- Advantages of Column Chromatography & TLC
- Why TLC is superior to Column Chromatography?
- Describe how HPLC differs from conventional LC?
- Explain commonly used detectors in LC and their basis of operation?
- Why are silica particles end capped in bonded reverse phase particles?
- Explain
a) How does temp effect in HPLC separation?
b) How fast LC differ from conventional HPLC?
a) Rf value
b) Retention time
c) Polarity of solvent
d) Slope phase
e) Selectivity factor
f) Elusion
g) Packing Material
· Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample.
· A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
· A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated.
· A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.
· Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), while the other (the mobile phase) moves in a definite direction.
· The eluate is the mobile phase leaving the column.
· The eluent is the solvent that carries the analyte.
· An eluotropic series is a list of solvents ranked according to their eluting power
· An immobilized phase is a stationary phase that is immobilized on the support particles, or on the inner wall of the column tubing.
· The mobile phase is the phase that moves in a definite direction. It may be a liquid (LC and Capillary Electrochromatography (CEC)), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In the case of HPLC the mobile phase consists of a non-polar solvent(s) such as hexane in normal phase or polar solvents in reverse phase chromotagraphy and the sample being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.
· Preparative chromatography is used to purify sufficient quantities of a substance for further use, rather than analysis.
· The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. See also: Kovats' retention index
· The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.
· The solute refers to the sample components in partition chromatography.
· The solvent refers to any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography.
· The stationary phase is the substance fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography
· The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation.
Chromatography is based on the concept of partition coefficient. Any solute partitions between two immiscible solvents. When we make one solvent immobile (by adsorption on a solid support matrix) and another mobile it results in most common applications of chromatography. If matrix support is polar (e.g. paper, silica etc.) it is forward phase chromatography, and if it is non polar (C-18) it is reverse phase.
Answer 1) –
Column chromatography in chemistry is the preparative application of chromatography. It is used to obtain pure chemical compounds from a mixture of compounds on a scale from micrograms up to kilograms using large industrial columns.
The classical preparative chromatography column is a glass tube with a diameter from 5 to 50 mm and a height of 50 cm to 1 m with a tap at the bottom. A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles. A solution of the organic material is pipetted on top of the stationary phase. This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent. Eluent is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the column.
The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent. At the end of the column they elute one at a time. During the entire chromatography process the eluent is collected in a series of fractions. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV absorption, or fluorescence. Colored compounds (or fluorescent compounds with the aid of an UV lamp) can be seen through the glass wall as moving bands.
Advantages of Column Chromatography & TLC:
The biggest advantage of column chromatography is that it can usually be scaled to the project at hand. This is especially useful if one is trying to separate and purify a reaction mixture preparing an intermediate in a sequence of reactions. The corresponding disadvantage is the column may take a long time to properly prepare and use.
Thin layer chromatography is usually great for initial analysis and determining the conditions needed for a good separation. Many plates are commercially available and the chromatograms usually do not take a long time to develop. The main disadvantage is the very small amounts of compounds handled. These can be recovered for instrumental analysis, but seldom is there enough for further reaction.
Answer 2) –Thin layer chromatography's advantages over Column Chromatography:
Answer 3) - HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50-350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC typical column dimensions are 2.1 - 4.6 mm diameter, and 30 - 250 mm length. Also HPLC columns are made with smaller sorbent particles (2- 5 micrometer in average particle size). This gives HPLC superior resolving power when separating mixtures, which is why it is a popular chromatographic technique.
The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature the separation is performed at.
Answer 4) – Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry.
Optical and Spectrophotometric Detectors
a. Differential refractometry
b. Ultraviolet spectrophotometry
c. Fluorescence detection
d. Infrared spectrophotometric detectors
e. Spectrophotometric detection with post-column chemical reaction
Some Miscellaneous Detection Systems
a) The mass spectrometer as an HPLC detector
b) Radioactivity detectors
c) Density, electrochemical and other detectors
Transport-flame Ionization Detectors
Evaporative Light-Scattering Detectors
Answer 5) - Silica is an amorphous polymer of silicon and oxygen. This polymer terminates at the surface of the particle as –Si-OH groups, commonly called “silanols.” These silanol groups serve as attachment sites for the bonded phase. A silane reagent, such as Cl(CH3)2SiC18H37 is reacted with the silanol to form a silyl ether (–Si-O-Si-). The bulk of the C18 group prevents bonding to all of the exposed silanols. This results in a surface that looks much like that on the left side of Figure 1, where there is a fairly high population of unbounded silanols, often termed “residual silanols.” The residual silanols are somewhat acidic and can be overly reactive with sample components, especially basic analytes, so it is preferable to reduce the population of residual silanols.
The end-capping process. Left, silica surface following bonding with the primary stationary phase. Right, reduced surface silanol population after end-capping.
To further deactivate the surface of the particle, a smaller reagent is used in the end-capping reaction. For example, Cl(CH3)2SiCH3 is one common end-capping reagent. You can see that substituting a methyl group for the large C18 group used above makes this reagent much smaller, allowing it to have access to some of the residual silanol groups on the surface. After this second reaction is completed, we say that the column is end-capped.
It is interesting to note that even when end-capping is done as thoroughly as possible, such as repeating the reaction (“double end-capping”), approximately half of the silanols are still unbonded. You might think that this is a problem, but on the contrary, I suspect that if we were to eliminate the silanols completely, we might be disappointed at the performance of the column. For example, if silica is replaced by a polymer, such as in the polymer reversed-phase materials, we often find that these columns lack adequate selectivity to get satisfactory separations. More important is to have the silanols well shielded so that their interactions with the sample and mobile phase are controlled.
If only the end-capping reagent is used (no C18), the surface is not very stable, with rapid loss of end-capping at low pH (e.g., pH<2). However, column stability under acidic conditions increases with the chain length of the bonded phase, so C8 and C18 phases are quite stable under conditions that would destroy an end-capped-only phase. When C8 or C18 phases are end-capped, the bulk phase serves to protect the end-capping group from hydrolysis and the end-capping reduces the reactivity of the unbounded surface – a win-win situation. As a result, most reversed-phase HPLC columns today are end-capped.
In addition to protecting and deactivating the silica surface, sometimes end-capping is used for other purposes. For example, if the end-capping reagent contains a polar moiety, such as a diol function, the reagents imparts some polar characteristics to the surface. This can be used to create an AQ or polar-embedded-phase column that can be used with 100% aqueous mobile phases to avoid phase dewetting.
Answer 6) –
A) – Effect of the temperature
Temperature effects in HPLC are not as significant as in gas chromatography. First, because we do not have same temperature range. Volatile solvents are not allowed to rise to higher temperatures too much, and the stability of the attached bonded ligands on the adsorbent surface may be influenced by the high temperature. So, the main temperature range is from ambient temperature to 60 or 70 C.
According the equation ,
increasing the temperature will decrease the value of K or k', thus the actual retention time will decrease. For most of the systems these decrease will not exceed 50% of the component reduced retention time at ambient temperature.
Picture below illustrate the influence of the column temperature on the HPLC retention.
There are two other significant effects of separation under the elevated temperature.
a) Stabilization of the column under the elevated temperature usually leads to the stabilization of the retention times. Origin of this effect is not well understood yet. Possible explanation is that the solvent viscosity decreased and more uniform stabilized temperature with absence of local temperature fluctuations due to the solvent friction lead to the more uniform adsorption-desorption process.
b) Another effect is the increase of the column efficiency. At the elevated temperature viscosity of liquids decrease and the diffusion coefficient increase. From the Van Deemter equation the second term will increase which will lead to the decrease of the efficiency at the very low flow rates (which is not important). The last term will decrease which will lead to the increasing of the efficiency at the common flow rates. It also widens the flow rate range with optimum efficiency.
