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Setting HPLC Chromatographic Parameters

Whether you work in a regulated environment or not, setting chromatographic performance targets can help to keep us focussed.  Let’s consider how to set these targets and examine some real life examples that may not always follow the rules.

Ultimately we need sensitive, reproducible, and robust chromatographic results, which are fit for purpose, according to our analytical requirements.  This may mean different things to different people, depending on the type of work you are carrying out.  Having chromatographic performance targets to work towards will not only result in more robust chromatography but they will be a great indicator of when we are heading down the wrong development path, or when there are underlying problems with the method or equipment.

There are many regulatory organisations that have set analytical method performance requirements.  Table 1 shows the current US Food and Drug Administration (FDA) values for the Validation of Chromatographic Methods which we will use for the purposes of this discussion.

Parameter Limit
Retention (Capacity) Factor k ≥ 2
Injection Precision RSD < 1% for n ≥ 5
Resolution Rs > 2
Tailing Factor T ≤ 2
Theoretical Plate (Efficiency) N > 2000

Download - FDA Validation of Chromatographic Methods

Download - ICH Harmonised Tripartite Guideline Validation Of Analytical Procedures: Text And Methodology Q2(R1)

Table 1: The current FDA values for the Validation of Chromatographic Methods.    


Retention Factor

Retention Factor  

It is suggested that values of k should be between 2 and 10, but will this work in all cases?  If k < 1 separations will be less stable and reproducible.  They will have a greater susceptibility to chromatographic interferences at the beginning of the chromatogram, with the chance of peaks being poorly resolved from unretained material at t0.  The retention of analytes with low k values will also be more sensitive to small changes in mobile phase composition.  However, sometimes a k value of between 1 and 2 may work well when faster chromatography is desired (high throughput), where samples do not contain a lot of endogenous/matrix components or where pH or buffer strength are not vital in controlling retention or selectivity.

For complex mixtures k values greater than 10 may be needed to resolve all peaks.  If complex mixtures are being analysed be aware of peak broadening of later eluting peaks that may reduce resolution.  

Real world example

An example in modern chromatography which demonstrates that sometimes larger k values are acceptable, and in this case necessary, in order to generate the required resolution of all 8 components is shown (Figure 1 – 3).  At 80 %B (Figure 1) the latest eluting peak has a k of 1.09 and only 3 analytes are fully resolved, reducing the %B to 70% increases the k value to 5, however, there are still only 7 of the 8 compounds resolved (Figure 2).  A k of 10 (60% B, Figure 3) is required to fully resolve all 8 compounds. 

Retention Factor  

Figure 1: Separation of 8 nitro compounds. 
Column: C18 10 cm x 0.21 cm x 2 µm. 
Eluent: Water:MeCN (80% B). 
Flow rate: 1.0 mL/min. 
Temperature: 35 °C 80% B. 
k = 1.09, N = 21629, and Rs (min) 0.05. 


Retention Factor  

Figure 2: Separation of 8 nitro compounds. 
Column: C18 10 cm x 0.21 cm x 2 µm. 
Eluent: Water:MeCN (70% B). 
Flow rate: 1.0 mL/min. 
Temperature: 35 °C 80% B. 
k = 5, N = 21182, and Rs (min) 0.29. 


Retention Factor  

Figure 3: Separation of 8 nitro compounds. 
Column: C18 10 cm x 0.21 cm x 2 µm. 
Eluent: Water:MeCN (60% B). 
Flow rate: 1.0 mL/min. 
Temperature: 35 °C 80% B. 
k = 10, N = 21030, and Rs (min) 2.03. 


Efficiency   Efficiency can be increased by increasing the column length, reducing the column internal diameter, or decreasing the particle size.  It is better to use a smaller diameter packing than increase the column length, which will increase analysis time.  However, a decrease in particle size will result in an increase in system backpressure.  The use of smaller particles and narrower column internal diameter both require minimized extra column dead volume in order to avoid efficiency losses.

The FDA stipulates a value for N > 2000 which is typically easily achieved with modern HPLC columns (Table 2).

Column Geometry Approximate Efficiency
(Plates, N/column) (Vεc – 50 µL)
250 x 4.6 mm, 5 µm 22,000
150 x 4.6 mm, 5 µm 13,000
150 x 4.6 mm, 3 µm 21,000
100 x 4.6 mm, 5 µm 9,000
100 x 4.6 mm, 3 µm 14,000
100 x 2.1 mm, 3 µm 11,000
100 x 4.6 mm, 1.7 µm 15,500
100 x 2.1 mm, 1.7 µm 23,500
50 x 4.6 mm, 3 µm 7,000
50 x 2.1 mm, 3 µm 5,500
50 x 4.6 mm, 1.7 µm 7,800
50 x 2.1 mm, 1.7 µm 11,700

Table 2:  Approximate column efficiencies for standard HPLC column geometries.


 A very practical measure of the relative separating power of columns of different dimension is the so-called Resolution Capacity (Table 3).

