HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HPLC stands for High Performance Liquid Chromatography. This is an analytical chemistry technique that is used to separate, quantify and identify every component that is in a mixture. The technique depends on pumps to push some constrain liquid solvent which contains a mixture of the sample through a column containing a solid absorbent material.
Each of the components in the sample will interact with the absorbent material in a slightly different way. This will cause different flow rates in the various components hence causing the components to separate as they flow out of the column. High Performance Liquid Chromatography is used mainly for manufacturing, medicine and research purposes.
High Performance Liquid Chromatography is used in separating and purifying compounds according to their polarity. To understand polarity, we can use a simple example of oil, water and ethanol. Ethanol is polar. Thus it will mix properly with water. On the contrary, oil is not polar, so it does not mix well with water.
The HPLC column is as well called the stationary phase. It is made from different substances, mostly silica, and it is highly compact in nature. Long carbon chains functionalize the silica particles. Carbon chains are non-polar. Thus, the longer the chain is, the more non-polar the column will become. C18 columns are the ones that are mostly used and contain 18 carbon chains.
High Performance Liquid Chromatography can be used in separating compounds in environmental samples, medical chemistry, microbiology and also environmental samples. The compounds are injected into the HPLC column. The mobile phase, which is practically a mixture of water and an organic solvent is then introduced. The organic solvent used is mostly methanol or acetonitrile. This is called the mobile phase since the mixture moves all through the HPLC column and still flushes out the compounds in the column at the same time. The process of flushing out is also known as eluting.
Tips for HPLC Analysis in Pharmaceuticals
1. Column Selection for HPLC Technique Development: The approach to selecting the right column varies. The toolkit approach offers an effective and wide range consideration of separation possibilities for the investigated application. Separation under development is driven by selectivity.
2. The profiles of the gradients, additives, modifiers and the nature of the stationary phase are the crucial parts of the selectivity. Characteristics and supports of a stationary phase which drives selectivity have to be put into consideration.
3. Visual Assessment Technique for Column Performance: Firstly, it is imperative to know a few things about the key relationships in chromatography when designing an experiment. You have to take the peak capacity so as to avoid an explosion.
4. How to Set up a UV Detector: UV detector is frequently used the in HPLC analysis, because they are considered easy to use and produce useful data. Sometimes the UV detector could be required to perform an unusual task to get better baselines, sensitivity and better reproduction from the detector
5. The type and volume of flow cell will impact the sensitivity of the technique and efficiency of the peaks. Setting the slit width to a narrow width will improve the resolution of the spectral, bandwidths and references. It is important to note that a wide bandwidth has an edge of noise reduction by middling over a diode with greater range.
6. How Sample Diluent affects HPLC: Sample diluents which are used in the preparation of HPLC samples can affect the peak shapes and retention times of HPLC. To annul overload effect, the volume of sample injected should be lower than the peak volume with about 15%. The values of the peak and resolution should be constantly noted to ensure no effects are happening.
7. Buffer Selection for HPLC Separations: A buffer is a solution that withstands a difference in the pH. It is an aqueous solution that comprises equilibrium of a mixture of a weak acid and strong base or vice versa. Many factors affect the selection of buffer; examples are the required pH of the mobile phase or the volatility of the buffer. If the buffer is chosen wrongly, it will be required to be added at a greater concentration so as to work well. This will tend to cause problems associated with the technique’s robustness.
8. Peak Tailing in HPLC Analysis: This is one of the usual peak shape distortions in chromatography. Once the asymmetry is higher than 1.2, it is referred to as peak. The main reason for peak tailing is because of occurrence of multiple reactions of analyte retention. To avoid peak tailing, ensure the system is working at a lower pH which will reduce secondary interactions when carrying out the separation in chromatography.
9. How to Solve Robustness Problems in HPLC: It is imperative to check the gradient profile or data system to know if the gradient has been inputted properly. Any alteration in the gradient dwell volume is taken care of. As soon as the checks are done, find out the eluent composition, this can be done by making a new set of eluent. The kind of organic modifier concentration sometimes has an effect on the selectivity technique, so as to know the exact organic solvent that is applied in the current proportion.
10. Retention Time Shift in HPLC: Wrongly prepared mobile phases bring about a swift change in retention time. So here a few tips on what to do and not to do.
