ and _______ information if coupled with liquid chromatography (LC). But there is a problem: LC creates a huge volume of ______ (as solvent evaporizes), which need to be removed before introduction into MS. Because MS only works in ________!
qualitative; quantitative; gas; Vacuum;
___________ (ESI) solving water problem which is very huge because now protein can be analyzed. ______ is supplied, and _____ charged sample droplets are generated. The droplets gets smaller and smaller till only ______.
electrospray ionization; high-voltage;positive;ions;
Strong ________ at nebulizer outlet combined with coaxial flow of ____ gas creates a fine _______of ______ particles. There is little _____.
electric filed; N2 (drying gas); aerosol; charged
fragementation;
Atmospheric pressure chemical ionization (APCI) contains _______ between the nebulizer and the MS
heater (~500 degree)
*fine mist heats up to ~125 C
extraction is useful for 3 reasons: _____, ______, ______.
isolating analyte; remove of the bad matrix; concentrate the sample;
solvent extraction: solvent with lower density (lighter) goes on ____ of aqueous phase.
top;
If partition coefficient is large in case of [S]2/[S]1 where 2 is the organic phase, then the solute would move into the _____.
organic phase;
If solute is acid or base, molecular form and charge may change with ____.
Charged species are _____ soluble in aqueous solution, and neutral and hydrophobic ones are _____ soluble in organic solvent.
pH;
more;
more;
Setting pH as pKa of the anaylte would cause 50% _____ of analyte.
Solution can be _____ pH and make the species _____ charged.
Therefore the species would stay in the _______ phase.
lose;
increase; neutral
organic;
Metal chelator separate _____ ions from each other where it _______ complex one ion with an ______ ligand and extract the complex into an organic solvent.
The metal ion become _____ charged.
metal; selectively; organic;
neutral;
Chelator are ______ .
weak acid;
Dithizone (green) + lead (colorless) -> metal complex (red)
Formation of _____ makes the complex extreme hydrophobic and _____ charged.
Then the addition of ______ solvent can extract the complex in to the organic solvent.
benzene rings;
neutral;
organic;
Frequently used chelators are 8-hydroxyquinoline (oxine) and Cupferron.
metal binds to N and Os in these chelators
The selectivity of the chelator can be achieved by controlling the _____.
The selected pH is where K (_______) is large for one metal and small for others.
pH; partition coefficient;
At pH ____ Cu2+ is completely extracted while Pb2+ and Zn2+ remain in the aqueous phase.
5
isolated form of plant pigments easily dissolved in petroleum ether (ligroin), but not from plant leaves.
Because there is a force between the molecules we call _____.
adsorption
In other words, the dissolving power of ligroin for the pigments is ______ than the adsorption force of the plant tissue.
lower
selected ______ (made of cellulose like plant leaves).
_______ are the solvent that pass through it.
_______ can overcome the adsorption force between the plant tissue.
Adsorption is observed by _______ of column effluent
filter paper;
adsorbent (*such as ethanol and ligorin);
ethanol;
color change;
1901, best separation of plant pigments mixture on a starch like inulin column (and CaCO3/Al2O3) using 10 % ______ in ligroin
ethanol;
The plant experiment discovered the presence of ____ pigments, chlorophyllin alpha and beta.
It is an example of ________(TLC)
two;
thin layer chromatography;
Partition chromatography separate similar substance by repeated _______ by two _______ liquids.
extraction; immiscible;
Chromatography operates on same principle as extraction, but one phase _____ in place while other ______ .
held in place; moves past it
The solute with greater affinity stays in the column ____ than others, which means it will elute _____.
longer; later
process of passing _____/___ through a chromatography column is _____.
liquid/gas; elution
separated a mixture into its components based on ______ of compounds.
partitioning
*partitioned between the stationary and mobile phase
________ chromatography (Tswett) is where solute adsorbed on surface of solid particles, the stronger the solute molecules adsorbed, the slower it travels.
adsorption;
- think about the lab did in organic chem
- functional group attached to the adsorbent (P124 check)
\_\_\_\_\_\_ chromatography (martin and synge) is liquid stationary phase bonded to solid surface (SiO2) solute partitions between stationary (liquid) and mobile phase (a gas in gas chromatography)
partition
\_\_\_\_\_\_ chromatography (adams and holmes) is anions (SO3-) or cations (N(CH3)3+) covalently attached to stationary phase (polymer resin). The mobile phase is the liquid
ion-exchange
mobile anions held near ______ that are covalently attached to stationary phase.
Anion-exchange resin is where only _____ can be attracted to it.
cations;
anion;
*difference between cation and anion exchange resin is important
_____ chromatography (porath) separates molecules according to their size, larger ones elute first followed by smaller ones.
size exclusion;
*good for baseline separation between proteins
______ chromatography is the most _____ kind, it employs specific interactions between one kind of solute molecule and a second one that is covalently attached (immobilized) to stationary phase.
affinity; selective;
example of affinity chromatography is the binding of ______. The technique is useful if only interested in ____ protein.
antigen (antibody) to the particles; one;
The speed of mobile phase passing through chromatography column is determined by two things.
1) volume flow rate (volume solvent/unit time; ml/min)
2) linear flow rate ( distance of solvent travelled/unit time)
There are two factors that determine how well compounds separated.
1) the farther apart the better their separation
2) the wider the peaks the poorer their separation
How to measure the efficiency of a separation?
by resolution!
- solute moving through column and spreads into a gaussian shape (bell shape)
The _____ solute resides in column the ______ the band/peak
longer; broader
For quantitative analysis resolution _____ is highly desirable
> 1.5
_______ causes longer time in column which leads to broader peak.
It is net transport of solute from region of high concentration to low concentration by random movement of molecules.
