About The HPLC Main Components And Its Column Maintenance And Use Notes

HPLC has been widely used in many fields such as food additives and pharmaceutical composition analysis due to its advantages of accurate quantitative analysis results, short analysis period, wide analysis range, and low detection limit.

Introduction of main components
Infusion system
The infusion system mainly includes an online degassing device, a high-pressure infusion pump, and a gradient elution device. The main function of the online degasser is to remove bubbles before the mobile phase enters the column. The packing particles of the liquid chromatography column are small, and the flow resistance through the 2 ~ 5 mm column is very large. Therefore, the mobile phase needs to be extracted by the high-pressure infusion pump and sent to the column.

High-pressure pumps can be divided into constant pressure and constant flow pumps according to infusion performance. Based on the mechanical structure, it can be divided into a hydraulic diaphragm pump, pneumatic amplification pump, screw injection pump, and reciprocating plunger pump.

Sampler
The sampler is a device that sends analytical samples to HPLC chromatographic column. It is divided into manual and automatic injectors. The manual sampler is generally equipped with a 20-100 μL quantitative loop.

HPLC chromatography column
The column uses liquid as the mobile phase and the solid phase in the broad sense as the stationary phase to separate the analytical samples. Chromatography columns can be roughly divided into normal-phase chromatography columns and reverse-phase chromatography columns according to the separation mode.

Detector
The detector converts the physical or chemical characteristics of the sample separated by the chromatographic column into measurable electrical signals, records them through the chromatogram, determines the separation effect from the peak shape in the chromatogram, and performs qualitative and quantitative analysis on the sample according to the peak time and peak area of the chromatogram of the standard and analytical sample.

HPLC column maintenance and use notes
Adjust the pH value of all the analyzed samples to the pH range suitable for the column. Avoiding overloading during injection is one of the important methods to maintain long-lasting and good column performance. If the manual injector is used for injection, the action should be fast and accurate during the injection. When the analyte composition is complex, a chromatographic protection column should be added in front of the column to protect the column. At the beginning of each analytical work, the chromatographic column should be equilibrated. The aqueous solution containing 10% of the organic solvent in the mobile phase is used for exhausting and flushing the chromatographic column and pipeline. Replace, and then equilibrate the column with a mobile phase of at least 10 column volumes until the baseline is equilibrated before injecting.

After each analysis, the buffer salt solution in the HPLC chromatographic column must be thoroughly rinsed with a certain amount of aqueous solution, and then the column should be rinsed with a solvent that has a strong elution capacity for the test substance. The amount of desolvation is at least 20 times the column volume. If the chromatographic column is not rinsed in time, impurities will accumulate in it for a long time, which will easily block the chromatographic column and reduce the efficiency of the column. In the long run, this will accelerate the reduction of column life. If the chromatographic column is not used for more than 4 days, it should be removed from the instrument and sealed tightly with a head nut. When storing the column, be careful not to vibrate or collide it strongly.

About The HPLC Main Components And Its Column Maintenance And Use Notes

The Specific Use Method Of SPE Cartridge

1. Select the SPE cartridge or filter membrane.

Firstly, according to the physical and chemical properties of the object to be measured, and the substrate of the sample, the fixed phase with strong retention ability should be selected. If the object to be tested has a negative charge, anion exchange packing can be used; otherwise, cation exchange packing can be used. If it is a neutral substance to be measured, it can be extracted with reverse phase packing. The size and specification of the SPE cartridge or membrane should depend on the concentration of the sample to be measured. For in vivo samples with low concentration, less fixed phase filler should be used to extract larger volume samples.

2. The activation
Before extraction, rinse the cartridge with a solvent filled with the cartridge or rinse the filter membrane with 5 ~ 10ml solvent. Generally, methanol and other water can be used first.
The soluble organic solvent rinses the filler because methanol wets the adsorbent surface and penetrates the non-polar silica gel bonding phase, making the silica gel easier.
Wet with water, then rinse with water or buffer. Before sample addition, SPE packing should be kept moist. If the packing is dry, the sample retention value will be reduced. However, the different drying degree of each cartridge will affect the reproducibility of the recovery rate.

