INTRODUCTION In gas chromatography, only about 20% of known compounds were lend themselves to analyse either because they are insufficiently volatile and cannot pass through the column or because they are thermally unstable and decomposes under the conditions of separation and one of the early problems with liquid chromatography was the slow rate at which the analysis took place. Early methods used gravity feed, and it was not uncommon for an analysis to take several days to complete. This led to great delay, but also the excessive time on the column inevitably led to loss of resolution by diffusion, and so on. Consequently, for a number of years liquid chromatography was not widely used as a mean of separating organic compounds. These problems were largely overcome by the advent of HIGH- PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) because it is not limited by sample volatility or thermal stability. High-performance liquid chromatography (HPLC) is a form of liquid chromatography to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by injecting a plug of the sample mixture onto the column and here in this system pressure is applied to the column, forcing the mobile phase through at much higher rate. The pressure is applied by using a pumping system. The action of the pump is critical, since it must not pulsate and mix up the sample being separated in the solvent, causing it to lose resolution. Development of pumps has proceeded quite quickly over the last several years, and now it is possible to achieve good resolution under the conditions required for HPLC. HPLC is able to separate macromolecules and ionic species, labile natural products, polymeric materials, and a wide variety of other high-molecular-weight polyfunctional groups. With an interactive liquid mobile phase, a parameter is available for selectivity in addition to an active stationary phase. Chromatographic separation in HPLC is the result of specific interactions between sample molecule with both the stationary and mobile phases. These interactions are essentially absent in the mobile phase of gas chromatography. HPLC offers a greater variety of mobile phases, which allow a greater variety of these interactions and more possibilities for separation. Sample recovery is

easy in HPLC. Separated fractions are easily collected by placing an open vessel at the end of the column. Recovery is usually is quantitative (barring irreversible absorption on a column), and separated sample components are readily isolated from the mobile phase solvent. In addition to usual type of organic compounds, liquid column chromatography can handle separations of ionic compounds, labile naturally occurring products, polymeric materials, and high molecular-weight polyfunctional compounds All the forms of liquid chromatography are migration processes where sample components are selectively retained by a stationary phase. High Performance Liquid Chromatography (HPLC) was developed in the late 1960s and early 1970s. Today it is widely applied for the separations and purifications in a variety of areas including pharmaceuticals, biotechnology, environmental, polymer and food industries. HPLC has over the past decade become the method of choice for the analysis of a wide variety of compounds. Inn HPLC the separation of a mixture into its components depends on different degrees of retention of each component in the column. The extent to which a component is retained in the column is determined by its partitioning between the liquid mobile phase and the stationary phase. In HPLC this partitioning is affected by the relative solute/stationary phase and solute/mobile phase interactions. Thus, unlike in gas chromatography, the ability to manipulate the character of both the stationary phase and the mobile phase decreases the need for a large number of stationary phases in liquid chromatography. Knowledge of the molecular structure of the sample components can be very helpful in the selection of a liquid chromatographic method.In general, HPLC is used for the following purposes- • Separation of organic, inorganic, biological compounds, and thermally labile compounds. • Qualitative and quantitative analysis of the compounds.

HPLC:-

High Pressure Liquid Chromatography;High Priced Liquid Chromatography;Hewlett-Packard Liquid Chromatography;High Performance Liquid Chromatography;Hocus Pocus Liquid Chromatography;High Patience Liquid Chromatography;

High Performance Liquid Chromatography (HPLC) is one mode of chromatography the most widely used analytical technique.HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.

PRINCIPLE OF HPLC:- Different types of HPLC act on the basis of two laws:- ● Adsorption law ● Partition law“When a solid surface is exposed to a gas or a liquid, molecules from the gas or the solution phase accumulated or concentrated at the surface of the solid.” this phenomenon is called as adsorption.The Partition Law states that –“The ratio of the partition or distribution of a compound between two immiscible phases remain constant.”The constant is called as “partition constant or distribution constant.” For a compound distributing itself between equal volumes of two immiscible solvents A and B, the value for this coefficient is a constant at a given temperature and is given by the expression:-

Kd= Concentration∈solvent AConcentration∈solvent B

The distribution of a compound can be described not only in of its distribution between two solvents, but also by its distribution between any two phases, such as solid/liquid or gas/liquid phases.

It is known that the resolving power of a chromatographic column increases with column length and the number of theoretical plates per unit length, although there are limit to the length of a column due to problem of peak broadening. As the number of theoretical plates is related to the surface area of the stationary phase it follows that the smaller the particle size of the stationary phase, better the resolution. Unfortunately, the smaller size, the greater the resistance to eluent flow. All of the forms of the column chromatography rely on gravity or lower pressure pumping systems for the supply of eluent to the column and called as low performance liquid chromatography.

