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chromatography
Analytical chemical chromatography mainly includes classic liquid chromatography, high performance liquid chromatography, gas chromatography, qualitative and quantitative analysis methods, etc.
Edited at 2024-01-19 17:01:40
- cáncer de pulmón
El cáncer de pulmón es un tumor maligno que se origina en la mucosa bronquial o las glándulas de los pulmones. Es uno de los tumores malignos con mayor morbilidad y mortalidad y mayor amenaza para la salud y la vida humana.
- diabetes
La diabetes es una enfermedad crónica con hiperglucemia como signo principal. Es causada principalmente por una disminución en la secreción de insulina causada por una disfunción de las células de los islotes pancreáticos, o porque el cuerpo es insensible a la acción de la insulina (es decir, resistencia a la insulina), o ambas cosas. la glucosa en la sangre es ineficaz para ser utilizada y almacenada.
- sistema digestivo
El sistema digestivo es uno de los nueve sistemas principales del cuerpo humano y es el principal responsable de la ingesta, digestión, absorción y excreción de los alimentos. Consta de dos partes principales: el tracto digestivo y las glándulas digestivas.
chromatography
- cáncer de pulmón
El cáncer de pulmón es un tumor maligno que se origina en la mucosa bronquial o las glándulas de los pulmones. Es uno de los tumores malignos con mayor morbilidad y mortalidad y mayor amenaza para la salud y la vida humana.
- diabetes
La diabetes es una enfermedad crónica con hiperglucemia como signo principal. Es causada principalmente por una disminución en la secreción de insulina causada por una disfunción de las células de los islotes pancreáticos, o porque el cuerpo es insensible a la acción de la insulina (es decir, resistencia a la insulina), o ambas cosas. la glucosa en la sangre es ineficaz para ser utilizada y almacenada.
- sistema digestivo
El sistema digestivo es uno de los nueve sistemas principales del cuerpo humano y es el principal responsable de la ingesta, digestión, absorción y excreción de los alimentos. Consta de dos partes principales: el tracto digestivo y las glándulas digestivas.
- Recommended to you
- Outline
Clinical Pharmacology Chapter 2 Research and Development of New Drugs and Clinical Research of Drugs
- 26
chromatography
Introduction
The essence of chromatography: substances have different distribution coefficients in the two phases, and they are separated from each other by repeatedly distributing in the two phases.
the term
Chromatographic peak
The number of chromatographic peaks determines the minimum number of components
baseline
Peak height
Peak area
Quantitative analysis
Chromatographic width
tailing factor
keep time
time
Dead time tM
The time required from injection to peak maximum for components that are not absorbed or dissolved by the stationary phase.
The flow velocity is similar to the mobile phase flow velocity
Calculate average linear velocity of mobile phase
keep time
Under the same chromatographic conditions, the same component has the same retention time
Adjust retention time
Retention time of a component minus dead time
Retention time of components in stationary phase
Retention volume: The volume of mobile phase required to remove a component from the chromatograph
dead volume
retention volume
Adjust retention volume
relative retention value
Chromatographic qualitative analysis parameters
It is only related to column temperature and stationary phase, and has nothing to do with column diameter, column length, and mobile phase flow rate.
