Liquid Chromatography

What is Liquid Chromatogaphy?

Separation, quantification, and identification of compounds in a mixture based on its retention time

If the analyte prefers the stationary phase, does it lead to longer or shorter retention time?

Longer retention time since it will elute out later

If the analyte prefers the mobile phase, does it lead to longer or shorter retention time?

Shorter retention time since it will elute out early

What is the isocratic mode of the pump?

Each injection will be the same mobile phase composition (remains constant)

What is the gradient mode of the pump?

The mobile phase composition will change with each injection

What is differential equilibrium in chromatography?

The different affinities a compound has for the mobile and stationary phase

What are the different types of liquid chromatography?

Adsorption, ion exchange, size exchange, partition

What is unique about partition chromatography?

Both the mobile and stationary phase are liquid

What is normal phase chromatography?

The stationary phase is made up of polar groups

What is reverse phase chromatography?

The stationary phase is made up of non-polar groups

In normal phase, do polar or non-polar analytes elute out first and why?

Non-polar analytes because the polar analytes prefer the stationary phase

In reverse phase, do polar or non-polar analytes elute out first and why?

Polar because non-polar prefer stationary ohase

Why is reverse group chromatography the most used techqniue?

Many biological compounds are polar and we want to elute them out first

What is the composition of the mobile phase?

Solvent and buffer. Maybe mobile phase modifiers

Why are methanol or acetonitrile chosen when using UV detectors?`

They have low UV cutoff and contribute less background noise

Why are buffers needed in the mobile phase?

Control pH, reduce peak trailing, and produce good peaks

What is the optimum pH range of the buffer?

It should be ±1 of pKa of buffer

Why are volatile buffers preferred in LC-MS/MS?

Minimizes ion suppression and maintains sensitivity

What is back pressure?

The force needed to move the mobile phase through the column

How is quantitation of samples done using UV detectors?

Beer’s Law A=abc

What is a limitation of using UV detectors?

Intense light sources are needed for light to get through the cell

What is the theory behind Fluorescence Detectors?

Based on the molecule’s ability to emit light after excition

What is the theory beind electrochemical detectors?

Analytes will flow past an electrode with voltage applied and it can either give up electrons (oxidized) or accept electrons (reduced). Net movement of electrons will produce a current that is proportional to analyte concentration

What is Retention Time (tR)?

The time it takes for the peak of an analyte on the chromatogram to form after the sample is injected

What is Void Time (t0)?

The time it takes to elute an unretained substance (does not interact with stationary phase)

What is Resolution (R)?

The degree of separate between two components on the chromatogram

What resolution signifies good separation?

Equal or greater than 1.25

What factors control resolution?

Capacity, selectivity, and efficiency

What is Capacity Factor (k)?

It is a measure of retention time

What is Selectivity (a)?

Measures the retention time of two peaks relative to each other (ratio)

What is Efficiency (N)?

The measure of the peak width

What order should the factors be adjusted in when wanting to improve resolution?

Capacity factor, selectivity, then efficiency

How can retention time be adjusted?

Changing the amount of organic solvent

How can selectivity be adjusted?

Composition of the mobile phase, the stationary phase, deriving sample chemistry, temperature

How can efficiency be maximized?

Increasing column length, increasing mobile phase flow rate, decrease injection volume

Why do samples have to be pre-treated before injection?

Prolongs the column life, helps detect analytes of interest, improves sensitivity