Due to consequences of this is the flow rates achieved are relatively low and this gives greater time for band broadening by simple diffusion phenomena. The use of faster flow rates is not possible because it creates a back-pressure problem which is sufficient to damage the matrix structure of the stationary phase, thereby actually reducing eluent flow and impairing resolution. In the past decade there has a dramatic development in column chromatographic technology, which has resulted in the availability of new particle size stationary phases which can withstand these pressures and of pumping system which can give reliable flow rates. These developments, which have occurred in adsorption, partition, ion-exchange, size exclusion and affinity chromatography, have resulted in faster and better resolution and explains why HPLC has emerged as the most popular, powerful and versatile form of the chromatography.

Pressure used normally range from 30 to 200 atm, depending on the type of column used. The pressure is varied to provide the optimum linear flow rate of the mobile phase. It is that pressure at which gives the smallest theoretical plate height. It is derived experimentally as in gas chromatography where the relationship H/V is developed.

TYPES OF HPLC:- There are many ways to classify liquid column chromatography. If this classification is based on the type of the instruments used for separation processes-

1. Classical HPLC can be classified on the nature of the stationary phase and the separation process, seven modes can be specified, which are— ● Adsorption chromatography; ●liquid-liquid chromatography; ●Ion-exchange chromatography; ●Size or steric exclusion chromatography; ●Affinity chromatography; ●Bonded phase chromatography; ●Ion pairing chromatography;

2. Advanced HPLC can also be classified on the nature of the stationary phase and the separation process, two main modes are—

●Chiral HPLC; ●Capillary HPLC; All above mentioned chromatographic techniques are briefly described below:-

ADSORPTION CHROMATOGRAPHY & LIQUID-LIQUID CHROMATOGRAPHY:- In adsorption chromatography the stationary phase is an adsorbent (like silica gel or any other silica based packings) and the separation is based on repeated adsorption-desorption steps. If the sample is water insoluble, possesses an aliphatic or aromatic character, and has a molecular weight less than 2000 Daltons for all of the sample components adsorption chromatography and liquid liquid chromatography are possibilities. Adsorption chromatography works best for class separation or for the separation of isomeric compounds. The technique of liquid liquid chromatography works better for the separation of homologous compounds. Functional groups that are capable of strong hydrogen binding are retained strongly in adsorption chromatography; however liquid-liquid chromatography provides an alternate method for the separation of such compounds. These will be samples that posses medium polarity and are soluble in weakly polar to polar organic solvents. In general the separation of non electrolytes in liquid-liquid chromatography is achieved by matching the polarities of the sample and stationary phase and using a mobile phase that has markedly different polarity. Concerning this type of chromatography, two modes are defined depending on the relative polarity of the two phases:- ● Normal-phase chromatography; ● Reversed-phase chromatography;

In normal-phase chromatography, the stationary bed is strongly polar in nature (e.g.; silica gel), and the mobile phase is nonpolar (such as n-hexane or tetrahydrofuran). Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.

In reversed-phase chromatography, is the inverse of this. The stationary bed is nonpolar (hydrophobic) in nature, while the

mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. Here the more nonpolar the material is, the longer it will be retained.

BONDED-PHASE CHROMATOGRAPHY:- The most widely used column packings for liquid-liquid partition chromatography are those with chemically bonded, organic stationary phases. They replace the classical packings in which the stationary liquid phase coated with a supporting material. Partition occurs between the bonded phase and a mobile liquid phase. Bonded –phase supports are made from silica by the covalent attachment of an organic hydrocarbon moiety to the surface. Supports include large porous silica gel, porous layer beads, and micro particles. Bonded-phase packings are quite stable because the stationary phase is chemically bound to the support and cannot be easily removed during use.

Si OH + ClSi (CH3)2R Si O Si(CH3)2R +HCl

The siloxane (Si O—Si C) type of bond has become the standard for commercial bonded phases. It is formed by the reactions of di-(or tri-) alkoxysilane with the surface of silanol groups of fully hydroxylated silica gel, as either pellicular or totally porous supports.

ION EXCHANGE CHROMATOGRAPHY:- In ion-exchange chromatography the stationary bed has ionically charge- bearing functional groups attached to a polymer matrix. The functional groups are permanently bonded ionic groups associated with counterions of the opposite charge. The most common retention mechanism is the simple exchange of sample ions and mobile-phase ions with the charged group of the stationary phase. This technique is

used almost exclusively with ionic or ionisable samples, when the sample is water soluble and molecular weights are less than 2000

Daltons. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. Some ion-exchange packings be negatively charged groups and are used for exchanging cationic species and positively charged packings are used for exchanging anionic species. The most commonly used functional groups are the sulphonate type exchangers for cation exchange & the quaternary amine type exchanger for anion exchange.

Sulfonic acids exchange resin for cation exchange.

Strong base (Quaternary amine) exchange resin for anion exchange. There are no any effect of pH of mobile phase on the exchange properties because they are strong acids or bases in the -H or –OH form respectively. These exchangers have considerable affinity for heavy metal cations and, to a lesser extent, for alkaline earth cations.

AminodiacetateAminoacetate exchanger fined its use as a column packing for ligand exchange.