An isocratic mobile phase should not be prepared in a measuring cylinder, do not apply organic to aqueous to get volume. A wrong solvent ratio will be derived.Ensure that the right buffer is used and also the correct pH.Ensure correct pH calibration.For mobile phases pre-mixed, do not separate under vacuum.
Some of the differences between C18 and C8 are discussed here.
Octadecylsilane (C18) has 18 carbon atoms. On the other hand, Octylsilane(C8) has only 8 carbon atoms on the column parking bonded to silica (Si). C18 will tend to retain more than C8. In that, if a similar compound was eluted on the two columns, it will elute faster on C8 and slower on C18. This is caused by the parking per unit surface area density of the column.
C18 is denser than C8. The denser parking will increase the surface area that the molecule in a mobile phase has to travel through per unit length. It will also increase the time of interaction within the elitues and stationary phase causing a greater separation ability for more complex molecules.
In other terms, C18 has Octadecyl chains which are usually hydrophobic and highly retain nonpolar compounds. The length of its carbon chain is longer. On the contrary, C8 has Octyl chains and hence it is less retained when used with the same compound as C18.
When a short retention time is required, C8 is more preferred. Its lower hydrophobicity will cause faster retention of nonpolar compounds. Therefore, nonpolar compounds will move down the column more rapidly with C8 than with C18.
C18 HPLC is extensively used in environmental science, pharmaceutical industries, radioanalytical laboratories and chemical analysis. They are used to analyze individual parts of chemical mixtures or radiolabeled/labeled molecules.
C18 is considered better for separating compounds such as long-chain fatty acids as compared to relatively small organic compounds. It is relatively cheap as it is produced in large amounts by most manufacturers.
C8 and C18 have a lot of elements in common. However, extensive research has helped to identify some of the distinctive differences of C8 and C18 used in HPCL. The two compounds have a structure that is almost similar. That is, for C18 is OH-Si-C18 and for C8 that is OH-Si-C8.
Considering the above-stated differences, it is easy to identify some of the major ones.
C18 has 18 carbon atoms while C8 has only 8 carbon atoms.C18 has a longer carbon chain, but C8 has a shorter one.C18 has higher retention while C8 has shorter retention.C18 has higher hydrophobicity, but C8 has a lower hydrophobicity.With the above-listed hplc column comparison, it is easier to know the appropriate column that one should use when performing different types of HPLC.
C18 HPLC Columns and Their Properties
Standards and success of any pharmaceutical company is determined by how professional chemical analysis are being undertaken. Chromatography remains the most important technique for identifying and analyzing a wide variety of chemical samples within pharmaceutical industries. The technique does not only help chemical analysis in scientifically analyzing compositions of various drugs during their development but also key in identifying compounds that may be unknown.
Chromatography, therefore, apart from helping in chemical identification, also ensures purity of chemical mixtures. Selection of the most suitable chromatography methods is of paramount importance for an effective, and efficient samples analysis. There are various types of separation systems. C18 is one of the most suitable systems you can consider for all your pharmaceutical analysis requirements. Exploring the working of C18 HPLC column will enable you to make a proper selection of an effective and efficient compounds analysis.
What are C18 Columns?
Also referred to as ODs or reversed-Phase C18 Columns. This is a separation system that uses high performance liquid chromatography (HPLC) with C18 as the main stationary phase substance. It is important to note that single stationary C18 phases will always be different from one C18 HPLC to the other.
The substance's molecule is composed of up to 18 carbon atoms. This implies that any variation on other types of atoms within the substance can easily point to a different substance.
C18 Column Specifications
Main substance: Hybrid silica which can be either organic or inorganicStationary component: C18 (in the form of USP-L1)Percentage of Carbon-20%Size of Particles that can pass through: 2, 3 and 5 micrometres (μm)Pore size: 120 ÅEndcapping technology used: Multi-stageType of bonding: PolymerRange of pH: 1 to 12
Package Material of C18 Column
There are diverse ranges to select from which include:
C18 silica bonded HPLC package Pro C18 Package- Hydrosphere C18 and Pro C18 RSODS series (ODS-M80, ODS-L 80, ODS-S80, ODS-1, ODS-AM, ODS-AQ, ODS-AL, PolymerC18 packages.
Which Package should You Select?
