High Performance Liquid Chromatography (HPLC)“High pressure liquid chromatography”, "High speed liquid chromatography", "High resolution liquid chromatography", "Modern column chromatography", etc.High performance liquid chromatography isChromatographyAn important branch ofmobile phaseHigh pressure infusion system is adopted, which will have differentPolaritySingle solvent or different proportionsMixed solvent, buffer and other mobile phases are pumped into thechromatographic columnAfter each component in the column is separated, it enters the detector for detection, so as to realizesampleAnalysis of.This method has become one of the key technologies in chemistry, medicine, industry, agricultureCommodity inspectionandForensic examinationApplication of important separation and analysis technology in such discipline fields as[1]。
1903 Russian phytochemistTswett （Tswett）"Chromatography" is proposed for the first time（Chromatography）And "Chromatogram"（Chromatogram）The concept of.Zwitt used chromatography (from Greek, chroma means color, and graph means record - literally translated into color record) to describe his color experiment.(Curiously, the Russian name Zwitt means color.) He wrote in his paper:
"(original) phytochromePetroleum etherThe solution is mainly composed ofcalcium carbonateAdsorbentAdd to the upper end of the glass tube, filter down along the tube, and then rinse with pure petroleum ether. The results show that their corresponding bands are observed in the tube according to the adsorption order of different pigments, just like the spectrum, calledchromatographicFigure. "
In 1952, British scholars Martin and Synge proposed a relatively complete theory and method of gas-liquid partition chromatography based on their research work in partition chromatography, which has pushed the chromatography technology forward a big step. This isGas chromatographyIn the following ten years, it developed very rapidly.
In 1958, based on the work of Moore and Stein, the instrumentalization of ion exchange chromatography led toAmino acid analyzerThis is an important attempt of modern liquid chromatography, but the separation efficiency is still not ideal.
In the middle and late 1960, the development of gas chromatography theory and practice, as well as mechanical, opticalElectronicsWith the development of technology, liquid chromatography has become active again.By the late 1960sHigh pressure pumpandChemical bondHPLC appeared when stationary phase was used in liquid chromatography.
After the middle of 1970, microprocessor technology was used in liquid chromatography, which further improved the automation level and analysis accuracy of the instrument.
After 1990, bioengineering andlife sciencesThe rapid development at home and abroad has raised more and newer topics of separation, purification and preparation for high performance liquid chromatography, such as the Human Genome Project,ProteomicsHPLC is used for pre separation.
application
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one
Separation of mixtures: High pressure liquid chromatography can efficiently separate various mixtures, including organic compounds, inorganic compounds, biological macromolecules, etc.
two
Quantitative analysis: The components in the sample can be quantitatively analyzed by measuring the peak area or peak height.
three
Purity detection: High pressure liquid chromatography can be used to detect the purity of the sample. The purity of the sample can be determined by comparing the peak of the sample with the peak of the standard.
four
Structure identification: The molecular structure of the sample can be inferred by combining with other technologies such as mass spectrometry.
High performance liquid chromatography has the characteristics of "four high and one wide":
① High pressure: the mobile phase is a liquid, and when flowing through the chromatographic column, the resistance is large. In order to pass through the chromatographic column quicklyCarrier fluidApply high pressure.
② High speed: The analysis speed is fast, the carrier liquid flow rate is fast, which is much faster than that of the classical liquid chromatography. Usually, the analysis of a sample can be completed in 15~30 minutes, and some samples can even be completed in 5 minutes, generally less than 1 hour.
③ High efficiency: high separation efficiency.The stationary phase and mobile phase can be selected to achieve the best separation effect, compared with the industrialDistillation towerAnd the separation efficiency of gas chromatography is many times higher.
④ High sensitivity:UV detectorIt can reach 0.01ng, and the injection volume is in the order of μ L.
⑤ Wide application range: more than 70%Organic compoundHigh performance liquid chromatography can be used for analysis, especially for separation and analysis of compounds with high boiling point, macromolecule, strong polarity and poor thermal stability, showing advantages.
