HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY. DETERMINATION OF CONTENTS OF ADDITION IN DRUGS.
CHROMATOGRAM AND ITS CHARACTERISTICS: RETENTION TIME, ADJUSTED TIME, PEAK WIDTH ON HALF OF HEIGHT.
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.
!!! adjusted time is proportional to chromatographic resolution of the given component of investigated mix.
adjusted time depends from:
§ The nature chromatographic compounds;
§ The nature of a mobile phase;
§ The nature and weight of stationary phase;
§ Speeds of mobile phase movement;
§ Column temperatures (in a gas chromatography);
§ Lengths of a column;
§ Partition coefficient (than it is more for substance, the more its retention time).
§ retention volume – the volume of mobile phase needed to move a solute from its point of injection to the detector (Vr).
§ baseline width – the width of a solute’s chromatographic band measured at the baseline (w).
§ Peak height h or h’ (from a point of crossing of tangents with a zero line).
§ Peak width µ0,5 – distance between peak points on half of height (or on any other mark on height).
Relative retention time tr¢ and relative adjusted time tr is calculated on equation:
tr¢=t/ts tr= (t – t0) / (ts – t0)
!!! Relative retention times:
• Less depend on external conditions, than retention time;
• Resolve the serial analysis without standard samples of defined substances.
§ Often retention time isn’t measured, but measure retention distance l – distance on chromatogram from a point which corresponds of introduction sample moment to an absciss which correspond of peak maximum.
§ The retention volume it depends on speed u of mobile phase movements
V = t × u
§ The retention factor (delay) R is a relation of moving speed w of the given component and the speed u of mobile phase movements:
R = w /u or R = t0 /t
The capacity factor k – is equal to ratio of relative retention time ¢t= t – t0 of given components to t0:
k = (t – t0) / t0
§ The more the capacity factor k, than more time of investigated component in stationary phase.
Nonideal asymmetrical chromatographic bands showing (a) fronting and (b) tailing. Also depicted are the corresponding sorption isotherms showing the relationship between the concentration of solute in the stationary phase as a function of its concentration in the mobile phase.
CHROMATOGRAPHIC PROCESS: THE THEORY OF THEORETICAL PLATES AND THE KINETIC THEORY.
Martin and Synge theory (theoretical plate)
At the beginning of a chromatographic separation the solute occupies a narrow band of finite width. As the solute passes through the column, the width of its band continually increases in a process called band broadening. Column efficiency provides a quantitative measure of the extent of band broadening.
In their original theoretical model of chromatography, Martin and Synge treated the chromatographic column as though it consists of discrete sections at which partitioning of the solute between the stationary and mobile phases occurs. They called each section a theoretical plate and defined column efficiency in terms of the number of theoretical plates, N, or the height of a theoretical plate, H
!!! A column’s efficiency improves with an increase in the number of theoretical plates or a decrease in the height of a theoretical plate.
The number of theoretical plates in a chromatographic column is obtained by combining equations:
Alternatively, the number of theoretical plates can be approximated as
where w1/2 is the width of the chromatographic peak at half its height.
Rate theory (kinetic theory)
Van Deemter equation is an equation showing the effect of the mobile phase’s flow rate on the height of a theoretical plate.
where:
A = Eddy-diffusion
B = Longitudinal diffusion
C = mass transfer kinetics of the analyte between mobile and stationary phase
u = Linear Velocity.
The constant A depends on the size of particles, their density or density of column filling. The constant B is connected with diffusion factor of molecules in a mobile phase. The constant C characterises kinetics of sorption – desorption process, material transfers and other effects.
The A factor is determined by a phenomenon called Eddy Diffusion. This is also called the multi-path term, as molecular particles of a certain compound have a multitude of options when it comes to finding a pathway through a packed column. The following figure helps in visualizing Eddy diffusion:
Because there is an almost infinite number of different paths that a particle can travel by through a column, some paths will be longer than others. The particles that find the shortest path through the column will be eluted more quickly than those that travel a longer way. In the figure, particle B will be eluted before particle C, and both will be eluted before particle A. Since it is improbable for all particles of one compound to find the shortest path, there will be fractions of the component that will behave like particles A, B, and C. This leads to the broadening of the band. There is little a scientist can do to minimize the Eddy Diffusion factor, as it is influenced by the nature of column being used and by the particles’ movement through that column. The A term is loosely affected by the flow rate of the mobile phase, and sometimes the affect of the flow rate is negligible. It is for this reason that sometimes the Van Deemter equation is written as such:
H = A + B/u + Cu
B/u is called the longitudinal diffusion term, and is caused by the components’ natural migration from a place of high concentration (the center of the band) to a place of lower concentration (either side of the band) within the column. Diffusion6 occurs because molecules in a place of high concentration will tend to spread out to areas of lower concentration to achieve equilibrium. Given enough time, diffusion will result in equilibrium of the diffusing fluid via random molecular motion. The figure below helps to visualize this phenomenon:
At time zero in the figure above, the particles of a compound are generally localized in a narrow band within the separating column. If the mobile phase flow rate is too small or if the system is left at rest, the particles begin to separate from one another. This causes a spread in the concentration distribution of that compound within the column, thus bringing about band broadening for the band of that particular compound. As the time that the system is left still approaches infinity, the compound reaches complete concentration equilibrium throughout the entire column. At this point, there is no definitive band for that component, as a single concentration of that compound is present throughout the entire column. Longitudinal diffusion is a chief cause of band broadening in Gas Chromatography, as the diffusion rates of gaseous species are much higher than those of liquids. It is for this reason that longitudinal diffusion is less of an issue in liquid chromatography. The magnitude of the term B/u can be minimized by increasing the flow rate of the mobile phase. Increasing the velocity of the mobile phase does not allow the components in the column to reach equilibrium, and so will hamper longitudinal diffusion. The flow rate of the mobile phase should not be increased in excess, however, as the term Cu is maximized when u is increased.
