Int. J. Pharm. Sci. Rev. Res., 55(2), March - April 2019; Article No.

09, Pages: 46 - 50 ISSN 0976 – 044X

Review Article

Fundamental Chromatographic Parameters

Panchumarthy Ravisankar*, S. Anusha, Kommanaboina Supriya, U. Ajith Kumar

Department of Pharmaceutical Analysis and Quality Assurance, Vignan Pharmacy College, Vadlamudi, Guntur, A.P, India.
*Address for Correspondence: E-mail: banuman35@gmail.com

Received: 14-02-2019; Revised: 25-03-2019; Accepted: 02-04-2019.

ABSTRACT
HPLC plays a vital role in product assessment, research and environmental monitoring. HPLC is suited to separate higher molecular
weight compounds in order to give quantitative and qualitative information. HPLC separatory systems, chromatographic separations
are characterized by the resolution, retention time of analyte peak, selectivity and efficiency (plate number). HPLC separations are
effected with liquid mobile phases following through a column packed with a solid stationary phase. This article briefly describes the
theory and equations behind many of the concepts that drive chromatography in pellucid and simple way of essential
chromatography concepts such as efficiency, retention factor, selectivity, resolution, pressure, Van Deemter curves and gradient
equation.
Keywords: Efficiency, retention factor, selectivity, resolution, chromatographic parameters.

INTRODUCTION
Retention time Peak width at half

H PLC is just the premier technique for trace analysis

of organic and inorganic compounds. If I think of
all the work which has being done in
pharma/biochem, environmental, forensics/toxicology,
industrial and food safety all these things are done
N = 5.54 (tR/W1/2)2
height

Equation 1b: Alternate equation for calculating efficiency

routinely and rapidly by HPLC. Columns with high plate numbers are more efficient. A
Essential chromatographic parameters column with a high N will have a narrower peak at a
given retention time than a column with a lower N
We start with fundamentals of performance: number.
1. Efficiency
2. Retention factor
3. Selectivity
4. Resolution
5. Pressure
These are all key to understand how to optimize results
and successfully develop methods.
We also explore a few more complex concepts:
1. Van Deemter curves
2. The gradient equation
These two topics are also important for method
development.
Figure 1: Chromatographic illustration of efficiency,
Efficiency (N) retention factor and resolution
Column efficiency is used to compare the performance of If we measure the distance tw here (Figure 1), by drawing
different columns. It is probably the most frequently cited tangent lines to approximate the four - sigma peak width,
parameter of column performance and is expressed as we can measure the theoretical plates for peak B, using
the theoretical plate number, N. Equation 1a, N = 16 (tR/tW)2. Sometimes the four - sigma
peak width is difficult to measure (e.g., with a noisy
Efficiency Retention time Peak width at base
baseline), so an alternate equation (Equation 1b) involves
N = 16 (tR/Wt)2 measuring the peak width at half - height (w1/2): N= 5.54
(tR/w1/2)2.
Equation 1a: Efficiency equation

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Int. J. Pharm. Sci. Rev. Res., 55(2), March - April 2019; Article No. 09, Pages: 46 - 50 ISSN 0976 – 044X

High column efficiency is beneficial since less selectivity between peaks A and B than between B and C.
is required to completely resolve narrow peaks. Column Calculations are provided to demonstrate.
efficiency is affected by column parameters (diameter,
Selectivity can be changed by changing the mobile
length, particle size), the type of eluent (especially its
phase constituents or changing the stationary phase.
viscosity), and flow rate or average linear velocity.
Temperature may also be a factor in adjusting
Efficiency is also affected by the compound and its
selectivity.
retention. When comparing columns, the number of
theoretical plates per meter (N/m) is often used. Resolution (Rs)
However, the same chromatographic temperature
Resolution describes the ability of a column to separate
conditions and peak retention (k) are required for the
the peaks of interest, and so the higher the resolution,
comparision to be valid. On stationary phases where α
the easier it is to achieve baseline separation between
is small, more efficient columns are beneficial.
two peaks. Resolution takes into consideration efficiency,
Retention Factor (k) selectivity and retention, as can be seen in Equation 4.
One can improve resolution by improving any one of
Formerly referred to as capacity factor or k´ (k prime), the
these parameters. Figure 3a shows the impact of
retention factor measures the period of time that the
efficiency, selectivity and retention on resolution. As a
sample component resides in a stationary phase relative
matter of fact selectivity may be the major effective tool
to the time it resides in the mobile phase. It is calculated
for optimizing resolution.
from the retention time divided by the time for an
unretained peak (t0). N (-1) k
Rs =
Retention time for the sample 4  (k+1)
peak
(tR-to) Equation 4: Resolution equation
K=
to

Retention Retention time for

factor unretained peak
Equation 2: Retention factor equation

Figure 3: Chromatographic illustration of resolution

Figure 2: Chromatographic illustration of retention factor

Selectivity or separation factor (α)
The separation factor is a measure of the time or distance
between the maxima of two peaks. If α = 1, the two peaks
have the same retention time and co-elute.