Qualitative analysis
System adaptability evaluation
Classification
The state of the two phases
Liquid Chromatography
liquid-solid chromatography
liquid-liquid chromatography
Gas chromatography
Gas-solid chromatography
Gas-liquid chromatography
supercritical fluid chromatography
Separation mechanism
Adsorption chromatography: components have different adsorption abilities on the stationary phase
Distribution chromatography: components have different solubilities (partition coefficients) in the stationary solution
Ion exchange chromatography: components have different affinities on ion exchangers
Size exclusion chromatography: selective permeation of molecules of different sizes in a porous stationary phase
Affinity chromatography: Separation of different components with high specific affinity to the stationary phase (solidified molecules) (commonly used for separation of proteins)
Operation form
column chromatography
packed column chromatography
capillary column chromatography
planar chromatography
PC
TLC
Polymer Thin Film Chromatography
Different instruments are used
Classical chromatography: column chromatography, TLC
Modern chromatography: GC, HPLC, SFC, CE
Advantages: high selectivity, high efficiency, high sensitivity, fast analysis speed, wide range of applications
Disadvantages: poor qualitative specificity for analysis of unknown substances
Classic liquid chromatography
Overview
Classic column chromatography: gravity transport of mobile phase; planar chromatography: capillary transport of mobile phase
Compare
Classic liquid chromatography
Stationary phase particles are larger and uneven
Transport mobile phase under normal pressure
Low separation efficiency and low sensitivity
Simple equipment, easy operation, large sample loading capacity
Classification
Classic liquid column chromatography: simple equipment and large sample loading capacity
Thin layer chromatography: simple, intuitive, fast and sensitive equipment, high resolution
Paper chromatography: good at separating highly polar compounds
modern liquid chromatography
The stationary phase particles are small and uniform
Deliver mobile phase under high pressure
High separation efficiency and high sensitivity
Requires special instruments and is more expensive
adsorption chromatography
Adsorbent as stationary phase
Adsorption: The phenomenon of concentration of solutes on the surface of solid substances
Adsorbent structure: porous material with many adsorption centers on the surface
Commonly used adsorbents and their properties
Commonly used adsorbents
Silica gel
structure
Internal-porous silicon-oxygen cross-linked structure External - silanol group, active adsorption center
Characteristics: Weakly acidic, separates acidic and neutral substances (organic acids, phenols, aldehydes, amino acids, etc.)
Activity: related to water content
Bound water >17% reduces adsorption capacity
105~110 degrees Celsius, can be removed in about 30 minutes
There are two forms of silanol groups: free hydroxyl group and bonded hydroxyl group.
Heat to 200 degrees
Silyl ether structure: non-polar, loses chromatographic activity
Alumina
Basic alumina (ph9~10): separation of alkaline and neutral substances
Neutral alumina (ph7.5): widely used to separate alkaloids, volatile oils, terpenes, steroids, anthraquinones and unstable substances in acids and bases
Acidic alumina (ph 4 ~ 5): acidic compounds and relatively stable substances
Adsorbent activity
related to moisture content
The higher the water content, the lower the adsorption activity, the weaker the adsorption force, and the greater the activity level.
The lower the water content, the higher the adsorption activity, the stronger the adsorption force, and the smaller the activity level.
Relationship between moisture content and activity level of silica gel and alumina
Activation: The process of heating to remove moisture at a certain temperature to enhance its activity.
Deactivation: Add a certain amount of water to reduce its activity
Try to use adsorbents of the same batch number and treated by the same method.
Fundamental
The cause of adsorption
Adsorption only occurs at the two-phase interface
Reason: The force on the molecules on the surface of the adsorbent is unbalanced. When there is residual gravity from the inside, the molecules outside the interface are pulled to the interface.
As the adsorbent surface increases, the adsorption capacity increases; the larger the specific surface area of the adsorbent, the stronger the adsorption capacity.
adsorption equilibrium
Adsorption is the interaction between adsorbent, solute and solvent
Elution protocol: the process of competitive adsorption between eluent molecules and adsorbed solute molecules, adsorption-desorption dynamic balance
Adsorption equilibrium constant K
K is large, adsorption is firm, solute molecules stay in the stationary phase for a long time and move slowly.
K=0, solute molecules are not adsorbed by the stationary phase and flow out quickly with the mobile phase.
The greater the difference in K, the easier it is for the components to separate from each other.
adsorption isotherm
At a certain temperature, after reaching adsorption equilibrium, the concentration of the component in the stationary phase is the ordinate and the concentration of the component in the mobile phase is the abscissa.
Line type: ideal state
Non-linear (actual situation) Reason: Inhomogeneity of the solid adsorbent surface
Convex shape: tailing peak
Concave: leading edge peak
Control the amount of solute and try to keep it within the linear range
Selection of chromatographic conditions (adsorbent and mobile phase)
Considerations
Polarity of mixture components (decisive factor)
The adsorption activity of the stationary phase
The strength of the elution effect of the eluent (mobile phase)
Elution: The essence is the process in which mobile phase molecules and molecules of the separated substance compete to occupy the active adsorption center on the surface of the adsorbent.
