Principle of elution of separated components in the column
â…¡ Basic concepts and theories
1. Basic concepts and terminology
1. Chromatogram and peak parameters
⊕chromatogram (chromatogram)-the sample flows through the chromatographic column and detector, the resulting signal-time curve, also known as chromatogram elution profile (elution profile).
⊕Base line—The mobile phase is flushed. After the column and mobile phase reach equilibrium, the detector measures the outflow curve for a period of time. Generally it should be parallel to the time axis.
⊕noise-the fluctuation of the baseline signal. Usually caused by poor power contact or transient overload, unstable detector, mobile phase containing bubbles or chromatographic column contamination.
⊕ The drift baseline changes slowly with time. Mainly due to the unstable operating conditions such as voltage, temperature, mobile phase and flow rate, the contaminants or stationary phase in the column will continue to elute and drift.
⊕ Chromatographic peak (peak)-the curve generated by the corresponding continuous signal when the component flows through the detector. The protruding part on the outflow curve. Normal chromatographic peaks approximate a symmetric normal distribution curve (Gaussian Gauss curve). There are two types of asymmetric chromatographic peaks: leading peak and tailing peak. The former is rare.
⊕ Tailing factor (T) —T = B / A, used to measure the symmetry of chromatographic peaks. Also known as symmetry factor (asymmetry factor) or asymmetry factor (asymmetry factor) "Chinese Pharmacopoeia" specifies T should be 0.95 ~ 1.05. T <0.95 is the leading peak, and T> 1.05 is the trailing peak.
⊕Bottom of the peak——The distance from the start point to the end point of the peak on the baseline.
⊕Peak height (Peak height, h) —— The distance from the highest point of the peak to the bottom of the peak.
⊕peak width (peak width, W)-the distance between the two intersections of the tangent line made at the inflection points on both sides of the peak and the baseline. W = 4σ.
⊕peak width at half-height (Wh / 2)-The peak width at half height. W h / 2 = 2.355σ.
⊕standard deviation (standard deviation, σ)-half of the peak width when the normal distribution curve x = ± 1 (inflection point). The inflection point of the normal peak width is 0.607 times the peak height. The size of the standard deviation indicates the degree of dispersion of the components during the flow out of the column. σ is small, the degree of dispersion is small, the pole concentration is high, the peak shape is thin, and the column efficiency is high; otherwise, the σ is large, the peak shape is fat, and the column efficiency is low.
⊕peak area (peak area, A)-the area enclosed by the peak and the bottom of the peak. A = × σ × h = 2.507σh = 1.064Wh / 2h
2. Qualitative parameters (reserved values)
⊕dead time (dead time, t0)-retention time without retaining components. That is, the time when the mobile phase (solvent) passes through the column. In reversed-phase HPLC, sodium benzenesulfonate can be used to determine the dead time.
⊕ Dead volume (V0)-the space occupied by the stationary phase from the inlet of the injector to the flow cell of the detector. It includes 4 parts: the volume of the pipeline from the injector to the chromatographic column, the gap of the stationary phase particles in the column (occupied by the mobile phase, Vm), the volume of the column outlet pipeline, and the volume of the detector flow cell. Among them, only Vm participates in the chromatographic equilibrium process, and the other three powders only play the role of peak expansion. In order to prevent peak expansion, the volume of these 3 parts should be reduced as much as possible. V0 = F × t0 (F is the flow rate)
⊕ retention time (retention time, tR)-the time from the start of injection to the maximum concentration of a component after the column appears.
⊕ retention volume (retention volume, VR)-the volume of solvent that flows out from the beginning of injection to the time when a certain component has a maximum concentration after the column. Also called elution volume. VR = F * tR.
⊕adjusted retention time (adjusted retention time, tR ')-retention time after deducting the dead time. Also called reduced retention time. When the experimental conditions (temperature, fixed equal) are constant, tR 'only depends on the nature of the component, so tR' (or tR) can be used for qualitative. TR '= tR-t0
⊕adjusted retention volume (adjusted retention volume, VR ')-the retention volume after deducting the dead volume. VR = VR-V0 or VR = F * tR '
3. Column efficiency parameters
⊕Theoretical plate number (N) is used to quantify the separation efficiency of column (referred to as column efficiency).
N depends on the type, nature (particle size, particle size distribution, etc.) of the stationary phase, packing status, column length, type and flow rate of the mobile phase, and the nature of the substance used to determine column efficiency. If the peak shape is symmetric and conforms to a normal distribution, N can be approximately expressed as:
N = (tR / σ) 2 = 16 (tR) 2 / W = 5.54 (tR / W1 / 2) 2
W: peak width; σ: half of the peak width at the inflection point of the curve, that is, half of the peak width at the peak height of 0.607.
When N is constant, W changes in proportion to tR. On a multi-component chromatogram, if the content of each component is equal, the peak eluting later is gradually wider than the previous peak, and the peak height is gradually decreasing.
