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spectrum analysis
This is a mind map about chromatographic analysis, including basics, retention values, theory, gas chromatography, Liquid chromatography, etc.
Edited at 2023-12-23 11:41:00
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spectrum analysis
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- Outline
spectrum analysis
Classification
According to the physical state of mobile phase
Gas Chromatography (GC)
Transmission system
assisting equipments
Purification device: activated carbon, molecular plug
Warm flow device: ensure the carrier gas flow rate is 30~100ml/min, and the variation fluctuation is <1%
Single column and single gas path: isothermal separation analysis
Double columns and double gas lines: Programmable temperature rise can be performed, which can compensate for the loss of fixative and stabilize the baseline. Commonly used
Sampling system
For gas sample injection, use a push-pull six-way valve or a rotary six-way valve. Injection should be fast and accurate to ensure reproducibility of the operation
separation system
The chromatographic column is the core part of the chromatograph.
packed column
Capillary column: The hollow column with coated wall, porous layer and coated carrier requires less sample and has fast analysis speed. However, the column capacity is low and requires high detector sensitivity.
Detection Systems
High sensitivity, low noise, wide linear range, fast response, insensitive to flow rate, temperature and pressure
Thermal Conductivity Detector (TCD)
Principle: Based on the different thermal conductivity of various substances and carrier gases, the resistance values of the measuring arm and the reference arm are not equal, and the bridge is unbalanced, so there is a signal output. The greater the difference between the thermal conductivity of the mixed gas and that of the pure carrier gas, the larger the output signal will be.
Features: stable, versatile, low sensitivity
Flame ionization detector (FID, mainly hydrogen flame detector)
The sensitivity is 10^3 times that of TCD, with small dead volume, fast response, wide linear range, stable and commonly used, but not universal, and only responds to carbon-containing organic compounds.
Mechanism: chemical ionization rather than thermal ionization. In the flame, carbon-containing organic matter first splits into free radicals, then reacts with oxygen to produce positive ions, and then reacts with water to produce H3O ions. The positive ions (CHO, H3O) and electrons produced by chemical ionization move directionally toward the polarizing pole and collecting pole respectively under the action of the electric field to form a current, which is then amplified and recorded.
Electron Capture Detector (ECD)
The electron capture detector is a highly selective detector with high sensitivity to electronegative substances (including halogen, sulfur, phosphorus, nitrogen, etc.)
The disadvantage is that the linear range is narrow, easily affected by experimental conditions, and reproducibility is poor. The disadvantage is that the linear range is narrow, easily affected by experimental conditions, and reproducibility is poor.
The greater the concentration of the sample component, the stronger the electronegativity, the greater the probability of capturing electrons, and the larger the inverted peak.
Flame photometric detector (FED)
A chemiluminescence detector that responds to the characteristic wavelengths of organic compounds containing sulfur and phosphorus
Principle: When sulfur and phosphorus compounds are burned in a hydrogen-rich flame, chemiluminescent substances are generated and emit light of characteristic wavelengths.
Stationary Phase
Carrier: Large specific surface area, inert, good thermal stability, uniform particle size, suitable pores and mechanical strength
Stationary solution: Better selectivity is measured by the relative adjusted retention value γ1,2. For packed column γ1,2 ＞1.15; for capillary column γ1,2 ＞1.08
Liquid Chromatography (LC)
Transmission system: high-pressure pump as the main component
Constant pressure pump: the pressure remains constant and the flow rate changes with the chromatographic resistance
Constant flow pump: stable retention time
Sampling system
Injection sampling: small injection volume, easy leakage, and poor reproducibility
High pressure six-way valve injection
separation system
Includes column tube and stationary phase
Detection Systems
Solute (selective) detector: only responds to the physical or physicochemical properties of the separated components, such as ultraviolet, fluorescence, electrochemical detectors, etc., and is selective
Overall (ordinary) detector: responds to the overall physical or physical and chemical properties of the sample and eluent, such as differential refraction and conductivity detectors, and is not selective.
Stationary Phase
Column temperature selection: column temperature ↑ → mass transfer resistance ↓ → tray height ↓ → column efficiency ↑ column temperature ↑ → longitudinal diffusion ↑ → column efficiency ↓ (it is better to use programmed temperature rise)
Carrier particle selection: Carrier particle size affects eddy current diffusion and mass transfer resistance. The carrier particle size is ↓→column efficiency↑, but if the particle size is too small, the resistance will significantly increase, which will also significantly increase the column pressure drop, causing inconvenience to the operation.