Strongly polar mobile phase, strong ability to occupy adsorption center and strong elution effect
Weakly polar mobile phase, weak ability to occupy the center of the adsorbent, and weak elution effect
The polarity of the components being separated
General rule: the greater the polarity, the stronger the adsorption
Nonpolar: saturated hydrocarbons The basic core is the same, the more polar the substituent, the stronger the polarity of the molecule; the greater the number of polar substituents, the stronger the polarity of the molecule.
The more double bonds, the stronger the adsorption capacity
The spatial arrangement of substituents in the molecule also affects
Polarity (adsorption capacity) of common compounds: Alkanes < Alkenes < Ethers < Nitro compounds < Dimethylamine < Esters < Ketones < Aldehydes < Amine < Amides < Alcohols < Phenol < Carboxylic acids
The relative polarity of the separated mixture
Polar size range for all components
can be estimated by the relative polarity of the extraction solvent
The polarity of the eluent
The greater the polarity, the stronger the elution ability, the smaller the K, the shorter the retention time.
Polarity order (smallest to largest): petroleum ether, cyclohexane, carbon disulfide, trichloroethane, benzene, toluene, dichloromethane, chloroform, diethyl ether, ethyl acetate, acetone, n-butanol, propanol, ethanol, methanol, pyridine, acid
S and M selection principles
operate
TLC
the term
origin
Expand
developing agent
developing agent frontier
Spots: spots formed after the sample is spread out and separated on a thin layer plate
TLC
adsorption thin layer chromatography
Features
Short expansion time
Strong separation ability
high sensitivity
Convenient color development
The instrument is simple and easy to operate
Can separate large amounts of samples as well as small amounts of samples
Operation (Chinese Medicine Chemistry Notes)
Board making (without adhesive, decking activation)
Spotting
Expand
Check out
Selection and activation of thin laminates
Common silicone board specifications
Silicone G-containing gypsum adhesive Silicone H - no adhesive Silica gel F254-contains fluorescent agent, emits light under 254nm UV light Silica gel F365-contains fluorescent agent, emits light under 365nm UV light
Spotting
Spotter: quantitative capillary
Spotting volume: round; diameter: 2~4mm
Spotting position: 10~15mm from the bottom edge, distance between spots >8nm
Note: Avoid multiple spots and do not damage the surface of the thin layer when spotting.
Expand
Double tank chromatography cylinder
Precautions
Presaturation, the purpose of presaturation
The developing agent should not be immersed into the lower end of the thin layer for more than 0.5cm, and should not be submerged beyond the origin.
Expand upwards 8 to 15cm
Two-way expansion method
Edge effect: For spots of the same substance, after unfolding, the ratio of spots at the edge of the thin layer is greater than the ratio of transplantation in the central area (concave shape)
Reason: The solvent vapor in the chromatographic cylinder has not reached saturation before development, and the evaporation rate of the developing agent is different between both sides of the thin layer plate and the middle part.
Ways to Reduce Edge Effects
Use a smaller expansion cylinder to pre-saturate Paste a filter paper strip soaked with developing agent on the inner wall of the expansion tank. If you use a narrow thin layer board of 3cm, only click 2 to 3 points.
Detection of spots
Optical detection method
Colored Compounds: Direct Targeting Under UV light Fluorescence chromatography: showing dark spots
actual colorimetric method
Spray color development Dip color development Steam detection method
Qualitative and quantitative analysis
Qualitative analysis: Transplantation Rf0.2～0.8
calculate
factors affecting transplantation
The nature of the adsorbent, the polarity and solubility of the developing agent
thin layer thickness
spread distance
Expand steam saturation
Spotting amount
The moisture content of each part of the thin layer is inconsistent
temperature and relative humidity
relative transplant
Quantitative analysis (rarely used)
Visual color comparison elution method thin layer scanning
R
Common terms
Adsorption adsorbent Solvent Elution solvent: eluent Eluent: the liquid that comes out of the end of a chromatographic column Elution: The process of passing a mobile phase through a chromatographic column
Operation process
Preparation, adding sample (loading), elution, detection
Adsorption column chromatography
Column Preparation
Cylinder: glass, quartz, nylon Inner diameter/column length stationary phase particle diameter Stationary phase dosage: alumina (20 to 50 times the sample dosage) Silica gel (30 to 60 times the sample weight)
Filling requirements: uniform, no bubbles
Dry filling
Wet filling
Add sample: When the eluent flows until it is equal to the surface of the column, you can add the sample.