ION-PAIRING CHROMATOGRAPHY:- Ion- pair chromatography, is considered as a subset of reverse-phase chromatography, can deal with ionized or ionisable species on reverse-phase columns. The method overcomes difficulty in handling certain samples by the other liquid chromatography methods; these are samples that are very polar, multiply ionized, and/or strongly basic. In ordinary reverse-phase HPLC, organic ions show poor peak shapes and inadequate retention. The ion suppression method is also limited to the pH range 2.0-7.5 by the instability of stationary bonded phases outside this pH range. Ion-exchange packings offer limited choices with little ability to vary selectivity by changing the column packing.In ion-pair chromatography an ion-pair reagent (a large organic counterion) is added low concentration (usually 0.005M) to the mobile phase. The ion-pair reagent is itself ionized. One ion of the reagent is retained by the stationary phase, thus providing the otherwise neutral stationary phase with its charge. This charged stationary phase can then retain and separate organic solute ions of the opposite charge by forming a reversible ion-pair complex (a columbic association species formed between two ions of opposite electrical charge) with the ionized sample as represented by the following equilibrium:

RCOO− + R4N+ [ R4N+,-OOCR]0 ion pair

Here it is assumed that the solute ion is a caboxylate anion, RCOO-

and that the counter ion is a quaternary ammonium ion, R4N+. Thus with a suitable counterion, ionic or nonionizable compounds can be converted to electrically neutral compounds that will partition between the mobile and nonpolar stationary phases. At the, same time, the stationary phase will not have lost any of its ability to retain and separate nonionized organic substances.There are two fundamental models have been proposed for the mechanism of the ion-pair chromatography-

The first postulate is that the solute molecule forms an ion pair with the counterion in the mobile phase. This uncharged ion pair then partitions into the lipophilic stationary phase(C-18).

Silica particles N+

The second is that the counterion partitions into the stationary

phase, or is “loaded” onto the bonded reverse-phase packing, with its ionic group oriented at the surface. This produces two possibilities for the material to be chromatographed. It can be attracted to the hydrocarbon partition in the usual revese-phase manner, or it can interact in an ion-exchange mode. It is quite likely that the true mechanism involves both postulates but is futher complicated by adsorption and micelle formation. Whatever the mechanism, ion-pair chromatography allows for unique separations not otherwise obtainable by either reverse phase or ion-exchange.

Silica particles Counterion (alkyl sulfonate) loaded onto stationary phase.

Fig:-Mode of selection of type of liquid chromatography for sample molecules having molecular weight less than 2000.

SIZE EXCLUSION OR GEL PERMEATION CHROMATOGRAPHY :- In size exclusion chromatography the column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according to its solvated molecular size. It is especially applied when it is known or suspected that the molecular weight exceeds approximately 2000 daltons for some or all of the sample components. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. Mainly for historical reasons, this technique is also called gel filtration or gel permeation chromatography although, today, the stationary phase is not restricted to a "gel". Much work in the food and beverage industry, or physiological samples in which most of the Krebs cycle acids (tricarboxylic acid cycle) are present, is easily done with an anion exclusion column. Wine, beer, fruit juices, and many dairy products are quickly analyzed with minimal sample preparation (usually only filtration or centrifugation). It also used in separation of oligosaccharides and sugar alcohols using an anion exclusion column in the calcium ionic form with water as eluent at temperature of 900C. Maltotriose and higher oligosaccharides are separated from mono- and disaccharides by steric exclusion effects.

AFFINITY CHROMATOGRAPHY:- The general scheme of affinity chromatography involves the covalent attachment of an immobilized biochemical (called an affinity ligand) to a solid support. When a sample is passed through the column, only the solute(s) that selectively binds to the complementary ligand is retained; the other sample components elute without retention. The separations exploit the “lock and key” binding that is prevalent in biological systems. The retained solute(s) can be eluted from the column by chaning the mobile-phase conditions. The major advantage of this technique is its tremendous specificity, which permits rapid isolation (in preparative work) with a good yield in a single step.

The column matrices used are organic gels such as beaded agarose (a purified form of the polysaccharide agar), cellulose, dextran, polyacrylamide, combinations of these polymers, and microporous glass beads. The matrix should be sufficiently hydrophilic to avoid the nonspecific binding of solutes and stable to most water-compatible organic solvents. A porous matrix permits a high degree of ligand substitution and is more accessible to large molecules. Preactivated agarose supports are available commercially or can be prepared as given below- NH C OH C O CN RNH2 C O C NHR BrCN C OH C OH C OH R=alkyl or aryl group

Agarose is a very popular matrix; its porous meshwork can be strengthened by cross-linking it with 1-chloro-2,3-epoxypropane.The affinity ligand can be antibodies, enzyme inhibitors, or other molecules that reversibly and bioselectively bind to the complementary analyte molecules in the sample. The affinity ligands are of two types; one is Ligand specific, and other is Group-specific. Group-specific ligands are used for the separation of ligands such as, proteins, including

RETENTION MECHANISM In general, HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous packing bead, tend to interact with the surface adsorption sites. Depending on the HPLC mode, the different types of the adsorption forces may be included in the retention process: ● Hydrophobic (non-specific) interactions are the main ones in reversed-phase separations. ● Dipole-dipole (polar) interactions are dominated in normal phase

mode. ● Ionic interactions are responsible for the retention in ion-exchange chromatography.