HPLC separation mode
Liquid-solid adsorption chromatography
Stationary phase: solid adsorbent such as silica gel, alumina, etc., the most commonly used is 5~10 μm silica gel adsorbent
Mobile phase: various single or multi-component solvents of different polarities. Generally, n-hexane and cyclohexane are used as the basic solvents, and dichloromethane, short-chain alcohols, and THF are added to adjust the elution strength.
Applicable objects: polar compounds, compounds or families containing different functional groups, isomer compounds
Peak order: alkyl group < halogen (F < Cl < Br < I) < ether < nitro compound < nitrile < tertiary amine < ester < aldehyde < primary amine < amide < carboxylic acid < sulfonic acid
Liquid-liquid partition chromatography
Stationary phase: early application of fixative will result in loss of fixative and is rarely used.
Chemically bonded stationary phase: various groups are bonded to free hydroxyl groups on the surface of silica gel (carrier) through chemical reactions
chemical bonding chromatography
Stationary Phase
Type: silicon-oxygen bond type, silicon-oxygen silicon-carbon bond type (the most widely used), silicon-carbon bond type, silicon-nitrogen bond type
preparation
Silicone esterification: Acid chlorination: Silanization
Si-O-R: Thermal instability, hydrolysis when exposed to water, ethanol, etc., and ester chain breakage Si-R (or Si-N): does not hydrolyze and has better thermal stability than silicate, but the Grignard reaction used is inconvenient Si-O-Si-R: Does not hydrolyze, has good thermal stability, and is stable to water in the pH range of 2-8
Advantages: long life, chemical bonding, no loss of stationary solution, resistance to mobile phase impact; fast mass transfer, no deep depressions on the surface, faster mass transfer than ordinary liquid stationary phases; good selectivity, can bond different functional groups to improve selectivity; available Convenient for gradient elution
The influence of mobile phase separation in reversed-phase bonding chromatography: the lower the polarity of the solvent, the stronger the elution ability; the higher the polarity, the weaker the elution ability. Water is the weakest solvent for reversed-phase chromatography.
Improve separation: mobile phase ratio, mobile phase type, gradient elution
Ion pair chromatography
Principle: Add "ion pair" reagents into the reversed-phase chromatography mobile phase to form "counter ions" with the ionizable components in the sample, and separate ionic compounds on the reversed-phase chromatography column.
Anion separation: Alkylammoniums, such as tetrabutylammonium hydroxide or cetyltrimethylammonium hydroxide, are often used as counter ions.
Cation separation: Alkylsulfonic acids, such as sodium hexanesulfonate, are often used as counter ions.
Ion exchange chromatography
principle
Separation is achieved based on differences in the affinity (or ion exchange capacity) of different ions to be measured for the stationary phase.
The ions to be measured undergo a reversible exchange reaction with the charged groups or free ions on the stationary phase of the ion exchange resin.
Stationary phase: anion and cation exchangers, grafted organic quaternary amine or sulfonic acid groups on organic polymers or silica gel
Classification: strong cation exchanger, weak cation exchanger, strong anion exchanger, weak anion exchanger
Mobile phase: salt buffer solution (with certain pH and ionic strength)
pH value: affects the dissociation balance of acid or base and controls the fraction of the component's molecular form. When a component exists in molecular form, it is not retained; the higher the ion fraction, the greater the retention value.
Ionic strength: has a greater impact on retention than pH. Component retention is controlled by the total concentration of salts in the flow term.
Ion chromatography
Among the basic components of ion chromatography, the most important and different from other high-performance liquid chromatography are the suppressor and conductivity detector.
Suppression column: converts a mobile phase with high background conductivity into a mobile phase with low background conductivity
The suppression column should be regenerated regularly
size exclusion chromatography
Principle: Separation is based on the volume of molecules in the solution, and large molecules are eluted first
Features: low separation efficiency, easy to predict chromatographic behavior
Stationary phase: soft gel, semi-hard gel, hard gel, inorganic gel, organic gel
Affinity chromatography
Principle: Based on biological specificity rather than relying on physical-chemical properties
Features: High selectivity, biocompatibility
Pre-treatment technology
Solid Phase Extraction
solid phase microextraction
subtopic
theory
tray theory
The number of theoretical plates n: one of the main indicators of column efficiency. The greater the number of plates, the stronger the separation ability of the chromatographic column (the effect of flow rate on the number of theoretical plates cannot be explained)
Theoretical plate height H
Effective number of plates neff: After adjusting the retention time tr′ instead of tr, the calculated number of plates is called the effective number of plates neff
Rate theory—chromatographic kinetic theory
In the concept of the theoretical plate height of the tray, the diffusion and mass transfer process of the components in the two phases is fully considered.
