1. Guaranteed reproducibility
2. Excellent bond phase stability
3. Interaction between hydrophobic and pentafluorophenyl "mixed mode"
Improving chromatographic resolution <br> The goal of chromatographic separation is to obtain sufficient resolution (Rs) of the target component in the shortest amount of time.
A resolution of 1.5 can achieve baseline separation, however the ideal resolution is 1.8-2.0 for a durable, repeatable method that can be easily converted between laboratories.
The resolution equation tells us what variables can affect the resolution:
 | Rs = resolution between target peaks N = efficiency - determined by theoretical plate number Î±ï€ = selectivity - retention ratio of two peaks (k value) k = retention factor - number of column volumes required to elute the peak |
Increasing the resolution Rs can be achieved by increasing N, α or k.
However, as shown in FIG. 1, it can be seen that increasing N or k to improve the return rate of Rs decreases rapidly.
For example, Rs only increases as the N square root increases.
N can be increased by increasing the length of the column or decreasing the particle size of the column packing material or some combination of the two.
Either way, the system back pressure increases as N increases, so a satisfactory separation by increasing N can be a very high "cost".
Similarly, increasing the retention value (k value) will increase Rs, but the rate of return will also drop rapidly.
Increasing k to more than 10 is usually an unfavorable trade-off between Rs and analysis time because only the marginal benefit of Rs is achieved as the retention time increases.
A graphical representation of this effect is shown in Figure 1 below.
Figure 1. Effect of N, α, and k on resolution (Rs) For a typical separation, where: N = 10,000, k = 4, α = 1.1
Increasing the N, α or k can increase the resolution (Rs).
However, as can be seen from these figures, an increase in N or k will quickly reduce the rate of return.
On the other hand, increasing the selectivity (α) does not have this problem, so it becomes the optimal optimization variable when developing the separation method.
Increasing α increases Rs, but unlike N and k, it is not constrained by a drop in the rate of return.
The change in α has no effect on the pressure and the effect on the separation time is negligible (see Figure 2).
Therefore, α is the most important variable when developing the separation method.
Optimizing alpha allows you to achieve satisfactory resolution across all target peaks while keeping system back pressure and separation time within acceptable limits.
Improve chromatographic resolution - selectivity or efficiency?
Selectivity (α) is controlled by the mobile phase, temperature, and stationary phase chemistry. Most method development strategies will explore all of these chromatographic variables.
If a "standard" 3 μm C18 phase is not used to achieve sufficient resolution, it is recommended to optimize the chromatographic selectivity of the separation rather than the separation efficiency, as shown in the examples below.
By simply changing the stationary phase chemistry (ie, the column) to a stationary phase chemistry with alternative chromatographic selectivity, it is easy to achieve the desired resolution on a standard HPLC system without the need for expensive UHPLC instruments.
In addition, complex mobile phase components, elevated temperatures and aggressive pH conditions can also be avoided.
Figure 2 uses selectivity to achieve fast, high resolution separation
Samples: 1) acetaminophen 2) hydrochlorothiazide 3) methylphenyl sulfoxide 4) methyl phenyl sulfone 5) aspirin 6) phenacetin 7) 1,3-dinitrobenzene
8) 1,2,4-Trimethoxybenzene 9) benzoic acid ethyl ester 10) Nimesulide 11) Ibuprofen 12) Indomethacin 13) Mefenamic acid Column size: 50 x 2.1 mm Flow rate :0.60 ml/min Temperature: 40°C Detection: UV, 254 nm Mobile phase: A = 5 mM formic acid (dissolved in water) and B = 5 mM (dissolved in methanol), gradient = 3-100 in 5 minutes % B
Comparing data does not represent all applications.
While maintaining the C18 bonded phase, reducing the particle diameter from 3 μm to 2 μm or less does not significantly improve the separation effect, and also causes a significant increase in pressure.
The ACE C18-PFP column provides better selectivity (α) for the three key pairs, so it provides excellent separation compared to C18 columns below 2μm, even for columns below 2μm Higher efficiency.
A better separation effect can be obtained by utilizing the ability of selectivity compared to the results obtained by attempting peak separation using a column having a high number of plates and a high pressure.
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