MLSCI 466 - Lec 5 Liquid Chromatography

components of chromatographic system

1. mobile phase solvents/buffers

2. delivery system (pump)

3. injector + sample

4. column

5. detector(s)

6. waste

7. data system

pump

moves the liquid mobile phase through the column

pump modes

gradient or isocratic mode

isocratic mode

mobile phase remains constant

gradient mode

mobile phase is changes in a stepwise or continuous fashion through the run

differential equilibrium

refers to the separation of analytes based on solubility/strength of interaction as it interacts with the mobile and stationary phase

liquid chromatography

separation technique causing mixtures of components to be resolved by exploiting differences in their chemical and physical properties

what is the stationary phase in LC

the column

what is the mobile phase in LC

solvents/buffers

types of LC

1. adsorption chromatography
2. ion exchange
3. size exclusion
4. partition chromatography

bonded stationary phases

prevents dissolution of stationary phase by mobile phase, created by covalently binding an organic moiety onto the surface of the support particle

examples of polar groups for stationary phase (normal phase)

1. cyanopropyl (CN)

2. aminopropyl (NH2)

3. glycidoxypropyl (diol) silanes

examples of nonpolar organic groups for stationary phase (reverse phase)

1. octadecylsilane (ODS or C-18)

2. octylsilane (C8)

normal phase

partition system when stationary phase is MORE POLAR than mobile phase

what analytes would elute first using normal phase

non-polar analytes elute first then polar

reverse phase

stationary phase is LESS POLAR than mobile phase

composition of mobile phase

1. solvents
2. buffers
3. sometimes mobile phase modifiers like triethylamine

examples of LC solvents

1. methanol
2. DMSO
3. Ethanol
4. Acetonitrile **
5. Tetrahydrofuran
6. Dioxane
7. Isopropanol

how are solvents chosen for LC

based on polarity or if using UV detector then consider UV cut-off

acetonitrile as a solvent

has increased ioniziation efficiency compared to methanol + lower sample viscosity (fine droplets produced)

purpose of buffers

1. control pH (affects selectivity)
2. reduce peak tailing
3. give well-shaped narrow peaks

optimal pH for buffer

+/- 1 pH unit of the buffer's pKa

optimal buffer for LC-MS/MS

volatile buffers to minimized ion suppression and maintain sens i.e. ammonium formate/formic acid or ammonium acetate/acetic acid-- using non-volatile buffers can lead to contamination of ion source

back pressure

force that pushes a liquid mobile phase through a tightly packed bed of tiny particles

how to troubleshoot back pressure

start at detector and work backwards up the flow path using systematic approach

criteria for choosing a detector

1. Sensitivity
2. Detectability
3. Linearity
4. Reproducibility
5. Peak Shape
6. Flow and temperature

examples of detectors

1. Ultraviolet
2. Fluorescence **
3. Electrochemical **
4. Mass Spectrometry **
5. Light Scattering
6. Radiochemical
7. Nuclear Magnetic Resonance
8. Infra Red

UV detector theory

follows Beer's Law (A=abc)

fluorescence detector theory

based on ability of a molecule to emit light after excitation by light radiation (longer wavelength is emitted after returning back to ground state)-- can also derivatize to achieve this

electrochemical detector theory

i.e. amperometric detector where column effluent flows past an electrode to which a voltage has been applied and if large enough, analytes can accept (reduction) or donate (oxidation) electrons -> net mvmt of e- causes current to flow and the current is proportional to concentration of analyte at the interface

retention time (tR)

time it takes from injection of sample into the chromatographic system to recording of peak max of the component of chromatogram

void time (t0)

time required to elute unretained substance

resolution (R)

degree of separation between 2 components by chromatography

good resolution

1.25 or greater

factors affecting resolution

peak retention (k and and α) width (N)

resolution troubleshooting

first adjust capacity factor (k') then selectivity (α) then efficiency (N)

equation for resolution

R = (tR2-tR1)/[(W1 + W2)/2]

equation for capacity factor

k´ = (tR - to)/to

equation for selectivity

α = k´2/k´1

equation for efficiency

N = 16(tR /W)2

capacity factor

measure of the degree of retention

how much organic solvent in water can cause 2- or 30fold change in k'

10% change in the fraction of organic solvent in water

selectivity factor

function of column packing, mobile phase, and solute chemistry

how do we change selectivity

1)the composition of the mobile phase (i.e. organic solvent, buffer and pH) 2) the stationary phase 3) the sample chemistry through derivatization 4) the separation temperature

efficiency

calculated # of theoretical plates for a column-- large N indicates narrow peaks/efficient column

theoretical plate

when perfect equilibrium is assumed to exist b/w the solute in the mobile and stationary phase

ways to max efficiency

1. use well packed column
2. increase column length
3. increase mobile phase flow rate
4. less volume of injection