Quiz 4 lec 12 material Flashcards
Can you use low quality mobile phase in HPLC?
No, impurities will ruin it
What six components do all HPLCS have?
solvent, injection, pump, chromatography column, detector, and recorder
How much of organic mlcls can be analyzed by GC?
Only about 20% of organic molecules can be analyzed by GC as 80% break down
What the difference between the analytes used in GC versus HPLC?
GC- need volatile analytes
HPLC- Need soluble analytes
The elute analyte what phases must be picked appropriately in HPLC?
Stationary phase and suitable mobile phase must be chosen
What are the three important differences between GC and HPLC?
Diffusion coefficient of the sample in the mobile phase is smaller in HPLC (gives faster analysis)
Viscosity of mobile phase higher in HPLC not a problem in GC as it uses gas.
the compressibility of mobile phase under pressure is small- HPLC safer to use than GC
How do we increase efficiency in chromatography?
ncrease the
rate at which solute molecules equilibrate between
stationary and mobile phase.
Why can’t we use OT columns in HPLC?
Since we use a liquid for the mobile phase and diffusion is much slower it will take forever for liquid molecules to interact with the stationary phase.
We used packed columns in HPLC but how does that effect the pump?
small particles give high efficeny but need a better pump cause you need greater pressure to force the solvent through the packed column.
what is the particle size used in columns in HPLC?
between 1.7-10 um
How do particles in column playa role in seperation?
particles are what do seperation through interactions with ur analyte
How does uniform particle size effect A term?
Decreases it
y do we want to minimize A term?
So we can decrease plate height in order to fit more theoretical plate sin the column
What was the first material used to make s phase of HPLC?
diatomacous earth as made of silica
IF you reduce particle size what happens to plate size? What happens to pressure?
You get a big increase in plates which causes 20x more pressure needed to push through the column
How can you make seperation in HPLC faster?
By using a stronger eluent (m phase)
What happens to van eter curve with smaller particle size?
It comes down as it decreases both the A term C term and plate height
What happens to pressure, run time, and dL when using small particles?
get higher pressure, shorter run time, lower DL ( as peaks are smaller and sharper)
What pressure porptional to?
flow rate, length of column
What is pressure inversely proportional to?
particle size
Up until 2004 what was the highest psi and flow rate we could achieve in an HPLC device?
6000 psi, 0.5-5.0 ml/min
After 2004, what was the highets pressyre and particle size we could use in HPLC?
Highest pressure is 15,000 psi and the particles are 1.5-2 um.
After 2004, UPLC pumps were created(Ulta perform LC), what did they do to reoslution, analysis time, and mobile phase consumption
Reduced analysis time and m phase consumtipn, increases resolution
As particle size decreases?
Plate number increases, retention time decreases, and pressure increases
What is a downside to small particle diamter?
increased frictional heating, center of column is 2 celsius warmer than outer wall causes band broadening.
How do address band broadening due to small particle sizes?
we address this by decreasing the diameter of the column(less than 2.1 mm)
How long are HPLC columns and what are they made of?
Are 3-30 cm and made of stainless steel
Why does mobile phase have to be filtered before entering HPLC column?
Because column is easily clogged
What is a guard column? Whens it used?
is acolumn that protects main column, has same s phase, if pressure of column goes up you remove it and replace w a new one
What column mm did we switch too an why?
Went from 4.6 mm Id column to 2.1 too have less sample used and less waste
What is the most common stationary phase in HPLC?
microporous silica particles
What is the surface area of the silica particles (s phase)
500-600 m2/g!
Why can’t silica particles be used over pH 8?
Because it dissolves in the base
hat do we use for s phase when ph is above 8?
use ethyelene bridged silica, resists hydrolysis upto ph 12
What is the s phase use din adsorption chromatography?
bare silica
What is adsorption?
When silanol groups in s phase form weka bonds with any mlcl in the vicinty
What are adsorption strengths based on?
functional group polarity
If a molecules has several functional groups, what detemrines the retention properties?
most polar one
How can a sample mlcl be adsorbed?
Can only be adsorbed if it interacts more strongly with s phase than m phase
How are sample mclls arranged on the silica surface?
all sample molecules arranged on silica surface so
their functional group/double bond close to silanol group
(cannot distinguish hexanol, heptanol + octanol)
strength of interaction also depends on steric factors
→ ideal to separate isomers
The more polar steric hindrance what happens to speed of elution?
gets slower
Why do we use type A silica over type B silica?
Type A has exposed silanol groups with strongly retained protnated bases leading to tailing, also has metallic impurities which also lead to tailing
What is the most commonly used s phase in liquid liquid chromatography?
Bonded s phases, these are s phases that ar ebonded to other mlcls through anyhroud rxns which changes the selectivity of the stationary phase
What is the most commonly used s phase bonded phase?
ODS (C 18 ocatdectyl stationary phase)
How do bulky isobutyl groups protect siloxane bonds?
They protect it from hydrolysis at a low ph <2 as it protects it form h30/
What is superficially porous C 18 silica?
Consists of a 0.25 um thick porous silica layer w stationary phase c18 bonded throughout it.
Why do we use superficillay porous c 18 silica?
Because they allow a higher diffusion much faster than a fully porous particle
How do superficially porous s phases behave like small packing material?
Can get the same seperation efficiency like if you used smller packing material but don’t need the high pressure associated w it
What is adsorption chromatography?
normal phase
chromatography
What is elution?
solvent
displaces solute
from stationary
phase
What has the higher eluting strenght? More or less polar solvent?
more polar
M phase must be less polar than s phase in order to?
in order to displace solute from stationary phase
What is eluent strength?
measure of solvent adsorption
if analyte viscous what more doe sit require to flow through HPLC?
Pressure
What is reversed phase chromatography?
Here s phase is nonpolar and m phase is polar
A hydrophobic part of the analyte reacts w the s phase and a less polar solvent replaces it for adsorption.
What does the mobile phase consist of in reversed phase chromatography?
consists of mixture sof water or aq buffer solutions with various water-miscible solvents
Why is super critical fluid chromtography “green”
It reduces organic solvent by 90% by provinding only co2, fast flow and resolution (bcuz of higher diffusion coefficients) and can also dissolve non volatile solutes.
def of isocratic elution?
elution with a single solvent mixture, only inject this as m phase
use for early eluting peaks or late eluting peaks
gradient elution def?
slowly increase m phase polarity by adding more of another solvent, this increases speed of seperation and allows for all analytes to be eluted.
ability of a column to seperate components is improved by?
decreasing plate height
What is the van demeter eqn?
The van Deemter equation is used to describe the factors that contribute to band broadening in chromatography. It states that H is equal to the sum of three terms, A, B, and C, which represent different mechanisms of band broadening. The A-term is proportional to flow rate and includes factors such as eddy diffusion and multiple flow paths that contribute to band broadening. The B-term is inversely proportional to flow rate and represents longitudinal diffusion, which causes band broadening due to analytes diffusing into areas of lower concentration. The C-term is independent of flow rate and represents the finite time required for solutes to equilibrate between the mobile and stationary phases.
The slower the linear flow rate in the column, the more complete the equilibration between the mobile and stationary phases, which results in less band broadening due to the C-term. Therefore, slower flow rates can lead to better separation efficiency by reducing band broadening. However, slower flow rates also lead to longer analysis times, so a balance between flow rate and separation efficiency must be achieved in practical chromatography applications.
What is H in the van demeter eqn?
Plate height