Selectivity Retention factor of

first peak


Figure 3a: Impact of selectivity, efficiency, and retention

Retention factor of second peak
on resolution
Equation 3: Selectivity equation In Figure 4, we see the different effects of each
Selectivity is defined as the ratio in capacity factors. In component on the separation process. All of these terms
Figure 1, you will see that there is better selectivity show a diminishing return. This means that the more you

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Int. J. Pharm. Sci. Rev. Res., 55(2), March - April 2019; Article No. 09, Pages: 46 - 50 ISSN 0976 – 044X

try to work on something to improve the separation, the A is distance from the leading edge of peak midpoint to
less effective it will become. the midpoint
If you double the column length, you will obtain more Asymmetry factor
theoretical plates, but your separation will take twice as
long; you will only get a square root of 2 or 1.4
improvement in the resolution.
A value of 1 is the minimum for a measurable separation
to occur and to allow adequate quantitation. A value of
0.6 is required to discern a valley between two equal -
height peaks. Values of 1.7 or greater generally are
desirable for rugged methods. A value of 1.6 is
considered to be a baseline separation and ensures the
most accurate quantitative result.

Figure 5b: Chromatographic illustration of asymmetry

factor
Where, AsF is peak asymmetry factor
B is distance from the point at the peak midpoint to the
tailing edge
A is distance from the leading edge of peak midpoint to
the midpoint
Pressure
The Pressure equation (Equation 5) identifies five key
factors that affect system pressure: solvent viscosity (h),
flow rate (F), column length (L), column radius (r) and
particle diameter (dp). It is a good idea to familiarize
Figure 4: Resolution as a function of selectivity, column
yourself with the pressure equation to understand these
efficiency or retention
key contributors to system pressure.
Tailing factor and Asymmetry factor
Change in Viscosity Flow rate
If the peak to be quantified is asymmetric, a calculation of pressure
the asymmetry would also be useful in controlling or Column length
characterizing the chromatographic system. Peak FL
P =
asymmetry arises from a number of factors. The increase
in the peak asymmetry is responsible for a decrease in
K0r2dp2
chromatographic resolution, and precision. The peak Particle
asymmetry can be calculated by using formula: Column Column diameter
permeability radius
Tailing factor
Equation 5: Pressure equation
As noted in the formula, even a small decrease in the
particle size (dp) has a significant impact on back
pressure.
Van Deemter Curves
The Van Deemter equation evaluates efficiency
(expressed as H, see Equation 6) as a function of linear
velocity (u) or flow rate. The H - called plate height, or
height of a theoretical plate is determined by dividing the
column length (L) by the calculated number of theoretical
Figure 5a: Chromatographic illustration of tailing factor plates. The goal is to get a small plate height. We can do
this most effectively with smaller particle columns,
Where, TF is tailing factor (measured at 5 % peak height) optimum linear velocities and low viscosity mobile phase.
B is distance from the point at the peak midpoint to the As particle size decreases, the optimum linear velocity
tailing edge increases.

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Int. J. Pharm. Sci. Rev. Res., 55(2), March - April 2019; Article No. 09, Pages: 46 - 50 ISSN 0976 – 044X

H = A + B/u + C u separated. For small molecules, the value of S is about

4 to 6. For peptides and proteins, S lies between 10
H=L/N and 1,000.
These days, it is common to change the dimension of
Equation 6: Van Deemter equation
the column, either to something shorter (e.g., for
higher throughput) or with a narrower internal
diameter (e.g. for mass spectrometric detection). Any
decrease in column volume must be offset by a
proportional decrease in gradient time (tG) or flow
rate (F). Any change in the gradient compositional
range (DF), using the same column, needs to be
adjusted by a proportional change in gradient time
(tG) or flow rate (F) if you want to maintain the same
gradient slope and k* value.
CONCLUSION
Figure 6: Illustration of the Van Deemter equation
An essential role of chromatography is the quality
We often plot Van Deemter curves to evaluate the control of the quality of drugs controlling the raw
performance of different columns, and to understand the materials, finished drugs ensuring the safety of the
optimum linear velocity (uopt) for a method. people we are so dependant in the world today on
The gradient equation synthetic chemicals made by chemists. So HPLC is the
best separation technique for the quantitative trace
Whenever your sample has a wide variety of components analysis of toxic chemical impurities. We all remember
present, it can be difficult to separate all of the the feeling we had in college as we learned math,
components in a reasonable time using isocratic elution wondering how it would actually come into practical
(e.g., constant mobile phase composition). Gradient use. Students, lecturers and scientists have to learn
elution is a process to increase the mobile phase strength more math than many professionals. One should
as a function of time, resulting in faster analysis, better understand the above said concepts which will help
peak shape and quantitation. With gradient elution, peak you to troubleshoot and get the best results if you
widths are typically more narrow and of constant width. encounter problems in HPLC.
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Source of Support: Nil, Conflict of Interest: None.

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