⑥ The column can be reused: different compounds can be separated with one column
⑦ Small amount of sample and easy recovery: the sample will not be damaged after passing through the chromatographic column, and can be collected as a single component or prepared.
In addition, high performance liquid chromatography has the advantages of reusability of chromatographic columns, non destruction of samples, and easy recovery, but it also has disadvantages. Compared with gas chromatography, it has its own advantages and complements each other.The disadvantage of high performance liquid chromatography is "out of column effect".If the flow pattern of the mobile phase changes in any dead space (injector, column connector, connecting tube, detection cell, etc.) other than the column from the injection to the detector, any diffusion and retention of the separated substance will significantly lead to broadening of the chromatographic peak,Column efficiencyLower.The sensitivity of HPLC detector is lower than that of gas chromatography.
It can be divided into "high-pressure infusion pump", "chromatographic column", "injector", "detector", "distillate collector" and "data acquisition and processing system".
Technical developments
The connection of liquid chromatography and mass spectrometry can add additional analytical capability, and can accurately identify and quantify trace compounds in complex sample matrices such as cell and tissue lysates, blood, plasma, urine and oral fluid.High performance liquid chromatography/mass spectrometry systems (ABSciex Eksigent LC/MS and LC/MS/MS) provide some unique advantages, including:
Minimum sample preparation required for rapid analysis and circulation
High sensitivity combined with the ability to analyze multiple compounds, even across the types of compounds
High precision, high resolution identification and quantification of target analytes
High pressure infusion pump
function
Drive mobile phase and sample through chromatographic separation column and detection system;
performance requirement
Stable flow (± 1), high pressure resistance (30~60Mpa), resistance to various mobile phases: for example:Organic solvent, water and buffer;
If the chromatographic separation is not performed for purechromatographic analysis It is necessary to collect fractions for other spectral identification or small preparation of a small amount of test samples;
method
① Manual operation, a few fractions, troublesome procedures, easy to make mistakes.
② Distillate collector is ideal for collection, and microcomputer control operation is accurate.
data
Acquisition and processing function
Display the signal detected by the detector.
Separation principle
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Liquid-liquid distribution
(Liquid liquid Partition Chromatography) andChemical bonded phase chromatography(Chemically Bonded Phase Chromatography)Both the mobile phase and the stationary phase are liquids.The mobile phase and the stationary phase should be mutually immiscible (different polarity, to avoid the loss of stationary liquid), with an obvious interface.When the sample enters the chromatographic column, the solute is distributed between the two phases.When balance is reached, submit toCalculation formula of high performance liquid chromatography：
[3]
Where, cs - concentration of solute in the stationary phase;Cm - concentration of solute in mobile phase;Vs - volume of stationary phase;Vm - volume of mobile phase.LLPC is similar to GPC, that is, the order of separation depends on KComponentsLarge retention value;However, there are also differences. In GPC, flow relative K has little influence, while LLPC flow relative K has greater influence.
a. Normal Phase liquid chromatography: The polarity of mobile phase is less than that of stationary phase.
b. Reverse Phase Liquid Chromatography: The polarity of mobile phase is greater than that of stationary phase.
c.Liquid-liquid partition chromatographyDisadvantages: Although the polarity requirements of the mobile phase and the stationary phase are completely different, the stationary solution is still slightly dissolved in the mobile phase;When the mobile phase passes through the chromatographic columnMechanical shockForce will cause the loss of fixed fluid.Developed in the late 1970sChemically bonded stationary phase(See below), the above shortcomings can be overcome.
Liquid solid
The mobile phase is liquid, and the stationary phase isAdsorbent(such as silica gelaluminaEtc.).This separation is based on the different adsorption of substances.The mechanism is that when the sample enters the chromatographic column, the solute molecule (X) and solvent molecule (S) competitively adsorb the active center of the adsorbent surface (when the sample is not injected, all the active centers of the adsorbent adsorb S), which can be expressed as follows: XmnSa=====XanSm
Where: Xm -- solute molecule in mobile phase;Sa -- solvent molecule in stationary phase;Xa - solute molecules in the stationary phase;Sm -- solvent molecule in mobile phase.