Cu is referred to as the mass transfer term. Mass transfer refers to when particles are so strongly adhered to the stationary phase that the mobile phase passes over them without carrying them along. This results is particles of a component being left behind. Since it is likely that more than a single particle of any given compound will undergo this occurrence, band broadening results. This results in a phenomenon called tailing, in which a fraction a component lags behind a more concentrated frontal band. Non-equilibrium effects can be caused by two phenomena: laminar flow and turbulent flow. Laminar flow7 occurs in tubular capillaries, and so is most prominent in Capillary Electrophoresis. Turbulent flow occurs as a result of particles becoming overwhelmed by the stationary phase and is more common in column chromatography. This occurrence can be visualized by observing the figure below:
In the above figure, particles of the adsorbent solid become occupied by particles of the sample. If too many particles of the adsorbent are occupied, particle A will have nothing hindering it from flowing through the column. So, the particles of a single compound separate from one another. Also, as the mobile phase moves through the column, particles of the sample leave the stationary phase and migrate with the mobile phase. However, if the flow rate of the mobile phase is too high, many of the sample particles are unable to leave the stationary phase and so get left behind. These occurrences result in band broadening, as the individual particles of a single compound become less closely packed. The high flow rate of the mobile phase makes it more difficult for the components within the column to reach equilibrium between the stationary and mobile phase. It is for this reason that the Cu term is also called the non-equilibrium factor. Minimization of this factor can be achieved by decreasing the flow rate of the mobile phase. Decreasing the flow rate of the mobile phase gives sample components more time to leave the stationary phase and move with the mobile phase, thus reaching equilibrium.
By observing the Van Deemter equation, it can be deduced that an ideal mobile phase flow rate must be determined to yield the best (lowest) value of H. Decreasing the flow rate too much will result in an increase of the longitudinal diffusion factor B/u, while exceedingly increasing the flow rate will increase the significance of the mass transfer term Cu. So, H can be minimized to a finite limit depending on the various parameters involved in the chromatography being performed.
There is some disagreement on the correct equation for describing the relationship between plate height and mobile-phase velocity.
§ At small mobile-phase velocity the height equivalent to a theoretical plate (HETP) decreases, and then starts to increase.
§ The optimum mobile-phase velocity corresponds to a minimum in a plot of H as a function of u.
§ Optimum velocity of division which gives us a considerable quantity of theoretical plates, and accordingly small HETP, is calculate:
So, the kinetic theory gives a basis for optimisation of chromatographic process.
CHARACTERISTICS OF DIVISION EFFICIENCY: SEPARATION DEGREE, RESOLUTION, NUMBER OF THEORETICAL PLATES, THE HEIGHT EQUIVALENT TO A THEORETICAL PLATE.
The goal of chromatography is to separate a sample into a series of chromatographic peaks, each representing a single component of the sample. Resolution is a quantitative measure of the degree of separation between two chromatographic peaks, 1 and 2, and is defined as
For two peaks of equal size, a resolution of 1.5 corresponds to an overlap in area of only 0.13%. Because resolution is a quantitative measure of a separation’s success, it provides a useful way to determine if a change in experimental conditions leads to a better separation.
Three examples of chromatographic resolution.
If peaks are mutually bridging, definition of peak width of each substance is impossible on separation degree Y :
In chromatography: the qualitative analysis is based on definition of retention time and quantitative – on definition of peak height or area.
ION-EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.
