Theory and application of High Performance Liquid Chromatography (HPLC)

Contents1. Introduction2. Theoretical Principles3. Types of HPLC

Normal Phase ChromatographyReversed phase chromatographySize exclusion chromatographyIon Exchange chromatographyBio-affinity chromatographyIsocratic flow and gradient elution

4. Pumps5. Columns and stationary phases6. Solvents and mobile phases7. Detectors8. Applications in Pharmacy

1. IntroductionWhat is HPLC?High performance liquid chromatography (HPLC) is a very efficient separation technique, that is, it yields excellent separation in a very short period of time. The inventors of modern chromatography, Martin and Synge, were aware as far back as 1941 that, in theory, the stationary phase requires very small particles and hence a high pressure is essential for forcing the mobile phase through the column. As a result, HPLC is also sometimes referred to as high-pressure liquid chromatography. High performance liquid chromatography (HPLC) is a form of column Chromatography. It is used frequently in biochemistry and analytical chemistry’ to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed (sample) and the constituent elements of column of HPLC.

Safety in HPLC handlingThere are some health risks inherent in HPLC handling. These are - 1. Toxic solvents - Short and long term risks of exposure to solvents and vapors are

generally known but too little attention is paid to them--Use perforated plastic lids to all feed and waste containers for filling and emptying purpose, so

that no toxic vapor can escape into the laboratory environment and no impurities can contaminant the highly pure solvents.

--A good ventilation system should be provided in the solvent handling areas2. Pulmonary irritation from the stationary phase - Particles of 5 micron

and less are used in HPLC. These particles are not retained by the bronchial tubes but pass straight through and the potential long-term risk to health has not yet been researched. However, these may enter into lungs, retained in the alveoli and may cause damage to lungs

-- As a safety measure, any operation involving possible escape of stationary phase dusts (dispensing and weighing) must be carried out in a fume cupboard

3. Dangers resulting from the high pressures - High pressure pump may be a risk factor but liquids are less compressible in comparison to gas. A jet of liquid may leak from a faulty fitting but there is no danger of explosion but this liquid may cause serious physical damage to the body.

4. Dangers from high voltage - In human body, the electricity works in milivolt level whereas we work with volt level (thousand times greater) in normal work. Any shock by electricity due to faulty electrical works may cause harm to the operator.

Apparatus for HPLC: (Draw and label the schematic diagram of HPLC machine)

Fig-1: Schematic diagram of a HPLC machine

1=solvent reservoir, 2=sintered metal frit, 3=high pressure pump, 4=pulse damper, 5=drain valve, 6=manometer, 7=pre-column, 8=injection syringe, 9=injection valve, 10=column, 11=thermostat oven, 12=detector, 13=data acquisition (recorder or integrator), 14=fraction collector

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2. Principle of HPLC

The principle of HPLC is to force the sample through the column of the stationary phase by pumping the mobile phase at high pressure. The sample to be analyzed is introduced in small volume to the stream of mobile phase and the sample molecules are retained by specific chemical and physical interactions with the materials of the stationary phase as it travels the length of the column. The amount of retention depends on- (1) the nature of the sample, (2) the nature of the composition of the stationary phase and (3) the nature of the composition of the mobile phase. The time taken by the sample to travel from the point of injection to the end of the column is called the retention time and is considered an unique identifying characteristic of a given sample because no two compounds will have the same retention time (like no two persons can sit in a single seat / No single person can have two heads). The use of pressure increases the linear speed giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used as mobile phase include any miscible combinations of water and various organic liquids (the most common are methanol and acetonitrile).

In HPLC separation, the compound that have higher affinity for the mobile phase, move more quickly than those that have strong affinity for the stationary phase. Phase preference can be expressed by distribution co-efficient, K:

Where,Cstat = Concentration of compound X in the stationary phaseCmob = Concentration of X in mobile phase.K = Partition coefficientThe stationary phase and mobile phase must be in intimate contact with each other in order to ensure a distribution balance. The various components must have different distribution coefficients for ease of separation.

mob

statx C

CK

2.3. Mathematical expression of principle of HPLCMathematically the principle of HPLC is based on van Deemter theory (The rate theory) expressed as van Deemter equation. This theory employs a kinetic approach and explains band broadening in terms of a number of rate factors. The van Deemter equation is: 2γDG 8 k’ df

2ū HEPT= 2λdp + ------------ + --- ---------- ------- ū π2 (1+k’)2 DL

Eddy Molecular Non-equilibrium effect diffusion diffusion Axial diffusionWhere,HEPT = Height Equivalent to Theoretical Platesλ = a measure of the packing irregularitiesdp= Particle diameter, γ= Tortuosity (Full of twists and turns) factorDG= Co-efficient of gaseous diffusionDL= Co-efficient of liquid diffusion ū =Average linear velocity (Flow rate)K’=ratio of the capacity of the liquid phase to that of the gas phase. For capillaries, k’ = 2kdf/r ; df = Average film thickness, r = radius of the capillary