When the adsorption competitive reaction reachesbalanceHour:
K=[Xa][Sm]/[Xm][Sa]
Where, K is the adsorption equilibrium constant.[Discussion: The larger the K, the larger the reserved value.]
The solute ions with the same charge in the mobile phase are exchanged reversibly, and these ions are separated according to the different affinity of the exchanger.withanionFor example, the exchange process of the exchanger can be expressed as follows:
X - (in solvent) (resin-R4N Cl -)==(resin-R4N X -) Cl - (in solvent)
[Discussion: Relationship between DX and reserved value]
Substances that can be ionized in solvents can usually be separated by ion exchange chromatography.
ion pair
(Ion Pair Chromatography)
Ion pair chromatographyIt is to add one (or more) ion (called pair ion or counter ion) opposite to the charge of the solute molecule to the mobile phase or the stationary phase, so that it can combine with the solute ion to form a hydrophobic ion pair compound, thus controlling the retention behavior of the solute ion.Its origin
Where: X aqueous phase -- organic ion (or cation) to be separated in mobile phase;Y-aqueous phase -- ion pair with opposite charge in mobile phase (such as tetrabutylammonium hydroxide, hexadecyl trimethylammonium hydroxide, etc.);X Y -- formed ion pair compound.
[Discussion: Relationship between DX and reserved value]
Ion pair chromatography (especially reverse phase chromatography) solves the separation problem of mixtures that are difficult to separate in the past, such as acid, base and ionic, non-ionic mixtures, especially some biochemical samples such as nucleic acid, nucleosidealkaloidAnd drug separation.
Ions
(Ion Chromatography)
Ion exchange resin is used as the stationary phase,electrolyte solutionIs the mobile phase.The conductivity detector is used as a general detector. In order to eliminate the interference of strong electrolyte background ions in the mobile phase to the conductivity detector, a suppression column is set up.The reaction principle of sample components on separation column and suppression column is the same as that of ion exchange chromatography.
withanionExchange resin (R-OH) is used as the stationary phase to separate anions (such as Br -).When the anion to be measured Br - enters the chromatographic column with the mobile phase (NaOH), the following exchange reaction occurs (the elution reaction is the reverse process of the exchange reaction):
Diagram of support
Suppress the reaction on the column:
R-H Na OH- === R-Na H2O
R-H Na Br- === R-Na H Br-
It can be seen that the eluent is transformed into water with very low conductivity value through the inhibition column, and the influence of background conductivity is eliminated;The sample anion Br - is converted into the corresponding acid H Br -, which can be detected sensitively by conductivity method.
Ion chromatographyIt is the best method for anion analysis in solution.It can also be used for cation analysis.
Space exclusion
(Steric Exclusion Chromatography)
Spatial exclusion chromatographyGel: Set & Match(gel) is the stationary phase.It is similar to the role of molecular sieve, but the pore size of gel is much larger than that of molecular sievenanometerTo hundreds of nanometers.The solute is separated according to the molecular size rather than the difference of the interaction force between the two phases.The separation is only related to the pore size distribution of the gel and the hydrodynamic volume or molecular size of the solute.After entering the chromatographic column, the sample flows along with the mobile phase in the external gap of the gel and near the hole.staysampleSome too large molecules can not enter the gel pores and are blocked, so they directly pass through the column. They first appear on the chromatogram. Some very small molecules can enter all the gel pores and penetrate into the particles. These components have the largest retention value on the column and finally appear on the chromatogram.
technological process
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Figure 1 Process
Process: As shown in Figure 1,Solvent containerThe mobile phase in (1) is sucked in by the pump (2), mixed by the gradient controller (3) according to a certain gradient, and then output. After measuring its pressure and flow through (4), it is introduced into (5) the sample injection valve (device) through (6) the protection column, (7) the separation column, and then to (8) the detector for detection. (10) the data processing equipment processes the data or (11) the recorder records the chromatogram, (12) the distillate collector collects the distillate, (13) is the waste liquid.