All these interactions are competitive. Analyte molecules are compete with the eluent molecules for the adsorption sites. So, the stronger analyte molecules interact with the surface and the weaker the eluent interaction, the longer analyte will be retained on the surface.SEC (size-exclusion chromatography) is a special case. It is the separation of the mixture by the molecular size of its components. In this mode any positive surface interactions should be avoided (eluent molecules should have much stronger interaction with the surface than analyte molecules). Basic principle of SEC separation is that the bigger the molecule, the less possibility for her to penetrate into the adsorbent pore space, so, the bigger the molecule the less it will be retained.

CHIRAL CHROMATOGRAPHY:- In chiral chromatography the following terms are used-Chiral stationary phase: - A stationary phase which incorporates a chiral selector. The term chiral stationary phase does not necessarily mean that the stationary phase itself is chiral (although in practice it usually is) but that the stationary phase is used to separate chiral substances. Two substances can only be separated if their standard energy of distribution differ, which means that their standard enthalpies and/or their standard entropies of distribution also differ. In general, the standard enthalpy reflects the net difference in the interactive forces on the molecule in the two phases (polar, dispersive and ionic interactive forces) whereas the standard entropy reflects their spatial disposition and, thus, their probability and proximity of interaction. Thus, for any chiral separation the stationary phase is chosen such that the spatial arrangement of its composite atoms results in the probability or proximity of interaction differing significantly between the two enantiomers to be separated. If not a constituent of the stationary phase as a whole, the chiral selector can be chemically bonded to (chiral bonded stationary phase)or immobilized onto the surface of a solid support or column wall (chiral coated stationary phase),or simply dissolved in the liquid stationary phase. Chiral selector: The chiral component of the separation system capable of interacting enantioselectively with the enantiomers to be separated. Chiral additive: The chiral selector which has been added as a component of a mobile phase or electrophoretic medium. Chiral mobile phase: A mobile phase containing a chiral selector. Chiral chromatography is highly dependent on the column, which has seen many recent improvements, and the detector.The chiral chromatography is specially used for the separation of enantiomeric, or diasteriomeric compounds (drugs). It is a relatively new technique of HPLC.

CAPILLARY HPLC:- Capillary HPLC uses smaller column internal diameters than conventional HPLC. Smaller ID columns, for fixed amounts of injected material, produce taller peaks. Taller peaks provide better detection

limits for mass spectrometry and other concentration sensitive detectors. For the same amount of material injected, the peak height is inversely proportional to the cross sectional area of the column. The use of smaller ID columns requires careful planning if you are used to normal 4.6 mm columns. Liquid chromatography/mass spectrometry, LC/MS, is a revolutionary tool in the chemical and life sciences. LC/MS is accelerating chemical research by providing a robust separations and identification tool for chemists and biologists in diverse fields. LC/MS is best done with capillary HPLC. This General Introduction to capillary HPLC is designed to provide a practical survey of the day-to-day issues in solving chemical problems using HPLC and to give you the necessary information to use the Agilent 1100 Capillary HPLC and Ion Trap SL.

TYPES OF ELUTIONS:- There are two elution types-

Isocratic elution. Gradient elution.

In the first type constant eluent composition is pumped through the column during the whole analysis. This is ISOCRATIC ELUTION.In the second type, eluent composition (and strength) is steadily changed during the run. This is GRADIENT ELUTION.

FACTORS INFLUENCING THE RETENTION TIME & SEPARATION WITH LIQUID CHROMATOGRAPHY:-There are several factors influencing the separation through HPLC are given below:

Column length, Sample distribution between stationary and liquid phases, The selection of the stationary and liquid phases, Temperature of the column, Pressure drop of the column, Viscosity of solvent, Particle diameter of stationary phase,

EFFECT OF TEMPERATURE OF COLUMN ON THE RETENTION TIME:-

In many critical HPLC analyses, variations in temperature can cause significant changes in retention times, making qualitative analysis difficult and affecting the precision of quantitative measurements. Elevated temperatures are advantageous because decreased mobile-phase viscosity, increased mass transfer, and increased sample solubility results in either better resolution or faster analysis. In the given below figure the same materials are separated at different temperatures while the mobile-phase flowrate and composition are held constant. As the temperature increases the k’ values decreases and the peaks become sharper. This observation is fairly general for most modes of HPLC excepting ion-exchange chromatography.