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Imagine if we had a tube whose surfaces were coated with an immobilized cation. These would have electrostatic attraction for anions. If a solution containing a mixture of positively and negatively charged groups flows through this tube, the anions would preferentially bind, and the cations in the solution would flow through
· This is the basis of ion exchange chromatography. The example above is termed “anion exchange” because the inert surface is interacting with anions
· If the immobile surface was coated with anions, then the chromatography would be termed “cation exchange” chromatography (and cations would selectively bind and be removed from the solution flowing through
· Strength of binding can be affected by pH, and salt concentration of the buffer. The ionic species “stuck” to the column can be removed (i.e. “eluted”) and collected by changing one of these conditions. For example, we could lower the pH of the buffer and protonate anions. This would eliminate their electrostatic attraction to the immobilized cation surface. Or, we could increase the salt concentration of the buffer, the anions in the salt would “compete off” bound anions on the cation surface.
Principle
Ion-exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. The ionic compound consisting of the cationic species M+ and the anionic species B- can be retained by the stationary phase.
Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group:
Anion exchange chromatography retains anions using positively charged functional group:
Note that the ion strength of either C+ or A- in the mobile phase can be adjusted to shift the equilibrium position and thus retention time.
The ion chromatogram shows a typical chromatogram obtained with an anion exchange column.
Ion Chromatography
Typical technique
A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. A buffered aqueous solution known as the mobile phase carries the sample from the loop onto a column that contains some form of stationary phase material. This is typically a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions. The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance.
In order to control an IC system, a chromatography data system (CDS) is usually needed. In addition to IC systems, some of these CDSs can also control gas chromatography (GC) and HPLC
Metrohm 850 Ion chromatography system
Separating proteins
Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange chromatography separates proteins according to their net charge, which is dependent on the composition of the mobile phase. By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively charged beads, whereas a negatively charged protein would not. By changing the pH so that the net charge on the protein is negative, it too will be eluted.
Preparative-scale ion exchange column used for protein purification.
Elution by changing the ionic strength of the mobile phase is a more subtle effect – it works as ions from the mobile phase will interact with the immobilized ions in preference over those on the stationary phase. This “shields” the stationary phase from the protein, (and vice versa) and allows the protein to elute.
PAPER CHROMATOGRAPHY
Paper chromatography is an analytical method technique for separating and identifying mixtures that are or can be coloured,especially pigments. This can also be used in secondary or primary colours in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Double-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.
Paper chromatography
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
Pigments and Polarity
Paper chromatography is one method for testing the purity of compounds and identifying substances. Paper chromatography is a useful technique because it is relatively quick and requires small quantities of material. Separations in paper chromatography involve the same principles as those in thin layer chromatography. In paper chromatography, like thin layer chromatography, substances are distributed between a stationary phase and a mobile phase. The stationary phase is usually a piece of high quality filter paper. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. Components of the sample will separate readily according to how strongly they absorb on the stationary phase versus how readily they dissolve in the mobile phase.
When a colored chemical sample is placed on a filter paper, the colours separate from the sample by placing one end of the paper in a solvent. The solvent diffuses up the paper, dissolving the various molecules in the sample according to the polarities of the molecules and the solvent. If the sample contains more than one colour, that means it must have more than one kind of molecule. Because of the different chemical structures of each kind of molecule, the chances are very high that each molecule will have at least a slightly different polarity, giving each molecule a different solubility in the solvent. The unequal solubilities cause the various color molecules to leave solution at different places as the solvent continues to move up the paper. The more soluble a molecule is, the higher it will migrate up the paper. If a chemical is very nonpolar it will not dissolve at all in a very polar solvent. This is the same for a very polar chemical and a very nonpolar solvent.
It is important to note that when using water (a very polar substance) as a solvent, the less polar the colour, the lower it will rise on the paper.
Types of Paper Chromatography
1. 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.
2. 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.
3. 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.
4. 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.
5.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.
Technique of paper Chromatography:
n Ascending Chromatography
n Descending Chromatography
n Circular Chromatography
n The qualitative analysis – as in TLC.
n The quantitative analysis:
ü Visual estimation – comparison of colouring intensity of stains;
ü Estimation of stain areas;
ü Cutting of stains and their weighing of investigated sample and a standard solution;
ü Eluating of substances from stains and the next definition by a physical and chemical method.
THIN LAYER CHROMATOGRAPHY
Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.
Thin layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Specific examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents.
A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantization. This method is referred to as HPTLC, or “high performance TLC”.
Plate preparation
TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at
Technique
The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.
To run a thin layer chromatography, the following procedure is carried out:
1. A small spot of solution containing the sample is applied to a plate, about
2. A small amount of an appropriate solvent (elutant) is poured in to a glass beaker or any other suitable transparent container (separation chamber) to a depth of less than
3. The TLC plate is then placed in the chamber so that the spot(s) of the sample do not touch the surface of the elutant in the chamber, and the lid is closed. The solvent moves up the plate by capillary action, meets the sample mixture and carries it up the plate (elutes the sample). When the solvent front reaches no higher than the top of the filter paper in the chamber, the plate should be removed (continuation of the elution will give a misleading result) and dried.