Related terms
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Chromatogram - the signal time curve obtained when the sample flows through the chromatographic column and detector, also known as the chromatography profile.
Base line - after mobile phase flushing, the column and mobile phase reach equilibrium, the detector will measure the outflow curve for a period of time.Generally, it should be parallel to the time axis.
Noise - fluctuation of baseline signal.It is usually caused by poor contact of power supply or transient overload, unstable detector, bubbles in mobile phase or contamination of chromatographic column.
Drift -- the gradual change of baseline with time.It is mainly caused by the instability of operating conditions such as voltage, temperature, mobile phase and flow rate, and the constant elution of pollutants or stationary phase in the column will also cause drift.
Peak the curve generated by the continuous signal that responds when the group splits through the detector.A projection on the outflow curve.Normal chromatographic peak is approximately symmetricalNormal distribution curve(Gaussian Gauss curve).There are two types of asymmetric chromatographic peaks:
Leading peak and tailing peak.The former is rare.Chinese Pharmacopoeian=0.95~1.05 are normal peaks, fn<0.95 is the forward peak, fn>1.05 is the trailing peak.[4]
Peak bottom the distance from the beginning of the peak to the end of the baseline.
Peak height (h) - the distance from the highest point of the peak to the bottom of the peak.
Peak width (W) - the distance between the intersection of two tangents made at the inflection points on both sides of the peak and the baseline.W=4σ
Peak width at half height, Wh/2.Wh/2=2.355σ
Peak area (A) - the area surrounded by the peak and the peak bottom.
Retention time (tR) - the time from the start of injection to the appearance of a maximum concentration of a component behind the column.
Number of theoretical trays(theoretical plate number, N) - used to quantitatively express the separation efficiency of the chromatographic column (column efficiency for short).
Resolution (R) - the ratio between the retention time difference of two adjacent peaks and the average peak width.Also called resolution, it indicates the degree of separation of two adjacent peaks.R ≥ 1.5 is called complete separation.
The Chinese Pharmacopoeia stipulates that R should be greater than 1.5.
Tailing factor (T) - T=, used to measure the symmetry of chromatographic peaks.Also called symmetry factor or asymmetry factor.
The Chinese Pharmacopoeia stipulates that T should be 0.95~1.05.
T1.05 is the trailing peak.
chromatographic column
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Composition of filler and mobile phase
The common chromatographic column packings are silica gel and chemically bonded silica gel according to the regulations under each category.The latter is represented byOctadecyl silaneOctyl bonded silica gel is the most commonly used, followed by cyano or amino bonded silica gel;Ion exchange packing is used for ion exchange chromatography;Gel orGlass microsphereFor molecular exclusion chromatography, etc.The sample injection volume is generally several microliters.Unless otherwise specified, the column temperature is room temperature and the detector is ultraviolet absorption detector.
When using the UV absorption detector, the flow used corresponds to the UVSpectrophotometryRequirements for solvents under.
Chemical bonded stationary phase reaction
The conditions specified under each category in the text, except that the type of stationary phase, mobile phase components and detector type shall not be changed arbitrarily, other conditions such as the diameter and length of chromatographic column, the brand of stationary phase, carrier particle size, mobile phase flow rate, the proportion of mixed mobile phase components, column temperature, sample injection volume, detector sensitivity, etc., can be changed appropriately.
To adapt to specific varieties and meet the requirements of system applicability test.Generally, the chromatogram shall be recorded within 20 minutes.
System suitability
The applicability test of the instrument shall be carried out according to the requirements of each variety, that is, the instrument shall be tested and adjusted with the specified reference substance to meet the specified requirements;Or specify the minimum theoretical plate number, resolution and tailing factor of the chromatographic column in the analytical state
chromatographic column
Application of chemically bonded stationary phase
Under the selected conditions, inject the test solution or the internal standard substance solution specified under each category, record the chromatogram, and measure the retention time t (R) andHalf peak widthW (h/2), calculate the theoretical plate number of the chromatographic column according to n=5.54 [t (R)/W (h/2)] ^ 2. If the measured theoretical plate number is lower than the minimum theoretical plate number specified under each category, change some conditions of the chromatographic column (such as column length, carrier performance, advantages and disadvantages of chromatographic column filling, etc.) to make the theoretical plate number meet the requirements.