Fig:- Influence of column temperature in liquid column chromatography.Mobile phase is 45%methnol/55%water. Peaks(1) 9,10-anthraquinone;(2) 2-methyl-9,10-anthraquinone;(3) 2-ethyl-9,10-anthraquinone;(4)1,4-dimethyl-9,10-anthraquinone; and (5) 2-t-butyl-9,10-anthraquinone.

PRESSURE DROP OF THE COLUMN:- N2h2Φη RT(tM) = ∆P ∆P = Pressure drop

From above equation, the pressure drop varies inversely with the retention time. Although an increase in pressure can increase the maximum attainable plate number, the generation of heat within the column as a result of the work done in forcing the mobile phase through the column at a very high pressure can seriously degrade column

performance. Pressures above 5000 psi (340 atm) do not appear worthwhile for most HPLC separations. PARTCLE DIAMETER OF STATIONARY PHASE:- The analytical performances improve dramatically when the particle diameter is reduced, particularly when the column is operated at the optimum velocity. Each time the particle diameter is halved, the pressure drop required is raised by approximately a factor of four. Often, however, the column length can be shortened significantly there are no significantly. There are no practical operating conditions when particles larger than 5 μm would be desirable from the point of view of achieving high plate numbers quickly. For a given column length, the plate counts increased 1.7 times for a 3 μmpacking material compared with a 5 μm packing material. Furthermore, within limits, the plate count is also directly proportional to the column length. Of course, one must always keep in mind that the resolution of the two peaks is proportional to only the square of the plate count. Eventually, however, the use of ever finer particles or longer column of the same size particles requires pressure that exceeds 5000 psi, the practical upper limit. Commercial columns are available with packing materials with particle diameters of 3, 5, and 10μm.

COLUMN LENGTH:- The ratio, Lλ = dp

is the number of particles to the column length and is called as reduced length.

Then, Φηλ

∆PO = TM

shows that when columns of the same reduced length are eluted with the same eluent to give the same elution

time for an unretained solute, the same pressure drop is required. Finerparticles are usually paired with shorter columns to achieve a goodseparation. This necessitates the use of higher inlet pressures to move

the mobile phase through the column at optimum velocity. Both effectseventually results in serious technical difficulties.

VISCOSITY:- A solvent with low viscosity is always preferred in HPLC. While maintaining constant pressure drop across the column, an increase in velocity of the solvent always decreases the flow rate of the mobile phase. The diffusion constant of the solutes are also affected by the viscosity of the mobile phase.

APPLICATIONS OF HPLC HPLC is still in its infancy, but with the further development of new support materials as well as new more sensitive detectors, it promises to become more and more important. HPLC offers the advantages of speed, resolution, and sensitivity it is especially useful for separating the high molecular compound which have either a low vapour pressure are under go pyrolysis when subjected to the heiger required temperatures of gas chromatography . The process have been applied to a wide variety of natural products such as nucleic acid, urine, serum, carbohydrates, lipids, amino acids bile acids and manufacture products such as pharmaceuticals, pesticides, herbicides, surfactants, and antioxidants.

HPLC IN PHARMACEUTICAL INDUSTRIES:- HPLC has wider application in the pharmaceutical industries for drug analysis. There are some applications of HPLC in pharmaceutical have shown below:-

1. HPLC IN IDENTIFYING AND QUATITATING THE COMPOUNDS IN DRUG SAMPLE:- Each elute comes at a specific location, measured by the elapsed time between the moment of injection [time zero] and the time when the

peak maximum elutes. By comparing each peak’s retention time [tR] with that of injected reference standards in the same chromatographic system [same mobile and stationary phase], a chromatographer may be able to identify each compound.

Figure-1: Identification

In the chromatogram shown in Figure-1, the chromatographer knew that, under these LC system conditions, the analyte, acrylamide, would be separated and elute from the column at 2.85 minutes [retention time]. Whenever a new sample, which happened to contain acrylamide, was injected into the LC system under the same conditions, a peak would be present at 2.85 minutes [see Sample B in Figure-2]. Once identity is established, the next piece of important information is how much of each compound was present in the sample. The chromatogram and the related data from the detector help us calculate the concentration of each compound. The detector basically responds to the concentration of the compound band as it passes through the flow cell. The more concentrated it is, the stronger the signal; this is seen as a greater peak height above the baseline.

Figure-2: Identification and Quantitation

In Figure-2, chromatograms for Samples A and B, on the same time scale, are stacked one above the other. The same volume of sample was injected in both runs. Both chromatograms display a peak

at a retention time [tR] of 2.85 minutes, indicating that each sample contains acrylamide. However, Sample A displays a much bigger peak for acrylamide. The area under a peak [peak area count] is a measure of the concentration of the compound it represents. This area value is integrated and calculated automatically by the computer data station. In this example, the peak for acrylamide in Sample A has 10 times the area of that for Sample B. Using reference standards, it can be determined that Sample A contains 10 picograms of acrylamide, which is ten times the amount in Sample B [1 picogram]. Note there is another peak [not identified] that elutes at 1.8 minutes in both samples. Since the area counts for this peak in both samples are about the same, this unknown compound may have the same concentration in both samples.

