Practical Aspects of HPLC Theory

Raymond J. Weigand

Alltech Associates, Inc.

2051 Waukegan Road • Deerfield, IL 60015
Phone: 1-800-ALLTECH • Web Site: www.alltechweb.com
Definition of Chromatography
Chromatography is a separation process in which physical means are
used to distribute sample components between a stationary and a
moving phase. The process occurs as a result of many sorption-
desorption steps during the movement of sample components through
the stationary phase. Separation is due to differences in the distribution
coefficiencts of the sample components.

1 2 3 4
Void Volume (Vo)

Let’s take a look at how theoretical aspects of HPLC can be used in a

practical way.

Concept of Void Volume:

• solutes are carried through the column by the mobile phase
• solute elution time is dependent on attraction to the stationary
phase
• solutes which are not attracted to the stationary phase move
down the column at the speed of the mobile phase
• these solutes elute when the mobile phase has been completely
replaced
• this volume is equal to the volume in the column not taken up by
the packing material (volume of void in the column)
• void volume serves as the reference point for two other often
measured parameters (capacity factor and selectivity)
What is the Practical Significance of Void
Volume?
• Void volume can be used to determine the integrity of the
packed bed
• Void volume is measured by injecting an unretained component and
measuring the volume at which it elutes
• As the void volume changes, peak symmetry will be affected
Capacity Factor (k’)
• While inside the column, a retained component spends part of its
time on the stationary phase and part time in the mobile phase
• When in the mobile phase, solutes move at the same speed as the
mobile phase
• this means that all solutes spend the same amount of time in the
mobile phase (to)
• the amount of time the solute spends on the stationary phase is
equal to tR- to(adjusted retention time, t’R)
• the ratio t’R/ to is the capacity of the column to retain the solute (k’)

tR
to t’R

k’ = (tr - t0) / t0
Inject

Unretained
Solute k’ = (t’r ) / t0
What does a Change in Capacity Factor Mean?

A change in capacity factor signifies at least one of the following:

• a change in the strength of the mobile phase

-can be caused by evaporation of one of the mobile phase components
• contamination of binding sites
-strongly bound sample components can reduce the number of binding
sites available and lower the column’s capacity to retain other solutes
-the use of guard columns will minimize this problem
-column regeneration can help restore column capacity
• loss of binding sites
-harsh mobile phases (low or high pH) can cause loss of bonded phase
or loss of endcapping
-this often causes a change in selectivity
Test Your Knowledge

These two chromatograms were generated on the same column several

weeks apart. Notice the reduction in Capacity Factor of the last peak.
What do you think happened to this column?

9261

Initial

Column: 150x4.6mm Alltima C18, 3um column

Mobile Phase: ACN:Water (58:42)
Flowrate: 1.0mL/min.
Column Temp: ambient
Detector: UV at 254nm

Final 1. Uracil
9262 2. Phenol
3. N,N-Diethyl-m-Toluamide
4. Toluene
Column Efficiency (N)
• Solutes are placed on an HPLC column in a narrow band
• Each solute band spreads as it moves through the column due to
diffusion and mass transfer affects
• The later eluting bands will spread more
• Peak shape follow a Gaussian distribution

to
t1
t2

Band spreading eventually causes peaks to merge into the baseline. We

want to minimize band spreading as much as possible.
The Chromatographic Process

Major reasons why bands spread when traveling through an HPLC column
Who Was Gauss Anyway??
• German mathematician that significantly affected
almost every branch of mathematics and physics

• Developed “Fundamental Theorem of Algebra”

• First to discover a non-Euclidian geometry
• Developed the “Prime Number Theorem” Karl Friederich Gauss 1777-1855

• Aided astronomers in rediscovering the asteroid, Ceres, and found method

for calculating astronomical orbits

• Performed extensive research on magnetism and had the basic unit of

magnetism, the gauss, named after him

• Developed the method of “Least Squares Analysis” and the fundamental

law of probability distribution. The normal distribution curve is named
after him
Who Was Gauss Anyway?

Found a new way to add consecutive numbers (before he was 10 years old!)

1
+2
+3
+4
+5
15
Who Was Gauss Anyway?

Found a new way to add consecutive numbers (before he was 10 years old!)