Each C18 package comes with its unique separation characteristics. It is, therefore, necessary that you keenly consider properties of compounds you need to be separated as a guide to making correct package selection. Details of your compounds' molecular weights will act as a guide in choosing a C18 package that suits your needs.
Reasons Why You Need C18
Your cost per analysis will be much lower compared to other alternatives. Being a hybrid silica formulated ODs column, it is durable with excellent performance. Durability property is due to its multi-layer structure which is composed of a non-organic silica-based layer, and an organic layer which is polymer-based It is highly suitable for use under low pressure conditions. Through the use of modern technology called microreactor, the substance is homogeneous with highly uniform particles. This allows you to effectively conduct your analysis under low pressures. They come in different sizes, and wide range of hydrophobicity making them suitable for separating, basic, and neutral substances. Your chemical tests and analysis is therefore not limited to PH levels They are of different pores for different types of particles. You can therefore use them with a diverse range of chemical substances. They display clear peak shapes for your quick analysis under all conditions. By identifying key properties of compounds to pass through your C18 columns, you can easily select the best C18 with ease. Compatible with both your current and older types of chromatography instruments. Enhanced lot to lot reproducibility irrespective of which compounds you use. You therefore able to effectively handle complex compounds that may ordinarily be difficult to chromatograph. It is user-friendly and will always work for you all the times!Conclusion
Selection of correct chromatography chemicals is a key decision for effective analysis of a wide range of chemicals. Understanding key characteristics of the compounds you are working with is necessary for correct selection. C18 HPLC column will not only provide you with precise result, but will also enable you to work with a wider range of compounds. This will save you both time and money, and enhance both the quality and standard of your analysis. With a wide range of packages to select from, you are assured of getting what suits you most.
Difference between GC and HPLC Techniques
Gas Chromatography and the High Performance Liquid Chromatography are separation techniques used in obtaining specific eluents from an analyte basing on the compounds in the analyte distribution between the two phases (Mobile phase and Stationary phase). The mobile phase is the part in chromatography which moves and it usually contains the sample while the stationary phase is one which does not move and contains adsorption material. Chromatography is a larger analytical technique in chemistry.
In modern-day analytical chemistry, Gas Chromatography and the High Performance Liquid Chromatography are the major techniques used in Analytical Chemistry. In earlier analysis, techniques such as the Ion Exchange Chromatography, Affinity Chromatography and Thin Layer Chromatography were used.Both these techniques are effective for separation of the analytes. The difference in between the two techniques are discussed in different subtopics below:
1. Mobile Phase: One of the major differences between these two methods is the difference in their mobile phases which eventually determines where each technique is used.
As the name suggests, gas chromatography uses gases as its mobile phase with the stationary phase either being liquid or solid. On the other hand, High Performance Liquid Chromatography uses a liquid as its mobile phase and a solid as its stationary phase in analysis.
2. Nature of Analyte: Compared to High Performance Liquid Chromatography, Gas Chromatography analyzes samples that are either volatile or semi-volatile thus it will analyze only few samples since volatile compounds are fewer than non volatile samples which the HPLC analyzes. Inadvertently, HPLC analyzes samples that are are only liquid in nature.
3. Instrumentation: Both these methods use chromatography columns, but there is differentiation in their sizes. The Gas chromatography uses columns which are long and thick usually 10 to 30 meters in length as compared to the chromatography column which is shorter and thin basically being 5 to 25 cm in length.
4. Detection: After chromatography is done on the analyte, the composition of the eluent has to be done. This calls for detection methods that are stringent to get the perfect sample type and volume. Each analytical technique has its own method of sample detection. For gas chromatography, two types of detectors are the most widely used i.e the Flame Ionization Detector (FID) that is only used for detection of hydrocarbons and the Thermal Conducting Detector (TCD) which is universal and detects any substance.
In the case of High Performance Liquid Chromatography, the only method use is via an Ultraviolet Spectrometric detector which uses the principle of absorption and transmittance to detect the different types of samples In spectrophotometry, a sample is placed between a source of light and a photometric reader. The amount of light passing through the sample is recorded as photons by a computer detector. Different samples have different absorbance rates.
5. Cost: Finally, the cost of each technique is a determining factor.The Gas Chromatography technique is less costly compared to the HPLC system that utilizes a lot of equipment including high pressure pumps to increase its accuracy and shorten the time required for analysis of the sample. This makes Gas Chromatography being used more compared to HPLC.