3. On the sample
Generally, the following measures can be taken: (1) using 0.1mol/L acid or base regulation, pH<3 or pH>9. Centrifugal extraction of upper liquid;(2) the protein was precipitated with methanol, acetonitrile, etc., then the supernatant was taken and diluted with water or buffer solution for extraction;(3) the supernatant was taken after the protein was precipitated with acid or inorganic salt, and the pH value was adjusted for extraction;(4) after ultrasound for 15min, add water and buffer solution, and extract with supernatant. The drug concentration in the urine sample is relatively high. Before adding the sample, dilute with water or buffer solution. When necessary, acid and alkali hydrolysis reaction can be used to destroy the combination of drug and protein, and then extract. The flow rate should be controlled to 1ml/min, and the fast flow rate is not conducive to the combination of the object to be measured and fixation.

4. The elution
The cleaning solvent of reversed-phase SPE is mostly water or buffer solution. A small amount of organic solvent, inorganic salt, or pH value can be added into the cleaning solution. The cleaning fluid added to the cartridge should not exceed the volume of one cartridge, and the SPE filter membrane is 5 ~ 10ml.

5. Elute the object under test
5 ~ 10ml eluent with weak ionic strength but capable of washing the substance to be measured should be selected. If higher sensitivity is required, the eluent can be dried before the mobile phase recombined residue is injected. In vivo samples, after elution contains more water, can choose the freeze-drying method. The SPE filler with weak retention ability can be washed with small volume and weak eluent, and then the eluent can be analyzed by the HPLC cartridge with strong polarities, such as C18 SPE cartridge.

If the substance to be measured can be ionized, the pH value can be adjusted to inhibit the sample ionization, so as to enhance the retention of the substance to be measured in the reverse SPE filler. During elution, the pH value can be adjusted to ionize the substance and elute it with a weak solvent. After collecting the eluent, the pH value can be adjusted to achieve the best separation effect in the HPLC analysis. In the process of elution, the flow rate should be slowed down, two small volume elution instead of a large volume elution, the recovery rate is higher.

The Specific Use Method Of SPE Cartridge

Structure And Design Feature of Roll Type Filter Membrane

Instruction to Structure of Roll Type Filter Membrane
The filter membrane is divided into three types according to the structure of membrane: roll membrane, ceramic membrane, and hollow fiber membrane. Roll type membrane plays an important role in membrane filtration technology. Now let’s focus on a roll film. By the guide screen, membrane and membrane support materials stacked in turn, with the adhesive along the three sides of the two layers of membrane bond seal, the other open side and the middle through the liquid collecting pipe connection, and then winding together. The liquid containing the material flows from one end into the diversion barrier and from the other end, flowing through the liquid along with the porous supporting material and drawn from the intermediate collecting pipe.

Advantages of Roll Type Filter Membrane
Roll type filter membrane is characterized in the wide distribution of filter aperture, a large area of the single membrane, high filtering accuracy, and relatively low energy consumption. In application, it can withstand higher pressures. The operating temperature is generally not higher than 45℃, and the operating temperature of the special film reaches 80℃.

Configuration of Roll Type Filter Membrane
Hawach discloses a roll type filter membrane, the inner part of which is provided with a central filter tube, the outer side of the diaphragm layer is provided with a membrane shell, the two ends of the filter are provided with a raw water seal washer, the other end of the original water seal washer is provided with an outlet pipe, the surface of the original water seal washer is provided with an inlet and outlet water hole, the diaphragm layer includes an inlet water screen, the outer side of the inlet water screen is provided with a water production screen, and the outer side of the water production screen is provided with graphene nanometre.

The function of Roll Type Filter Membrane
The roll type filter membrane can greatly accelerate the water production speed by setting a ceramic central filter tube, and the graphene nano-film built into the diaphragm layer effectively improves the purification ability and water quality purification effect of the membrane layer. At the same time, the filter ends are buckled with raw water seal washer, which is convenient to disassemble, easy to clean up the fouling on the membrane surface, reduce the fouling plugging of the diaphragm layer, avoid the secondary pollution of the water source, and ensure our healthy life.