1 1 + 2 + 3 + 4 +…+ 50
+2 100 + 99 + 98 + 97 +…+ 51
+3
+4
+5
15
Who Was Gauss Anyway?

Found a new way to add consecutive numbers (before he was 10 years old!)

1 1 + 2 + 3 + 4 +…+ 50
+2 100 + 99 + 98 + 97 +…+ 51
+3 101 + 101 + 101 + 101+…+101
+4
+5
15
Who Was Gauss Anyway?

Found a new way to add consecutive numbers (before he was 10 years old!)

1 1 + 2 + 3 + 4 +…+ 50
+2 100 + 99 + 98 + 97 +…+ 51
+3 101 + 101 + 101 + 101+…+101
+4
+5 101
15 X 50
5050
Addition of the numbers from 1 to 100 equals 5050!!
Column Efficiency (N)
The sharpness of a chromatographic peak is an indication of the quality
of the chromatographic column.

• Peak sharpness is determined by measurement of the peak width

• Peak width is dependent on flow rate so measurement of the width
alone is not enough
• a good measure of column efficiency is tR/Wb

Actual equation for column efficiency is N = 16(tR/Wb)2

Practical Significance of Measuring Column
Efficiency

A reduction in the efficiency of an HPLC system is caused by:

• Column voids or channels

• Contamination of the packing material binding sites (this can also
cause selectivity and capacity factor changes)
• Extra-column dead volume

A loss of system efficiency points to physical changes in your system.

The column may be at fault (voids, channels, or contamination) or you
may have unknowingly introduced extra-column dead volume into
your system. Monitoring system efficiency can help you find problems
before they get out of hand.
Peak Tailing (As)

A properly packed HPLC column will give symmetrical or gaussian peak

shapes. Changes in either the physical or chemical integrity of the
column bed can lead to peak tailing.

As = W0.05/ 2f

f
W0.05

Tailing can be caused by:

• Column voids, channels, or extra-column dead volume
(affects early eluting peaks most)
• stripping of the bonded phase (affects late eluting peaks most)
Test Your Knowledge

What is the problem with a system that shows:

• increase in void volume (V0)

• decrease in column efficiency (N) for all compounds
• increase in tailing (As) for early eluting compounds?

How about a system that shows:

• no change in void volume

• decrease in capacity factor (k’) of neutral solutes
• decrease in column efficiency (N) for polar analytes
• increase in tailing (As) for late eluting compounds?
Separation Factor ( )

Because we are usually dealing with samples that contain more than
one sample component, a term describing the separation of peaks is
needed.

• The separation factor describes the relative position of two peak

maxima
• It is equal to the ratio of the time each component spends on the
packing material
t’R(B) / t’R(A)

This equation is more often seen as the ratio of the capacity factors
k’(B) / k’(A)
Separation Factor ( )

The separation factor, also referred to as column selectivity, is affected

by changes in the chemistry of the chromatographic method such as:

• a change in the choice of solvents for the mobile phase

• a change in the packing material in the column

Under strongly acidic mobile phase conditions, standard C18 bonded

phases are slowly hydrolyzed. What does this do to column
selectivity?

What happens to neutral analytes?

What happens to polar analytes?
Resolution (Rs)

Resolution is the goal of every chromatographic method. The equation

below shows how resolution (Rs) is dependent on the Capacity Factor
(k’), Column Efficiency (N), and the Separation Factor (  ).

Rs = 1/4 ( k
k +1 )( N )( -1
 )
Resolution (Rs)

Capacity Factor,
Efficiency, and
Selectivity are the
Initial Separation
three factors that
control resolution.
Understanding the
factors that influence
Vary k
each of these will
help solve any
separation problem.

Vary N

Vary alpha

time
How Smart Are You??
Is this a Capacity Factor, Efficiency, or Selectivity problem? What
happened to the system to cause this change? How can it be fixed?

9263

Column: 150x4.6mm Spherisorb ODS-2 5um

Mobile Phase: ACN:20mM Potassium Phosphate
buffer pH 7.0 (65:35)
Flowrate: 1.0mL/min.
Column Temp: 30C
Detector: UV at 254nm

1. Uracil
2. Phenol
3. N,N-Diethyl-m-Toluamide
4. 3-Butylpyridine
9264 5. 4-Phenylbutylamine
6. Propylbenzene
7. Butylbenzene