2.4. The simplified form of the Van Deemter equation for the plate height is :

B HEPT= A+ ---------+ Cū

ū                         WhereA = Eddy-diffusionB =Longitudinal diffusion C = Resistance to mass transfer ū= Flow rate

A is equal to the multiple paths that occur in packed columns where there are several paths exist in the column packing, which results in band spreading. In open tubular capillaries this term will be zero as there are no multiple paths. B/ū is equal to the longitudinal diffusion of the particles of the compound.Cū is equal to the equilibration point. In a column, there is an interaction between the mobile and stationary phases, Cū accounts for this.A = Eddy-diffusion, B = Longitudinal diffusion , C = Resistance to mass transfer

2.2: Band Broadening (Causes and remedies of band broadening)First Cause : Eddy Diffusion - The column is packed with small stationary phase particles. The mobile phase pass through the stationary phase particles and transports the samplemolecules with it (Fig 5). Some molecules are fortunate and leave the column before mostothers by traveling an almost straight line path. Other sample molecules undergo severaldiversions along the way.

Second Cause: Flow distribution - The mobile phase passes in a laminar flow between thestationary phase particles (Fig 6). The flow is faster in the channel center than it is near theedge of the particle. Eddy diffusion and flow distribution may be reduced by packing the columnwith evenly sized particles (First remedy).

Third Cause: Sample molecule diffusion in the mobile phase - It is also called longitudinaldiffusion effect. Sample molecules spread out in the solvent without any external influence (Justlike salt or sugar dissolves slowly in water). The longitudinal diffusion has a harmful effect if -(i) small stationary phase particles (<10 micron) (ii) low mobile phase velocity and (iii) large sample diffusion co-efficient etc

coincide in the HPLC system. This can be overcome by following second remedy.

Fig-5: Eddy diffusion Fig 6: Flow distribution

Fourth cause: Mass transfer between mobile, stagnant mobile and stationary phase - Pore structure of a stationary phase particle (Fig 2.4) show us that the channels are both narrow and wide, some channels pass through the whole of the particle while others are closed off (half way open and then closed). Half way open pores are filled with mobile phase and are stagnated. The recovery process may occur in two ways - (a) The molecules may diffuse back to the mobile flux phase. It depends on the size of pores and viscosity of the solvents used, (b) The molecules may interact with stationary phase and is adsorbed and then after sometime desorbed. Hence mass transfer takes place. In both cases band broadening decrease with increasing mobile phase flow velocity. These effects can be overcome by Third and Fourth principles.

Remedy:First remedy: The packing should be composed of particles with as narrow a size distribution as possible (5:7.5=1:1.5)Second remedy: The mobile phase flow velocity should be selected so that longitudinal diffusion has no adverse effectThird remedy: Small particles or those with a thin, porous surface layer should be used as the stationary phaseFourth remedy: Low viscosity solvents should be used

3. HPLC separation modes (Types of HPLC )

i. Normal Phase HPLC (Adsorption chromatography)ii. Reversed phase HPLC chromatographyiii. Liquid-liquid partition chromatographyiv. Chromatography with chemically bonded v. Ion-exchange chromatographyvi. Ion-pair chromatographyvii. Ion chromatographyviii. Size exclusion chromatographyix. Affinity chromatography

3.1. Isocratic flow and gradient elution

i. Normal phase HPLC (Adsorption HPL Chromatography)Normal phase HPLC (NP-HPLC) separates samples based on polarity. This method uses (i) a polar stationary phase(ii) a non-polar mobile phase, and (iii) is used when the samples is polar in nature.

The polar samples dissolves in polar stationary phase and is retained by the polar stationary phase. Adsorption strength increases with increase in samples polarity, and the interaction between the polar samples and the polar stationary phase (relative to the mobile phase) increases the elution time. NP-HPLC had fallen out of favor in the 1970's with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina as chromatographic media.