2. SEPARATION OF THE METABOLITES OF VITAMINS D2

AND D3 ON SMALL-PARTICLE SILICA COLUMNS:- The separation of all of the known metabolites of vitamin D2 and vitamin D3 found in biological fluids and the analysis of vitamin D mixtures, purification of vitamin D metabolites, and identification of radioactive peaks by applying HPLC.

Zorbax-SIL is a small-particle silica column packing that has strong adsorptive affinity for the hydroxyl group(s) of vitamin D and its metabolites.

Figs.1 and 2 illustrate the resolution of vitamin D and its metabolites. Although it is difficult to devise a single solvent system that will elute 1,25-(OH)D3 in a convenient time and yet will resolve vitamin D from the solvent front of the column, 10% isopropanol in Skellysolve B (using 3000 psi pressure) provides a reasonable compromise. Obviously, an increase in the number of hydroxyl groups on the vitamin D molecule increases the interaction with the silica adsorbent as reflected by increased retention. The 1α-hydroxyl groups apparently interacts much more strongly with the silica than do the side-chain hydroxyls. This is best illustrated by the retention of the dihydroxylated 1α-OH-D(2or3) compounds over the trihydroxylated 24,25-(OH)D(2or3) compounds.Thus, high-pressure liquid chromatography on silica allows for a dramatic resolution of the naturally made1,25-(OH)2D3 and 25,26-(OH)2D3 in normal lipid extracts, a resolution impossible on

conventional Sephadex LH-20 column chromatography or ordinary silicic acid column chromatography. However, the interaction between the side-chain hydroxyls and the silica is more than adequate to provide an impressive separation of 24, 25-(OH)2D3 from 25,26-(OH)2D3 and a separation of 25-OH-D3 from vitamin D3.

Of some importance is the resolution of vitamin D2 compoundsfrom vitamin D3 compounds. The silica columns do not permit the resolution of vitamin D2 from vitamin D3(Fig. 3) or 1α-OH-D2 from 1α-OH-D3 (Fig. 4), suggesting that the side chain without hydroxyls does not interact significantly with the silica. However, the introduction of hydroxyls on the side-chain positions of 24 or 25 permits a clear resolution of the vitamin D2 and D3 analogs (Fig. 3 and Fig. 5). A partial separation of 24,25-(OH) & from 24,25-(OH)2D3 is also achieved (Fig. 6). In all cases the D2 analog elutes before its corresponding D3 analog. These results suggest that the methyl group

on (C-24 must shield or reduce the interaction of either the 24-OH or the 25-OH, with the silica making such compounds less tightly held than their D3 counterparts.

Base-line resolution of vitamin D, 24-OH-D, and 25-OH-D is achieved only by use of a less polar solvent system (2.5% isopropanol is Skellysolve B), as depicted in Fig. 3. Again, side-chain hydroxylation is necessary to provide a significant effect of the 24-methyl group on the interaction with the silica adsorbent.

To illustrate the analytical usefulness of this system for biological materials, Figs. 7 and 8 have been included. Fig.7 represents the radioactivity and absorbance profiles of a blood plasma extract of vitamin D-deficient rats given two 5-IU doses of 26,27-3H-labeled 25-OH-D3 36 and 12 hr. before being killed. The extract was first chromatographed on a Sephadex LH-20 column (1 X 60 cm) using a solvent system of chloroform-Skellysolve B 65:35, the 1,25-(OH)2D3 region was combined with standard nonradioactive 25,26-(OH)2D3 and 1,25-(OH)2D3 compounds, and an aliquot was applied to the high-

pressure liquid column. Note that the presence of other tissue lipids did not change the resolution or the elution position of the metabolitesappreciably.

Fig. 8 represents a profile from an extract of liver homogenate from vitamin D-deficient chicks incubated with 3~x-~H-labelevdi tamin D2 according to the procedure of Tucker, Gagnon, and Haussler and prepurified on hydroxyalkoxypropyl Sephadex (1 X 60 cm; 10% chloroform in Skellysolve B;). Aliquots of the 25-0H-D~region were then chromatographed with marker vitamin D2,24-OH-D2, and 25-OH-D2. Thus, the HPLC system is a powerful tool for the separation of all the known metabolites of either vitamin D2 or vitamin D3. There is a rough correlation between the number of hydroxyl groups and elution position, illustrating the more hydroxyls, the more tightly held is the compound. However, the position of the hydroxyl on the molecule is also of great

importance. This is best illustrated by the fact that 1α-OH-D3 (a synthetic analog of 1,25-(OH)2D3), which is a dihydroxy compound, is more tightly held than 24,25-(OH)2D3, a trihydroxy compound. The strong interaction of the 1α-OH group undoubtedly is responsible for the fact that 1α,25-(OH)2D3 is held tightly to the column and elutes very late in the profile. This interaction is also responsible for the impressive and highly desirable separation of 25,26-(OH)2D3 from 1, 25-(OH)2D3.