Chromatogram of 10 essential oils coloured with vanillin reagent.
Separation Process and Principle
Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be adjusted. Also, the separation achieved with a TLC plate can be used to estimate the separation of a flash chromatography column.
Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the more polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound moves higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. It is commonly said that “strong” solvents (elutants) push the analyzed compounds up the plate, while “weak” elutants barely move them. The order of strength/weakness depends on the coating (stationary phase) of the TLC plate. For silica gel coated TLC plates, the elutant strength increases in the following order: Perfluoroalkane (weakest), Hexane, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile, Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest). For C18 coated plates the order is reverse. Practically this means that if you use a mixture of ethyl acetate and hexane as the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will normally not result in reversed order of running of the compounds on the TLC plate. An eluotropic series can be used as a guide in selecting a mobile phase. If a reversed order of running of the compounds is desired, an apolar stationary phase should be used, such as C18-functionalized silica.
Analysis
As the chemicals being separated may be colorless, several methods exist to visualize the spots:
1. Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.
2. Iodine vapors are a general unspecific color reagent
3. Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate:
Ø Potassium permanganate – oxidation
Ø Iodine
Ø Bromine
4. In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis, for example mass spectrometry, a technique known as Far-Eastern blotting.
Once visible, the Rf value, or retardation factor, of each spot can be determined by dividing the distance the product traveled by the distance the solvent front traveled using the initial spotting site as reference. These values depend on the solvent used and the type of TLC plate and are not physical constants.
or relative Retention factor:
Factors which influence on value Rf:
v Quality and activity of sorbent;
v Sorbent moisture;
v Thickness of a layer of a sorbent;
v Quality of solvent
The number of theoretical plates n
lb – distance from start line to a stain bottom edge;
ll – distance which shows length of a stain in direction of movement of solvent front.
the height of a theoretical plate
separation factor
Sorbents in ТLС:
– Silica gel;
– Aluminium oxide;
– Starch;
– Cellulose and some other substances with high adsorptive ability
TLC:
n Ascending Chromatography
n Descending Chromatography
n Circular Chromatography
Substrates for a sorbent (plate):
– Glass;
– Aluminium foil;
– Polyester film.
The choice of solvents depends from:
n The sorbent nature;
n Properties of investigated substances.
Zones on chromatogram are displayed physical or chemical methods after termination of carrying out chromatography.
n Physical method – UV-light or radio autographies (photographic materials – a paper or films).
n Chemical method – chromatogram are displayed by reactant solution spraying
Applications
In organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a capillary tube are placed on the plate: a spot of starting material, a spot from the reaction mixture, and a “co-spot” with both. A small (3 by
As an example the chromatography of an extract of green leaves (for example spinach) in 7 stages of development. Carotene elutes quickly and is only visible until step 2. Chlorophyll A and B are halfway in the final step and lutein the first compound staining yellow.
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
In one study TLC has been applied in the screening of organic reactions for example in the fine-tuning of BINAP synthesis from 2-naphthol. In this method the alcohol and catalyst solution (for instance iron(III) chloride) are placed separately on the base line, then reacted and then instantly analyzed.
Equipment for ТLС:
– A sealed container with suitable solvents;
– Chromatographic plates (Merck, Silufol, Sorbfil and others);
– Micro syringes, TLC Manual Sampler or Micropipettes;
– TLC Viewing
nm;
– TLC SPRAYER (ATOMIZER) and TLC Spray Cabinet;
– TLC Plate Heater;
The PLATE HEATER is intended for heating to a given temperature a TLC plate at different analysis stages. Heating at the stage of application samples and standards provides compact spots and correspondingly, increases plate effectiveness. The device is provided with a removable ruler with
The temperature of 200×100 mm heating area is selectable between 35 and 125°Ñ and is uniformly maintained over the entire heating surface. Programmed and actual temperature are digitally displayed.
The qualitative analysis in ТСХ at identification (pharmacopoeia):
n Comparison stain of investigated substance and standard substance. They must be identical on colouring (colour of fluorescence), retention factor and size on chromatogram.
!!! Check of operability of TLC-PLATES: the standard mix of indicators –
n suitable solvents: methanol – toluene (20:80).
n When the front of solvents will pass ⅔ of plate length, it is considered suitable if on chromatogram 4 divided stains are accurate:
– Stain of bromcresol green with Rf is no more 0,15;
– Stain of methyl orange with Rf from 0,1 to 0.25;
– Stain of methyl red with Rf from 0,35 to 0,55;
– Stain of
Quantitative analysis:
n Direct definition on a plate by means of measurement transmission of light or reflexions of light, fluorescence.
n Directly on a plate or on the separate cut strips of plates by means of radio-activity counters.
n After removal of stationary phase, its dissolution in corresponding solvent and measurements of a suitable physical indicator or a radio-activity of the received solution.