Wet and dry dosing
Elution: isocratic and gradient elution The eluent should be added continuously to maintain a certain liquid level.
Check out
HPLC
Overview
Based on the classic liquid phase chromatography, the theory and technology of GC are introduced, using high liquid pump, high efficiency stationary phase and high sensitivity detector.
Compared
mobile phase
GC: Inert gas, few types HPLC: liquid, many types, wide range of choices
Stationary Phase
GC: carrier➕fixative HPLC: chemically bonded stationary phase
Application scope
GC: suitable for volatile, thermally stable substances, accounting for 15 to 20% of organic matter HPLC: Substances that are soluble in solvents and can be detected
High performance liquid chromatography
High pressure infusion system Sampling system Chromatographic separation system Detection Systems Data recording and processing systems
Infusion system
Mobile phase pretreatment: remove impurities, degassing
Hazards of gases in the mobile phase
Gas forms bubbles, causing pressure fluctuations
Affects baseline stability
Dissolved oxygen: causes fluorescence quenching, oxidation of components, and reaction with the stationary phase to cause degradation.
Degassing methods: ultrasonic vibration degassing (commonly used), vacuum degassing, heating reflux degassing, etc.
High-pressure infusion pump (core)
Constant flow pump (commonly used plunger reciprocating pump): small volume, easy to clean and change the mobile phase, suitable for gradient elution
Constant pressure pump
gradient elution device
Isocratic elution: mobile phase composition remains constant
Gradient elution: mobile phase components change with the program
High-pressure gradient (two infusion pumps), high precision, expensive, high failure rate
Low pressure gradient: low cost, easy to use, prone to bubbles
Sampling system
Six-way injection valve
Injection method
Full valve injection
Injection without full valve
Chromatographic separation system
Guard column: prevent insoluble particles from the sample from entering the analytical column, and intercept strongly retained components on the pre-column
Chromatographic column: The direction of the mobile phase during use should be consistent with the direction of the arrow on the column.
Column oven
Column evaluation
Common samples and operating conditions
Silica gel column: sample (benzene, naphthalene, biphenyl, phenanthrene) Mobile phase: n-hexane
Alkyl bonded column: sample is the same as silica gel column Mobile phase: methanol-water (83:17)
Chromatography system adaptability test
Number of theoretical plates n
Resolution R
Tailing factor T
RepeatabilityRSD
Detection Systems
Universal
Evaporative light scattering detector ELSD
Sugars, higher fatty acids, saponins
Refractive Index Detector RID
Selectivity
UV detectorUVD
The most widely used
Advantages: high sensitivity, wide linear range, no damage to samples, insensitive to temperature and flow rate fluctuations, and can be used for gradient elution
Disadvantages: Not suitable for samples that have no absorption of ultraviolet light. The mobile phase is selective (the cutoff wavelength is smaller than the sample detection wavelength)
Category: Variable Wavelength Detector Photodiode Array Detector (PDAD): multi-channel, obtains chromatographic-spectral three-dimensional spectrum
Fluorescence detector FD
enzymes, steroids, vitamins, amino acids
Main types of HPLC methods and their principles
Liquid-solid adsorption chromatography LSC
Distribution mechanism: polarity difference
Suitable for fat-soluble components with medium relative molecular weight, isomers
Outflow order: components with less polarity come out first
LLC (liquid-liquid partition chromatography)
Separation mechanism: components have different solubilities in the two phases
Stationary phase: carrier➕stationary solution Chemically bonded phase: solving the problem of fixative loss
Partition Chromatography Classification
Normal phase chromatographyNPLC Stationary phase polarity>Mobile phase polarity Separates polar to moderately polar compounds
Reversed Phase ChromatographyRPLC Stationary phase chromatography <mobile phase polarity Non-polar to moderately polar compounds
Ion exchange chromatography IEC
Stationary phase: ion exchanger Mobile phase: buffer solution Principle: The forces between ions and ion exchange groups are different Application: amino acids, nucleic acids, etc.
