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Liquid Chromatography Cheat Sheet (DRAFT) by

Theory of liquid chromatography

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Basic Theory

Liquid Chroma­tog­raphy
MP Liquid
Actively partic­ipate in equili­brium process
SP Quasi/­porous solid
Most common do revers­e-phase chroma­tog­raphy SP
Film thickness Very small (monol­ayer)
Dm ~ 10Ds
B/U ~ 0
df ~ 0
CsU ~ 0
Always carrie­d-out in packed columns
More versatile than GC
Adaptable to needs
Much less efficient than GC
Diffusion coeffi­cient of analyte is orders of magnitude smaller than in GC
Bounced into other molecules Diffusion rate is small
In liquid phase (not gas)
Improve efficiency
Use small particle Narrow range velocity
Dependent on particle diameter (dp)
Smaller particle size = smaller plate height at any given velocity
Compensate for travel distance of analyte to reach surface of SP
Minimize the space between the particles that the analyte have to diffuse across
Analytical LC < 5μm
HPLC >3μm
UHPLC < 3μm

Theory Equations

LC Velocity Range


Packed Column
Packed full of particles
Usually composed of stainless steel, etc.
Large column Use for prepar­ative scale
Small column Packaged inside a capillary (75-100um diameter) Couple effici­ently to MS
Efficiency (N)
Dramat­ically lower than GC
Rule of thumb (GUEST­IMATE ONLY): N~ 3500 *L(cm) / dp(um)
Sample Capacity
Depending on size of column and packing
~5mg/g {[fa-a­rro­w-r­ight}} C18/silica
~10mg "­van­ill­a" column
Thickness of SP
~ 1-2nm
Resistance to mass transfer in MP and multipath terms dominate

HPLC Column

HPLC System

MP reservoirs
Stores MP in inert glass bottles
Platic coated Pyrex bottle (common) $300/pc
Degas solvent
Add element for filter­ing­/degas
Minimize amount of oxygen dissolved into MP Oxygen reactive in high pressure (increase oxidation of analyte)
Small bubbles can form Result intensive undesi­rable peaks
Connected to a comput­er(­pump)
Control mixing value to produces desired MP mixture
Analytical Column
Wide variety
Diameter ~0.5cm (general)
Length 10-20cm (general)
Syringe with sample
Inject needle into port and release
Liquid flow into loop (at atmosp­heric pressure)
When rotate lever to 60 degree Rearrange injector (set of valves)
Switches loop into flow path = swept down into analytical column
Operates at very high pressure
If inject sample into septum Shatter syringe
Prepare in vials Tightly seal for sample to not evaporate
Program computer
Runs separation overnight
~100 samples
Record data and integrate peak area
High-P­ressure Pump
Direct MP through system
Use high pressure
Analytical column is filled with fine particles
Dynamic Mixer
Needed to blend the different fluids (MP)
Provides the correct percen­tages of fluids dynami­cally as the separation goes
Guard Column
Avoid column killers
Species that strongly store in SP Never eluted
Contam­inated column
Can change separation Destroy separation in terms of its analytical quality
Very small column
Contains same type of SP as analytical column
Contam­ination is trapped inside
Period­ically replace cartridges Preserves analytical column
Narrow Bore Tubing
Tubes that connects components
Has to be rated for HPLC
Can handle high pressure
Has to be narrow boreDon't want MP to mixing + dilute sample peaks
Use short length as much as possible
If fitting not installed correctly
Can result in dead volume Analyte that gets trapped in dead volume = gets broaden
Thermostat Oven
Constant temper­ature
30-45 degrees
For a given MPEquili­brium is constant
Fraction Collector
Robots that period­ically move tubes of the eluted species from detector
Collect in vials
Sophis­ticatedDeposit 1 peak per vial
Less sophis­ticated Period­ically move from one vial to the next