B) – Many laboratory budgets do not allow the purchase of new ultrahigh-pressure liquid chromatography (UHPLC) systems, and workloads typically are not declining.
a) Fast LC incorporates the use of faster mobile phase flow rates and smaller particles to achieve separations in less time and with equivalent resolution to traditional high performance liquid chromatography (HPLC).
b) Fast LC is one way to get more productivity out of existing HPLC technology and prepare for the next generation of UHPLC systems with more-efficient separation schemes.
Fast liquid chromatography (LC) has been discussed since the 1980s, but there had been only modest interest in it until the advent of ultrahigh-pressure liquid chromatography (UHPLC) systems. Fast LC affords fast flow rates through shorter columns, which are often packed with smaller particles. Some reasons for developing fast LC methods now include
By learning to develop fast LC methods, you will be better prepared to develop methods with UHPLC systems.
If fast LC saves time and money and has all of these benefits, then why do not more chromatographers adopt this technology?
I have discussed this issue with a number of colleagues and have found there are six reasons that they give for not converting their methods to fast LC. I will list them here and discuss them throughout this review:
They do not know how to implement the technology.
An effort to transfer an existing method to a fast LC method resulted in worse resolution.
They simply cannot afford to lose resolution for decreased cycle time.
The stability of the columns will not be as good as 5-µm packing.
The need to quantitate peaks with reliable retention times and peak areas is critical.
A method used in a GMP environment would need to be revalidated.
Although there are numerous articles explaining that fast LC is useful, there are not many that explain how to implement the technology. Even when there is an explanation, some of the significant elements of fast LC are ignored. Most discussions of fast LC begin with band-broadening theory and a review of the Knox or van Deemter equations. While band broadening is a major driver in the observed speed and resolution, other areas also contribute to the benefits of the method. I will discuss two: system volume and the geometry of the particles and columns.
For those who have tried to implement fast LC and found poorer resolution, there can be a number of reasons. Following is one anecdote that I have heard more than once. A scientist related to me that he had purchased a 50 mm × 2.1 mm column packed with sub-2-µm C18 packing material. To run solvent through the column, the flow rate had to be below 0.5 mL/min. The resulting peaks were broad and the back pressure was a little over 300 bar. There were two factors working against this chromatographer's separation. The low flow rate contributed to a larger plate height, and the system volume was not minimized. These causes are explained below, under system volume and theory.
Answer 7) –
a) Rƒ value
The retention factor (Rƒ) may be defined as the ratio of the distance traveled by the substance to the distance traveled by the solvent. Rƒ values are usually expressed as a fraction of two decimal places but it was suggested by Smith that a percentage figure should be used instead. If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front. To calculate the Rƒ value, take the distance traveled by the substance divided by the distance traveled by the solvent (as mentioned earlier in terms of ratios). For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, (2.1/2.8) the Rƒ value = 0.75
b) - Polar protic and polar aprotic
Solvents with a relative static permittivity greater than 15 can be further divided into protic and aprotic. Protic solvents solvate anions (negatively charged solutes) strongly via hydrogen bonding. Water is a protic solvent. Aprotic solvents such as acetone or dichloromethane tend to have large dipole moments (separation of partial positive and partial negative charges within the same molecule) and solvate positively charged species via their negative dipole. In chemical reactions the use of polar protic solvents favors the SN1 reaction mechanism, while polar aprotic solvents favor the SN2 reaction mechanism.
Physical properties of common solvents
Properties table of common solvents
The solvents are grouped into non-polar, polar aprotic, and polar protic solvents and ordered by increasing polarity. The polarity is given as the dielectric constant. The properties of solvents that exceed those of water are bolded.
Hansen solubility parameter values (HSPiP)
There's another powerful way to look at these same solvents. By knowing their Hansen solubility parameter values, which are based on δD=dispersion bonds, δP=polar bonds and δH=hydrogen bonds, you know important things about their inter-molecular interactions with other solvents and also with polymers, pigments, nanoparticles etc. so you can do two things. First, you can create rational formulations knowing, for example, that there is a good HSP match between a solvent and a polymer. Second, you can make rational substitutions for "good" solvents (they dissolve things well) that are "bad" (for the environment, for health, for cost etc.). The following table shows that the intuitions from "non-polar", "polar aprotic" and "polar protic" are put numerically – the "polar" molecules have higher levels of δP and the protic solvents have higher levels of δH. Because numerical values are used, comparisons can be made rationally by comparing numbers. So acetonitrile is much more polar than acetone but slightly less hydrogen bonding.
Consider a simple example of rational substitution. Suppose for environmental reasons we needed to replace the chlorinated solvent, chloroform, with a solvent (blend) of equal solvency using a mixture of two non-chlorinated solvents from this table. Via trial-and-error, a spreadsheet or some software such as HSPiP we find that a 50:50 mix of toluene and 1,4 dioxane is a close match. The δD of the mixture is the average of 18.0 and 17.5 = 17.8. The δP of the mixture is the average of 1.4 and 1.8 = 1.6 and the δH of the mixture is the average of 2.0 and 9.0 = 5.5. So the mixture is 17.8, 1.6, 5.5 compared to Chloroform at 17.8, 3.1, 5.7. Because Toluene itself has many health issues, other mixtures of solvents can be found using a full Hansen solubility parameter data set.
An important property of solvents is the boiling point. This also determines the speed of evaporation. Small amounts of low-boiling-point solvents likediethyl ether, dichloromethane, or acetone will evaporate in seconds at room temperature, while high-boiling-point solvents like water or dimethyl sulfoxide need higher temperatures, an air flow, or the application of vacuum for fast evaporation.
Density
Most organic solvents have a lower density than water, which means they are lighter and will form a separate layer on top of water. An important exception: most of the halogenated solvents like dichloromethane or chloroform will sink to the bottom of a container, leaving water as the top layer. This is important to remember when partitioning compounds between solvents and water in a separatory funnel during chemical syntheses.
Often, specific gravity is cited in place of density. Specific gravity is defined as the density of the solvent divided by the density of water at the same temperature. As such, specific gravity is a unitless value. It readily communicates whether a water-insoluble solvent will float (SG < 1.0) or sink (SG > 1.0) when mixed with water.
C) – Retention Time: The retention time of a solute is taken as the elapsed time between the time of injection of a solute and the time of elution of the peak maximum of that solute. It is a unique characteristic of the solute and can be used for identification purposes. The corrected retention time of a solute is the retention time minus the retention time of a completely unretained solute. For gas chromatography an air peak of often used as a non-retained peak. For liquid chromatography (HPLC) many substances have been used including salts, deuturated solvents, uracil (reversed phase) and benzene (normal phase), read the Dead Volume topic for more information. By multiplying the corrected retention time of a solute by the exit flow rate then the corrected retention volume can be obtained. If the mobile phase is compressible (i.e. the mobile phase is a gas) a pressure correction must be applied which is a function of the column inlet-outlet pressure ratio. Values of the corrected retention volume per ml of stationary phase for a solute measured over a range of temperatures can provide the standard energy of distribution, the standard enthalpy of distribution and the standard entropy of distribution for the solute concerned.
D) – Selectivity Factor: In order to separate two compounds, their respective retention factors must be different, otherwise both compounds would be eluted simultaneously; the selectivity factor is the ratio of the retention factors.
E) – Elution: Elution is a term used in analytical and organic chemistry to describe the process of extracting one material from another by washing with a solvent (as in washing of loaded ion-exchange resins to remove captured ions).