______ peak in short time vs _____ peak in longer time.
diffusion; sharper and higher; shorter and wider;
Brownian motion
bead bouncing around
the bead is pushed by water molecules moving with random directions and speeds.
change in the x and y coordinates of the bead in successive intervals follow a Gaussian distribution.
_________ measures rate at which molecules move from region with high concentration to one with low concentration
diffusion coefficient
____ (J) = mol per m^2 per second (mol/m^2 * s) of molecules diffusing across a plane of unit area is proportional to _________ gradient and diffusion coefficient.
J = - D (dc/dx)
flux; concentration;
Diffusion in liquids ____ times slower than in gases.
Macro-molecules diffuse _____times slower than small molecules.
10^4;10-100
plate height (=2D/Ux=variance/x) is the measure of ________ . It is the length of column required for one ______. It is named from theory of ________.
The _____ plate height = ______ band width. Prefer the band width to be as small as possible.
column efficiency; equilibration; distillation
smaller; narrower; * GC 0.1 -1.0 mm, HPLC 10Um, CE<1Um
*If solute travelled a distance x at linear flow rate Ux (m/s), time on column t=x/Ux (distance divide by flow rate)
The ability of column to separate components is improved by _____ plate height, where more number of theoratical plates=N
Number of plates in entire column: N = _______ if plate height is small enough.
decreasing; dimensionless
*more number of plates, more thinner slices and shorter bands, equilibration established more often
Different solutes passing through the same column result in different plate heights because they each have different D (_______)
diffusion coefficient
Why does bands spread?
band of solute spreads as it travels through column and emerges at detector with standard deviation of sigma.
each individual mechanism that contributes to broadening produces a standard deviation of sigma*
*standard deviation itself is not added.
Plate height is proportional to _______ of chromatographic band (H=sigma^2/x)
variance
3 band-broadening mechanisms that are: (the van deemter equation, Ux =flow rate)
1) proportional to flow rate
2) inversely proportional to flow rate
3) independent of flow rate
1) C; 2) B ; 3) A constants
*they are constants for a given column and stationary phase.
thus changing the column and stationary phase changes A,B,C.
increase flow rate will ______ diffusion
decrease
Longitudinal diffusion is (____)
zone of solute after longer time on column.
B/Ux;
it is much faster in gas than in liquid thus the optimum linear flow rate in gas C is higher than in LC.
A term arise from multiple effects for which theory murky
______ .
(Eddy diffusion)
multiple flow path;
some flow path are longer than others, molecules entering the column at the same time on the left are eluted at different times on the right.
finite equilibration time between phases
_____ comes from finite time required for solute to equilibrate between mobile and stationary phase.
The slower the linear flow, more complete equlibiration and less broadening.
C-term;
P556
Electrospray ionization (ion spray) For positive ion mass spectrometry, the nebulizer is held at \_\_ V and the spray chamber is held at \_\_\_\_ V. It is reversed for negative. It requires \_\_\_\_ buffer concentration so the ions in buffer do not overwhelm analyte ions in the MS. For protein, it is common to find \_\_\_\_\_\_ ions such as [M +nH] n+ and sometimes, [M +nNa]n+ or [M +nNH4]n+. There is \_\_\_\_\_ fragmentation. It can be intentially increased by \_\_\_\_\_\_\_\_\_. Adjusting the skimmer cone \_\_\_\_\_\_ controls the degree of fragmentation; Where N2 molecules is collided with positive ions under low pressure.
0, -3500;
low; multiply charged; little; collisionally activated dissociation (collision-induced dissociation) occurs between capillary and skimmer; voltage (large -> fragmentation)
*water is involved.
Atmospheric pressure chemical ionization
____ and coaxial flow of ____ convert eluate into a fine mist from which solvent and analyte _____.
The distinct feature is _____ is applied to a metal needle in the path of the aerosol. electric _____ needle creates electric discharge under ATM pressure.
Analyte must be capable of forming the _____ MH+.
It produce ______ charged ions and is ______ for macromolecules such as proteins.
heat; N2; evaporates;
High voltage; corona (a plasma containing charged particles);
protonated ion;
single; unsuitable;
*fragmentation is little but can be adjusted with skimmer cone voltage difference.
LC creates a huge volume of _____ when solvent vaporizes at the ______ between the column and the mass spectrometer.
Most of these gas must be ______ before ion separation.
gas; interface; removed
MS requires high vacuum to prevent _____ during ion separation.
Chromatography is a ______ technique.
The problem of combining the two together is to _______ the huge _____ of _____ between the chromatograph and the spectrometer.
Some buffer additives that are ______ must be avoided for MS. There are other compatible ones.
molecular collisions; high pressure;
remove; excess; matter (in context to LC and MS is gas);
nonvolatile ( such as phosphate buffer);
*avoid additive concentration >20 mM and detergent concentration >10Um
There is no attractive interaction between the stationary phase and the solute in the _______ chromatography.
molecular exclusion
The linear flow rate corresponds to 0.30mL/min which is _____.
1 cm of column length contains 0.0565mL of mobile phase, 0.30mL would occupy 5.3 cm of column length.
0.30mL/min(volume flow rate) ; 5.3 cm/min
The ______ phase or an unretained solute travels through the column in the ______ possible time (tm)
mobile; minimum;
The van deemter equation tells how the ______ and _____ affect plate height.
column; flow rate
Advantage of open tubular columns than the packed columns are:
higher resolution, shorter analysis time, increased sensitivity and lower sample capacity.
Allows increased linear flow rate or a longer column or both;
decreased plate height, thus higher resolution.