HPLC System Diagram

Stationary Phase

Control Retention
Control retention Control distri­bution consta­nt(K)
Control by:
Adjust type of MP and SP
Adjust "­str­eng­th" of MP and/or SP
Add additives to MP Interact specif­ically with analyte, SP, MP
MP velocity Does not alter retention (K or K')
Stationary Phase
Most use silica support particles
Not great in high pHUse alumin­a(high pH resist­ance)
Low pH SP can come off of support (hydro­lyzed) Use polymera support
Almost always a "­pur­e" SP
Not mixed
Use chemistry reaction to anchor SP to wall/s­urface
Almost always a "­mon­ola­yer­" SP
Wide range of polarities
Use chemical reactions to adjust the SP
Can use chiral SP
Separate enanti­omers
Reasonable enviro­nment conditions
Silane Reaction
Use to anchor­/bond silicones to silica surfaces
In packing materials (parti­cles)
FS capill­aries
Use to deactivate silanols
Very reactive
Highly polar
Expose on surface of silica
Deactivate silanol
Use chloro silane
Ex: C18
Result in silani­zation of surface
Residual silanol
SP is usually of a different polarity (non-polar)
Results in tailing of analytical peak
After reacting surface with SP
Use a short chain alkyl
Take care of residual silanol
If silanol peaks present
Column is old
Molecules of SP are desorbs or removed from surface
Silanol Intera­ctions
“Standard” silica SP support particles
Has silanols on surface Si-OH
~50% of Si-OH are reacted to Si-O-S­i-C18
Residual Si-OH
When close to a metal in the silica
Are “acidic” and deprot­onate easily leaving a Si-O- on the surface
Act as ion-ex­change sites for basic analyte
Revers­e-Phase MP is not suited to ion-ex­change separation
Very poor peaks are obtained for basic analyte Tailing peak
Altern­ative options
Use a high purity silica column Less acidic silanol
Purchase a deacti­vated silica column
Affects only basic compounds
Neutral and acidic compounds does not show tailing
Particle and Surface Area
Terms that dominate:
Overall plate height
Overall plate number
dp VD eq.: A and CmU
Spherical Particle
Surface area/V­olume scales with 1/dp
A/V = Retention
More SP packed = Retention (K') = Resolving Power (R')
Nearly all SP are um scale silica particles
Impacts VD equations
Porous Particles
Surface area per particle
Amount of SP inside column
Retention and sample capacity = Better R'
Smaller the pore the larger the surface area/g of support
Diffus­ional Trap of small pores
Loss of analyte Tailing peaks
Large MW analyte go into small pores Never gets eluted
Separation molecules
Small molecules ~ 80Å
Large proteins ~120-300Å
Normal Phase (NP)
Developed initially
Used raw silica as SP Polar silanol
Separation based
Polar-­polar intera­ctions with silanol
Non-polar elute earlier
Polar analyte elute later
In general
MP is opposite polarity to SP
Works well for polar species only
Reverse Phase (RP)
Use non-polar SP Silane reaction
Use polar MP (Water based)
Separation based
Nonpol­ar-­Non­polar intera­ctions
Reverse separation of normal phase
Polar species elute first
Non-polar species elute later
More popular
Organic solvents used for MP Expens­ive­/da­ngerous
Most analytes are made our of biological origin Soluble in water-­based MP

Contro­lling Retention

SP Polarities
Retention depends on
Mass of SP
Type of SP
Control by chain length
Density of SP on silica % of silanol reacted
Surface area Porisity
Select­ivity depends on
Type of SP
Chain length
Linker type/l­ength
Chain Length
Chain length and/or % organic (carbon) load of SP = Retention (K')
C4 C8 chain
Double chain length Double volume of SP Double retention
If within the same type of SP
Ex: Alkyl chains
No signif­icant changes in select­ivity
Only shrink­ing­/ex­panding the chroma­togram about tm
Shrink c-gram = reduce carbon
Stretch c-gram = Increase carbon load
If analyte is not retained
Increase % carbon of SP/chain length
If analyte is excess­ively retained
Reduce % carbon
Signif­icant changes in select­ivity Resolving power
By changing the type of SP
Overall retention should remain roughly constant
Keeping the % carbon constant
Effect of MP Strength
MP plays an active role in retention
Distri­bution constant
Common solvents can be sorted according to their polarity
Polarity of MP
Main factors of contro­lling K K'
When changing MP strenght
Can calculate the retention under new MP
Equation (Only for RP):
K'new/K'old= 10((P'new-P'old)/2)
Equation (Polarity) P'MP= Weighted Polarity = %A*PIA + %B* PIB
PI= Polarity
Equation (Only for NP):
K'old/K'new= 10((P'old-P'new)/2)
1. Look at K' of first and last peak
K'old = (last peak - first peak)/­first peak K' = (2.8-1.8)/1.8 = 0.5
K'new Want it at 10
2. Replace terms in equation
K'new/K'old = 10/0.5 = 20
3. Old MP polarity
If old MP is 20% water/80% Acetonitrile
P'old= (0.2)(­10.2­)+­(0.8­)(5.8) = 6.68
4. New MP polarity
P'new = 2log(K'new/K'old) + P'old
P'new= 2log(20) +6.68 = 9.28
5. Solve new MP components
P'new = (x)(10.2) + (1-x)(5.8) = 9.98
79.1% water and 20.9% ACN
Rule of 3
Tool to check/­est­imate results (not used in calcul­ations)
Change of 20% water ~ 3x change in K'
MP Gradient
Dynami­cally adjust MP
Some sample contain wide range of analytes
Low or high retention
no single MP that will elute them all in a satisf­actory range of k’
MP gradient
MP strength is initially "­wea­k"Analyte well retained
Those with low retentionElute at reasonable K'
Strengthen MP over the course of separation
Strongly retained species can be elutedAt a reasonable K" and R'
MP≠con­stant Changing strength
K and K' ≠ constant
Can no longer be predicted
MP Select­ivity
Alter select­ivity (α) by changing type of solvent
Resolving power
R= (α-1)(K'/(1+K'))(√𝑁/4)
Sensitive to select­ivity Critical pairs in peaks
Depends on nature of MP
Change select­ivity = change type of MP
Try and keep
Overall retention is roughly the same
Select­ivity of peaks change
Select­ivity changes cannot be predicted