Rule of thumb (Normal state of things): Polar compounds are eluted later than non-polar compounds

Polar=Water soluble, Hydrophilic, Non-polar=Fat Soluble, Lipophilic, Protic= Produces H+ ions (Able to donate protons)

ii. Reversed phase HPLC chromatographyIn reversed phase HPLC (RP-HPLC) following applies:(a)The stationary phase is very non-polar(b) The mobile phase is relatively polar(c) A polar solvent such as water elutes more slowly than a less polar solvent such as

acetonitrile

Rule of Thumb: Non-polar compounds are eluted later than polar compounds

iii. Liquid-liquid partition chromatography-The porous support material is loaded with a liquid that acts as the stationary phase. This liquid is insoluble in mobile phase. The molecules of the sample are distributed between liquid stationary phase and mobile phase according to the distribution law (as extraction in separating funnel). If a sample is more soluble in the liquid stationary phase, it will stay more time in the stationary phase and will be eluted latter (that is it takes long time to elute).

iv. Chromatography with chemically bonded phase -The stationary phase is not applied to porous support material in a liquids film form, instead, the stationary phase material is covalently bonded to the support material by chemical reaction (e.g. as in case of RP-HPLC)

v. Size exclusion chromatographySEC can be subdivided into gel permeation chromatography (with organic solvents) and gel filtration chromatography (with aqueous solutions). SEC separates particles on the basis of molecular size, i.e. according to molecular mass. The largest molecules are eluted first and the smallest molecules last. 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, and is the primary technique for determining the average molecular weight of natural and synthetic polymers (Molecular mass difference must be about 10%).

vi. Ion-pair chromatography - Molecules of the ionic samples are masked by a suitable counter ion. The advantages are (i) RP-HPLC system can be used, so no ion-exchange is needed, (ii) Acids, base and neutral products can be analyzed simultaneously.

vii. Ion exchange chromatography (IEC)The stationary phase contains ionic groups (e.g. NR3+ or SO3- ) which interact with counter ionic groups of the sample molecules. The method is suitable for separating amino acids, ionic metabolic products and organic ions.In IEC, retention is based on the attraction between solute ions and charged ions bound to the stationary phase. Ions of the same charge are excluded.

In general, ion exchangers favor the binding of ions of higher charge and smaller radius.

This form of chromatography is widely used in the following applications: In purifying water, pre-concentration of trace components, Ligand-exchange chromatography, Ion-exchange chromatography of proteins, High-pH anion-exchange chromatography of carbohydrates and oligosaccharides, etc.

Some types of Ion Exchangers include: (1) Polystyrene resins- allows cross linkage which increases the stability of the chain. (2) Cellulose and dextran (gels)-These possess larger pore sizes and low charge densities making them suitable for protein separation, (3) Controlled-pore glass or porous silica.

viii. Ion chromatography- is used for separating the ions of strong acids and bases (Cl-, NO3

+, Na+, K+). It is an special case of ion exchange chromatography.

ix. Bio-affinity chromatographyThis 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 Waal's interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, bio-specific bond is formed by a simultaneous and concerted action of several of these forces in the complementary binding sites.

Antigen AntibodyEnzyme InhibitorHormone Carrier

Highly specific biochemical interaction is the basis of separation. Stationary phase contains specific groups of molecules which can only adsorb the sample (as in antigen-antibody reaction)

Fig 4: Affinity chromatography

3.1. Isocratic flow and gradient elution

With regard to the mobile phase, a composition that remains constant throughout the procedure is termed isocratic.Gradient elution is a separation technique where the mobile phase changes its composition during a separation process. One example is a gradient in 20 min starting from 10% Methanol and ending up with 30% Methanol. Such a gradient can be increasing or decreasing. The benefit of gradient elution is that it helps speed up elution by allowing components that elute more quickly to come off the column under different conditions than components which are more readily retained by the column. By changing the composition of the solvent, components that are to be resolved can be selectively more or less associated with the mobile phase as a result at equilibrium they spend more time in the solvent and less in the stationary phase therefore they elute faster.

4. PumpsHPLC uses 400 - 1000 amt pressure to force the mobile phase through the stationary phase

and only gravity can not create such a high pressure. The stationary phase particles are very small and they create a very strong resistance to flow. Therefore, pumps for HPLC must be capable of producing high pressures. This is essential if the mobile phase is to be forced quickly enough through the chromatographic bed, the small particles of which offer a high resistance to flow. A high-flow rate is essential for preparative work and column packing.

1. Gas-driven discontinuous displacement system2. Discontinuous displacement pumps3. Membrane piston pumps4. Short-stroken piston pump5. Pneumatic amplifier pumps

5. Columns to be used as Stationary phases How to make column itself? -Steel, Glass, Tantalum, peek, PE tubes etc.

Most HPLC columns are made of 316-grade stainless steel, which is chromium-nickel-molybdenum (16:10:2-Typical Applications -Because of its superior corrosion and oxidation resistance, good mechanical properties and fabricability, SX 316 has applications in many sectors of industry.  Some of these include: Tanks and storage vessels for corrosive liquids. Specialised process equipment in the chemical, food, paper, mining, pharmaceutical and petroleum industries. Architectural applications in highly corrosive environments) steel, resistant to usual HPLC pressure and also relatively inert to chemical corrosion. The inside of the column should have no rough surfaces, grooves or micro porous structure, so the steel tubes must be either precision drilled or polished or electro-polished after common manufacturing e.g. by drawing. Rough surfaces can lead to up to ten times fewer plate numbers.

