Chapter 2 - Basic terms and concepts Flashcards
The term “sample component” is used interchangeably with
“analyte” and “solute”.
The most common stationary phase in RPC is
hydrophobic C18-bonded phase on a porous silica support.
The term “sorbent” refers to
the bonded phase.
The term “support” refers to the
unbonded silica material.
Retention time (tR)
The time between the sample injection and the peak maximum.
Void time (tM).
The total time spent by any unretained component in the mobile phase within the column.
The adjusted retention time (tR’) is equal to
the time the solute resides in the stationary phase (tR - tM).
wb
Peak width measured at the base.
w½
Peak width measured at the peak’s half-height.
For Gaussian peaks, wb is approximately equal to
four times the standard deviation.
The height or area of a peak is proportional to
the amount of analyte component present in the sample.
Peak area is more commonly used to perform
quantitative calculations.
The retention volume (VR) is
the volume of mobile phase needed to elute the analyte at a given flow rate (F).
Retention volume equation
VR = tRF
Void volume equation
VM = tMF
The void volume is the
total volume of the mobile phase contained in the column.
For most columns, the void volume can be estimated by
VM = 0.65Vc = 0.65(pi)r2L
Peak volume equation
Peak volume = wbF
Retention factor (k) is
the degree of retention of the analyte in the column.
Retention factor is defined as
the time the solute resides in the stationary phase (tR’) relative to the time it resides in the mobile phase (tM).
Retention factor equation
k = tR’/tM
Retention volume equation
VR = VM(1 + k)
A peak with k = 0 means that
a component is not retained by the stationary phase and elutes with the solvent front.
A value of k > 20 indicates that
the component is highly retained.
The desired k values for most analyses are between
1 and 20.
Separation factor or selectivity (alpha) is
a measure of the relative retention (k2/k1) of two sample components.
Selectivity must be ______ for physical peak separation to occur.
> 1.0
Selectivity is a measure of
differential migration.
Changing alpha is the most effective way to
separate two closely eluting solutes.
The number of theoretical plates or plate number (N) is a measure of
the efficiency of the column.
N is defined as
the square of the ratio of the retention time divided by the standard deviation of the peak.
Number of theoretical plates equation
N = 16(tR/wb)2
For a Gaussian peak, w½ is equal to
2.355 sigma
N can only be measured under _______ conditions.
isocratic
Peak volume is the
volume of the mobile phase, or eluent, containing the eluting peak.
Peak volume is proportional to
k and VM.
The height equivalent to a theoretical plate (HETP or H) is equal to
the length of the column (L) divided by the plate number (N)
In HPLC, the main factor controlling H is the
diameter of the particles (dp) used as the packing material.
For a well-packed column, H is roughly equal to
2dp
Resolution (Rs) definition
The degree of separation of two adjacent peaks.
Rs is defined as
the difference in retention times of the two peaks divided by the average peak width.
Resolution equation
delta (tR)/wb
Rs = 0 indicates
complete co-elution or no separation.
Rs = 0.6 indicates
that a shoulder is discernible or slight separation has been achieved.
Rs = 1 indicates
partial separation, allowing for a rough quantitation.
Rs = 1.5 indicates
that a baseline separation of the two components has been achieved.
The goal of most HPLC methods is to achieve separation of
Rs = 1.5 for all key analytes
The asymmetric factor (As) is used to
measure the degree of peak symmetry.
Asymmetry factor equation
As = B/A
Tailing factor (Tf) equation
Tf = W0.05/2f
Tf = 1.0 indicates
a perfectly symmetrical peak.
Tf > 2 indicates
a tailing peak that is less acceptable due to difficulty in integrating the peak area precisely.
With a Tf between 0.5 and 2.0, the values of As and Tf are
similar.
Peak tailing is typically caused by
adsorption, strong interaction with the stationary phase, or extra-column band broadening of the solute.