Chiral Separation

Basic Theory
Important to bioana­lyses and pharma­ceu­tical separation
Separation of chiral species
Enantio selective
Chiral SP or chiral additives to MP Separation of enanti­omers
Possible to separate structural isomers Strength of intera­ction changes as a function of isomer


Ionic Species
Small "­har­d" ions
Inorganic ions
Cannot use ion pairing
Ions can interact with approp­riate SP Ionic SP
Basic Theory
Equili­bri­um-­based separation
Discreet soption and displa­cement process
Carry throughout column
Does not use silica particles
Use polymer resin
Attach with a strong anion or cation
Using a strong anion SP
Cation exchange column
1. SP sulfonic acid (IEX resin) Anion surface particle (SO3-)
Wash column with acid solution {{fa-arrow-right} Cations (H+)
Sulfonic acid is protonated (SO3H)
2. Inject sample with cation analytes Metal ion (M2+)
Metal ions interact with SP
Metal ions displace some of H+ from resin
3. Unbind analyte from SP
Introduce a higher concen­tration of protons behind analytes MP gradient

H+ displace weakly bound analytes the move onto strongly bound analyte (cation) Exchange process
Cation analytes is displace off of surface and solubilize in MP
4. Analytes move down the column in strength of MP
Each type of analyte elute as a peak
MP ahead of each analyte is too weak Bound
MP behind each analyte is too strong Fully displace
Equili­brium Constant
If it behaves like an equili­brium There is an equili­brium constant
Expect to behave like an LC Produce peaks
Ion-ex­change equili­brium constant would behave like a distri­bution constant
Obtain similar result of chroma­togram peaks
MP controls retention
Kiex=[exch­ang­e&­ana­lyte]s/[analyte]m = Cs/Cm


Process of Separation
1. Carry out initial separation
Choose a strong MP
Ensure everything is eluted and fast separation
2. Adjust MP strength
Retention of last peak is within the right region
Depending on the complexity of sample
Simple sample K' ~ 10
Complex sample K'~ 20
Do calcul­ations for an estimate adjusting needed
3. Examine if peaks are within the acceptable region
Examine if all analytes are well resolved
4. Consider if a gradient is required
Presence of large area of empty baseline
5. If needed
Switch MP type to alter select­ivity
Gradient to reach acceptable retention and resolution
6. Consider using additives in MP
Help alter select­ivity
If MP type/mix strength does not achieve required separation
Change SP type
May consider type of separation
Summary of MP Effects
Very powerful tool Versatile
Control retention and select­ivity
Directly affects distri­bution constant
MP strength = K'
MP "­str­eng­th" is polarity
Effect are opposite in RP vs NP
RP Non-polar solvent (organic) = Stronger solvent
NP Polar solvent = Stronger solvent
Ramped MP Gradient
Helps dynami­cally adjust K'
Useful to make separation less intuitive R = K'
Gradient runs R = K'