Resolution
In quantitative analysis, in order to facilitate accurate measurement, it is required to have a good separation between the quantitative peak and other peaks or internal standard peaks.The calculation formula of resolution (R) is: R=2 (tR2-tR1)/(W1+W2), where t (R2) is the retention time of the last of the two adjacent peaks;T (R1) is the retention time of the first of the two adjacent peaks;W1 and W2 are the peak widths of two adjacent peaks.Unless otherwise specified, the resolution shall be greater than 1.5.
Trailing factor
To ensure the measurement accuracy, especially when the peak height method is used for measurement, check whether the trailing factor (T) of the peak to be measured meets the requirements under various varieties or whether the error of the correction factor of different concentration injection meets the requirements.The calculation formula of trailing factor is:
T (tailing factor)=W0.05h/2d1, where W (0.05h) is the peak width at 0.05 peak height;
D1 is the distance from the peak maximum to the peak front.Unless otherwise specified, T shall be between 0.95 and 1.05.
Alternatively, under the item of calibration factor determination for each variety, prepare the reference solution equivalent to 80%, 100% and 120%, add the specified amount of internal standard solution, prepare three solutions with different concentrations, inject samples three times respectively, calculate the average calibration factorrelative standard deviation It shall not be greater than 2.0%.
determination
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In quantitative determination, the peak area method or peak height method can be used according to the specific situation of the sample.However, when the total amount of impurities is determined by normalization method or internal standard method, the peak area method must be used.
Area normalization method
The peak area normalization method without correction factor is used to determine the total amount limit of impurities and impurities in the test article (or the derivative treated test article).Calculate the peak area of each impurity and its sum, and calculate the percentage of the total peak area.However, the solvent peak is not included.The recording time of the chromatogram shall be determined according to the retention time of the impurities contained in each variety. Unless otherwise specified, it can be a multiple of the retention time of the main components under this variety.
Principal component self contrast method
When the impurity peak area is significantly different from the component peak area, usePrincipal component self contrast method。Before determination, dilute the test sample into a solution with a certain concentration as the reference solution according to the impurity limit specified under each category. Inject the sample, adjust the sensitivity of the detector or the injection volume, so that the peak area of the main component chromatogram in the reference solution meets the requirements for accurate measurement.Then take the test solution, inject the sample, and record the time. Unless otherwise specified, it should be multiple of the retention time of the main component.Calculate the impurity limit according to the measured peak area of each impurity in the test solution and its sum, and compare it with the peak area of the main component of the reference solution.
Determination of the total amount limit of impurities in the test sample by internal standard method
The peak area method without correction factor is adopted.Take the test sample, prepare the test sample solution without internal standard substance according to the method specified under each category, inject it into the instrument, and record the chromatogram I;Then prepare the test solution containing the internal standard substance, inject the sample under the same conditions, and record the chromatogram II.Unless otherwise specified, the recording time shall be a multiple of the retention time of the internal standard peak specified under this item, and the height of the internal standard peak on the chromatogram shall be more than 30% of the recorder's full scale, otherwise the sample injection volume orDetector sensitivity。
If there is no impurity peak in chromatogram I with the same retention time as the internal standard peak on chromatogram II, the sum of the impurity peak areas in chromatogram II should be less than the peak area of the internal standard substance (excluding solvent peak).If there is an impurity peak in the chromatogram I with the same retention time as the internal standard peak on the chromatogram II, the peak area of the internal standard substance on the chromatogram II should be subtracted from the peak area of this impurity in the chromatogram I, which is the correction area of the internal standard substance peak;The total area of each impurity peak in chromatogram II plus the area of this impurity peak in chromatogram I is the total corrected area of each impurity peak. The total corrected area of each impurity peak should be less than the corrected area of the internal standard substance peak.