Different Types of HPLC Detectors
HPLC detectors are used in the detection of the solute present in the eluent coming from the HPLC column. They are capable of determining the identity and concentration of eluting compounds in the mobile phase. There are specific detectors which respond to a specific compound and their response is not dependent on mobile phase composition.
However, the response of the bulk-property detectors is dependent on the collective changes in the composition of the mobile phase and sample. There is a broad range of detectors available for different sample requirements. Some of the crucial features required of a detector include sensitivity, a linear response and either a universal or selective response.
Some of the desirable features required in HPLC detectors are:
1. It should have either specific or general response to compounds in a mixture.
2. Sensitivity towards solute over mobile phase.
3. It should have a response to solute and not the mobile phase.
4. It should have a low cell volume for memory effects minimization.
5. It should respond linearly to solute concentration.
6. Low detector noise and detection limits.
7. It should not contribute to zone spreading.
8. It should be not be affected by temperature variation and flow rate.
The different types of detectors are categorized into two, i.e. specific and bulk property detectors.
A) Specific HPLC Detectors
1. Mass Spectroscopic HPLC Detectors
Mass spectroscopy detectors offer very high selectivity and sensitivity and their detection is based on molecular fragmentation by electric fields and separation based on the mass to charge ratio of fragmented molecules. New application areas have been introduced by the LC-MS technique because of its advantages in terms of sensitivity and resolution.
2. UV/VIS HPLC Detectors
They are of three types, i.e. fixed wavelength detectors, variable wavelength detectors and the diode array detectors. UV-VIS Detectors have mostly used detectors due to its specific response to the class of compounds or particular compounds depending on the functional groups of elutingmolecules that absorb light, although some compounds with no light absorbing groups give suitable response after post-column derivatization to introduce light absorbing entities. A beam of the electromagnetic radiation is passed through the detector flow cell and due to this interaction, it experiences a change in intensity and the measurement of this particular changes is the basis of most optical detectors.
3. Photo Diode Array (PDA) HPLC Detectors
A large number of diodes serving as detector elements makes it possible for simultaneous monitoring of many absorbing components at differing wavelengths, with little time and reduced cost spent on expensive solvents.
4. Fluorescence HPLC Detectors
They are most specific, sensitive and selective, and offers greater sensitivity than the UV-VIS detector. Naturally fluorescent compounds are less as compared to the light absorbing compounds, a limitation solved by post-column derivatization. There are three types of fluorescent detectors, namely the single wavelength fluorescent detector, multi-wavelength fluorescent detector and laser-induced fluorescent detector.
B) Bulk Property HPLC Detectors
1. Electrical Conductivity HPLC Detectors
They provide reproducible, universal and high sensitivity detection of the charged species and surfactants. The detectors measure the conductivity of the mobile phase, therefore are classified as bulk property detectors. Their sensors consist of the flow-through cell with few microliters of the volume containing 2 electrodes, usually made of stainless steel, platinum or a noble metal used in the determination of alkali and alkali earth metals.
2. Refractive Index HPLC Detectors
There are various types of RI detectors which include, the Christiansen effect detector, interferometer detector, thermal lens detector and the dielectric constant detector. They have limited sensitivity and detection depends on the changes in refractive index of eluting molecules in the mobile phase. These detectors are used for detecting non-ionic compounds that neither fluoresce nor absorb in the UV region. Temperature control is required due to high-temperature sensitivity.
3. Electrochemical HPLC Detectors
The detectors can be classified into two, which are the equilibrium and the dynamic detectors. They respond to oxidizable or reducible substances and are sensitive to changes in flow rate or composition of the mobile phase. The reaction takes place on the surface of the electrode producing electrical signals. Their suitability will depend on the volumetriccharacteristics of the solute molecules in the aqueous or organic mobile phase. They require the working electrode, reference electrode and the auxiliary electrode.
4. Light Scattering HPLC Detectors
Light scattering detectors are of two types which include low angle laser light scattering detector and the multiple angle laser light scattering detector. They are based on measuring the scattered light and are useful for detection of large molecular weight molecules present in the eluent, which is examined by being passed through an appropriate sensor cell depending on molecular sizes.