In a liquid chromatography experiment, for example, an analyte is generally adsorbed, or "bound to", an adsorbent in a liquid chromatography column. The adsorbent, a solid phase (stationary phase), is a powder which is coated onto a solid support. Based on an adsorbent's composition, it can have varying affinities to "hold" onto other molecules—forming a thin film on its outside surface (or on its internal surface if there are cavities within the compound). Elution then is the process of removing analytes from the adsorbent by running a solvent, called an "eluent", past the adsorbent/analyte complex. As the solvent molecules "elute", or travel down through the chromatography column, they can either pass by the adsorbent/analyte complex or they can displace the analyte by binding to the adsorbent in its place. After the solvent molecules displace the analyte, the analyte can be carried out of the column for analysis. This is why as the mobile phase passes out of the column, it typically flows into a detector or is collected for compositional analysis.
Predicting and controlling the order of elution is a key aspect of column chromatographic methods.
F) Packing Material: (will be added later)
Answer 8) – Names (any five)
1. LC1200 Variable Wavelength UV-Vis Detector
2. LC1205K Programmable Variable Wavelength Detector
3. LC1210K Programmable Scanning Dual Wavelength UV-Vis Detector
4. LC1246K Refractive Index Indicator
5. LC1255S Programmable Scanning Fluorescence Detector
Discuss any one;
The high performance LC1200 is designed for standard HPLC applications requiring UV-Vis detection, and most suitable for QC and educational laboratories, offering a level of performance and versatility one would expect from much higher priced models. Flexible operation at single wavelengths within the range of 190 - 600 nm makes for versatility. For example, rather than being restricted to one or two wavelengths, you can use the LC1200 at 254 nm for the detection of aromatic compounds, improve sensitivity and selectivity for proteins, peptides, phenols and catecholamines at 280 nm, detect analytes of interest in the visible range, or monitor nitrate/nitrite and carboxylic acids at 200 nm.
Answer 9) – High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC (or just LC), is achromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture. HPLC is considered an instrumental technique of analytical chemistry (as opposed to a gravitimetric technique). HPLC has many uses including medical (e.g. detecting vitamin D levels in blood serum), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. during the production process of pharmaceutical and biological products).
HPLC has contributed to analytical solutions in diverse fields such as pharmaceuticals, foods, life sciences, environment, forensics, etc. In the present module we shall discuss some application areas in pharmaceuticals and foods.
Pharmaceuticals
High Performance Liquid Chromatography provides reliable quantitative precision and accuracy along with a high linear dynamic range to allow determination of API and related substances in a single run. A convenient method for sample preparation for solid dosage forms is dispersion in water or aqueous media modified with acetonitrile or methanol .HPLC offers several possibilities for separation of chiral molecules into their respective enantiomers.These include precolumn derivatization to form diastereomers. Alternately, specialty columns prepared with cyclodextrins or special chiral moieties as stationary phases maybe used .In short HPLC, particularly reverse phase HPLC is the most popular choice for quantitative analysis in the pharmaceutical industry.
Common application areas in pharmaceutical analysis are:
• Assay
• Related Substances
• Analytical Method Validation
• Stability Studies
• Compound Identification
• Working Standards
Foods
High Performance Liquid Chromatography has brought desirable advantages in the field of food analysis. Food matrices are generally complex and extraction of analytes is not an easy task. To further complicate matters both desirable and undesirable components are often found in trace levels and classical extraction and analysis does not provide the required levels of accuracy and precision. HPLC offers viable solutions due to vast choice of stationary phases and mobile phase options. Common applications in foods are :
• Fat soluble vitamins (A,D,E and K)
• Water soluble vitamins (B-complex vitamins such as B1, B2, B3, B6, Folic acid, Pantothenic acid, B12, VitaminC)
• Residual pesticides such as 2, 4-D and Monochrotophos.
• Antioxidants such as TBHQ, BHA and BHT.
• Sugars: Glucose, Fructose, Maltose and other saccharides.
• Cholesterol and sterols
• Dyes and synthetic colours.
• Mycotoxins such as Aflatoxins B1,B2,G1,G2,M1,M2and ochratoxin
• Amino acids
• Residual antibiotics
• Steroids and flavanoids
• Aspartame and other artificial sweeteners.
• Active ingredients of farm produce such as allin in garlic and catachin in tea extracts.
Answer 10) – HPLC vs GC
HPLC and GC are both methods of separation of compounds from a mixture. Whereas HPLC applies to constituents that are fluids, GC is used when the compounds are gaseous or can be vaporized during separation process. Both have the same underlying principle of heavy molecules flowing slower than lighter ones. While HPLC refers to High Pressure Liquid Chromatography, GC is simply Gas Chromatography. This article will highlight the differences between these two separation techniques.
HPLC
The technique of HPLC is made heavy use of in analytical chemistry to identify and analyze the individual components of a mixture. In HPLC, columns and high pressure are used. High pressure ensures movement of constituents in a mobile phase. It also moves the analyte in a densely packed column. Smaller particle size helps in increasing the density of the constituents that helps in better separation when columns of shorter length are used.
GC
Gas Chromatography on the other hand is mainly used to check the purity of a substance, and in some cases, also helps in identifying a substance. There are two phases, the mobile, and the stationary phase in the process. In the mobile phase, the carrier is an inert gas such as helium. Sometimes, nitrogen is used as it is unreactive. The stationary phase involves using a polymer or a layer of liquid on an inert solid base.
What is the difference between HPLC and GC?
The major difference between GC and HPLC lies in the phases used. While in GC, compounds of a mixture are separated using a liquid (stationary) phase and a gas (mobile) phase, in case of HPLC the stationary phase is a solid while liquids make up mobile phase. Another difference lies in temperature control during the process. In GC, there is an oven to contain the column consisting of gas phase and it can control the temperatures when the gases are passing through the column. On the other hand, there is no such provision of temperature control in HPLC. The last difference pertains to the concentration of the compounds. In GC, it is the vapor pressure of the gases that decide the concentration of compounds whereas it is possible to increase or decrease the concentration of the compounds in HPLC.
Answer 11) – Paper Chromatography
Types of Paper Chromatography
a) Descending Paper Chromatography- In this type development of paper is done by allowing the solvent to travel down the paper is called Descending Chromatography. Here the mobile phase is present in the upper portion.
b) Ascending Paper Chromatography- Here the solvent travel upward direction of the Chromatographic paper. Both the Descending and Ascending Paper Chromatography are used for separation of Organic and Inorganic substances.
c) Ascending-Descending Paper Chromatography- It is the hybrid of both the above technique. The upper part of the Ascending chromatography can be folded over a rod and allowing the paper to become descending after crossing the rod.
d) Radial Paper Chromatography- It is also called as Circular chromatography. Here a circular filter paper is taken and the sample is given at the center of the paper. After drying the spot the filter paper tied horizontally on a Petridish containing solvent. So that Wick of the paper is dipped inside the solvent. The solvent rises through the wick and the component get separated in form of concentrate circular zone.
e) Two-Dimensional Paper Chromatography- In this technique a square or rectangular paper is used. Here the sample is applied to one of the corner and development is performed at right angle to the direction of first run.
A) – Types of Papers used in PC: Paper chromatography uses paper as the stationary phase. The exact type of paper used is important. Filter paper is one of the best types, although paper towels and even newspaper can also be used. Writing paper is coated so that ink does not run and because of this is less satisfactory. Of course, wax paper, not being absorbant, is unsatisfactory.
B) – Developer:
C) – Capillary action (sometimes capillarity, capillary motion, or wicking) is the ability of a liquid to flow in narrow spaces without the assistance of, and in opposition to, external forces like gravity. The effect can be seen in the drawing up of liquids between the hairs of a paint-brush, in a thin tube, in porous materials such as paper, in some non-porous materials such as liquified carbon fiber, or in a cell. It occurs because of intermolecular forcesbetween the liquid and solid surrounding surfaces. If the diameter of the tube is sufficiently small, then the combination of surface tension (which is caused by cohesion within the liquid) and adhesive forces between the liquid and container act to lift the liquid
D) – Partition equilibrium chromatography is a type of chromatography that is typically used in gas chromatography (GC) and high performance liquid chromatography (HPLC). The stationary phase in GC is a high boiling liquid bonded to solid surface and the mobile phase is a gas.