THEORETICAL BASE OF LIQUID CHROMATOGRAPHY
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane.
Precondition of occurrence and application:
– Substances with big molar weight
– Nonvolatile substances
– thermally unstable substances
– Ionic substanses
– LC is based on adsorption from a solution.
– Adsorptive balance between solution and adsorbent will be co-ordinated with the equation of Langmuir isotherm, in the range of the diluted solutions the isotherm is linear.
– Selectivity of adsorption depends by nature forces of interaction between adsorbed substances and adsorbent.
H = 2Rr(1 – Rr)Uts,
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic 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).
Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with a sorbent, leading to the separation of the sample components. The active component of the column, the sorbent, is typically a granular material made of solid particles (e.g. silica, polymers, etc.), 2-50 micrometers in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the sorbent particles. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as “mobile phase”. Its composition and temperature plays a major role in the separation process by influencing the interactions taking place between sample components and sorbent. These interactions are physical iature, such as hydrophobic (dispersive), dipole-dipole and ionic, most often a combination thereof.
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 –
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.
HPLC apparatus ((1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in “inject position”, (6′) Switching valve in “load position”, (7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector)
Operation
The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are function of specific physical interactions with the sorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time measured under particular conditions is considered an identifying characteristic of a given analyte. Many different types of columns are available, filled with sorbents varying in particle size, and in the nature of their surface (“surface chemistry”). The use of smaller particle size packing materials requires the use of higher operational pressure (“backpressure”) and typically improves chromatographic resolution (i.e. the degree of separation between consecutive analytes emerging from the column). In terms of surface chemistry, sorbent particles may be hydrophobic or polar iature. Common mobile phases used include any miscible combination of water with various organic solvents (the most common are acetonitrile and methanol). Some HPLC techniques use water-free mobile phases (see Normal-phase chromatography below). The aqueous component of the mobile phase may contain acids (such as formic, phosphoric or trifluoroacetic acid) or salts to assist in the separation of the sample components. The composition of the mobile phase may be kept constant (“isocratic elution mode”) or varied (“gradient elution mode”) during the chromatographic analysis. Isocratic elution is typically effective in the separation of sample components that are not very dissimilar in their affinity for the stationary phase.
In gradient elution the composition of the mobile phase is varied typically from low to high eluting strength. The eluting strength of the mobile phase is reflected by analyte retention times with high eluting strength producing fast elution (=short retention times). A typical gradient profile in reversed phase chromatography might start at 5% acetonitrile (in water or aqueous buffer) and progress linearly to 95% acetonitrile over 5–25 minutes. Periods of constant mobile phase composition may be part of any gradient profile. For example, the mobile phase composition may be kept constant at 5% acetonitrile for 1–3 min, followed by a linear change up to 95% acetonitrile.
The chosen composition of the mobile phase (also called eluent) depends on the intensity of interactions between various sample components (“analytes“) and stationary phase (e.g. hydrophobic interactions in reversed-phase HPLC). Depending on their affinity for the stationary and mobile phases analytes partition between the two during the separation process taking place in the column. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/acetonitrile gradient, more hydrophobic components will elute (come off the column) late, once the mobile phase gets more concentrated in acetonitrile (i.e. in a mobile phase of higher eluting strength).
The choice of mobile phase components, additives (such as salts or acids) and gradient conditions depend on the nature of the column and sample components. Often a series of trial runs are performed with the sample in order to find the HPLC method which gives adequate separation.
!!! The choice of mobile phase is always more important, than a choice of stationary phase.
n The stationary phase should retention divided substances.
n The mobile phase (solvent or more often mix of solvents) should provide different capacity of a column and effective separation in optimum time.
n The stationary phase is porous finely dispersed materials with a specific surface is more 50 m2/g
Stationary phase
Polar: SiO2, Al2O3, MexOy, and also with graft polar groups (–NH2, – ОН, diols)
Non-polar: dag, kieselguhr, diatomite.
Polar stationary phases is used for separation:
– non-polar, low-polar and mean polar substance
Non-polar stationary phases is used for separation:
– they don’t have selectivity to polar molecules
These sorbents are put on superficial-porous carriers.
Mobile phase
From it depends:
n Selectivity of separation;
n Efficiency of column;
n velocity of chromatographic zone movement.
Requirements to a mobile phase:
ü Should dissolve investigated sample;
ü Small viscosity (diffusion factors D of sample components are high enough);
ü Possible excretion from it divided components;
ü It must be inertness in relation to materials of all parts of chromatograph;
ü Meets the requirements of the chosen detector;
ü The safe;
ü The cheap.