3. USE OF HPLC IN CHIRAL DRUGS ANALYSIS:- The necessity to analyse and purify chiral compounds has increased. This has generated demand for chiral stationary phases (CSP) with good chiral selectivity and high loadability. Kromasil CelluCoat is a silica based CSP coated with tris-(3,5-dimethylphenyl)-carbamoyl cellulose as the chiral selector. The chiral interaction between analyte and a CSP like CelluCoat in normal phase mode is a combination of steric, H-bond, π–π, and dipole interactions. Two of the most predominant factors in analytical chiral HPLC are selectivity leading to enantiomer separation, and hereafter the speed of the separation. For analytical separations it is ideal to use small particle CSP, which gives high chromatographic resolution and makes it possible to run fast separations at high flow-rates without loosing significant column efficiency. As demanded by the Food and Drug Administration (FDA) chiral pharmaceuticals should be developed and reach the market as single enantiomer products.3 This fact makes robust chiral separation methods essential for product quality control analysis, because the unwanted enantiomer is often the most critical product impurity.In the field of chiral chromatography preparative purification needs range from semi-prep purification in the lab during early development to full scale manufacturing of enantiopure drugs; e.g.

(i) Atenolol:-

Sample Name Atenolol

Injection Volume 10 µl

Detector UV (235 nm)

Mobile Phase CO2/MeOH + 0.2% IPHH (80:20 v/v)

Temperature 40 °C

Flow Rate 4 ml/min

Column Name OD

Length 25 cm

Diameter 0.46 cm

Particle Size 10 µm

Observed Pressure 200 bar

4. APPLICATION OF HPLC IN MONITORING THE PROGRESS OF A REACTION IN DRUG SYNTHESIS:- A facile method was established to enzymatically synthesize rhapontigenin from the glycosylated parent compound rhaponticin. A novel and simple high-performance liquid chromatographic method was used for the determination of rhapontigenin prepared.

INTRODUCTION:- Rhapontigenin, (3, 3’, 5 –trihydroxy-4’-methoxy-stilbene) C15H16O4, MW: 258, is a stilbene found in Korean rhubarb rhizomes, and is most abundant in the Rhei undulatum species. Rhaponticin, the glycosylated parent compound of rhapontigenin has long been employed as an oral haemostatic agent and to treat and prevent allergies. Rhapontigenin, the aglycone of rhaponticin, has been suggested to be the active molecule. Recent research has shown rhapontigenin to be a potent anti-allergic, anti-coagulant, and anti-inflammatory compound. Rhapontigenin also possesses potent anti-cancer activity. Structures of Stilbenes-

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Rhapontigenin OH OH H OCH3 OH

Rhaponticin O-Glucose

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Synthesis of rhapontigenin from commercially available rhaponticin is carried out. Furthermore, a selective, isocratic reversed-phase HPLC method for the determination of rhapontigenin and its metabolites in rat serum and its application to in vitro and in vivo kinetic studies is in detailed.

CHROMATOGRAPHIC SYSTEM AND CONDITIONS:-The HPLC system used was a Shimadzu HPLC (Kyoto, Japan),

consisting of an LC-10AT pump, a SIL-10AF auto injector, a photodiode-array SPD-10A VP UV/VIS spectrophotometric detector and an SCL-10A system controller. Injection volume was 150 μL.

The analytical column used was an amylose tris 3, 5- dimethylphenylcarbamate (150 ´ 4.6 mm, ID, 5 mm). The mobile phase consisted of acetonitrile and 0.1% phosphoric acid (30:70, v/v), filtered and degassed under reduced pressure prior to use. Separation was carried out isocratically at ambient temperature, and a flow rate of 1.0 mL/min, with UV detection at 324 nm. Daidzein with methnol is used as an internal standard.

ENZYMATIC SYNTHESIS OF RHAPONTIGENIN:- A 0.01M tetraethylammonium acetate buffer was made by adding 261 mg tetraethylammonium acetate to 100 mL HPLC water in a volumetric flask. The pH was adjusted to 5.0 using 1M HCL. 4 mL buffer was filtered and added to a clean glass test tube. 20 mg rhaponticin was weighed carefully and added to the prepared buffer. The rhaponticin solution was sonicated and vortexed until dissolved. The rhaponticin solution was then placed in a 37 oC shaking water bath. Next, an enzyme solution was prepared by adding 1 mL buffer to 6 mg β-glucosidase. The enzyme solution was shaken gently and directly added to the rhaponticin solution. The resulting solution was incubated for 72 hours. 200 μL aliquots of the incubate were taken every 24 hours and the reaction progression was monitored via HPLC.

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Figure: HPLC-monitored enzymatic reaction of rhaponticin into rhapontigenin via β-glucosidase.