E) -- Ascending & Descending:
Answer 12) – in paper chromatography n-butanol:acetic acid:water mixture in the ratio 4:1:1 is used as a solvent whereas in case of thin layer chromatography(TLC) petroleum ether and acetone in the ratio 9:1 is used as a solvent...also in some cases DCM(dichloromethane is used as a solvent.
Answer 13) – Precautions in PC:
Use a pencil to draw a thin line near the bottom of the paper. Make sure it is not too near the bottom.
Mark a cross for each mixture on the line.
Mark a spot of color/dye/mixture on each respective crosses and allow to dry.
Put chromatography paper in solvent. Ensure top of solvent does not cover the mixture.
Observe as spots move up the chromatography paper.
Use a pencil to draw the thin line as a pen may have ink that is soluble in the solvent. The ink may move up the chromatography paper and ruin the experiment.
Allow the color/dye/mixture to dry first before putting the chromatography paper in the solvent. This is to ensure that the ink will not totally dissolve in the solvent while it is still wet.
You must make sure that the solvent does not cover the mixture as the mixtures may dissolve totally in the solvent,
Answer 14) – Partition Chromatography is a liquid liquid extraction which involves two solvents while adsorption chromatography is a liquid solid extraction which involves a solid stationary phase & a liquid mobile phase
The partition chromatography involves separation between liquids while adsoption chromatography involves solid and liquid separations.
Partition PC:
The tendency for a compound to divide its time between two immiscible solvents (solvents such as hexane and water which won't mix) is known as partition. Paper chromatography using a non-polar solvent is therefore a type of partition chromatography.
Paper chromatography is a classical example of partition chromatography as the separation of the analyte occurs by the process of partition between the water molecules (present in the interstices of the cellulose of which the paper is made of) serving as liquid stationary phase and any solvent used as mobile phase.
Partition chromatography a method using the partition of the solutes between two liquid phases (the original solvent and the film of solvent on the adsorption column).
Partition chromatography
§ Stationary phase = non-volatile liquid film supported on an inert solid
§ Mobile phase = liquid or gas
§ Mixture of components are partitioned between the liquid film and the mobile phase (Used partition coefficient / distribution coefficient, K)
§ Interpretation: high value K = the component (X) dissolves more readily in the mobile phase and the component moves rapidly along the stationary phase.
§ Interpretation: low value K = the component (X) remain largely adsorbed on the stationary phase and the components moves slowly along the stationary phase.
§ Example: paper chromatography, gas-liquid chromatography (GLC)
Adsorption PC:
adsorption chromatography that in which the stationary phase is an adsorbent.
Adsorption chromatography
§ Stationary phase = solid
§ Mobile phase = liquid or gas
§ Mixture of components are adsorbed on the surface of the stationary phase
§ Example: column chromatography, thin layer chromatography (TLC)
b) How fast LC differ from conventional HPLC?
- Define
a) Rf value
b) Retention time
c) Polarity of solvent
d) Slope phase
e) Selectivity factor
f) Elusion
g) Packing Material
- Detectors of HPLC, Name any five and Explain any one.
- Applications of HPLC
- Comparison of HPLC with GC
- DiscussType of paper used in PC
- Developer
- Capillary force
- Partition equilibrium
- Ascending & Descending development
- Choice of solvent for PC
- Precautions of PC
- Partition PC and Absorption PC
Chromatography terms
· The analyte is the substance to be separated during chromatography.· Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample.
· A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
· A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated.
· A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.
· Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), while the other (the mobile phase) moves in a definite direction.
· The eluate is the mobile phase leaving the column.
· The eluent is the solvent that carries the analyte.
· An eluotropic series is a list of solvents ranked according to their eluting power
· An immobilized phase is a stationary phase that is immobilized on the support particles, or on the inner wall of the column tubing.
· The mobile phase is the phase that moves in a definite direction. It may be a liquid (LC and Capillary Electrochromatography (CEC)), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In the case of HPLC the mobile phase consists of a non-polar solvent(s) such as hexane in normal phase or polar solvents in reverse phase chromotagraphy and the sample being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.
· Preparative chromatography is used to purify sufficient quantities of a substance for further use, rather than analysis.
· The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. See also: Kovats' retention index
· The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.
· The solute refers to the sample components in partition chromatography.
· The solvent refers to any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography.
· The stationary phase is the substance fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography
· The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation.
Chromatography is based on the concept of partition coefficient. Any solute partitions between two immiscible solvents. When we make one solvent immobile (by adsorption on a solid support matrix) and another mobile it results in most common applications of chromatography. If matrix support is polar (e.g. paper, silica etc.) it is forward phase chromatography, and if it is non polar (C-18) it is reverse phase.
Answer 1) –
Column chromatography in chemistry is the preparative application of chromatography. It is used to obtain pure chemical compounds from a mixture of compounds on a scale from micrograms up to kilograms using large industrial columns.
The classical preparative chromatography column is a glass tube with a diameter from 5 to 50 mm and a height of 50 cm to 1 m with a tap at the bottom. A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles. A solution of the organic material is pipetted on top of the stationary phase. This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent. Eluent is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the column.
The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent. At the end of the column they elute one at a time. During the entire chromatography process the eluent is collected in a series of fractions. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV absorption, or fluorescence. Colored compounds (or fluorescent compounds with the aid of an UV lamp) can be seen through the glass wall as moving bands.
Advantages of Column Chromatography & TLC:
The biggest advantage of column chromatography is that it can usually be scaled to the project at hand. This is especially useful if one is trying to separate and purify a reaction mixture preparing an intermediate in a sequence of reactions. The corresponding disadvantage is the column may take a long time to properly prepare and use.
Thin layer chromatography is usually great for initial analysis and determining the conditions needed for a good separation. Many plates are commercially available and the chromatograms usually do not take a long time to develop. The main disadvantage is the very small amounts of compounds handled. These can be recovered for instrumental analysis, but seldom is there enough for further reaction.
Answer 2) –Thin layer chromatography's advantages over Column Chromatography:
- Apparatus is smaller (TLC just needs a 2 by 0.5 inch glass plate)
- Quantity of material required is lesser
- Faster procedure
- Above points make the overall procedure cheaper
- The combinations of solvents that can be used is more
- The Rf value can be measured easily, once a suitable visualizing technique (like an iodine gas chamber for carbohydrate mixtures) is used
- All these makes it better suited for analytical purposes (but not for separation purposes which is actually one of its disadvantages)
Answer 3) - HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50-350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC typical column dimensions are 2.1 - 4.6 mm diameter, and 30 - 250 mm length. Also HPLC columns are made with smaller sorbent particles (2- 5 micrometer in average particle size). This gives HPLC superior resolving power when separating mixtures, which is why it is a popular chromatographic technique.
The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature the separation is performed at.
Answer 4) – Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry.
Optical and Spectrophotometric Detectors
a. Differential refractometry
b. Ultraviolet spectrophotometry
c. Fluorescence detection
d. Infrared spectrophotometric detectors
e. Spectrophotometric detection with post-column chemical reaction
Some Miscellaneous Detection Systems
a) The mass spectrometer as an HPLC detector
b) Radioactivity detectors
c) Density, electrochemical and other detectors
Transport-flame Ionization Detectors
Evaporative Light-Scattering Detectors
Answer 5) - Silica is an amorphous polymer of silicon and oxygen. This polymer terminates at the surface of the particle as –Si-OH groups, commonly called “silanols.” These silanol groups serve as attachment sites for the bonded phase. A silane reagent, such as Cl(CH3)2SiC18H37 is reacted with the silanol to form a silyl ether (–Si-O-Si-). The bulk of the C18 group prevents bonding to all of the exposed silanols. This results in a surface that looks much like that on the left side of Figure 1, where there is a fairly high population of unbounded silanols, often termed “residual silanols.” The residual silanols are somewhat acidic and can be overly reactive with sample components, especially basic analytes, so it is preferable to reduce the population of residual silanols.