Glass tubes are smooth, chemically inert and the main advantage is that dirt, cracks and the presence of air can all be easily monitored. Up to 1.5 times more plates may be obtained than with steel tubes due to smooth interior walls. They are also corrosion resistant and for this are strongly recommended when aqueous buffers are the components of the mobile phase.

Tantalum (Ta), Peek (plastic) and flexible polyethylene tubes are rarely used as columns.

5.1. Parameters of Columns5.1.1. Internal diameter and LengthThe internal diameter (ID) of an HPLC column is a critical aspect that determines quantity of sample that can be loaded onto the column and also influences sensitivity. Columns of i.d. 2-5mm are generally used for analytical purposes. Wider columns of i.d. 10-25mm are used for preparative work. Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller (MICRO- AND CAPILLARY) columns are rapidly gaining popularity. They are used in traditional quantitative analysis of samples and often use a UV-VIS absorbance detector. Narrow i.d. 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 LC-MS. Capillary columns (under 0.3 mm) which are used almost exclusively with alternative detection means such as Mass spectrophotometry. They are usually made from fused silica capillaries, rather than the stainless steel tubing that larger columns employ.

Length: Columns 5, 10, 15 or 25 cm long are common if micro-particulate stationary phase of 10μm or less are used. For preparative purposes columns up to 1m in length are used.

5.1.2. Particle sizeMost traditional HPLC is performed with the stationary phase of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5μm beads being the most common. Smaller particles provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared (Pα1/d2). This means that changing to particles that are half as big in the same size of column will double the performance, but increase the required pressure by a factor of four (Pα1/(d/2)2).

5.1.3. Pore sizeMany stationary phases are porous (3, 5 or 10 μm in size) to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics especially for larger sample. For example a protein which is only slightly smaller than a pore might enter the pore but not easily leave once inside.

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5.1.4. Pump pressurePumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressures may reach as high 400 atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore be able to use much smaller particle sizes in the columns (< 2 μm). These "Ultra High Performance Liquid Chromatography" systems or UHPLCs can work at up to about 1000 atmospheres.

5.1.5. Injection Volume -For a fixed volume auto-sampler or manual valve, this is set by changing the loop. For a variable volume auto-sampler, it is set in the auto-sampler method. 10 micro liter

5.1.6. Flow rate -Usually around 1 ml/min for an analytical column, slower if using polymer based columns

5.1.7. Run time -Usually from 2-60 min. This is a little longer than it takes for the last peak to elute. For a gradient run we have to allow for the system to return to the starting conditions after the run is over and then re-equilibrate before the next injection can be made.Column packing material, particle size, length, diameter and manufacturer are all important. More than one column type may work, but an HPLC method must specify a column

5.2. Pre-columns and Guard columnsPre-columns placed prior to the sample injector are used for mobile phase

conditioning. Any solvent dissolves silica to give silicic acid. A scavenger column filled with coarse silica is advisable when silica or silica-based chemically bonded phases are used as stationary phases. This ensures that the mobile phase is already saturated with silicic acid on entering the column and increases its life time accordingly. Moreover, even pH of 10-11 can then be tolerated for chromatography with no problems.

On the other hand, short pre-columns (known as guard columns) are used as a protection between the injector and the column. They are filled in exactly the same way as the main column and prevent impurities such as water or pigments from contaminating it. This is particularly important when biological fluids are involved or for direct beverage analysis. Guard columns must be changed frequently to ensure that the life time of the main column (several hundred hours running time) is not shortened.

5.3. General information on column packing:(1) Large number of theoretical plates can be obtained by ensuring short diffusion

paths in the stationary phase pores, hence HPLC favors micro-particles. Shorter column length, higher flow rate(2) The particle size distribution should be as narrow as possible , e.g., 1:1.5 or 1:2(3) Smallest particles determines the column permeability whereas largest particles fix

the plate numbers(4)Small particles produce a high flow resistance but large particles are responsible for a high degree of band broadening.(5) Particle size distribution is achieved by sieving, air separation, sedimentation,

optical methods etc.(6) Two groups of packing materials are used (i) Fully porous particle (3,5,or 10 micron) (ii) Those with a porous layer and a non-porous core, also known as porous layer bed (PLB, 1-3 micron thick)

5.4. Some packing materials

1. Silica2.Chemically modified silica3. Styrene –divinylbenzene4.Alumina5. Mg-silicate7. Controlled-pore glass8. Hydroxyalkylmethacrylate9. Hydroxylapatite10. Agarose11. Porous graphitic carbon12. Restricted surface access phases