LC Detectors

Ideal Detectors
High sensit­ivity
Steep slope
High stability
Minimal drift
Minimal noise on baseline
Very low DL
Long LDR
Can accept MP over wide range
Need reference to null out MP gradients
Fast response
Indepe­ndent of MP
Easy to use, maintain and repair
Can be either depending on properties
Non destru­ctive
Can collect fractions
1λ: UV- Vis Detector
~1-10 uL (very small)
If V is too large, the signal becomes constant and we see a square shaped peak
Longer = better
Bigger absorbance for same concen­tration (beer-­lambert law)
Window material
D2 lamps
Good broad UV source
Spectr­ometer to isolate narrow band of wavelength
More simple than FAA
UV does not have to compensate for a flame
2 sensors
Sample diode (I)
Intensity coming through the sample
Reference diode (Io)
Intensity from the light source
A= -log(I/Io)= -log(T)
Abs vs time
Use peak area for quanti­tation
Many λ: Photodiode array (PDA) Detector
Collect many chroma­tograms across many wavelength (a spectrum)
Can choose/use chroma­togram that provides the greatest sensit­ivity for each analyte
Find wavelength where analyte has least interf­erence from neighb­ouring peaks
Useful to verify which peaks is which when MP is changed
Refractive Index (RI) Detector
Uses refractive index of analyte compared to MP
Snell’s law
The rays will bend if there is a mismatch in refractive indices of the outside and the inside
When refractive indices match
Rays not refracted
If RI match (only MP)
Full intensity reaches sensor
If RI does not match (analyte eluting)
Reduced intensity reaches sensor
Plot signal vs time
~3 orders of magnitude less sensitive than UV
Optically silent
Reference flow
Limited gradient capability
Evapor­ative Light Scatter (ELS) Detector
How it works
Uses nebulizer to produce aerosol
MP evaporates
Leaves behind analyte fine crystals
Scattering of light (usually laser)
Only when crystals are present
Needs to produce crystals
Very low volatility
Can work for non-ab­sorbing analytes
Response is nearly uniform for all analytes
Buffers (MP)
Must be volatile
Restricts choices
Can’t use inorganic buffers: Leads to buffer salts
Better than RI detector
Higher sensit­ivity
Longer LDR

UV-Vis Detector Diagram

Photodiode Array Chroma­togram

Refractive Index Detector Diagram


Sample goes through nebulizer
High voltage is applied Produces charged droplets
Fine metal capillary tube
Connected to the outlet
Charged with high voltage
MP is pumped
Charged droplets are attracted to MS interface
Droplets dry down in flight
charge density until charge repulsion causes coulombic explosion
Single Quadrupole MS
Mass spectrum
MP evaporates away
Leaves [M+H]+ ionsno fragments
Difficult for definitive ID
Potential m/z overlap
Triple Quadrupole MS
Allows the production of fragments
Contains Q1, Q2(CID) and Q3
Q1 Parent ions are selected
Q2(CID) Collision induced dissoc­iationSelected ions collide with Ar/He/N2 (Creates fragments)
Q3Fragment ions are filter­ed/­scannedDetected to produce mass spectrum
Mass spectrum
Scanning modeProduce full spectrum For method develo­pment
Multiple reaction monitoring (MRM)Only selected fragments are measuredFor quanti­tation
Better than single quadrupole
Lower DL
Less interferences
Longer LDR
Allows positive ID of analyte
Better select­ivity with MRM
Q3 scans across m/z range pretty slowly (1-30 spectra/s)
Lowering resolution allows faster scanningCan’t get a full detailed spectrum
Quadrupole time of flight MS
Can scan 10000 spectra/s
Many are averaged together to improve quality (better than QQQ)
Allows more analytes to be measured simult­ane­ously
Higher mass accuracies and resolution
Permits greater ID power
LDR>5 orders of magnitude
Not as precise as QQQ

Triple Quadrupole Detector Diagram

QTOF MS Detector Diagram

Summary and Applic­ations

MP plays a critical role in contro­lling separation
Retention "­Str­eng­th"
Select­ivity "­Typ­e"
Wide range of MP available
Diverse set of separation conditions
Within the same SP and column
Allow to quickly try different separation conditions
Allow to quickly arrive to a newer optimi­zation separation
No requir­ements of volatile analyte
Needs to be soluble in MP
Wider range of SP available
Can choose type
Can change particle size
Can choose the amount of SP/unit of column
Easy to collect purified analyte
Much lower N compared to GC-FSOT
Degrades R and Overla­pping peaks
Many LC have low N 1000-5000
Detector Comparison
Selective or universal
Sample capacity
Immune from MP gradients?
Amendable to using IS?
Key Factors if LC is useful
1. Analytes soluble in liquid MP
2. Concen­tration of analytes are high enough
Can load larger volume­s/c­onc­ent­raton on columns
Combine with sensitive detectors
3. Does sample require a high R' separation
GC favored over LC
4. Need to recover analyte
5. Slower than GC
Anti-d­opping and forensics
Process control
Quality control
Food and Beverages
Industrial materials
Organic synthesis