Size exclusion chromatography SEC
Stationary phase: gel (distribution of gaps of a certain size) Mobile phase: can dissolve the sample, wet the stationary phase, and has low viscosity Principle of separation: molecular size Small molecules elute the slowest, while large molecules are excluded and elute the fastest. Separate macromolecules
stationary phase and mobile phase
Stationary Phase
Silica gel
Category: Surface porous silica gel Fully porous silica gel: spherical shape can be used as bonding phase carrier Stacked silica beads (ideal for high efficiency fillers) Application: Polar to weakly polar molecular compounds
chemical bonding phase
Advantages: No stationary phase loss Stable chemical properties High column efficiency Large sample loading capacity Suitable for gradient elution
Classification
carrier type
Silica gel carrier
The most widely used
Principle: Distribution, adsorption
End sealing: trimethylchlorosilane Purpose: Reduce adsorption, reduce tailing, and increase stability
Mobile phase pH: 2～8 Less than 2, chemical bonds are hydrolyzed and fall off Greater than 8, the carrier silica gel is dissolved
Non-silica gel carrier
functional group
Non-polar: reversed phase chromatography
Reversed Phase ChromatographyRPLC Stationary phase chromatography <mobile phase polarity Non-polar to moderately polar compounds
Group: non-polar hydrocarbon group
Commonly used: octadecyl bonded phase ODS or C18
Separation principle: solvophobic theory
Reserved value
Molecular structure of components: the weaker the polarity, the stronger the hydrophobicity, and the greater the retention value
The larger the contact area with the alkyl-bonded stationary phase, the greater the retention value.
Effect of Alkyl Bonded Stationary Phase
The number of alkyl groups directly determines the capacity factor k The longer the carbon chain, the greater the hydrophobicity, the retention value and the separation selectivity.
Effect of mobile phase
The greater the surface tension of the mobile phase, the stronger the polarity, the weaker the elution strength, and the greater the retention value.
Weak polarity
Group: ether group, glycol group bonded phase
Normal phase or reversed phase chromatography
Polarity: normal phase chromatography
Normal phase chromatographyNPLC Stationary phase polarity>Mobile phase polarity Separates polar to moderately polar compounds
Group: Amino bonded phase (strongly polar) Cyano bonded phase (medium polarity)
Ion exchange
mobile phase
Requirements: high purity, good chemical inertness To match the detector Suitable solubility for the sample Have lower viscosity, appropriately low boiling point, and high purity low toxicity
polarity
Elution capacity Normal phase chromatography: the greater the polarity, the greater the elution ability Reversed phase chromatography: the greater the polarity, the smaller the elution ability
Ways to improve separation
Adjust mobile phase polarity and selectivity: Normal phase: saturated alkane is used as the basic solvent, and ether, dichloromethane, chloroform, etc. are added to adjust the polarity. Reverse phase: water as the basic solvent, add methanol, acetonitrile, and tetrahydrofuran
Add modifier
Elution method
Isocratic elution: mobile phase polarity, ionic strength, constant pH Suitable for analyzing a small number of components and little difference in component properties Not suitable for complex samples with wide polarity range and many components
Gradient elution: the mobile phase composition is changed by the program Suitable for analyzing complex samples with many components and large differences in polarity Disadvantages: baseline drift, reduced reproducibility
Selection of HPLC analysis conditions
gas chromatography
Overview
Principle: Separation is achieved by utilizing differences in boiling point, polarity and adsorption properties of substances
Classification
Stationary Phase
Gas-solid chromatography GSC
Gas-Liquid ChromatographyGLC
principle
adsorption chromatography
Partition chromatogram
column
fill in chromatogram
capillary column chromatography
use
analytical chromatography
preparative chromatography
Features
High selectivity
High sensitivity: trace analysis
High column efficiency
Fast analysis
Wide range of applications
Advantages: Can separate mixtures and perform quantitative and qualitative analysis
limitation
Without pure samples, it is impossible to conduct qualitative and quantitative analysis of unknown substances.
