The Chromatogram Terms:

Retention time Peak area Peak width (at half height, at base) Peak height Void time/volume Adjusted retention time Leading edge, tailing edge

The chromatogram

TERMS - Retention time, Peak Area, Void Time (volume), Adjusted Retention TimePeak Width ( at half height, at baseline), Peak Height, Leading Edge, Tailing Edge

Chromatographic Terms Retention volume

Volume of mobile phase needed to carry component through column to detector

VR

Retention volume Not convenient to measure volume directly Measure time taken from injection to

appearance of signal peak Record as retention time tR

-maintain a constant flow rate

VR = tRFc where Fc = flow rate

Retention time

Void volume/time A compound cannot possibly exit the column

in “zero seconds” Takes time to travel through a column even at

“top speed” Fastest a component can travel is at the speed

of the eluting solvent – ie the same speed as the mobile phase

Void volume/time Have a flow rate of for example 1 mL/min Column and tubing occupy a volume of 2

mLs of liquid (excluding volume occupied by solid phase)

Then fastest a component could travel would be 2 minutes – as fast as the MP

Void Volume/time The fastest speed an unretained component could

travel through the column and “system” is represented by the void volume/time

No peak can be seen before the void time If a column has a void time of 2 minutes, then no

peak can occur at <2 minutes

Void volume/time To measure void time, inject a component

known not to stick or be retained on your column

The peak you see will represent the void

Why is this important? i.e.Why is it important to an analyst to know the void time of their system when performing an analysis?

The adjusted retention time Retention time = the time taken for a

component to travel through the column However, this cannot be less than the void

time Therefore the adjusted retention time is the

actual retention time minus the void time

Adjusted retention time V’R = VR – Vo

t’R = tR – t - equation for calculating adjusted RT

Retention times and volumes are dictated by the distribution coefficient K

K= Cs/Cm

The chromatogram

TERMS - Retention time, Peak Area, Void Time (volume), Adjusted Retention TimePeak Width ( at half height, at baseline), Peak Height, Leading Edge, Tailing Edge

Capacity Factor k – describes the ability of the stationary

phase to retain components

Ratio between number of molecules in SP compared to MP

k=Cs/Cm

Capacity Factor k1= (V1-Vo)/Vo

k1 = (tR –to)/to

= t’R/to

The longer a component is retained by the column, the larger the capacity factor

Capacity factor Large capacity factors favour good separation

but increased elution times Capacity factors > 1 and < 5 are favoured If over time the capacity factor changes,

usually indicates degradation of the column

Separation FactorSelectivity A purpose of chromatography is to separate

compounds from each other If want to separate A from B, they must have

different retention times (ie different capacity factors)

Retention time

Selectivity Separation factor (alpha) aka selectivity - for

two peaks:

Alpha (a) = kB/kA

= t’R(B)/t’R(A)

For separation to occur, alpha must be greater than 1

Which peak must have the longer retention time?

Selectivity Separation factor depends on type and

properties of stationary phase used, the composition and properties of mobile phase, the interactive forces of the analyte and the column temperature

Must be optimized for each separation If maximize alpha, get good separation BUT

analysis times are too long

Separating efficiency of a column Every component should show up as a nice

narrow peak Must then move in a narrow band through the

column Peak width is an indication of the efficiency

of a column

Efficiency Observation: the longer a component stays

on the column, the wider the peak The band is dispersed as it travels through the

column The longer it is on the column, the more time

it has to disperse Dispersion leads to band broadening and wide

peaks

Peak width Simple measurement: peak width Gives an estimation of the efficiency of the

column when combined with retention time The number of theoretical plates, or the plate

count, N is used as a quantitative measure of efficiency

Plate theory (again) Chromatography is a continuous process Separation occurs on all particle surfaces Think of in terms of imaginary segments of

the column called plates – remember the molecular “hurdles”?

Each plate represents a surface at which the separation occurs (“separation event” – ie the analogy of the hurdles in a race)

Plates The more plates (hurdles) the better the

separation and the efficiency of the column Abstract concept used to compare the

efficiencies of different columns To give it a value, need two easy

measurements, retention time and peak width

Plate count N Relationship between Rt and W As the retention time increases, so does the

peak width If look at two columns, compare peak width

of peaks having the same retention time Can use W or W1/2

Formula for N – Theoretical Plates N = 16(VR/W)2

= 16(tR/W)2

Where both tR and W are in the same units (eg seconds or minutes)

Remember, N is a measure of the separation power (efficiency) of the chromatographic system

Measurements for N