Other types of HPLC detectors are the evaporativelight scattering detectors, IR detector, transport detectors, aerosol-based detectors, chiral detectors and the pulsed amperometric detectors.
Each of the components in the sample will interact with the absorbent material in a slightly different way. This will cause different flow rates in the various components hence causing the components to separate as they flow out of the column. High Performance Liquid Chromatography is used mainly for manufacturing, medicine and research purposes.
High Performance Liquid Chromatography is used in separating and purifying compounds according to their polarity. To understand polarity, we can use a simple example of oil, water and ethanol. Ethanol is polar. Thus it will mix properly with water. On the contrary, oil is not polar, so it does not mix well with water.
The HPLC column is as well called the stationary phase. It is made from different substances, mostly silica, and it is highly compact in nature. Long carbon chains functionalize the silica particles. Carbon chains are non-polar. Thus, the longer the chain is, the more non-polar the column will become. C18 columns are the ones that are mostly used and contain 18 carbon chains.
High Performance Liquid Chromatography can be used in separating compounds in environmental samples, medical chemistry, microbiology and also environmental samples. The compounds are injected into the HPLC column. The mobile phase, which is practically a mixture of water and an organic solvent is then introduced. The organic solvent used is mostly methanol or acetonitrile. This is called the mobile phase since the mixture moves all through the HPLC column and still flushes out the compounds in the column at the same time. The process of flushing out is also known as eluting.
Tips for HPLC Analysis in Pharmaceuticals
1. Column Selection for HPLC Technique Development: The approach to selecting the right column varies. The toolkit approach offers an effective and wide range consideration of separation possibilities for the investigated application. Separation under development is driven by selectivity.
2. The profiles of the gradients, additives, modifiers and the nature of the stationary phase are the crucial parts of the selectivity. Characteristics and supports of a stationary phase which drives selectivity have to be put into consideration.
3. Visual Assessment Technique for Column Performance: Firstly, it is imperative to know a few things about the key relationships in chromatography when designing an experiment. You have to take the peak capacity so as to avoid an explosion.
4. How to Set up a UV Detector: UV detector is frequently used the in HPLC analysis, because they are considered easy to use and produce useful data. Sometimes the UV detector could be required to perform an unusual task to get better baselines, sensitivity and better reproduction from the detector
5. The type and volume of flow cell will impact the sensitivity of the technique and efficiency of the peaks. Setting the slit width to a narrow width will improve the resolution of the spectral, bandwidths and references. It is important to note that a wide bandwidth has an edge of noise reduction by middling over a diode with greater range.
6. How Sample Diluent affects HPLC: Sample diluents which are used in the preparation of HPLC samples can affect the peak shapes and retention times of HPLC. To annul overload effect, the volume of sample injected should be lower than the peak volume with about 15%. The values of the peak and resolution should be constantly noted to ensure no effects are happening.
7. Buffer Selection for HPLC Separations: A buffer is a solution that withstands a difference in the pH. It is an aqueous solution that comprises equilibrium of a mixture of a weak acid and strong base or vice versa. Many factors affect the selection of buffer; examples are the required pH of the mobile phase or the volatility of the buffer. If the buffer is chosen wrongly, it will be required to be added at a greater concentration so as to work well. This will tend to cause problems associated with the technique’s robustness.
8. Peak Tailing in HPLC Analysis: This is one of the usual peak shape distortions in chromatography. Once the asymmetry is higher than 1.2, it is referred to as peak. The main reason for peak tailing is because of occurrence of multiple reactions of analyte retention. To avoid peak tailing, ensure the system is working at a lower pH which will reduce secondary interactions when carrying out the separation in chromatography.
9. How to Solve Robustness Problems in HPLC: It is imperative to check the gradient profile or data system to know if the gradient has been inputted properly. Any alteration in the gradient dwell volume is taken care of. As soon as the checks are done, find out the eluent composition, this can be done by making a new set of eluent. The kind of organic modifier concentration sometimes has an effect on the selectivity technique, so as to know the exact organic solvent that is applied in the current proportion.
10. Retention Time Shift in HPLC: Wrongly prepared mobile phases bring about a swift change in retention time. So here a few tips on what to do and not to do.
An isocratic mobile phase should not be prepared in a measuring cylinder, do not apply organic to aqueous to get volume. A wrong solvent ratio will be derived.Ensure that the right buffer is used and also the correct pH.Ensure correct pH calibration.For mobile phases pre-mixed, do not separate under vacuum.