Adsorption Mechanism of Membrane Filter
According to the adsorption mechanism and the pore characteristics of the membrane layer and the supporting layer, it can be explained why the membrane filtration is easy to block the oily sewage for the clean water. For general solid suspended matter, particles larger than the membrane pore are intercepted (screened) on the surface of the membrane, and particles smaller than the membrane pore pass through the membrane pore, which cannot be adsorbed on these particles because of the thin film as the filter layer. Although the supporting layer is relatively thick, its pore is larger than the membrane pore and is not sufficient to produce adsorption.

Structure And Design Feature of Roll Type Filter Membrane

Several Points Should Be Considered In The Selection Of Filter Paper

Filter paper on the market can generally be divided into qualitative and quantitative. In the application of analytical chemistry, when inorganic compounds are separated by filtration to precipitate, the residue collected on the filter paper can be used to calculate the loss rate during the experiment. Qualitative filter paper produces more cotton fibers after filtration, which is an ideal choice for qualitative analysis; quantitative filter paper, especially ash-free filter paper, undergoes special processing procedures and can effectively resist chemical reactions, so the resulting impurities Less, can be used for quantitative analysis.

In addition to filter papers used in general laboratories, there are many applications of filter papers in daily life and engineering. Coffee filter paper is one of the widely used filter papers. The filter paper on the outer layer of tea bags provides high softness and high wet strength. Other air filter papers for testing suspended particles in the air, and fiber filter papers for different industrial applications.

The technical indicators of filter paper can be divided into two aspects, one is the filter characteristics of the filter paper, and the other is the physical characteristics. Filtration characteristics include air permeability, air resistance, maximum pore size, and average pore size. Physical properties include basis weight, thickness, stiffness, corrugation depth, burst resistance, resin content, etc.

Quantity: refers to the mass of filter paper per square meter, unit: g / m?
Thickness: refers to the thickness of the filter paper, excluding the corrugated depth. Unit: mm.
Air resistance: the resistance of filter paper to airflow. It is represented by the pressure drop value obtained by passing 100 liters of filter paper through 85 liters of air in one minute. The unit is mbar. Or the water column height (mm).
Corrugation depth: the depth of the groove pressed to strengthen the longitudinal stiffness of the filter paper, the unit is mm. In general, the value is 0.2mm.
Air permeability: the amount of air passing through the filter paper per unit time under a certain area and certain pressure (20 mm water column). The unit is L / m2 · s.
Nominal filtration accuracy: refers to the retention capacity of the filter paper to particles of a certain size, and the particle size of the tiny spheres when 50% can be retained or filtered out Unit: μm.
Maximum pore size: the void size calculated by the pressure when the first bubble emerges from the filter paper sample during the test. Unit: μm.
Average pore diameter: The pore diameter calculated by pressure when “dense” bubbling is called the average pore diameter. The unit is: μm.
Resin content: The percentage of resin in the weight of filter paper. Generally 10% ~ 30%.
Stiffness: the deformation resistance of filter paper. Unit: mg.
Burst: the maximum pressure that the filter paper can withstand per unit area. Unit: kPa.

1. The effective area is large, that is, the use area of the filter paper is large, the dust holding capacity is large, the resistance is small, the service life is long, and of course, the cost increases accordingly.
2. The finer the fiber diameter, the better the interception effect, and the correspondingly higher filtration efficiency.
3. The content of the binder in the filter material is high, the tensile strength of the paper is high, the filtration efficiency is high, the lint phenomenon is less, the dust background of the filter material is small, the resistance is good, but the resistance increases.

Several Points Should Be Considered In The Selection Of Filter Paper

Syringe Filters Increase Sensitivity And Accuracy Of Analysis Results

Brief introduction

Syringe filters can be found in the chromatographic analysis to filter mobile phases and samples. The presence of impurities in the sample and solvent will not only cause damage to the chromatographic instrument and column, but will also greatly affect the analysis results. Organic phase syringe filters have a good effect on protecting the column and the chromatographic system from contamination, and can improve the sensitivity and accuracy of the analysis results. It is widely used in pharmaceutical analysis, food testing, environmental monitoring, protein removal and sterility testing.