It is more convenient and commonly used to calculate the theoretical plate number with half-peak width than with peak width, because half-peak width is easier to determine accurately, especially for slightly trailing peaks.
N is proportional to the length of the column. The longer the column, the greater the N. When N is used to indicate column efficiency, the column length should be indicated. If not, it indicates the theoretical plate number when the column length is 1 meter. (Generally, the N of the HPLC column is above 1000.)
If the adjusted retention time (tR ') is used to calculate the theoretical plate number, the value obtained is called the effective theoretical plate number (N effective or Neff) = 16 (tR' / W) 2
⊕ The variance of the theoretical plate height (H) per unit column length. H =. In practical application, the column length L and the number of theoretical plates are often used for calculation: H = L / N
4. Phase equilibrium parameter (K)-at a certain temperature, when the compound reaches the distribution equilibrium between the two phases, the ratio of the concentration in the stationary phase and the mobile phase. K = [xs] / [xm]
Cs-solute concentration in stationary phase
Cm-solvent concentration in mobile phase
The partition coefficient is related to the thermodynamic properties of the components, mobile phase and stationary phase, as well as temperature and pressure. In different chromatographic separation mechanisms, K has different concepts: adsorption chromatography is the adsorption coefficient, ion exchange chromatography is the selectivity coefficient (or exchange coefficient), and gel chromatography is the permeation parameter. But the general situation can be expressed by the distribution coefficient.
Under certain conditions (mobile phase, stationary phase, temperature, pressure, etc.), when the sample concentration is very low (Cs, Cm is very small), K depends only on the nature of the components, and has nothing to do with the concentration. This is just the chromatographic conditions under ideal conditions. Under these conditions, the resulting chromatographic peak is a normal peak; in many cases, as the concentration increases, K decreases, and the chromatographic peak is a trailing peak; sometimes As the concentration of the solute increases, K also increases, and the chromatographic peak is the pre-extension peak. Therefore, only when the injection volume is as small as possible to reduce the concentration of the component in the column and the K is constant, a normal peak can be obtained.
Under the same chromatographic conditions, the component with a large K value in the sample has a long residence time in the stationary phase and then flows out of the column; the component with a small K value has a short residence time and flows out of the column first. The greater the difference in the partition coefficients of the components in the mixture, the easier the separation. Therefore, the different partition coefficients of the components in the mixture are the premise of chromatographic separation.
In HPLC, after the stationary phase is determined, K is mainly affected by the nature of the mobile phase. In practice, it is mainly by adjusting the composition ratio and PH value of the mobile phase to obtain the difference in the distribution coefficient between the components and the appropriate retention time to achieve the purpose of separation.
⊕capacity factor (capacity factor, K)-the ratio of the amount of the compound in the stationary phase and the mobile phase when the compound reaches equilibrium between the two phases. K = (tR-t0) / t0 = tR '/ t0 (or the amount of solute in the stationary phase / the amount of solute in the mobile phase). Therefore, the capacity factor is also called the mass distribution coefficient.
{K = Cs / Cm = K'Vm / Vs k = (tR-t0) / t0 = K * Vs / Vm Vs: volume of stationary phase in the column; Vm: volume of mobile phase in the column. ï½
The distribution coefficient, capacity factor and retention time have the following relationship: k === K =, tR '= kt0. The above formula illustrates the physical meaning of the capacity factor: it means that the residence time (tR ') of a component in the stationary phase is several times the retention time (t0) of the unretained component. When k = 0, all the compounds exist in the mobile phase and are not retained in the stationary phase, tR '= 0; the larger the k, the larger the capacity of the component relative to the stationary phase, the slower the column, and the longer the retention time.
The difference between the capacity factor and the partition coefficient is that K depends on the nature and temperature of the components, mobile phase and stationary phase, and has nothing to do with the volume Vs and Vm; k is related to Vs and Vm in addition to the nature and temperature. Since tR 'and t0 are easier to measure than Vs and Vm, the capacity factor is more widely used than the distribution coefficient.
⊕selectivity factor (selectivity factor, α)-the ratio of the distribution coefficient or capacity factor of two adjacent components. α == (Let k2> k1). Because k = tR '/ t0, then α = k2 / k1, so α is also called relative retention time ("United States Pharmacopoeia").
To separate the two components, α ≠1 must be achieved. α is related to the distribution properties of the compound in the stationary phase and the mobile phase, the column temperature, and not to the column size, flow rate, and packing. Essentially, the size of α represents the difference in the thermodynamic properties of the equilibrium distribution of the two components between the two phases, that is, the difference in the interaction force between the molecules.
5. Separation parameters
⊕ Resolution (resolution, R)-the ratio of the difference between the retention time of two adjacent peaks and the average peak width. Also called resolution, it indicates the degree of separation between two adjacent peaks. R = (tR2-tR1) / [(W1
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