Not suitable for substances with high boiling point, poor thermal stability, corrosiveness and high reactivity
Basic structure of chromatograph
Air system
Carrier gas: high purity hydrogen, nitrogen, helium, air
Purification device
constant flow rate
Sampling system
Sampling device and vaporization chamber
Injection method
direct injection
Liquid: Microsyringe
Gas: gas injection valve
Top hole injection
Separation system (heart)
column
Column oven
Detection system (eyes)
Temperature Control System
Directly affects the selectivity, separation efficiency, detector sensitivity and stability of the chromatographic column
Vaporization chamber: ensure instant vaporization of liquid samples
Detector chamber: ensures that the separated components are not condensed when passing through
Chromatographic column chamber: accurately controls the temperature required for separation
Data processing system (recorder, integrator, chromatography workstation)
column
Stationary Phase
Solid stationary phase (solid adsorbent, gas-solid adsorption chromatography)
adsorbent
Silica gel (strongly polar)
Aluminum oxide (medium polarity)
Activated carbon, graphitized carbon black (non-polar)
Molecular sieve (strongly polar special adsorbent)
Polymer porous microspheres GDX
The most widely used stationary phase in GC
Can be used directly (adsorption), can be used as a carrier to apply fixative (distribution mechanism)
chemically bonded stationary phase
Silica gel is the matrix, and silicone hydroxyl groups are chemically bonded with organic reagents.
More widely used in HPLC
Liquid stationary phase (carrier stationary solution, gas-liquid distribution chromatography)
fixative
basic requirements
The vapor pressure should be low, and the thermal and chemical stability should be good
When the operating temperature is higher than the minimum operating temperature of the fixative, it will be in a liquid state
No loss or decomposition when the stationary phase is lower than the maximum operating temperature.
Does not react chemically with carrier gas, carrier, or components
High solubility and high selectivity for each component in the sample (large difference in K)
Can form a uniform liquid film on the surface of the carrier
Classification
relative polarity classification
Relative polarity P
Chemical structure classification
Similarity dissolves principle
Hydrocarbons: alkanes, aromatics. Weak polarity
Silicones: various polarities
Alcohols and ethers: highly polar
Esters and polyesters: medium to strong polarity
Nitriles and nitrile ethers: highly polar
Assignment by selectivity constant
Choice of fixative
Similar Compatible Selection
polar similarity selection
Non-polar components: non-polar fixative (dispersion force) The column is discharged in order of boiling point, with the lower boiling point being discharged first and the higher boiling point being discharged later.
Medium polarity components: medium polarity fixative (dispersion power, induction power) Exit the column in order of boiling points. If the boiling points are the same, non-polar components will exit the column first.
Strongly polar components: Strongly polar stationary phase (orientation force) Columns are removed in order of polarity, non-polar columns are removed first.
Select based on similarity of chemical functional groups
Components capable of forming hydrogen bonds (water, alcohols, phenols, amines)
Use polar or hydrogen-bonding fixatives
Those that are less likely to form hydrogen bonds flow out first, and those that are easier to form hydrogen bonds flow out later.
Select according to the difference in component properties
The boiling point difference is mainly: non-polar stationary liquid, the lower boiling point flows out first
The main difference is polarity: polar fixatives, less polar ones flow out first.
Normal phase chromatography efflux pattern
Difficult to separate components: mixed fixative
mixed coating, mixed installation, series connection
Prepare fixative
carrier
Chemically inert porous immobilized particles
Function: Carrying fixative
Require
Large specific surface area
Chemically inert, non-adsorbent, and has good wettability to fixatives
Have certain mechanical strength
Classification
Diatomite type (natural calcined diatomite)
Red carrier (diatomaceous earth and binder calcined)
Same as non-polar stationary solution, can separate non-polar or weakly polar compounds
White carrier (diatomaceous earth mixed with 20% sodium carbonate and calcined)
Used for polar stationary solutions and analysis of polar compounds
Non-diatomite type (fluorine carrier, glass microspheres, polymer beads)
Carrier surface treatment
Chemical treatment is performed before use to passivate the surface, reduce adsorption, reduce tailing, and improve efficiency.
Approach
Acid washing: removes inorganic impurities and is used to analyze acidic and ester compounds
Alkaline washing: remove impurities such as alumina and analyze alkaline compounds
Silanization: remove surface silanol groups and analyze components that easily form hydrogen bonds
Surface glazing: shields or inerts the adsorption polar center of the carrier to increase mechanical strength
Detector
Concentration type
Thermal Conductivity Detector TCD
Basis: thermal conductivity (thermal conductivity)
Features: simple structure, stable performance, good general performance, no damage to components, wide linear range
The most extensive and mature
Low sensitivity and loud noise
Electron Capture Detector ECD
Advantages: Trace analysis of electronegative organic compounds
Disadvantages: low response value to alkanes, alkenes, and alkynes organic compounds, narrow linear range, reproducibility affected by operating conditions and radioactive contamination
Application: Organochlorine pesticide residues
Note: Gas selection: high purity carrier gas Carrier gas flow rate: 40～100ml/min The detector contains a radioactive source
quality type
Hydrogen flame ionization detector FID
Advantages: high sensitivity, fast response, wide linear range
Disadvantages: Can only detect C-containing substances The sample is destroyed during testing and cannot be recycled.