Components

The shell is made of polypropylene (PP) with excellent chemical stability, made of different colors, which is convenient for users to distinguish between products with different specifications and avoid confusion. The filter membrane adopts high-performance filter membrane with stable product quality and good reproducibility, which is an ideal choice for laboratory filtration samples. The standard interface is convenient to connect with various syringes, and it can also be directly connected to the syringe of the valve, which can be directly injected after filtering.

Membrane materials determine the effectiveness of the filtration, and Hawach always implements high quality standard and humanized services, different syringe filters are available according to customer demand. The consistency between the production filter batches is good, from the raw materials to the production process and to the incoming and outgoing warehouse delivery management, the whole process has strict quality control, to the greatest extent to ensure the quality and use value of the product. Commonly membranes of various specifications such as PES、PTFE、Nylon、MCE、GF、PVDF、CA are provided, and the pore size range from 0.1μm to 5μm, the diameter includes 4 mm、13mm、17mm、25mm、30mm、47mm etc.

Syringe filters made of PTFE membrane has excellent chemical resistance, high temperature resistance, strong acid resistance, strong alkali resistance, strong hydrophobicity. Besides, hydrophilic membrane and hydrophobic membrane are all provided to meet different liquid filtration requirements. Polyvinylidene fluoride membrane is hydrophobic, which is featured in low moisture absorption, easy constant weight, and oxidation resistance. It is also design of thread interface.

Main features

The pore size is relatively uniform, the porosity is high, there is no medium shedding, the texture is thin, the resistance is small, the filtration speed is fast, the adsorption is very small, the price is low, but it is not resistant to organic solutions and strong acid and alkali solutions.

Uses: The pharmaceutical industry needs autoclaved water injection to filter out particles during large infusion.

For sterilization of heat-sensitive drugs (biochemical preparations such as insulin ATP and coenzyme A), use a 0.45 micron filter (or 0.2)

Medicine and health, using the thin film method to test the C-AmP, C-GmP and bloodworm of human body fluids, the determination of E. coli groups in drinking water, surface water and well water, and the detection of industrial and mining dust 0.8-5.0 microns.

Syringe Filters Increase Sensitivity And Accuracy Of Analysis Results

Description Of Chromatographic Column Installation Of Color Harmonic Column

Flush the pumps and piping of the chromatograph thoroughly with the filtered and degassed mobile phase (without buffer salts) so that the system is free of bubbles. According to the flow direction of the mobile phase marked on the chromatographic column, connect the inlet of the chromatographic column with the pipe at the pump end and the outlet of the column is not connected.

Set the flow rate of the pump to 0.1mL/min or lower, and the flow rate slowly rises to the normal flow rate (t > 5min). When the solvent flows freely from the exit of the column, set the flow rate as 0, and connect the column exit with the pipeline at the detector end.

The chromatographic column was balanced with the mobile phase and normal flow rate of 10-30 times column volume. Amino column, cyano column can be used in the positive phase environment, but also in the inverse phase environment. When it is necessary to change from the reverse phase into the positive phase or the positive phase into the reverse phase, it is necessary to use 20-30 times the column volume of THF as the transition solvent to flush the system, otherwise, it is easy to make the one-way valve of the pump blocked, abnormal pressure phenomenon.

Notes for chromatographic column use
Mobile phase: the solvent must be HPLC grade and the reagent should be as pure as possible. There is no chemical reaction with the fixed phase, the viscosity is small, and the sample has an appropriate solubility, k is required in the range of 1 ~ 10 (available range) or 2 ~ 5(the best range), k value is too small, is not conducive to the separation of k value. Too large and may precipitate the sample in the mobile phase. The mobile phase must be filtered and degassing d before use to ensure that the solvents are miscible with each other. The mobile phase must match the detector. For example, when the UV detector is used, the solvent whose cut-off wavelength is greater than the detection wavelength cannot be used.