Add correction factor to determine the content of main components of the test article
According to the regulations under each variety, accurately weigh (measure) the reference substance and internal standard substance, prepare solutions respectively, precisely measure each solution, prepare the reference solution for calibration factor determination, inject a certain amount into the instrument, record the chromatogram, measure the peak area or peak height of the reference substance and internal standard substance, and calculate the calibration factor according to the following formula:
As/ms] correction factor f=- Ar/mr Where As is the peak area or peak height of the internal standard substance, Ar is the peak area or peak height of the reference substance;Ms is the amount of added internal standard substance mr is the amount of added reference substance.Then take the test sample solution containing internal standard substance under each item, inject it into the instrument, record the chromatogram, measure the peak area or peak height of the test sample (or its impurities) and the internal standard substance, and calculate the content according to the following formula: Ax content (mx)=f × - As/ms, where Ax is the peak area or peak height of the test sample (or its impurities);Mx is the amount of the test article (or its impurities).f. As and ms have the same meaning.
When the same internal standard substance solution is used for preparing the reference solution for calibration factor determination and the test solution containing internal standard substance, it is not necessary to accurately weigh (measure) the internal standard substance solution.
Determination of content in test sample by external standard method
According to the regulations under each variety, weigh (measure) the reference substance and test substance precisely, prepare a solution, take a certain amount precisely, inject the instrument, record the chromatogram, measure the peak area (or peak height) of the components to be measured of the reference substance and test substance, and calculate the content according to the following formula:
A<[x]>content (mx)=mr × - Ar The meaning of each symbol in the formula is the same as above
Since it is not easy to accurately control the injection volume of the microinjector, when using the external standard method to determine the content of an impurity or main component in the test sample, quantitative ring injection is preferred.
overview
To select chromatographic separation methods correctly, first of all, we must understand the relevant properties of samples as much as possible, and then we must be familiar with the main characteristics and application range of various chromatographic methods.The selection of chromatographic separation method is mainly based on the samplerelative molecular massSize, solubility in water and organic solvents, polarity and stability, andChemical structureAnd other physical and chemical properties.
relative molecular mass
For samples with low relative molecular weight (generally below 200), good volatility, and difficult decomposition when heated, you can chooseGas chromatographyConduct analysis.Compounds with relative molecular weight of 200~2000 can be used by liquid-solid adsorption, liquid-liquid partition and ion exchange chromatography.If the relative molecular weight is higher than 2000, space exclusion chromatography can be used.
solubility
For water-soluble samples, it is better to use ion exchange chromatography and liquid-liquid partition chromatography;Compounds that are slightly soluble in water but can be ionized well in the presence of acid or alkali can also be usedIon exchange chromatography；Oil soluble sample or relatively non-polar mixture, availableLiquid-solid chromatography。
Chemical structure
If the sample contains ionic or ionizable compounds, or compounds that can interact with ionic compounds (such as ligands and organic chelating agents), ion exchange chromatography can be considered first, but space exclusion chromatography and liquid-liquid partition chromatography can also be successfully applied toIonic compound；The isomers can be separated by liquid-solid chromatography;Compounds and homologues with different functional groups can be determined by liquid-liquid partition chromatography;aboutPolymer, spatial exclusion chromatography can be used.
equipment
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overview
The emergence of HPLC has not lasted for more than 30 years, but this separation and analysis technology has developed rapidly and been widely used.Its instrument structure and process are various.typicalHigh performance liquid chromatographyThe structure and process can be represented by the following block diagram (See Fig. 3-4).High performance liquid chromatograph generally has the main components of reservoir, high pressure pump, gradient elution device (with double pumps), injector, chromatographic column, detector, thermostat, recorder, etc.
High performance liquid chromatography is more suitable for separating and analyzing substances with high boiling point, poor thermal stability, physiological activity and relatively large molecular weight, so it is widely used in nucleic acids, peptides, lactones, polycyclic aromatic hydrocarbonshigh polymerAnalysis of drugs, human metabolites, surfactants, antioxidants, pesticides, rust removers and other substances.