Note: Gas flow ratio: nitrogen: hydrogen: air = 1:1:10 Mass detector, quantification based on peak area A
Flame photometric detector FPD
Application: Atmospheric trace pollutants (sulfides), organic sulfur, and organophosphorus pesticide residue detection Disadvantages: narrow line range
Nitrogen phosphorus detector NPD
Application: Detection of nitrogen-containing and organophosphorus pesticides
Classification by detection selectivity
Universal type:TCD
Selective type: ECD, FID
Performance
NoiseN
Drift
Influencing factors: Detector stability Carrier gas purity and stability Column temperature stability Voltage fluctuations
Sensitivity (response value, response value)
Concentration detector sensitivity Sc
Mass detector sensitivity Sm
Detection detection limit (sensitivity) D
Line type range (closely related to quantitative analysis)
The smaller N and d, the better the stability The higher the sensitivity, the smaller the detection limit and the better the performance Ideal detector: high sensitivity, small detection limit, fast response, wide linear range, and good stability
Condition selection
Chromatographic conditions
Fixative selection
Similarity dissolves principle
Main differences according to component properties
Ratio (mass ratio of fixative to carrier)
High boiling point components: carrier with small specific surface area, low mixing ratio (1~3%), low column temperature
Low boiling point components: high proportion (10~25%)
Capillary column for difficult to separate components
Choice of column length
Principle: Choose a column as short as possible to shorten the separation time on the basis of meeting the separation conditions.
Column temperature
Principle: Keep the column temperature as low as possible under the premise of satisfying resolution and suitable analysis time.
High column temperature: components volatilize quickly, gas phase Dm increases, K decreases, retention time is short, resolution decreases, which is not conducive to separation.
Low column temperature: K increases, enhancing stationary phase selectivity, Dm decreases, and resolution improves
If the column temperature is too low, the peaks will be broadened and the analysis time will be delayed.
High boiling point 300~400: column temperature is 100~200 lower than the boiling point Boiling point <300: The column temperature is lower than the average boiling point and within the range of 50 to the lower average boiling point. Gases, gaseous hydrocarbons and other low-boiling point mixtures: the column temperature is at or above the boiling point Wide boiling range, multi-component: temperature programmed
Programmed temperature rise: The column temperature increases linearly or nonlinearly with time according to a preset program. Advantages: Improve separation effect, shorten analysis cycle, improve peak shape, and increase detection sensitivity
Vaporization temperature and detection chamber temperature
Vaporization temperature: generally slightly higher than the highest boiling point of the component
Detection chamber temperature: 20 to 50 degrees higher than the column temperature or equal to the vaporization temperature
Carrier gas and flow rate
Considerations
Column efficiency Column pressure Detector sensitivity
Carrier gas flow rate: generally 20~80ml/min
Injection volume
The larger the injection volume, the wider the chromatographic peak will be and the deformation and tailing will affect the separation. The injection volume is too small and the detector is not sensitive enough to produce peaks.
Maximum allowable injection volume: gas 0.5 ~ 3ml, liquid 0.1 ~ 0.2 microliters
Injection requirements: fast speed, time period
Qualitative and quantitative analysis methods (GC, HPLC)
Qualitative analysis
Reserved value r
known matter comparison method
Retention time qualitative
Known substance peak height increasing method
Relative retained value qualitative method
Qualitative instrumentation
Quantitative analysis
Calculation of correction factors
external standard method
standard curve method
external mark one point method
two-point method of appearance
Disadvantages: The injection volume must be accurate and consistent, and the operating conditions must be stable.
internal standard method
correction factor method
standard curve method
Internal standard method (when the correction factor is unknown)
Disadvantages: high sample preparation requirements, troublesome finding of internal standards
normalization method
Disadvantages: Each component has to peak, and the correction factor of the component must be known.