!!! As it has been told, eluting force of a mobile phase – solvent influences on seperation.
Eluting force of solvent shows, in how many time energy of eluent sorption more than energy of eluent sorption, chosen as the standard, for example, n-heptane.
Solvents (eluents) divide on weak and strong.
n Weak eluents are a little adsorbed by stationary phase therefore distribution factors D of sorbate is high.
n Strong eluents are strongly adsorbed by stationary phase, therefore distribution factors D of sorbed substances (sorbate) is low.
For example: SiO2 – stationary phase.
Force of solvents increases in range:
pentane (0) < CCl4 (0,11) < C6H6 (0,25) < CHCl3 (0,26) < CH2Cl2 (0,32) < acetone (0,47) < dioxane (0,49) < acetonitrile (0,5)< ethanol < methanol.
Sequence of solvents according to their increase eluting forces is named eluotropic series.
In liquid adsorptive chromatography eluotropic Snyder’s series:
pentane (0) < n-hexane (0,01) < cyclohexane (0,04) < CCl4 (0,18) < benzene (0,32) < CHCl3 (0,38) < acetone (0,51), ethanol (0,88) < water, СН3СООН (very big).
Eluotropic series for reverse phase chromatography on С18:
methanol (1,0) < acetonitrile (3,1), ethanol (3,1) < isopropanole (8,3) < n-propanole (10,1) < dioxane (11,7).
TYPES
Partition chromatography
HILIC Partition Technique Useful Range
Partition chromatography was the first kind of chromatography that chemists developed. The partition coefficient principle has been applied in paper chromatography, thin layer chromatography, gas phase and liquid-liquid applications. The 1952 Nobel Prize in chemistry was earned by Archer John Porter Martin and Richard Laurence Millington Synge for their development of the technique, which was used for their separation of amino acids. Partition chromatography uses a retained solvent, on the surface or within the grains or fibers of an “inert” solid supporting matrix as with paper chromatography; or takes advantage of some coulombic and/or hydrogen donor interaction with the stationary phase. Analyte molecules partition between a liquid stationary phase and the eluent. Just as in Hydrophilic Interaction Chromatography (HILIC; a sub-technique within HPLC), this method separates analytes based on differences in their polarity. HILIC most often uses a bonded polar stationary phase and a mobile phase made primarily of acetonitrile with water as the strong component. Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences. HILIC bonded phases have the advantage of separating acidic, basic and neutral solutes in a single chromatographic run.
The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. The stronger the interactions between the polar analyte and the polar stationary phase (relative to the mobile phase) the longer the elution time. The interaction strength depends on the functional groups part of the analyte molecular structure, with more polarized groups (e.g. hydroxyl-) and groups capable of hydrogen bonding inducing more retention. Coulombic (electrostatic) interactions can also increase retention.
Use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times.
Normal-phase chromatography
It was one of the first kinds of HPLC that chemists developed. Also known as normal-phase HPLC (NP-HPLC), or adsorption chromatography, this method separates analytes based on their affinity for a polar stationary surface such as silica, hence it is based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase, and works effectively for separating analytes readily soluble ion-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on steric factors. The effect of steric hindrance on interaction strength allows this method to resolve (separate) structural isomers.
The use of more polar solvents in the mobile phase will decrease the retention time of analytes, whereas more hydrophobic solvents tend to induce slower elution (increased retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention. This behavior is somewhat peculiar to normal phase chromatograhy because it is governed almost exclusively by an adsorptive mechanism (i.e. analytes interact with a solid surface rather than with the solvated layer of a ligand attached to the sorbent surface; see also reversed-phase HPLC below). Adsorption chromatography is still widely used for structural isomer separations in both column and thin-layer chromatography formats on activated (dried) silica or alumina supports.
Partition- and NP-HPLC fell out of favor in the 1970s with the development of reversed-phase HPLC because of poor reproducibility of retention times due to the presence of a water or protic organic solvent layer on the surface of the silica or alumina chromatographic media. This layer changes with any changes in the composition of the mobile phase (e.g. moisture level) causing drifting retention times.
Recently, partition chromatography has become popular again with the development of HILIC bonded phases which demonstrate improved reproducibility, and due to a better understanding of the range of usefulness of the technique.
Displacement chromatography
The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.[3] There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentration.
Reversed-phase chromatography (RPC)
Reverse phase partition chromatography
n Stationary phase: non-polar solvent is fixed on firm carrier
n Mobile phase: polar solvent (water, alcohol, buffer solutions, strong acids)
Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been surface-modified with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis). An investigator can increase retention times by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is often incorrectly referred to as “HPLC” without further specification. The pharmaceutical industry regularly employs RP-HPLC to qualify drugs before their release.