Column Length, L (mm) Particle Size, dp (µm) Resolution Capacity L/dp
300 10 30,000
150 5 30,000
100 3 33,300
50 1.7 29,500
100 1.7 58,820
150 1.7 88,230

Table 3: Resolution Capacity of standard HPLC column geometries.

Tailing Factor

Tailing Factor   Tailing peaks create issues with resolution, quantitation (integration), and reproducibility.  Peak shape is often the controlling factor when optimising complex separations, especially when components are present in very different concentrations.


Tailing Factor   Figure 4: TF = (a) 1.24, (b) 1.42, (c) 1.58
and (d) overlay of a, b, and c.


Real world example

The analysis of a stability indicating sample at different buffer concentrations demonstrates the importance of the tailing factor (Figure 5 and 6).  At a lower buffer concentration (13.4 mM) the degradent peak in the sample has a relatively high tailing factor of 1.72, which although it is in the FDA recommended range, results in poor resolution (Rs = 0.98) between the degradent and the proceeding peak.  Increasing the buffer concentration (23.6 mM) not only improves the peak tailing of the degradent peak (TF = 1.43) but also results in resolution of the two peaks (Rs = 1.69).  Note: The retention time of the peaks does not change. 

Retention Factor   Figure 5: Stability indicating sample (no ionisable compounds), separation previously optimised for selectivity. 
Column: C18 10 cm x 0.21 cm x 2 µm. 
Eluent: Phosphate buffer (aq):MeOH. 
Flow rate: 1.0 mL/min. 
Temperature: 35 °C. 
Buffer concentration = 13.4 mM.  Rs = 0.98. 
Degradent TF = 1.72.


Retention Factor  

Figure 6: Stability indicating sample (no ionisable compounds), separation previously optimised for selectivity. 
Column: C18 10 cm x 0.21 cm x 2 µm. 
Eluent: Phosphate buffer (aq):MeOH. 
Flow rate: 1.0 mL/min. 
Temperature: 35 °C. 
Buffer concentration = 23.6 mM.  Rs = 1.69. 
Degradent TF = 1.43.


Precision   Injection precision is important for reproducible chromatographic results and should be estimated in the same way for each analysis.  It is indicative of performance of the plumbing, column, and environmental conditions at the time of analysis and assessment of injection reproducibility can be used to aid in the diagnosis of potential system problems such as leaks (Table 4).  It is expressed as RSD (relative standard deviation) and is measured by multiple injections of a homogeneous sample (Table 5).  Modern autosamplers injection precision is in the range 0.15 – 0.9% (depending on the sample volume).


Sample Rt Peak Area Δ Rt Δ Peak Area
A1 A2 5.62 5.66 2155699 2120466 0.04 35233
B1 B2 5.87 6.13 2205659 2288355 0.26 82696
C1 C2 6.21 6.48 2227066 2265279 0.27 38213
D1 D2 6.73 6.99 2581888 2602016 0.26 20128

Table 4: Injection repeatability data for an HPLC system that developed a leak during sampling.


n Mean ± SD RSD
10 1993162 ± 5029 0.25%

Table 5: Representative data for 10 injections


There are several factors that will affect precision which should be considered to minimise errors.

Chemical factors

  • pH/buffer type
  • O2 (sample oxidation)
  • Light (sample photo-degradation)
  • Heat (sample thermal degradation)
  • Organic components (contaminants, degradation products)
  • Mobile phase stability



Facile/Sample handling Issues

  • Adsorption to sample containers, syringes, sample loops etc.
  • Accurate dilutions
  • Thorough sample mixing
  • Homogeneous sampling
  • Over filled vials (cavitation)
  • Emulsions settling over time
  • Use of internal standard to estimate loss during sample pre-treatment



handbook for thursday   Before setting a value for resolution it is important to ask a couple of questions that relate to the specific separation first:
  1. What value is acceptable?
  2. What value is required for reliable quantitation?

Resolution is a function of retention (k), selectivity (α), and efficiency (N) which can all be modified, as has been seen, to improve resolution.  Rs > 1.5 can often be easily obtained for samples containing 5 or less components, however, for complex mixtures Rs > 1.8 is required for rugged performance.  ‘Real world’ setting of resolution specifications requires experience in HPLC and the method under consideration.


Real world example 1

Although values of Rs > 2 are recommended this is sometimes not practical with very complex samples or depending on the type or stage of analysis.  The early stage analysis of pharmaceutical impurities (Figure 7) was carried out with a minimum Rs value of 1.2 which, for this type of analysis, gave reliable, reproducible quantitation of all impurities.  

Early phase pharmaceutical impurity analyses Figure 7:  Early phase pharmaceutical impurity analyses

Real world example 2

Conversely, late stage pharmaceutical impurity analyses (Figure 8) require much more stringent Rs values (> 4) to give reliable quantitation of impurities on the tail of the API.  Any integration differences will result in issues with method robustness.

tailing peak api

Figure 8: Late phase pharmaceutical impurity analyses.

Discrimination factor

With complex samples it may be worth considering the use of a more descriptive measure such as the Discrimination Factor (d0, Figure 9).

Discrimination Factor

tailing peak api




Figure 9: Schematic representation of discrimination factor d0.

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