6. Mobile phasesThe mobile phase must obviously be chosen for its chromatographic properties: it must interact with asuitable stationary phase to separate a mixture as fast and as efficiently as possible. As a general rule, arange of solvents is potentially able to solve any particular problem, so selection must be based ondifferent criteria:1. Viscosity: a low-viscosity solvent produces a lower pressure drop than a solvent with higher viscosity

for a specific flow-rate. It also allows faster chromatography as mass transfer takes place faster. 2. UV transparency: if a UV detector is used, the mobile phase must be completely transparent at the

required wavelength, e.g. ethyl acetate is unsuitable for a detection level of 254 nm as it does not become sufficiently optically transparent until 275 nm (less than 10% absorption). The UV transparency of buffer salts, ion-pair reagents and other additives must also be considered.

3. Refractive index: only important if a refractive index detector is used. The difference between the refractive indices of the solvent and the sample should be as great as possible when working at the detection limits.

4. Boiling point: a lower boiling point of the mobile phase is required if the eluate is to be recovered and further processed; this means less stress during evaporation of the mobile phase for heat-sensitive compounds. On the other hand, solvents with a high vapor pressure at the operating temperature tend to produce vapor bubbles in the detector.

5. Purity: this criterion has a different meaning depending on intended use: absence of compounds that would interfere with the chosen mode of detection; absence of compounds that disturb gradient elution (see Fig. 19.5); absence of non-volatile residues in the case of preparative separations (see Problem 32 in Section 21.4). Hexane for HPLC does not necessarily need to be pure n-hexane but may also contain branched isomers because the elution properties are not influenced by this (but it may not contain benzene, even in traces, if used for UV detection).

6. Inert with respect to sample compounds: the mobile phase must not react at all with the sample mixture (peroxides!). If extremely oxidation-sensitive samples are involved, 0.05% of the antioxidant 2,6-di-terf-butyl-/?-cresol (BHT) may need to be added to the solvent. BHT is readily removed from the eluent by vaporization, but it absorbs in the UV region below 285 nm. 7. Corrosion resistance: light promotes the release of HC1 from chlorinated solvents. The ever-present traces of water combine with this to produce hydrochloric acid, which attacks stainless steel. Corrosion is intensified by the presence of polar solvents; mixtures such as tetrahydrofuran-carbon tetrachloride or methanol-carbon tetrachloride are particularly reactive. All iron complex-forming compounds, e.g. chloride, bromide, iodide, acetate, citrate or formate ions (buffer solutions!) have a corrosive effect. Lithium salt buffers are also very corrosive at low pH (pH 2). Steel may even corrode with methanol or acetonitrile. Perhaps a passivation of the apparatus with nitric acid or a change in instrument design is necessary. In any case a wash with a halogen- and ion-free solvent after a chlorinated mobile phase or a salt solution has been used is recommended.8. Toxicity: here the onus is on each individual laboratory to avoid toxic products as far as possible. Chlorinated solvents may release the highly poisonous phosgene gas. Toluene should always replace benzene (carcinogenic) wherever possible.9. Price!

6.2. Preparation of Mobile PhaseCautions to be exercised during selection and preparation of mobile phase: (1) Only HPLC-grade products should be used(2) Stabilizers may alter the solvent polarity completely ,e.g. Chloroform + Ethanol(3) Additives may dramatically reduce the UV transparency or may gradually accumulate in the column and give rise to ghost peaks (4) Fractional distillation is used to purify large amount of eluents and for recycling(5) Adsorption CC is used for treating smaller amounts and removes stabilizers, traces of UV absorbing compounds peroxides and water. Column filled with alumina and silica give best level of purification.

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CuCl2 or CoCl2 as indicator

Adsorbent, Safety zone

Cotton wool

Fig 2: Column for solvent purification by adsorption

(6)The first few mililiters of the eluate should be discarded as there may still be some water left. A layer of anhydrous CuCl2 or CoCl2 at the bottom of column provides a visual detection of the presence of water. When the water front breaks through, the colorless salt becomes bright blue or pink, respectively. The accumulated impurities move through the column ahead of the water-hence the need for a safety zone at the very bottom of the pipe as seen in the Figure.(7) Cooling is the useful facility as the risk of catalytic changes becomes less and cleaning capacity greater at lower temperature levels. The stopcock of the column must not be greased.(8) A volume of 150-600ml of solvent can be cleaned with a packing of 100g of column filling, depending on polarity, purity and original Water content. A pre-CaCl2 drying stage is an advantage. Solvents that are more polar than ethyl acetate can not be cleaned by this Method.(9) The pure solvent is stored in dark bottles over a desiccant.(10) The solvent should be filtered and degassed before use in HPLC*(11) Degassed solvents is kept under helium. Water and lower alcohols may dissolve large amount of airDegassing is done by (i) reflux boiling, (ii) Helium expulsion, (iii) Ultrasonic bath(12) The solvent level in the supply vessel must be permanently controlled to a certain level. The pump must not be allowed to dry.*Reasons for filtering and degassing:►Debris in solvents would damage pump valves, block the capillaries, reduce column performance► Air must not be allowed► Gas bubbles-gives ghost peak► Dissolved O2-absorb UV light, quenches fluorescence