High pressure pump
The chromatographic column used in HPLC is very thin (1~6 mm), and the particle size of the stationary phase used is also very small (several μ m to dozens of μ m), so the mobile phase flow in the column is subject to great resistance. Under normal pressure, the mobile phase flow rate is very slow, and the column efficiency is low and time-consuming.In order to achieve rapid and efficient separation, a great deal of pressure must be applied to the mobile phase to accelerate its flow speed in the column.To this end,RequiredThe high-pressure pump carries out high-pressure infusion.High pressure and high speed is one of the characteristics of high performance liquid chromatography.The high-pressure pump used by HPLC shall meet the following conditions:
a. The flow is constant, without pulsation, and has a large adjustment range (generally 1~10 mL/min);
b. Resistant to solvent corrosion;
c. High infusion pressure;For general separation, the pressure of 60 × 10 ^ 5Pa is enough, and for efficient separation, it is required to reach 150~300 × 10 ^ 5Pa.
⑴reciprocating piston pump
When the plunger is pushed into the cylinder block, the check valve at the pump head outlet (upper part) is opened, and the check valve (lower part) where the mobile phase enters is closed, then a small amount of fluid is output.On the contrary, when the plunger is pulled outward, the check valve at the inlet of the mobile phase opens, and the check valve at the outlet closes at the same time, and a certain amount of mobile phase is sucked into the cylinder by its reservoir.The characteristic of this pump is that it is not affected by the slight change of resistance in the rest of the whole chromatographic system, and it continuously supplies a constant volume of mobile phase.
Its working principle is that the low-pressure gas with pressure p1 pushes the large area (SA) piston A, and then the small area (SB) piston B outputs the liquid whose pressure increases to p2.The multiple of pressure increase depends on the area ratio of A and B pistons. If the area ratio of A and B is 50: 1, the output liquid with pressure of 250 × Pa can be obtained from the gas with pressure of 5 × Pa.This is a constant pressure pump.
gradient elution
Pneumatic amplification pump
It is similar to the temperature programmed in GC.It has become an indispensable part of modern high performance liquid chromatography.Gradient elution, that isCarrier fluidIt contains two (or more) solvents with different polarity. During the separation process, the ratio and polarity of the solvent in the carrier solution are continuously changed according to a certain procedure. The polarity of the carrier solution is changed to change theSeparation factorTo improve the separation effect.Gradient elution can be divided into two types:
a.low pressureGradient (also called external gradient): under normal pressure, mix two or more solvents with different polarity according to a certain procedure in advance, and then input them into the chromatographic column with a high-pressure pump.
b. High pressure gradient (or internal gradient system): two sets of high pressure infusion pumps are used to send two solvents with different polarity into the gradient mixing chamber according to the set proportion. After mixing, they enter the chromatographic column.
Sample injection device
(1) Syringe sample injection device: the microinjector and sample injection method used for sample injection are the same as those of GC method.When the injection pressure is 150 × 10 ^ 5Pa, stop flow injection must be adopted. ⑵High pressure quantitative injection valve: similar to the flow method used in GC method, it can inject samples under high pressure.
chromatographic column
Chromatographic column is the most important component (heart) of chromatograph.It is usually made of thick wall glass tube or stainless steel tube with polished inner wall. For some corrosive samples and high pressure resistance is required, copper tube, aluminum tube orPTFE pipe。The inner diameter of the column is generally 1~6 mm.The commonly used standard column type is a straight stainless steel column with an inner diameter of 4.6 or 3.9 mm and a length of 15~30 cm.The particle size of the packing is 5~10 μ m, and the column efficiency is about 7000~10000 based on the theoretical number of trays.
The development trend is to reduce the particle size of filler and column diameter to improve column efficiency.
detector
⑴Ultraviolet photometric detector
Its working principle is based on the effect of the analyzed sample components on the specific wavelength of ultraviolet lightselectivityAbsorption, relationship between component concentration and absorbanceBill's law。The most commonly used detector has the widest application and is responsive to most organic compounds.
characteristic:
a. High sensitivity: its minimum detection amount is 10-9g · mL-1, so it can be detected even for substances with weak UV absorption;
b. Wide linear range;(Bill's Law)
c. The flow cell can be very small (1mm × 10mm, volume 8 μ L);
d. It is insensitive to the flow rate and temperature of mobile phase and can be used for gradient elution;
e. Wavelength optional, easy to operate: for example, use the visible with flow cellUltraviolet spectrophotometer(Variable wavelength detector).
