Liquid Chromatography Flashcards
What is special about liquid chromatography
The mobile phase is a liquid mixture of solvents
The stationary phase is a silica solid or a bonded phase liquid (all packed there’s no open tubular)
What are the types of liquid chromatography determined by
The types of stationary phases focus liek:
The polarity (default)
The analyte molecular size
The ion activity
The specific binding to targets (antibody or chiral)
What type of chromatography is default HPLC
It’s partition charmatogeay based on polarity
Why do we need higher pressure in HPLC
We need tiny particles because the tinier particles means lower plate heights and fewer multiple paths (the A term in the equation)
This causes narrower peaks and more efficient separation
If the particles were larger in HOLC what would happen
The peaks would get broader and slightly slower
QUESTIONS IN NOTRS APP
What is special about the columns for HPLC
They’re 2x longer and made of stainless steel to manage the pressure
The diameter of the column determines the flow rate
It has porous silica stationary phase inside that allows the participles to go in and out of (they aren’t trapped)
The efficiency of the column changes as it ages
What is special about the HPLC machine
it has a pump that mixes up to 4 solvents to make the eluent which allows us to change the polarity
The column is in an oven that’s about 10 degrees above room temp (east temp to maintain because it needs a constant temp)
In an oven to make high pressure
Made of a guard column and an inline filter
What is the guard column in an HPLC machine
A disposable column that collects and filters the permanently binding contaminants that bind to the analyte
What is the in-line filter in a HOLC machine
It filters out stuff that’s physically in the solution that could block the column
Comes before the optional guard column
If it’s partition chromatography what does it mean
If adsorption
partition: the stationary phase is liquid and the solute partitions/dissolved into it
adsorption: the stationary phase is solid silica and the solute sticks to it
Both a determined by the strength of intermolecular forces
Slide 8
What is normal phase liquid chromatography
What is the order of elution
The stationary phase is polar
The mobile phase is less polar (or non polar)
The order of elution is that less polar analytes come out first, more polar last because more interaction with polar stationary phase
What is reverse phase liquid chromatography
What is the order of elution
The stationary phase is non polar (alkanes ex C18)
The molar phase is more polar (range of polar like 2-propanol (less polar) or water (more polar))
Most polar elutes first
Less polar elutes last
What solvents are normally used in reverse phase liquid chromatography
What about normal phase
Water and methanol
More polar mobile phase
Alkanes and tert butyl methyl ether
Less polar mobile phase
If something more polar elites out faster than something less polar what does this mean
We’re using more polar mobile phase
Reverse phase
What are isocratic runs
When you use one single mobile phase concentrstion/composition throughout the entire hplc run
How do we adjust the composition of the mobile phase why
We make a solvent gradient
If normal phase, the ending solvent should be more polar to get the most out of the column (same polarity as column)
So we first use a less polar solvent then at the end use a more polar solvent (water)
What affects separation in HPLC
Temp (effect the kinetics of how thing move off the column and the viscosity of the elunt)
PH (if analyte is protonated/deprotonated it’s interactions with the column change, use buffer to keep constant ph)
Ionic strength (if the solvent supports the analyte breaking into ions, the strength of the analyte affect its interactions of ions with everything)
How do we keep the ionic strength constant
Add a salt or buffer
In liquid chromatography what is pulse prevention
As the pump pushes the liquid through, pulses occur where the analyte has pulse in pressure
This causes uneven flow of the analyte
To prevent this, the solvent is pushed in and out to keep a constant pressure with no pulse
In liquid chromatography what thisgs actually helps is pulse prevention
The valves and pistons in the machine
Is the liquid for liquid chromatography injected directly on to the column
What do we use
No
An injection loop
What is an injection loop
It allows for smooth insertion of the liquid and helps deliver a reproducible volume of injection
How does the injection valve work in the load position
We inject more volume than the loop can hold to fill and rinse it through the sample loop
This solvent is kept in the loop until the inject position
How does the injection valve work in the inject position
New solvent is sent in and
the pump pushes the stuff in the previously filled sample loop through to the column
The syringe isnt playing a role in it anymore
What are the 4 types of detectors in HPLC
refractive index
Absorbance
Electrochemical
Mass spectrometry
What is the refractive index detector
How does it work
Is detects changes in light refraction of the sample
Has the lowest sensitivity but is most common
The solvent (mobile phase) is sent through the two triangular compartments toward a PDA
There is collimated light coming in
When the solute with a different refractive index enters, the light beam shifts and this is detected by the PDA.
What is an absorbance detector
The path length is small (1cm) in the detector meaning the sensitivity is increased
It becomes smaller by having a crooked path
It’s selective since the response at each different wavelength can be different for the same molecule
Universal because is has a wavelength range that most compounds absorb
Can an absorbance detector also be a fluorescence detector?
Yes we use the same detector just no light source
What a light source the gives uv light
Deuterium lamp
What is an electrochemical detector
It measures the conductivity/redox of the Eluate
Only works for things that are ionized or match a specific value of E
Used electrodes where the sample hits the working electrode
What is mass spec detection in HPLC
it’s the most specific and sensitive detector
Uses electro spray ionization