6.3. Gradient systemThe composition of the mobile phase may need to be changed as the substance migrate through the column when complex mixtures are involved. Changes must be accompanied by an increase in mobile phase elution strength so that peaks which would otherwise be eluted late or not at all are accelerated. Two different gradient systems are available:(1) Low-pressure gradient system and (2) High-pressure gradient system6.4. Sample injectorSample feed is one of the critical aspects of HPLC. Even a best column produces poor separation if injection is not carried out carefully. Usually very small volume of sample mixture is placed at the centre of the columnhead and care is taken so that air does not enter at the same time. Ways for sample injection are(i) With syringe and septum injector(ii) With a loop valve(iii) With a automated injection system (autoinjector)6.5. sample solution and sample volumeSample preparation is a difficult problem. General procedure for sample preparation are (i) filtration, (ii) Solid phase extraction with disposable catridge, (iii) protein precipitation and (iv) desalting.The sample solution should pass through a preliminary filtration step to ensure that it contains no solids. It is best to dissolve the sample in mobile phase solvent. If the sample solvent is weaker than the mobile phase the plate number of the column may be increased. If the sample solvent is stronger than the mobile phase this can lead to band broadening or strange peak shapes. If the sample solution is higher in acetonitrile, then the effect is band broadening and then distorted peak shape. The sample volume should be kept as small as possible. It may be preferable to dissolve the sample in a relatively large volume to prevent mass overload at the column inlet.

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1a=Solvent-I reservoir1b=Solvent-II reservoir2=Mixing chamber3=Magnetic stirrer4a=Valves4b=Valves

Fig 3: Components of low pressure gradient systems

7. Detectors Definition: The detector is defined as that device and associated equipment used to

sense and measure the small amounts of the components present in the sample. It is important that the detector should have high sensitivity, good stability and a satisfactory response to a wide variety of substance. A detector in any instrument is compared with human brain. The detector should be able to recognize when a substance zone is eluted from the column. Therefore, it has to monitor the changes in mobile phase composition, convert this into an electrical signal and then convey this signal to the monitor or display it as a deviation from the baseline.

An ideal detector should have the following properties:(i) It should either be equally sensitive to all eluted peaks or record only those of

interest(ii) It should not be affected by changes in temperature or in mobile phase

composition (as in gradient elution)(iii) It should be able to monitor small amounts of compounds (trace analysis)(iv) It should not contribute to band broadening, hence cell volume should be small(v) It should react quickly to pick up correctly narrow peaks which pass rapidly through

the cell(vi) It should be easy to manipulate, robust and cheap.

7.1. Types of detector

1. UV -Visual 2. Refractive Index (RI)3. Electrochemical4. Fluorescence5. Other detectors

(i) Conductivity- Anions or cations, with or without chemical suppression (dual column technique).

(ii) Photoconductivity(iii) IR(iv) Radioactive (v) Transport(vi) Light scattering

UV DETECTORSThis is the most commonly used type of detector as

-it can be rather sensitive-it has a wide linear range-it is relatively unaffected by temperature fluctuations and -it is also suitable for gradient elution.

It records compounds that absorb ultraviolet or visible light. Absorption takes place at a wavelength above 200 nm (range 200-800nm).

Molecule must have show following properties to absorb at UV-VIS range:

1. a double bond adjacent to an atom with a lone electron pair, X=Y—Z (e.g. vinyl ether);2. bromine, iodine or sulphur;3. a carbonyl group, C=O; a nitro group NO2;4. two conjugated double bonds, X=X—X=X;5. an aromatic ring and6. inorganic ions: Br-, I-, NO3, NO2

These groups do not absorb to the same extent or at the same wavelength. The absorption intensity and the wavelength of maximum absorption are also affected by neighboring atom groups in the molecule. The molar absorptivity, e, which is found in tables in many handbooks for a large number of compounds, is a measure of the light absorption intensity. Aromatic molecules have higher and ketones (with the functional group C=O) comparatively smaller molar absorptivities.