The end-capping process. Left, silica surface following bonding with the primary stationary phase. Right, reduced surface silanol population after end-capping.
To further deactivate the surface of the particle, a smaller reagent is used in the end-capping reaction. For example, Cl(CH3)2SiCH3 is one common end-capping reagent. You can see that substituting a methyl group for the large C18 group used above makes this reagent much smaller, allowing it to have access to some of the residual silanol groups on the surface. After this second reaction is completed, we say that the column is end-capped.
It is interesting to note that even when end-capping is done as thoroughly as possible, such as repeating the reaction (“double end-capping”), approximately half of the silanols are still unbonded. You might think that this is a problem, but on the contrary, I suspect that if we were to eliminate the silanols completely, we might be disappointed at the performance of the column. For example, if silica is replaced by a polymer, such as in the polymer reversed-phase materials, we often find that these columns lack adequate selectivity to get satisfactory separations. More important is to have the silanols well shielded so that their interactions with the sample and mobile phase are controlled.
If only the end-capping reagent is used (no C18), the surface is not very stable, with rapid loss of end-capping at low pH (e.g., pH<2). However, column stability under acidic conditions increases with the chain length of the bonded phase, so C8 and C18 phases are quite stable under conditions that would destroy an end-capped-only phase. When C8 or C18 phases are end-capped, the bulk phase serves to protect the end-capping group from hydrolysis and the end-capping reduces the reactivity of the unbounded surface – a win-win situation. As a result, most reversed-phase HPLC columns today are end-capped.
In addition to protecting and deactivating the silica surface, sometimes end-capping is used for other purposes. For example, if the end-capping reagent contains a polar moiety, such as a diol function, the reagents imparts some polar characteristics to the surface. This can be used to create an AQ or polar-embedded-phase column that can be used with 100% aqueous mobile phases to avoid phase dewetting.
Answer 6) –
A) – Effect of the temperature
Temperature effects in HPLC are not as significant as in gas chromatography. First, because we do not have same temperature range. Volatile solvents are not allowed to rise to higher temperatures too much, and the stability of the attached bonded ligands on the adsorbent surface may be influenced by the high temperature. So, the main temperature range is from ambient temperature to 60 or 70 C.
According the equation ,
increasing the temperature will decrease the value of K or k', thus the actual retention time will decrease. For most of the systems these decrease will not exceed 50% of the component reduced retention time at ambient temperature.
Picture below illustrate the influence of the column temperature on the HPLC retention.
There are two other significant effects of separation under the elevated temperature.
a) Stabilization of the column under the elevated temperature usually leads to the stabilization of the retention times. Origin of this effect is not well understood yet. Possible explanation is that the solvent viscosity decreased and more uniform stabilized temperature with absence of local temperature fluctuations due to the solvent friction lead to the more uniform adsorption-desorption process.
b) Another effect is the increase of the column efficiency. At the elevated temperature viscosity of liquids decrease and the diffusion coefficient increase. From the Van Deemter equation the second term will increase which will lead to the decrease of the efficiency at the very low flow rates (which is not important). The last term will decrease which will lead to the increasing of the efficiency at the common flow rates. It also widens the flow rate range with optimum efficiency.
B) – Many laboratory budgets do not allow the purchase of new ultrahigh-pressure liquid chromatography (UHPLC) systems, and workloads typically are not declining.
a) Fast LC incorporates the use of faster mobile phase flow rates and smaller particles to achieve separations in less time and with equivalent resolution to traditional high performance liquid chromatography (HPLC).
b) Fast LC is one way to get more productivity out of existing HPLC technology and prepare for the next generation of UHPLC systems with more-efficient separation schemes.
Fast liquid chromatography (LC) has been discussed since the 1980s, but there had been only modest interest in it until the advent of ultrahigh-pressure liquid chromatography (UHPLC) systems. Fast LC affords fast flow rates through shorter columns, which are often packed with smaller particles. Some reasons for developing fast LC methods now include
- You can run more samples in the same time frame.
- It is where the field of high performance liquid chromatography (HPLC) is going.
- You can develop fast methods with the existing HPLC systems in your laboratory.
- You can save time and solvent by coverting to fast LC.
- You can get better resolution and equivalent quantitation to conventional HPLC.
- You can have more time to analyze your data and provide a value added dimension to your results.
By learning to develop fast LC methods, you will be better prepared to develop methods with UHPLC systems.
If fast LC saves time and money and has all of these benefits, then why do not more chromatographers adopt this technology?
I have discussed this issue with a number of colleagues and have found there are six reasons that they give for not converting their methods to fast LC. I will list them here and discuss them throughout this review:
They do not know how to implement the technology.
An effort to transfer an existing method to a fast LC method resulted in worse resolution.
They simply cannot afford to lose resolution for decreased cycle time.
The stability of the columns will not be as good as 5-µm packing.
The need to quantitate peaks with reliable retention times and peak areas is critical.
A method used in a GMP environment would need to be revalidated.
Although there are numerous articles explaining that fast LC is useful, there are not many that explain how to implement the technology. Even when there is an explanation, some of the significant elements of fast LC are ignored. Most discussions of fast LC begin with band-broadening theory and a review of the Knox or van Deemter equations. While band broadening is a major driver in the observed speed and resolution, other areas also contribute to the benefits of the method. I will discuss two: system volume and the geometry of the particles and columns.
For those who have tried to implement fast LC and found poorer resolution, there can be a number of reasons. Following is one anecdote that I have heard more than once. A scientist related to me that he had purchased a 50 mm × 2.1 mm column packed with sub-2-µm C18 packing material. To run solvent through the column, the flow rate had to be below 0.5 mL/min. The resulting peaks were broad and the back pressure was a little over 300 bar. There were two factors working against this chromatographer's separation. The low flow rate contributed to a larger plate height, and the system volume was not minimized. These causes are explained below, under system volume and theory.
Answer 7) –
a) Rƒ value
The retention factor (Rƒ) may be defined as the ratio of the distance traveled by the substance to the distance traveled by the solvent. Rƒ values are usually expressed as a fraction of two decimal places but it was suggested by Smith that a percentage figure should be used instead. If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front. To calculate the Rƒ value, take the distance traveled by the substance divided by the distance traveled by the solvent (as mentioned earlier in terms of ratios). For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, (2.1/2.8) the Rƒ value = 0.75
b) - Polar protic and polar aprotic
Solvents with a relative static permittivity greater than 15 can be further divided into protic and aprotic. Protic solvents solvate anions (negatively charged solutes) strongly via hydrogen bonding. Water is a protic solvent. Aprotic solvents such as acetone or dichloromethane tend to have large dipole moments (separation of partial positive and partial negative charges within the same molecule) and solvate positively charged species via their negative dipole. In chemical reactions the use of polar protic solvents favors the SN1 reaction mechanism, while polar aprotic solvents favor the SN2 reaction mechanism.