RP-HPLC operates on the principle of hydrophobic interactions, which originates from the high symmetry in the dipolar water structure and plays the most important role in all processes in life science. RP-HPLC allows the measurement of these interactive forces. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand on the stationary phase. This solvophobic effect is dominated by the force of water for “cavity-reduction” around the analyte and the C18-chain versus the complex of both. The energy released in this process is proportional to the surface tension of the eluent (water: 7.3×10−6 J/cm², methanol: 2.2×10−6 J/cm²) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent (methanol, acetonitrile) into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.
Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) is retained longer because it is non-interacting with the water structure. On the other hand, analytes with higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH2, COO– or -NH3+ in their structure) are less retained as they are better integrated into water. Such interactions are subject to steric effects in that very large molecules may have only restricted access to the pores of the stationary phase, where the interactions with surface ligands (alkyl chains) take place. Such surface hindrance typically results in less retention.
Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C-C-bonds elute later than those with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond.
Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5×10−7 J/cm² per Mol for NaCl, 2.5×10−7 J/cm² per Mol for (NH4)2SO4), and because the entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC).
Another important factor is the mobile phase pH since it can change the hydrophobic character of the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. Buffers serve multiple purposes: control of pH, neutralize the charge on the silica surface of the stationary phase and act as ion pairing agents to neutralize analyte charge. Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of analyte-ammonium adducts. A volatile organic acid such as acetic acid, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column effluent. Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is a fairly strong organic acid. The effects of acids and buffers vary by application but generally improve chromatographic resolution.
Reversed phase columns are quite difficult to damage compared with normal silica columns; however, many reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as these will destroy the underlying silica particle. They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. RP-HPLC columns should be flushed with clean solvent after use to remove residual acids or buffers, and stored in an appropriate composition of solvent. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is a mixture of 2,2′- and 4,4′- bipyridine. Because the 2,2′-bipy can chelate the metal, the shape of the peak for the 2,2′-bipy will be distorted (tailed) when metal ions are present on the surface of the silica.
Size-exclusion chromatography
Size-exclusion chromatography (SEC), also known as gel permeation chromatography or gel filtration chromatography, separates particles on the basis of molecular size (actually by a particle’s Stokes radius). It is generally a low resolution chromatography and thus it is often reserved for the final, “polishing” step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.
This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weight heparins.
Bioaffinity chromatography
This chromatographic process relies on the property of biologically active substances to form stable, specific, and reversible complexes. The formation of these complexes involves the participation of common molecular forces such as the Van der Waals interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and concerted action of several of these forces in the complementary binding sites.
Aqueous normal-phase chromatography
Aqueous normal-phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reversed-phase solvents.
ISOCRATIC AND GRADIENT ELUTION
Methods of elution
1. When a separation uses a single mobile phase of fixed composition it is called an isocratic elution. It is often difficult, however, to find a single mobile-phase composition that is suitable for all solutes.
2. gradient elution is the process of changing the mobile phase’s solvent strength to enhance the separation of both early and late eluting solutes.
A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic (meaning constant composition). The word was coined by Csaba Horvath who was one of the pioneers of HPLC.
The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution. One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed “A” and “B”; A is the “weak” solvent which allows the solute to elute only slowly, while B is the “strong” solvent which rapidly elutes the solutes from the column. In reversed-phase chromatography, solvent A is often water or an aqueous buffer, while B is an organic solvent miscible with water, such as acetonitrile, methanol, THF, or isopropanol.
In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that late-eluting peaks get very flat and broad. Their shape and width may keep them from being recognized as peaks.
Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks “sharper”), which is important in trace analysis. The gradient program may include sudden “step” increases in the percentage of the organic component, or different slopes at different times – all according to the desire for optimum separation in minimum time.
In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change – that is, the peaks elute in the same order. In gradient elution, the elution order may change as the dimensions or flow rate change.
The driving force in reversed phase chromatography originates in the high order of the water structure. The role of the organic component of the mobile phase is to reduce this high order and thus reduce the retarding strength of the aqueous component.
Rules of thumb help us at choice of eluent. Sorption increases at increase number of olefinic linkage and ОН-groups in compounds.
Sorption decrease (for organic compound): acids > alcohols > aldehydes > ketones > esters > unsaturated hydrocarbon > saturated hydrocarbon.
PARAMETERS
Theoretical
HPLC separations have theoretical parameters and equations to describe the separation of components into signal peaks when detected by instrumentation such as by a UV detector or mass spectrometer. The parameters are largely derived from two sets of chromatagraphic theory: plate theory (as part of Partition chromatography), and the rate theory of chromatography / Van Deemter equation. Of course, they can be put in practice through analysis of HPLC chromatograms, although rate theory is considered the more accurate theory.