Trace impurities can greatly degrade the performance of the column, ensuring that solvents (buffers) are miscible with each other when the mobile phase needs to be replaced. If the solvent used and the column of the solvent is not miscible, the use of transition solvent, otherwise the column will produce permanent damage. The separation of salt or buffer from the mobile phase can also permanently damage the column. The solubility of the sample in the mobile phase should be checked before each sample injection, and the sample should be dissolved in the mobile phase whenever possible.

Fixed phase: the pH value of the mobile phase should be kept between 2.0 and 8.0 (unless otherwise specified); Use pre-saturated column and guard column if necessary; When using an amino column, avoid aldehydes and ketones in mobile phase and sample.

Column packing can be divided into the following three: high pH (>8.0), silica gel dissolution. If the pH of the mobile phase is close to 2.0 or 8.0, a saturated column is required. Silica gel column: high column efficiency, high mechanical strength, but sensitive to pH, low pH (<2.0) will cause the bonding phase to hydrolyze (bond removal of functional groups).

Organic polymer packing: in the wide range of stable residual silica hydroxyl effect is small, but its column efficiency is low. Mechanical strength is not high and may shrink or swell in different solvents. In the application process, the pressure must not exceed its maximum bearing range. And mobile phase loyalty, organic solvents such as methanol. The content of isopropanol ethanol should not exceed 10%.

A new type of organic-inorganic hybrid filler with two high – purity monomers. Synthetic high purity filler which combines the advantages of silica gel and an organic polymer. High efficiency, high mechanical strength, good stability, wide ph range.  The silicon hydroxyl group can be effectively shielded for the most part. So that it has better retention of alkaline substances.

Back pressure and flow rate
Keep the back down to 3,500 psi. Avoid any sudden changes in stress. If the back pressure is high, the column can recoil, but the flow rate should be 1/5 to 1/2 of the normal flow rate. If the test pool needs to be degassed, use the back pressure governor. In order to extend the service life of the chromatographic column to the greatest extent, the velocity of flow can be adjusted according to the particle size of packing and the inner diameter of the chromatographic column. So that the back pressure does not exceed the limit.

Column temperature
Most of the separation is done at room temperature, and if the temperature in the lab stays the same, there will be no problems. But most work environments have temperature fluctuations. The retention time of temperature equivalence separation has an effect. Even under the condition of the constant temperature of day and night, the room temperature will change greatly due to the difference in temperature between day and night. Especially when large quantities of samples are analyzed, there will be large differences. The separation of compounds is achieved by controlling the temperature. The retention time and selectivity of the column change with the change of temperature, and the peak width also change.

Preservation of chromatographic column
The preservation condition of the column will affect the life of the column. The storage solution should contain at least 20% organic solvent to prevent the growth of microorganisms. Never use buffer to store chromatographic column, wash chromatographic column with 5 times column volume of mobile phase without a buffer to remove buffer and salt.

Description Of Chromatographic Column Installation Of Color Harmonic Column

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This new type large LCD screen 

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To

 

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4. Triple-layered coil condenser equipped with the dripping point increases condensation efficiency.
5. Positive and negative rotate function designed for drying of powder.
6. And the safest protection function of auto-lifting in over-temperature or outage, makes Hawach RE1-OW-T Series rotating evaporator the most reliable to buy.

Features of RE1-OW-T Series rotating evaporator

• The rotating speed can reach 310rmp/min for crystalization experiment.
• The temperature of the rotating evaporator can reach 180/210°C for both oil and water baths, which is controlled by PID with Temp. The precision of ±2°C.
• Big LCD touch screen displaying both temperature and rotating speed.
• Effective 3-layered condenser and dripping point design prevent backflow of evaporation and increased evaporation process.
• Auto-lifting design in overheat or stop prevents the rotating bottle from breakage; Integral bath with a safety handle which is safe and easy to clean.
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310rpm Oil & Water Bath, Color/Touch Screen Rotary Evaporator