Physical properties of common solvents
Properties table of common solvents
The solvents are grouped into non-polar, polar aprotic, and polar protic solvents and ordered by increasing polarity. The polarity is given as the dielectric constant. The properties of solvents that exceed those of water are bolded.
|
Solvent
|
|||||
|
CH3-CH2-CH2-CH2-CH3
|
36 °C
|
1.84
|
0.626 g/ml
|
0.00 D
|
|
|
C5H10
|
40 °C
|
1.97
|
0.751 g/ml
|
0.00 D
|
|
|
CH3-CH2-CH2-CH2-CH2-CH3
|
69 °C
|
1.88
|
0.655 g/ml
|
0.00 D
|
|
|
C6H12
|
81 °C
|
2.02
|
0.779 g/ml
|
0.00 D
|
|
|
C6H6
|
80 °C
|
2.3
|
0.879 g/ml
|
0.00 D
|
|
|
C6H5-CH3
|
111 °C
|
2.38
|
0.867 g/ml
|
0.36 D
|
|
|
/-CH2-CH2-O-CH2-CH2-O-\
|
101 °C
|
2.3
|
1.033 g/ml
|
0.45 D
|
|
|
CHCl3
|
61 °C
|
4.81
|
1.498 g/ml
|
1.04 D
|
|
|
CH3-CH2-O-CH2-CH3
|
35 °C
|
4.3
|
0.713 g/ml
|
1.15 D
|
|
|
CH2Cl2
|
40 °C
|
9.1
|
1.3266 g/ml
|
1.60 D
|
|
|
/-CH2-CH2-O-CH2-CH2-\
|
66 °C
|
7.5
|
0.886 g/ml
|
1.75 D
|
|
|
CH3-C(=O)-O-CH2-CH3
|
77 °C
|
6.02
|
0.894 g/ml
|
1.78 D
|
|
|
CH3-C(=O)-CH3
|
56 °C
|
21
|
0.786 g/ml
|
2.88 D
|
|
|
H-C(=O)N(CH3)2
|
153 °C
|
38
|
0.944 g/ml
|
3.82 D
|
|
|
CH3-C≡N
|
82 °C
|
37.5
|
0.786 g/ml
|
3.92 D
|
|
|
CH3-S(=O)-CH3
|
189 °C
|
46.7
|
1.092 g/ml
|
3.96 D
|
|
|
C4H6O3
|
240 °C
|
64.0
|
1.205 g/ml
|
4.9 D
|
|
|
Polar protic solvents
|
|||||
|
H-C(=O)OH
|
101 °C
|
58
|
1.21 g/ml
|
1.41 D
|
|
|
CH3-CH2-CH2-CH2-OH
|
118 °C
|
18
|
0.810 g/ml
|
1.63 D
|
|
|
CH3-CH(-OH)-CH3
|
82 °C
|
18
|
0.785 g/ml
|
1.66 D
|
|
|
CH3-CH2-CH2-OH
|
97 °C
|
20
|
0.803 g/ml
|
1.68 D
|
|
|
CH3-CH2-OH
|
79 °C
|
24.55
|
0.789 g/ml
|
1.69 D
|
|
|
CH3-OH
|
65 °C
|
33
|
0.791 g/ml
|
1.70 D
|
|
|
CH3-C(=O)OH
|
118 °C
|
6.2
|
1.049 g/ml
|
1.74 D
|
|
|
CH3-NO2
|
100–103 °C
|
35.87
|
1.1371 g/ml
|
3.56 D
|
|
|
H-O-H
|
100 °C
|
80
|
1.000 g/ml
|
1.85 D
|
|
Hansen solubility parameter values (HSPiP)
There's another powerful way to look at these same solvents. By knowing their Hansen solubility parameter values, which are based on δD=dispersion bonds, δP=polar bonds and δH=hydrogen bonds, you know important things about their inter-molecular interactions with other solvents and also with polymers, pigments, nanoparticles etc. so you can do two things. First, you can create rational formulations knowing, for example, that there is a good HSP match between a solvent and a polymer. Second, you can make rational substitutions for "good" solvents (they dissolve things well) that are "bad" (for the environment, for health, for cost etc.). The following table shows that the intuitions from "non-polar", "polar aprotic" and "polar protic" are put numerically – the "polar" molecules have higher levels of δP and the protic solvents have higher levels of δH. Because numerical values are used, comparisons can be made rationally by comparing numbers. So acetonitrile is much more polar than acetone but slightly less hydrogen bonding.
|
Solvent
|
δD Dispersion
|
δP Polar
|
δH Hydrogen bonding
|
|
|
Non-polar solvents
|
||||
|
CH3-CH2-CH2-CH2-CH2-CH3
|
14.9
|
0.0
|
0.0
|
|
|
C6H6
|
18.4
|
0.0
|
2.0
|
|
|
C6H5-CH3
|
18.0
|
1.4
|
2.0
|
|
|
CH3CH2-O-CH2-CH3
|
14.5
|
2.9
|
4.6
|
|
|
CHCl3
|
17.8
|
3.1
|
5.7
|
|
|
/-CH2-CH2-O-CH2-CH2-O-\
|
17.5
|
1.8
|
9.0
|
|
|
Polar aprotic solvents
|
||||
|
CH3-C(=O)-O-CH2-CH3
|
15.8
|
5.3
|
7.2
|
|
|
/-CH2-CH2-O-CH2-CH2-\
|
16.8
|
5.7
|
8.0
|
|
|
CH2Cl2
|
17.0
|
7.3
|
7.1
|
|
|
CH3-C(=O)-CH3
|
15.5
|
10.4
|
7.0
|
|
|
CH3-C≡N
|
15.3
|
18.0
|
6.1
|
|
|
H-C(=O)N(CH3)2
|
17.4
|
13.7
|
11.3
|
|
|
CH3-S(=O)-CH3
|
18.4
|
16.4
|
10.2
|
|
|
Polar protic solvents
|
||||
|
CH3-C(=O)OH
|
14.5
|
8.0
|
13.5
|
|
|
CH3-CH2-CH2-CH2-OH
|
16.0
|
5.7
|
15.8
|
|
|
CH3-CH(-OH)-CH3
|
15.8
|
6.1
|
16.4
|
|
|
CH3-CH2-CH2-OH
|
16.0
|
6.8
|
17.4
|
|
|
CH3-CH2-OH
|
15.8
|
8.8
|
19.4
|
|
|
CH3-OH
|
14.7
|
12.3
|
22.3
|
|
|
H-C(=O)OH
|
14.6
|
10.0
|
14.0
|
|
|
H-O-H
|
15.5
|
16.0
|
42.3
|
|
Consider a simple example of rational substitution. Suppose for environmental reasons we needed to replace the chlorinated solvent, chloroform, with a solvent (blend) of equal solvency using a mixture of two non-chlorinated solvents from this table. Via trial-and-error, a spreadsheet or some software such as HSPiP we find that a 50:50 mix of toluene and 1,4 dioxane is a close match. The δD of the mixture is the average of 18.0 and 17.5 = 17.8. The δP of the mixture is the average of 1.4 and 1.8 = 1.6 and the δH of the mixture is the average of 2.0 and 9.0 = 5.5. So the mixture is 17.8, 1.6, 5.5 compared to Chloroform at 17.8, 3.1, 5.7. Because Toluene itself has many health issues, other mixtures of solvents can be found using a full Hansen solubility parameter data set.
|
Solvent
|
Boiling point (°C)
|
|
ethylene dichloride
|
83.48
|
|
pyridine
|
115.25
|
|
methyl isobutyl ketone
|
116.5
|
|
methylene chloride
|
39.75
|
|
isooctane
|
99.24
|
|
carbon disulfide
|
46.3
|
|
carbon tetrachloride
|
76.75
|
|
o-xylene
|
144.42
|
An important property of solvents is the boiling point. This also determines the speed of evaporation. Small amounts of low-boiling-point solvents likediethyl ether, dichloromethane, or acetone will evaporate in seconds at room temperature, while high-boiling-point solvents like water or dimethyl sulfoxide need higher temperatures, an air flow, or the application of vacuum for fast evaporation.