Disadvantages: It can not detect the sample that does not absorb ultraviolet light;At the same time, the choice of solvent is limited.
The important progress of ultraviolet detector;The array consists of 1024 photodiode arrays, each photodiode is only 50 μ m wide, and each photodiode detects a narrow wavelength.In the detector, the ultraviolet or visible light emitted by the light source passes through the liquid chromatography flow cell, where each component in the mobile phase is characteristic absorbed, then passes through the slit, enters the monochrome for light separation, and finally is detected by the photodiode array to obtain the absorption signal of each component.The three-dimensional spectrogram was obtained by computer rapid processing.
The structure and working principle of the fluorescent detector are similar to those of the fluorescent photometer.
⑷Differential refractometer
The most widely used detector except UV detector.
The differential refractive detector is a detector that measures the sample concentration by continuously measuring the refractive index of the solution in the flow cell.The refractive index of solution is pure solvent (mobile phase) and pure solute（sample）The refractive index is multiplied by the sum of the concentrations of each substance.Therefore, the difference in refractive index between the mobile phase with the sample dissolved and the pure mobile phase indicates the concentration of the sample in the mobile phase.
Its working principle is to measure the content of ionized substances according to the conductivity change of substances after ionization in some media.
Positive and negative chromatography
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Normal phase chromatography
Use polar stationary phase (such as polyethylene glycol, amino and nitrile bonded phase);The mobile phase is a relatively non-polar hydrophobic solvent (alkanes such as n-hexane, cyclohexane), and ethanol, isopropanolTetrahydrofuran、TrichloromethaneTo adjust the retention time of components.It is often used to separate compounds with medium polarity and strong polarity (such as phenols, amines, carbonyls and amino acids).
Reversed phase chromatography
Generally, non-polar stationary phases (such as C18, C8) are used; the mobile phase is water or buffer solution, and methanol, acetonitrile, isopropanol, acetone, tetrahydrofuran and other organic solvents that are miscible with water are often added to adjust the retention time.It is suitable for separating non-polar and weak polar compounds.RPC is the most widely used in modern liquid chromatography. According to statistics, it accounts for about 80% of the total application of HPLC.
example
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High performance liquid chromatography only requires that the sample can be made into solution without gasification, so it is not limited by the volatility of the sample.In principle, the organic substances with high boiling point, poor thermal stability and large relative molecular weight (more than 400) (these substances almost account for 75%~80% of the total organic substances) can be separated and analyzed by high performance liquid chromatography.According to statistics, among known compoundsGas chromatographic analysisAbout 20% of them can be analyzed by liquid chromatography, and about 70-80% can be analyzed by liquid chromatography.
1. Analysis of organochlorine pesticide residues in the environment
Stationary phase: thin shell silica gel (37 ～ 50mm)
Seven anions were basically completely separated within 20 minutes, and the content of each component was 3~50 ppm.
Theory of liquid chromatography
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Development profile
Liquid chromatographyAt the beginning, a large diameter glass column is used to transport the mobile phase with liquid level difference at room temperature and normal pressure, which is called classic liquid chromatography. This method has low column efficiency and takes a long time (often several hours).High performance liquid chromatography (HPLC) is based on the classic liquid chromatography, which was rapidly developed by introducing the theory of gas chromatography in the late 1960s.
It is different from the classical liquid chromatography in that the packing particles are small and uniform, and the small particles have high column efficiency, but will cause high resistance, so the mobile phase needs to be transported under high pressure, so it is also called High Pressure Liquid Chromatography (HPLC). It is also called High Speed Liquid Chromatography (HSLP) because of its fast analysis speed. Also called modern liquid chromatography[2]。