Eddy current diffusion term (A), the width and length of the channels that the component molecules pass through in the column vary. Mass transfer resistance term (C), high-concentration components spontaneously distribute along the longitudinal concentration gradient, resulting in band broadening Molecular diffusion term (B), the difference in mass transfer rate of components in the mobile phase and stationary phase broadens the chromatographic peaks
u is the average linear velocity of the mobile phase
When the linear velocity u of the mobile phase is constant, H can be small and the column efficiency will be high only when A, B, and C are all small.
For gas spectroscopy, when longitudinal diffusion of molecules dominates, N2 and Ar with larger relative molecular masses should be selected as carrier gases to reduce the diffusion of components in the mobile phase; when mass transfer resistance dominates, H2 with smaller relative molecular masses should be selected , used as carrier gas, so that the components have a larger diffusion coefficient, improve the mass exchange rate, and ensure column efficiency.
Flow rate (u) ~ plate height (H) curve graph
Minimum column height and optimal linear speed
Reserved value
Dead time (to): the time from the beginning of injection to the peak of a component that is not retained in the stationary phase.
Retention time (tr): refers to the time from the injection of the component to the maximum point after the column.
Adjusted retention time (tr’): refers to the component retention time after deducting the dead time to. Adjusted retention time is the total time that a component remains in the stationary phase
Dead volume (Vo): The volume of mobile phase consumed when components not retained by the stationary phase flow out from the post-column. Dead volume is the sum of the spaces between the stationary phase particles in the column, the space between the joints in the chromatograph tubing, and the space in the detector.
Retention volume (Vr): The volume of mobile phase that passes through from the beginning of injection to the point when the concentration of the measured component appears at the maximum value after the column.
Adjusted retention volume (Vr’): component retention volume after deducting dead volume
Relative retention value (γ2,1): Relative retention value is the basis for qualitative chromatography. It is related to column temperature and stationary phase properties (dimensionless)
Selection factor: In qualitative analysis, a certain chromatographic peak is usually used as the standard peak (S), and then the relative retention values of other component peaks (i) to the standard peak (S) are found. In this case, it is represented by the symbol α (generally > 1)
Capacity factor (k): When the temperature and pressure are constant and the distribution of components between the two phases reaches equilibrium, the mass ratio of the components in the two phases
kAdjust the ratio of retention time to dead time
Retention equation: After the chromatographic column is determined, the volume of the stationary phase Vs and the volume of the mobile phase Vm are both fixed values. The component with a larger distribution coefficient K will have a longer residence time in the chromatographic column.
basic
Chromatographic elution curve
Distribution coefficient (K) (large ones move slowly on the column, small ones move quickly)
Isothermal distribution curve: Under isothermal conditions, the distribution coefficient changes with component concentration.
baseline
Concept: Only pure mobile phase flows through, and the signal-time curve (usually a horizontal line) is detected
Baseline drift: Unstable operating conditions or changes in the working status of the detector and its accessories cause the baseline to slowly change in a certain direction.
Noise: Occasional factors make the baseline fluctuate
Chromatographic peak
Peak height: distance from peak top to baseline
Zone width: measures the efficiency of the chromatographic column and reflects the kinetic factors of chromatographic separation.
standard deviation
Peak base width
half width
Peak area
Separation
The ratio of the difference between the retention values of two adjacent chromatographic peaks to half of the sum of the base widths of the two peaks. R=1.5 is usually used as an indicator of complete separation of adjacent chromatographic peaks. If α=1, then R=0, no matter how high the column efficiency n is It is also impossible to separate the two groups. Only the larger α deviates from 1, the better the selectivity.
Note: The selection factor α is an evaluation of the performance of the stationary phase, and the resolution R is an evaluation of the degree of component separation.
For a column with a certain theoretical plate height, the square of the resolution is proportional to the column length. A longer column can improve the resolution, but at the same time prolongs the separation time (peak broadening). Therefore, the best way to improve resolution is to reduce the plate height. A good chromatographic separation column should have a higher number of theoretical plates.
Qualitative: determined based on the chromatographic peak retention value and comparison with standard materials under the same conditions.
Quantification: ① External standard method: The external standard method is a simpler method in chromatographic quantitative analysis, that is, the standard curve method. ② Internal standard method: add a certain amount of pure substances as internal standards