Some of the differences between C18 and C8 are discussed here.
Octadecylsilane (C18) has 18 carbon atoms. On the other hand, Octylsilane(C8) has only 8 carbon atoms on the column parking bonded to silica (Si). C18 will tend to retain more than C8. In that, if a similar compound was eluted on the two columns, it will elute faster on C8 and slower on C18. This is caused by the parking per unit surface area density of the column.
C18 is denser than C8. The denser parking will increase the surface area that the molecule in a mobile phase has to travel through per unit length. It will also increase the time of interaction within the elitues and stationary phase causing a greater separation ability for more complex molecules.
In other terms, C18 has Octadecyl chains which are usually hydrophobic and highly retain nonpolar compounds. The length of its carbon chain is longer. On the contrary, C8 has Octyl chains and hence it is less retained when used with the same compound as C18.
When a short retention time is required, C8 is more preferred. Its lower hydrophobicity will cause faster retention of nonpolar compounds. Therefore, nonpolar compounds will move down the column more rapidly with C8 than with C18.
C18 HPLC is extensively used in environmental science, pharmaceutical industries, radioanalytical laboratories and chemical analysis. They are used to analyze individual parts of chemical mixtures or radiolabeled/labeled molecules.
C18 is considered better for separating compounds such as long-chain fatty acids as compared to relatively small organic compounds. It is relatively cheap as it is produced in large amounts by most manufacturers.
C8 and C18 have a lot of elements in common. However, extensive research has helped to identify some of the distinctive differences of C8 and C18 used in HPCL. The two compounds have a structure that is almost similar. That is, for C18 is OH-Si-C18 and for C8 that is OH-Si-C8.
Considering the above-stated differences, it is easy to identify some of the major ones.
C18 has 18 carbon atoms while C8 has only 8 carbon atoms.C18 has a longer carbon chain, but C8 has a shorter one.C18 has higher retention while C8 has shorter retention.C18 has higher hydrophobicity, but C8 has a lower hydrophobicity.With the above-listed hplc column comparison, it is easier to know the appropriate column that one should use when performing different types of HPLC.
C18 HPLC Columns and Their Properties
Standards and success of any pharmaceutical company is determined by how professional chemical analysis are being undertaken. Chromatography remains the most important technique for identifying and analyzing a wide variety of chemical samples within pharmaceutical industries. The technique does not only help chemical analysis in scientifically analyzing compositions of various drugs during their development but also key in identifying compounds that may be unknown.
Chromatography, therefore, apart from helping in chemical identification, also ensures purity of chemical mixtures. Selection of the most suitable chromatography methods is of paramount importance for an effective, and efficient samples analysis. There are various types of separation systems. C18 is one of the most suitable systems you can consider for all your pharmaceutical analysis requirements. Exploring the working of C18 HPLC column will enable you to make a proper selection of an effective and efficient compounds analysis.
What are C18 Columns?
Also referred to as ODs or reversed-Phase C18 Columns. This is a separation system that uses high performance liquid chromatography (HPLC) with C18 as the main stationary phase substance. It is important to note that single stationary C18 phases will always be different from one C18 HPLC to the other.
The substance's molecule is composed of up to 18 carbon atoms. This implies that any variation on other types of atoms within the substance can easily point to a different substance.
C18 Column Specifications
Main substance: Hybrid silica which can be either organic or inorganicStationary component: C18 (in the form of USP-L1)Percentage of Carbon-20%Size of Particles that can pass through: 2, 3 and 5 micrometres (μm)Pore size: 120 ÅEndcapping technology used: Multi-stageType of bonding: PolymerRange of pH: 1 to 12
Package Material of C18 Column
There are diverse ranges to select from which include:
C18 silica bonded HPLC package Pro C18 Package- Hydrosphere C18 and Pro C18 RSODS series (ODS-M80, ODS-L 80, ODS-S80, ODS-1, ODS-AM, ODS-AQ, ODS-AL, PolymerC18 packages.
Which Package should You Select?
Each C18 package comes with its unique separation characteristics. It is, therefore, necessary that you keenly consider properties of compounds you need to be separated as a guide to making correct package selection. Details of your compounds' molecular weights will act as a guide in choosing a C18 package that suits your needs.