Principle of UV-VIS detector: The degree of absorption resulting from passage of the light beam through the cell is a function of the molar absorptivity (a), the molar concentration (c ) of the compound and length of the cell, (b). The product of a, b and c is known as the absorbance, A, and is given by the simplified form of Lambert-Beers law:

A = abc

A UV detector measures the absorbance of the sample solution (eluate) placed in a suitable cell. The mobile phase should be selected for optical transparency at the detector lamp wavelength, i.e. its absorbance should be zero or at least be adjustable to zero electronically. If this is the case the detector signal itself is also zero, and the recorder is set to the required position and produces the baseline.

According to the above equation, peak height is a function of the molar absorptivity and the concentration of a substance passing through the detector cell. Compounds with a higher molar absorptivity produce larger peaks than those with a small molar absorptivity when identical amounts of a compound are injected.As the signal amplitude is a function of light path, b, the flow cells in UV detectors should be as long as possible. However, they must also be thin in order to keep cell volume small. Light travels in a longitudinal direction through the cells. Figure 5.5 shows one such type of design.

Source of light

The same lamp transmits light through the measurement and reference cells. Two photodiodes measure the light intensity in both cells; the electronic system compares these signals and fluctuations in illumination intensity are balanced accordingly. The reference cell must not contain solvent as it is generally inaccessible, even if used at all (the light from the lamp then being used as a direct reference). However, there are detectors in which the reference cell can be filled with liquid and this is a definite asset when the mobile phase itself is prone to specific absorption or its refractive index changes during the course of gradient elution. However, it is difficult to obtain this change in the reference cell exactly in time with that taking place in the measuring cell, so a good detector will not be significantly affected by fluctuations in refractive index.Source of Radiation: Low-pressure mercury vapor lamps or cadmium, zinc, deuterium and tungsten lamps are all

suitable light sources.Low-pressure mercury vapor lamps emit mostly 253.7 nm light. Weaker emissions at other

wavelengths must be carefully filtered out, otherwise the linear range would be reduced. This type of fixed-wavelength detector is up to 20 times more sensitive than one with a variable wavelength. All aromatic molecules absorb strongly at 254 nm; even compounds containing carbonyl and similar groups and multiple conjugated double bonds can be detected, provided that their absorption maximum is not too far from 254 nm.

The 229 nm cadmium lamps and 214 nm zinc lamps can be used as a supplement for the shorter wavelengths, and longer wavelengths can be covered by incorporating phosphorescent layers into the radiation path of the lamp.

Deuterium lamps (H or D2 lamp) emit a continuous UV spectrum (@ 340 nm). The wavelength at which detection actually occurs can be adapted to suit the problem. This type of detector is less sensitive than the fixed-wavelength variety, but this is partly or wholly compensated for by the fact that the wavelength can be adapted to the absorption maximum of the compound in question and a suitable choice also permits the removal of interfering peaks, thus improving the accuracy of quantitative determination. Figure 5.6 shows an example of this: at 266 nm the vitamin B peak is partly overlapped by that of another compound which is eluted at nearly the same time. The co-eluting peak does not absorb at 300 nm. Detectors with time-dependent wavelength control are of great help to the analyst.

Tungsten lamps emit in the near-UV and visible ranges (ca. 340-850 nm) and make an ideal supplement to deuterium lamps.

The spectral band width in both of these models must not be as narrow as that in recording spectrophotometers, a value of 10 nm being the norm (i.e. the filtered light is not monochromatic but covers a range of about 280-290 nm for a chosen wavelength of 285 nm). This has the advantage of supplying a greater light intensity, which in turn increases the sensitivity. However, the band width should not be too wide, otherwise the linear range of the signal would be restricted.

B. Classification of Detectors1. Integral and differentialThe integral detector, as the term implies, measures the total amount of the component. The resulting chromatogram, as shown in Figure 10-3A, is a series of steps, each step being a sigmoid curve. Where the integral detector is applicable, it gives a record which is suitable for quantitative analysis, since the step height corresponds to the total amount of solute. The nitrometer used by Janak (45) and the recording titration buret of James and Martin (40) are examples of integral detectors. The most serious disadvantages of these detec tors are lack of versatility and sensitivity.The differential detector measures some property related to the concentration of the components in the carrier gas stream, and the resulting chromatogram is the familiar series of bell-shaped peaks, Figure 10-3B. With the pure carrier gas flowing, the signal (base line) is constant. As the component emerges in the gas stream, the signal increases to a maximum as the concentration reaches a maximum, after which the signal falls to the original base line value. The differ ential detectors have a decided advantage over the integral types in detecting trace components as a blip on the base line or on the shoul der of a major component. The peak which is obtained permits meas urement of both the retention value (at peak maximum) and the amount of solute (peak area) for each component.

2. Destructive and nondestructiveA further distinction in detectors can be made by classifying them as destructive or nondestructive. This classification depends pri marily upon whether the operation of the detector makes the sep arated components available for collection and further study. De tectors such as Scott's flame detector are clearly destructive in operation, since the effluent stream is burned at a jet. The use of de structive detectors, however, is not a serious handicap in gas chroma-tography. Usually sufficient sample is available for replicate analy ses, since only a few microliters are required for each chromatogram.