- Low boilers: boiling point below 100 °C (boiling point of water)
- Medium boilers: between 100 °C and 150 °C
- High boilers: above 150 °C
Density
Most organic solvents have a lower density than water, which means they are lighter and will form a separate layer on top of water. An important exception: most of the halogenated solvents like dichloromethane or chloroform will sink to the bottom of a container, leaving water as the top layer. This is important to remember when partitioning compounds between solvents and water in a separatory funnel during chemical syntheses.
Often, specific gravity is cited in place of density. Specific gravity is defined as the density of the solvent divided by the density of water at the same temperature. As such, specific gravity is a unitless value. It readily communicates whether a water-insoluble solvent will float (SG < 1.0) or sink (SG > 1.0) when mixed with water.
|
Solvent
|
Specific gravity
|
|
Pentane
|
0.626
|
|
Petroleum
ether
|
0.656
|
|
Hexane
|
0.659
|
|
Heptane
|
0.684
|
|
Diethyl
amine
|
0.707
|
|
Diethyl
ether
|
0.713
|
|
Triethyl
amine
|
0.728
|
|
Tert-butyl
methyl ether
|
0.741
|
|
Cyclohexane
|
0.779
|
|
Tert-butyl
alcohol
|
0.781
|
|
Isopropanol
|
0.785
|
|
Acetonitrile
|
0.786
|
|
Ethanol
|
0.789
|
|
Acetone
|
0.790
|
|
Methanol
|
0.791
|
|
Methyl
isobutyl ketone
|
0.798
|
|
Isobutyl
alcohol
|
0.802
|
|
1-Propanol
|
0.803
|
|
Methyl
ethyl ketone
|
0.805
|
|
2-Butanol
|
0.808
|
|
Isoamyl
alcohol
|
0.809
|
|
1-Butanol
|
0.810
|
|
Diethyl
ketone
|
0.814
|
|
1-Octanol
|
0.826
|
|
p-Xylene
|
0.861
|
|
m-Xylene
|
0.864
|
|
Toluene
|
0.867
|
|
Dimethoxyethane
|
0.868
|
|
Benzene
|
0.879
|
|
Butyl
acetate
|
0.882
|
|
1-Chlorobutane
|
0.886
|
|
Tetrahydrofuran
|
0.889
|
|
Ethyl
acetate
|
0.895
|
|
o-Xylene
|
0.897
|
|
Hexamethylphosphorus
triamide
|
0.898
|
|
2-Ethoxyethyl
ether
|
0.909
|
|
N,N-Dimethylacetamide
|
0.937
|
|
Diethylene
glycol dimethyl ether
|
0.943
|
|
N,N-Dimethylformamide
|
0.944
|
|
2-Methoxyethanol
|
0.965
|
|
Pyridine
|
0.982
|
|
Propanoic
acid
|
0.993
|
|
Water
|
1.000
|
|
2-Methoxyethyl
acetate
|
1.009
|
|
Benzonitrile
|
1.01
|
|
1-Methyl-2-pyrrolidinone
|
1.028
|
|
Hexamethylphosphoramide
|
1.03
|
|
1,4-Dioxane
|
1.033
|
|
Acetic
acid
|
1.049
|
|
Acetic
anhydride
|
1.08
|
|
Dimethyl
sulfoxide
|
1.092
|
|
Chlorobenzene
|
1.1066
|
|
Deuterium
oxide
|
1.107
|
|
Ethylene
glycol
|
1.115
|
|
Diethylene
glycol
|
1.118
|
|
Propylene
carbonate
|
1.21
|
|
Formic
acid
|
1.22
|
|
1,2-Dichloroethane
|
1.245
|
|
Glycerin
|
1.261
|
|
Carbon
disulfide
|
1.263
|
|
1,2-Dichlorobenzene
|
1.306
|
|
Methylene
chloride
|
1.325
|
|
Nitromethane
|
1.382
|
|
2,2,2-Trifluoroethanol
|
1.393
|
|
Chloroform
|
1.498
|
|
1,1,2-Trichlorotrifluoroethane
|
1.575
|
|
Carbon
tetrachloride
|
1.594
|
|
Tetrachloroethylene
|
1.623
|
D) – Selectivity Factor: In order to separate two compounds, their respective retention factors must be different, otherwise both compounds would be eluted simultaneously; the selectivity factor is the ratio of the retention factors.
α =kB/kA
Where B is the compound that is retained more strongly by the column and A is the compound with the faster elution time.E) – Elution: Elution is a term used in analytical and organic chemistry to describe the process of extracting one material from another by washing with a solvent (as in washing of loaded ion-exchange resins to remove captured ions).
In a liquid chromatography experiment, for example, an analyte is generally adsorbed, or "bound to", an adsorbent in a liquid chromatography column. The adsorbent, a solid phase (stationary phase), is a powder which is coated onto a solid support. Based on an adsorbent's composition, it can have varying affinities to "hold" onto other molecules—forming a thin film on its outside surface (or on its internal surface if there are cavities within the compound). Elution then is the process of removing analytes from the adsorbent by running a solvent, called an "eluent", past the adsorbent/analyte complex. As the solvent molecules "elute", or travel down through the chromatography column, they can either pass by the adsorbent/analyte complex or they can displace the analyte by binding to the adsorbent in its place. After the solvent molecules displace the analyte, the analyte can be carried out of the column for analysis. This is why as the mobile phase passes out of the column, it typically flows into a detector or is collected for compositional analysis.
Predicting and controlling the order of elution is a key aspect of column chromatographic methods.
F) Packing Material: (will be added later)
Answer 8) – Names (any five)
1. LC1200 Variable Wavelength UV-Vis Detector
2. LC1205K Programmable Variable Wavelength Detector
3. LC1210K Programmable Scanning Dual Wavelength UV-Vis Detector
4. LC1246K Refractive Index Indicator
5. LC1255S Programmable Scanning Fluorescence Detector
Discuss any one;
The high performance LC1200 is designed for standard HPLC applications requiring UV-Vis detection, and most suitable for QC and educational laboratories, offering a level of performance and versatility one would expect from much higher priced models. Flexible operation at single wavelengths within the range of 190 - 600 nm makes for versatility. For example, rather than being restricted to one or two wavelengths, you can use the LC1200 at 254 nm for the detection of aromatic compounds, improve sensitivity and selectivity for proteins, peptides, phenols and catecholamines at 280 nm, detect analytes of interest in the visible range, or monitor nitrate/nitrite and carboxylic acids at 200 nm.
Answer 9) – High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC (or just LC), is achromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture. HPLC is considered an instrumental technique of analytical chemistry (as opposed to a gravitimetric technique). HPLC has many uses including medical (e.g. detecting vitamin D levels in blood serum), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. during the production process of pharmaceutical and biological products).
HPLC has contributed to analytical solutions in diverse fields such as pharmaceuticals, foods, life sciences, environment, forensics, etc. In the present module we shall discuss some application areas in pharmaceuticals and foods.
Pharmaceuticals
High Performance Liquid Chromatography provides reliable quantitative precision and accuracy along with a high linear dynamic range to allow determination of API and related substances in a single run. A convenient method for sample preparation for solid dosage forms is dispersion in water or aqueous media modified with acetonitrile or methanol .HPLC offers several possibilities for separation of chiral molecules into their respective enantiomers.These include precolumn derivatization to form diastereomers. Alternately, specialty columns prepared with cyclodextrins or special chiral moieties as stationary phases maybe used .In short HPLC, particularly reverse phase HPLC is the most popular choice for quantitative analysis in the pharmaceutical industry.