Reasons Why You Need C18
Your cost per analysis will be much lower compared to other alternatives. Being a hybrid silica formulated ODs column, it is durable with excellent performance. Durability property is due to its multi-layer structure which is composed of a non-organic silica-based layer, and an organic layer which is polymer-based It is highly suitable for use under low pressure conditions. Through the use of modern technology called microreactor, the substance is homogeneous with highly uniform particles. This allows you to effectively conduct your analysis under low pressures. They come in different sizes, and wide range of hydrophobicity making them suitable for separating, basic, and neutral substances. Your chemical tests and analysis is therefore not limited to PH levels They are of different pores for different types of particles. You can therefore use them with a diverse range of chemical substances. They display clear peak shapes for your quick analysis under all conditions. By identifying key properties of compounds to pass through your C18 columns, you can easily select the best C18 with ease. Compatible with both your current and older types of chromatography instruments. Enhanced lot to lot reproducibility irrespective of which compounds you use. You therefore able to effectively handle complex compounds that may ordinarily be difficult to chromatograph. It is user-friendly and will always work for you all the times!Conclusion
Selection of correct chromatography chemicals is a key decision for effective analysis of a wide range of chemicals. Understanding key characteristics of the compounds you are working with is necessary for correct selection. C18 HPLC column will not only provide you with precise result, but will also enable you to work with a wider range of compounds. This will save you both time and money, and enhance both the quality and standard of your analysis. With a wide range of packages to select from, you are assured of getting what suits you most.
Difference between GC and HPLC Techniques
Gas Chromatography and the High Performance Liquid Chromatography are separation techniques used in obtaining specific eluents from an analyte basing on the compounds in the analyte distribution between the two phases (Mobile phase and Stationary phase). The mobile phase is the part in chromatography which moves and it usually contains the sample while the stationary phase is one which does not move and contains adsorption material. Chromatography is a larger analytical technique in chemistry.
In modern-day analytical chemistry, Gas Chromatography and the High Performance Liquid Chromatography are the major techniques used in Analytical Chemistry. In earlier analysis, techniques such as the Ion Exchange Chromatography, Affinity Chromatography and Thin Layer Chromatography were used.Both these techniques are effective for separation of the analytes. The difference in between the two techniques are discussed in different subtopics below:
1. Mobile Phase: One of the major differences between these two methods is the difference in their mobile phases which eventually determines where each technique is used.
As the name suggests, gas chromatography uses gases as its mobile phase with the stationary phase either being liquid or solid. On the other hand, High Performance Liquid Chromatography uses a liquid as its mobile phase and a solid as its stationary phase in analysis.
2. Nature of Analyte: Compared to High Performance Liquid Chromatography, Gas Chromatography analyzes samples that are either volatile or semi-volatile thus it will analyze only few samples since volatile compounds are fewer than non volatile samples which the HPLC analyzes. Inadvertently, HPLC analyzes samples that are are only liquid in nature.
3. Instrumentation: Both these methods use chromatography columns, but there is differentiation in their sizes. The Gas chromatography uses columns which are long and thick usually 10 to 30 meters in length as compared to the chromatography column which is shorter and thin basically being 5 to 25 cm in length.
4. Detection: After chromatography is done on the analyte, the composition of the eluent has to be done. This calls for detection methods that are stringent to get the perfect sample type and volume. Each analytical technique has its own method of sample detection. For gas chromatography, two types of detectors are the most widely used i.e the Flame Ionization Detector (FID) that is only used for detection of hydrocarbons and the Thermal Conducting Detector (TCD) which is universal and detects any substance.
In the case of High Performance Liquid Chromatography, the only method use is via an Ultraviolet Spectrometric detector which uses the principle of absorption and transmittance to detect the different types of samples In spectrophotometry, a sample is placed between a source of light and a photometric reader. The amount of light passing through the sample is recorded as photons by a computer detector. Different samples have different absorbance rates.
5. Cost: Finally, the cost of each technique is a determining factor.The Gas Chromatography technique is less costly compared to the HPLC system that utilizes a lot of equipment including high pressure pumps to increase its accuracy and shorten the time required for analysis of the sample. This makes Gas Chromatography being used more compared to HPLC.