Classification of the properties of an ideal detector: Two types (i) Functional and (ii) non-functional

Functional Non Functional1 Sensitivity 1 Simplicity23

Stability 2 Cost and availabilityVersatility 3 Robustness

4 Proportionality 4 Safety5 Reactivity6 Response Time7 Signal Recording

One should distinguish between those necessary to do the required detecting job as opposed to those that do not affect the technical functioning of the detector, such as cost, availability, etc. On this basis, the first seven characteristics can be termed functional, and the last four nonfunctional. The vari ous characteristics of detectors are discussed in more detail below:

Sensitivity and limits of detection- These are clearly the most important properties of any detecting system. High sensitivity detectors permit the determination of trace components directly in small samples of complex mixtures. Some detectors are inherently sensitive, while others can be improved by modifications in the detecting element or in physical geometry, e.g., reduced detector cell volume.

Stability- We recognize here the desirability of freedom from two types of instability: a short-term instability, or noise, and a long-term instability, or drift. Noise limits the reliability of low level signals and is detrimental where amplification is required for trace analysis.

Slow variations or drift in the base line are less serious, but do limit the utility for separations that require extended periods of time. Erratic noise, such as would result if the detector is sensitive to vibration, also is undesirable. Instability may result from sensitivity to fluctuations in gas flow rate, pressure, or temperature, although design features can be incorporated to minimize such adverse effects. Stability will determine reproducibility and the need and frequency of recalibration.

Versatility- Since different HPLC columns can be conveniently prepared for separating many different types of compounds, it is desirable that the detector not be the limiting factor in obtaining maximum utility from the technique. The detecting system should not exclude certain compounds or classes of compounds, unless for convenience or other reasons a detector possessing specificity is advantageous. The detector should not only have versatility for a wide range of materials, but also should be operable with suitable sensitivity over a wide range of conditions. For example, at the elevated temperatures required for high temperature HPLC, the materials of construction assume particular importance. Operation over a wide pressure range is usually of less importance.

Proportionality - A linear response of the detector with concentration is desirable over a practical concentration range. Nonlinearity may be overcome by calibration. The ideal detector would not require calibration, the response being calculable from known solute properties.

Reactivity - It is desirable that the composition of the eluate should not be affected on passing through the detector. The materials of construction, including gaskets, may exhibit adsorption - desorption effects producing instability. Corrosive samples may attack the cell, while metals, particularly at higher temperatures, may catalyze cracking or rearrangement of labile organic substances. An all-glass or quartz system might be required in such cases.

Response time - The time constant of the detector must be small enough so that the separated components are detected individually when they arrive at the detection point. The response time becomes especially important in high speed HPLC where a component may be eluted in one second or less. For conventional use, a time constant of one second is adequate.

Signal recording - The requirement of speed in the analyses of complex samples containing a number of components makes it almost imperative that the detector produce a signal which can be recorded with an automatic potentiometer. The strip chart with the chromatogram is a useful permanent record of the analysis. Some recorders are equipped to produce automatic integration of differential peaks. Integral-type detectors offer integration simultaneously with indication.

Simplicity- Although simplicity is a desirable characteristic of detectors, this advantage is not to be sought at the expense of functional characteristics. This is equally true of the necessary auxiliary equipment for the detector.

Cost and availability - Low cost and availability are definitely characteristics of a nonfunctional nature. Detector parts such as the model glow plugs or modified neon indicator lamps cost less than $1.00. Such low cost and easily available detectors are only a small part of the monetary outlay for a HPLC apparatus, because a recorder and the accessory power supply commit the user to a sizable investment. Robustness - This requirement is dictated by use conditions and servicing or maintenance

problems. The detector must withstand the ordinary vibrations and shocks encountered in the laboratory. Safety - The item of safety can be handled by proper design and care in use. -If hydrogen is used in detector operation, precautions must be taken against explosion

hazards. -Adequate shielding can be arranged for radioactive materials used in radiological detectors.

Necessary protective measures also must be taken in the assembly and maintenance of such detectors. -The high voltage hazards associated with circuits of some detectors are also to be taken into

consideration.

7.2. Description of UV –VIS detector:

Figure 7- Schematic diagram of UV-VIS detector

Sample Holder/SampleDevice

Source of radiation

MonochromatorWavelength AnalyzerFrequency AnalyzerPrismGrating

Detector with polygraph(MonitorRecorderPrinter)

M

RP

D

A HPLC. From left to right: A pumping device generating a gradient of two different solvents, a steel enforced column and an apparatus for measuring the absorbance.