Common application areas in pharmaceutical analysis are:
• Assay
• Related Substances
• Analytical Method Validation
• Stability Studies
• Compound Identification
• Working Standards
Foods
High Performance Liquid Chromatography has brought desirable advantages in the field of food analysis. Food matrices are generally complex and extraction of analytes is not an easy task. To further complicate matters both desirable and undesirable components are often found in trace levels and classical extraction and analysis does not provide the required levels of accuracy and precision. HPLC offers viable solutions due to vast choice of stationary phases and mobile phase options. Common applications in foods are :
• Fat soluble vitamins (A,D,E and K)
• Water soluble vitamins (B-complex vitamins such as B1, B2, B3, B6, Folic acid, Pantothenic acid, B12, VitaminC)
• Residual pesticides such as 2, 4-D and Monochrotophos.
• Antioxidants such as TBHQ, BHA and BHT.
• Sugars: Glucose, Fructose, Maltose and other saccharides.
• Cholesterol and sterols
• Dyes and synthetic colours.
• Mycotoxins such as Aflatoxins B1,B2,G1,G2,M1,M2and ochratoxin
• Amino acids
• Residual antibiotics
• Steroids and flavanoids
• Aspartame and other artificial sweeteners.
• Active ingredients of farm produce such as allin in garlic and catachin in tea extracts.
Answer 10) – HPLC vs GC
HPLC and GC are both methods of separation of compounds from a mixture. Whereas HPLC applies to constituents that are fluids, GC is used when the compounds are gaseous or can be vaporized during separation process. Both have the same underlying principle of heavy molecules flowing slower than lighter ones. While HPLC refers to High Pressure Liquid Chromatography, GC is simply Gas Chromatography. This article will highlight the differences between these two separation techniques.
HPLC
The technique of HPLC is made heavy use of in analytical chemistry to identify and analyze the individual components of a mixture. In HPLC, columns and high pressure are used. High pressure ensures movement of constituents in a mobile phase. It also moves the analyte in a densely packed column. Smaller particle size helps in increasing the density of the constituents that helps in better separation when columns of shorter length are used.
GC
Gas Chromatography on the other hand is mainly used to check the purity of a substance, and in some cases, also helps in identifying a substance. There are two phases, the mobile, and the stationary phase in the process. In the mobile phase, the carrier is an inert gas such as helium. Sometimes, nitrogen is used as it is unreactive. The stationary phase involves using a polymer or a layer of liquid on an inert solid base.
What is the difference between HPLC and GC?
The major difference between GC and HPLC lies in the phases used. While in GC, compounds of a mixture are separated using a liquid (stationary) phase and a gas (mobile) phase, in case of HPLC the stationary phase is a solid while liquids make up mobile phase. Another difference lies in temperature control during the process. In GC, there is an oven to contain the column consisting of gas phase and it can control the temperatures when the gases are passing through the column. On the other hand, there is no such provision of temperature control in HPLC. The last difference pertains to the concentration of the compounds. In GC, it is the vapor pressure of the gases that decide the concentration of compounds whereas it is possible to increase or decrease the concentration of the compounds in HPLC.
Answer 11) – Paper Chromatography
Types of Paper Chromatography
a) Descending Paper Chromatography- In this type development of paper is done by allowing the solvent to travel down the paper is called Descending Chromatography. Here the mobile phase is present in the upper portion.
b) Ascending Paper Chromatography- Here the solvent travel upward direction of the Chromatographic paper. Both the Descending and Ascending Paper Chromatography are used for separation of Organic and Inorganic substances.
c) Ascending-Descending Paper Chromatography- It is the hybrid of both the above technique. The upper part of the Ascending chromatography can be folded over a rod and allowing the paper to become descending after crossing the rod.
d) Radial Paper Chromatography- It is also called as Circular chromatography. Here a circular filter paper is taken and the sample is given at the center of the paper. After drying the spot the filter paper tied horizontally on a Petridish containing solvent. So that Wick of the paper is dipped inside the solvent. The solvent rises through the wick and the component get separated in form of concentrate circular zone.
e) Two-Dimensional Paper Chromatography- In this technique a square or rectangular paper is used. Here the sample is applied to one of the corner and development is performed at right angle to the direction of first run.
A) – Types of Papers used in PC: Paper chromatography uses paper as the stationary phase. The exact type of paper used is important. Filter paper is one of the best types, although paper towels and even newspaper can also be used. Writing paper is coated so that ink does not run and because of this is less satisfactory. Of course, wax paper, not being absorbant, is unsatisfactory.
B) – Developer:
C) – Capillary action (sometimes capillarity, capillary motion, or wicking) is the ability of a liquid to flow in narrow spaces without the assistance of, and in opposition to, external forces like gravity. The effect can be seen in the drawing up of liquids between the hairs of a paint-brush, in a thin tube, in porous materials such as paper, in some non-porous materials such as liquified carbon fiber, or in a cell. It occurs because of intermolecular forcesbetween the liquid and solid surrounding surfaces. If the diameter of the tube is sufficiently small, then the combination of surface tension (which is caused by cohesion within the liquid) and adhesive forces between the liquid and container act to lift the liquid
D) – Partition equilibrium chromatography is a type of chromatography that is typically used in gas chromatography (GC) and high performance liquid chromatography (HPLC). The stationary phase in GC is a high boiling liquid bonded to solid surface and the mobile phase is a gas.
E) -- Ascending & Descending:
Answer 12) – in paper chromatography n-butanol:acetic acid:water mixture in the ratio 4:1:1 is used as a solvent whereas in case of thin layer chromatography(TLC) petroleum ether and acetone in the ratio 9:1 is used as a solvent...also in some cases DCM(dichloromethane is used as a solvent.
Answer 13) – Precautions in PC:
Use a pencil to draw a thin line near the bottom of the paper. Make sure it is not too near the bottom.
Mark a cross for each mixture on the line.
Mark a spot of color/dye/mixture on each respective crosses and allow to dry.
Put chromatography paper in solvent. Ensure top of solvent does not cover the mixture.
Observe as spots move up the chromatography paper.
Use a pencil to draw the thin line as a pen may have ink that is soluble in the solvent. The ink may move up the chromatography paper and ruin the experiment.
Allow the color/dye/mixture to dry first before putting the chromatography paper in the solvent. This is to ensure that the ink will not totally dissolve in the solvent while it is still wet.
You must make sure that the solvent does not cover the mixture as the mixtures may dissolve totally in the solvent,
Answer 14) – Partition Chromatography is a liquid liquid extraction which involves two solvents while adsorption chromatography is a liquid solid extraction which involves a solid stationary phase & a liquid mobile phase
The partition chromatography involves separation between liquids while adsoption chromatography involves solid and liquid separations.
Partition PC:
The tendency for a compound to divide its time between two immiscible solvents (solvents such as hexane and water which won't mix) is known as partition. Paper chromatography using a non-polar solvent is therefore a type of partition chromatography.
Paper chromatography is a classical example of partition chromatography as the separation of the analyte occurs by the process of partition between the water molecules (present in the interstices of the cellulose of which the paper is made of) serving as liquid stationary phase and any solvent used as mobile phase.
Partition chromatography a method using the partition of the solutes between two liquid phases (the original solvent and the film of solvent on the adsorption column).
Partition chromatography
§ Stationary phase = non-volatile liquid film supported on an inert solid
§ Mobile phase = liquid or gas
§ Mixture of components are partitioned between the liquid film and the mobile phase (Used partition coefficient / distribution coefficient, K)
§ Interpretation: high value K = the component (X) dissolves more readily in the mobile phase and the component moves rapidly along the stationary phase.
§ Interpretation: low value K = the component (X) remain largely adsorbed on the stationary phase and the components moves slowly along the stationary phase.
§ Example: paper chromatography, gas-liquid chromatography (GLC)
Adsorption PC:
adsorption chromatography that in which the stationary phase is an adsorbent.
Adsorption chromatography
§ Stationary phase = solid
§ Mobile phase = liquid or gas
§ Mixture of components are adsorbed on the surface of the stationary phase
§ Example: column chromatography, thin layer chromatography (TLC)
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