Different Types of HPLC Detectors
HPLC detectors are used in the detection of the solute present in the eluent coming from the HPLC column. They are capable of determining the identity and concentration of eluting compounds in the mobile phase. There are specific detectors which respond to a specific compound and their response is not dependent on mobile phase composition.
However, the response of the bulk-property detectors is dependent on the collective changes in the composition of the mobile phase and sample. There is a broad range of detectors available for different sample requirements. Some of the crucial features required of a detector include sensitivity, a linear response and either a universal or selective response.
Some of the desirable features required in HPLC detectors are:
1. It should have either specific or general response to compounds in a mixture.
2. Sensitivity towards solute over mobile phase.
3. It should have a response to solute and not the mobile phase.
4. It should have a low cell volume for memory effects minimization.
5. It should respond linearly to solute concentration.
6. Low detector noise and detection limits.
7. It should not contribute to zone spreading.
8. It should be not be affected by temperature variation and flow rate.
The different types of detectors are categorized into two, i.e. specific and bulk property detectors.
A) Specific HPLC Detectors
1. Mass Spectroscopic HPLC Detectors
Mass spectroscopy detectors offer very high selectivity and sensitivity and their detection is based on molecular fragmentation by electric fields and separation based on the mass to charge ratio of fragmented molecules. New application areas have been introduced by the LC-MS technique because of its advantages in terms of sensitivity and resolution.
2. UV/VIS HPLC Detectors
They are of three types, i.e. fixed wavelength detectors, variable wavelength detectors and the diode array detectors. UV-VIS Detectors have mostly used detectors due to its specific response to the class of compounds or particular compounds depending on the functional groups of elutingmolecules that absorb light, although some compounds with no light absorbing groups give suitable response after post-column derivatization to introduce light absorbing entities. A beam of the electromagnetic radiation is passed through the detector flow cell and due to this interaction, it experiences a change in intensity and the measurement of this particular changes is the basis of most optical detectors.
3. Photo Diode Array (PDA) HPLC Detectors
A large number of diodes serving as detector elements makes it possible for simultaneous monitoring of many absorbing components at differing wavelengths, with little time and reduced cost spent on expensive solvents.
4. Fluorescence HPLC Detectors
They are most specific, sensitive and selective, and offers greater sensitivity than the UV-VIS detector. Naturally fluorescent compounds are less as compared to the light absorbing compounds, a limitation solved by post-column derivatization. There are three types of fluorescent detectors, namely the single wavelength fluorescent detector, multi-wavelength fluorescent detector and laser-induced fluorescent detector.
B) Bulk Property HPLC Detectors
1. Electrical Conductivity HPLC Detectors
They provide reproducible, universal and high sensitivity detection of the charged species and surfactants. The detectors measure the conductivity of the mobile phase, therefore are classified as bulk property detectors. Their sensors consist of the flow-through cell with few microliters of the volume containing 2 electrodes, usually made of stainless steel, platinum or a noble metal used in the determination of alkali and alkali earth metals.
2. Refractive Index HPLC Detectors
There are various types of RI detectors which include, the Christiansen effect detector, interferometer detector, thermal lens detector and the dielectric constant detector. They have limited sensitivity and detection depends on the changes in refractive index of eluting molecules in the mobile phase. These detectors are used for detecting non-ionic compounds that neither fluoresce nor absorb in the UV region. Temperature control is required due to high-temperature sensitivity.
3. Electrochemical HPLC Detectors
The detectors can be classified into two, which are the equilibrium and the dynamic detectors. They respond to oxidizable or reducible substances and are sensitive to changes in flow rate or composition of the mobile phase. The reaction takes place on the surface of the electrode producing electrical signals. Their suitability will depend on the volumetriccharacteristics of the solute molecules in the aqueous or organic mobile phase. They require the working electrode, reference electrode and the auxiliary electrode.
4. Light Scattering HPLC Detectors
Light scattering detectors are of two types which include low angle laser light scattering detector and the multiple angle laser light scattering detector. They are based on measuring the scattered light and are useful for detection of large molecular weight molecules present in the eluent, which is examined by being passed through an appropriate sensor cell depending on molecular sizes.
Other types of HPLC detectors are the evaporativelight scattering detectors, IR detector, transport detectors, aerosol-based detectors, chiral detectors and the pulsed amperometric detectors.
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