They are analogous to the calculation of retention factor for a paper chromatography separation, but describes how well HPLC separates a mixture into two or more components that are detected as peaks (bands) on a chromatograms. The HPLC parameters are the: efficiency factor(N), the retention factor (kappa prime), and the separation factor (alpha). Together the factors are variables in a resolution equation, which describes how well two components’ peaks separated or overlapped each other. These parameters are mostly only used for describing HPLC reversed phase and HPLC normal phase separations, since those separations tend to be more subtle than other HPLC modes (e.g. ion exchange and size exclusion).
Ø Void volume is the amount of space in a column that is occupied by solvent. It is the space within the column that is outside of the column’s internal packing material. Void volume is measured on a chromatogram as the first component peak detected, which is usually the solvent that was present in the sample mixture; ideally the sample solvent flows through the column without interacting with the column, but is still detectable as distinct from the HPLC solvent. The void volume is used as a correction factor.
Ø Efficiency factor (N) practically measures how sharp component peaks on the chromatogram are, as ratio of the component peak’s area (“retention time”) relative to the width of the peaks at their widest point (at the baseline). Peaks that are tall, sharp, and relatively narrow indicate that separation method efficiently removed a component from a mixture; high efficiency. Efficiency is very dependent upon the HPLC column and the HPLC method used. Efficiency factor is synonymous with plate number, and the ‘number of theoretical plates’.
Ø Retention factor (kappa prime) measures how long a component of the mixture stuck to the column, measured by the area under the curve of its peak in a chromatogram (since HPLC chromatograms are a function of time). Each chromatogram peak will have its own retention factor (e.g. kappa1 for the retention factor of the first peak). This factor may be corrected for by the void volume of the column.
Ø Separation factor (alpha) is a relative comparison how well two neighboring components of the mixture were separated (i.e. two neighboring bands on a chromatogram). This factor is defined in terms of a ratio of the retention factors of a pair of neighboring chromatogram peaks, and may also be corrected for by the void volume of the column. The greater the separation factor value is over 1.0, the better the separation, until about 2.0 beyond which an HPLC method is probably not needed for separation.
Resolution equations relate the three factors such that high efficiency and separation factors improve the resolution of component peaks in a HPLC separation.
Internal diameter
The internal diameter (ID) of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution. It also determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Low-ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.
Ø Larger ID columns (over
Ø Analytical scale columns (
Ø Narrow-bore columns (1–2 mm) are used for applications when more sensitivity is desired either with special UV-vis detectors, fluorescence detection or with other detection methods like liquid chromatography-mass spectrometry
Ø Capillary columns (under
Particle size
Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5 µm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.
This means that changing to particles that are half as big, keeping the size of the column the same, will double the performance, but increase the required pressure by a factor of four. Larger particles are used in preparative HPLC (column diameters
Pore size
Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.
Pump pressure
Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressure may reach as high as 40 MPa (6000 lbf/in2), or about 400 atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns (<2 μm). These “Ultra High Performance Liquid Chromatography” systems or RSLC/UHPLCs can work at up to 100 MPa (15,000 lbf/in2), or about 1000 atmospheres. The term “UPLC” is a trademark of the Waters Corporation, but is sometimes used to refer to the more general technique.
Types of detector:
n Refractive index – generalpurpose detector (sensitivity ~ 10–6 g);
n UV/Vis-detector (sensitivity 10–9 g) (254 nm);
n photometric і spectrophotometric;
n Photodiode array;
n Fluorescent detector for definition toxines, microbiological objects, vitamins;
n Conductometric (0,01 µg/mL – 100 mg/mL).
The qualitative analysis (pharmacopoeia):
n Comparison of retention times of analyzed substance in the investigated sample and a comparison solution (a standard solution of the same substance) – is used more often;
n Comparison of relative retention times of analyzed substance the investigated sample and a comparison solution (a standard solution of the same substance) – is applied, when is possible non-reproducible conditions of chromatography;
n Comparison of chromatogram of the investigated sample with chromatogram of comparison solution or with chromatogram, resulted in separate article (for preparations of plant and animal material).
For quantitative definition to define the peak areas or peak height (pharmacopoeia):
– Heights are considered only at isocratic elution and at factor of asymmetry 0.8-1.2.
– Peak areas are considered only at gradient elution.
The quantitative analysis (pharmacopoeia) for main component:
– Absolute calibration
– Method of the internal standard.
The quantitative analysis (pharmacopoeia) of impurities:
n Quantitative definition of impurity with usage of comparison solution (known concentration of impurity);
n Method of internal normalisation;
n Comparison with the diluted solution of the main substance;
n Method of standard additives (for accuracy increase use of a method of the internal standard is possible).
At a technique necessarily mark:
– Column parametres (the sizes, a sorbent, commercial mark, the size of particles of stationary phase);
– Column temperature;
– Speed and structure of mobile phase;
– Detector type.