5. DETERMINATION OF PHARMACOKINETICS OF RHAPONTIGENIN IN RATS VIA HPLC:- The HPLC method has been applied to the determination of rhapontigenin in pharmacokinetic studies in rats. Following administration of rhapontigenin there was an apparent terminal elimination half-life of ~6h for the parent compound.

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Figure: Mean intravenous pharmacokinetics of rhapontigenin in male Sprague Dawley rats.

One previously unidentified metabolite was detected with a retention time of 4 minutes in the solvent front. The metabolite was measured indirectly by treating samples with β-glucuronidase and measuring the increase in parent compound. The pharmacokinetics of rhapontigenin appears to be qualitatively very similar to resveratrol in the rat where a glucuronide metabolite is also present in plasma.

METABOLISM OF RHAPONTIGENIN IN RAT LIVER MICROSOMES UNDER A UGT GENERATING SYSTEM:- The HPLC method has been applied to the determination of rhapontigenin and its metabolic products in the phase II metabolic kinetic study of rhapontigenin in rat liver microsomes. Rhapontigenin was added individually to microsomes in a concentration of 10 mg/mL. Following the incubation of rhapontigenin as parent drug at 37 °C in rat liver microsomes with the UGT enzyme, a rapid and significant decrease in rhapontigenin was detected. A metabolic peak was observed, eluting at 4 minutes, which could not be resolved from the solvent front.

Due to the fact that the metabolite eluted with the solvent front, the quantification of metabolite was determined indirectly using β-glucuronidase to hydrolyze the metabolite back to parent compound. This technique has been extensively used in metabolism and pharmacokinetic research to determine metabolite concentration over time. β-glucuronidase was added to a set of microsomal samples instead of acetic acid/acetonitrile stop solution. These samples were analyzed via HPLC along side of original microsomal samples exposed to the stop solution. HPLC analysis confirmed the absence of the glucuronidated metabolite. This same peak at the same retention time was also apparent in the rat serum.

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Figure : Phase II Microsomal metabolism of Rhapontigenin in rat liver microsome.

6.SEPARATION OF ALKALOIDAL EXTRACT FROM Aspidosperma ramiflorum:- The alkaloidal extract from A. ramiflorum has Anti-leishmanial activity. For HPLC analysis, the crude extract was dissolved in CH2Cl2:MeOH (80:20) and 10 µl were injected onto a Waters µ-Bondapak RP-18 (reverse phase, 4.6 mm x 250 mm) column at 40؛. Solvent A was 100 mmol/lit. ammonium formate in 0.12% octanesulfonic acid (v/v)/, formic acid and acetonitrile (88:4:8, v/v), while solvent B consisted of 100 mmol l-1 aqueous ammonium formate containing 0.12% octanesulfonic acid (v/v)/formic acid/acetonitrile (64:4:32, v/v). The separation was carried out using a mixture of

solvent A and, a progressively increasing amount of B (0, 10, 40, 90, 100%) during 60 min. The flow rate was 1.3 ml min-1. The effluent was monitored with a photodiode-array detector with windows at 222 nm and 254 nm and also by mass spectral analysis of isolated eluates. The major constituents of A. ramiflorum alkaloidal extracts are: ramiflorine A (1) and ramiflorine B (2), whose presence was monitored by HPLC.

Fig. A: high performance liquid chromatography (HPLC) chromatogram of standards mixture isolated from Aspidosperma ramiflorum;

B: HPLC chromatogram of alkaloidal extract. Peaks - 1: internal standard (tryptophol); 2: 10-methoxy-geissoschizol; 3: ramiflorine A;4 : ramiflorine B.

7. INVESTIGATION OF THE SOLID STATE PROPERTIES OF AMOXICILLIN TRIHYDRATE AND THE EFFECT OF POWDER pH:-

The research is carried out to investigate some physicochemical and solid-state properties of amoxicillin trihydrate (AMT) with different powder pH within the pharmacopoeia-specified range. AMT batches prepared using Dane salt method with the pH values from 4.39 to 4.97 were subjected to further characterization studies. Optical and scanning electron microscopy showed that different batches of AMT powders were similar in crystal habit, but the length of the crystals increased as the pH increased. Further solid-state investigations using powder x-ray diffraction (PXRD) demonstrated the same PXRD pattern, but the intensity of the peaks raised by the powder pH, indicated increased crystallinity. Differential scanning calorimetry (DSC) studies further confirmed that as the powder pH increased, the crystallinity and, hence, thermal stability of AMT powders increased. Searching for the possible cause of the variations in the solid state properties, HPLC analysis showed that despite possessing the requirements of the United States Pharmacopoeia (USP) for purity/impurity profile, there was a direct relationship between the increase of the powder pH and the purity of AMT, and also decrease in the impurity I (α-Hydroxyphenylglycine) concentration in AMT powder.

Amoxicillin trihydrate or AMT is a commonly used β-lactam antibiotic, which is highly active against a broad spectrum of bacteria. High solubility, high rate of absorption, and stability of AMT under acid conditions are among the most important advantages of this antibiotic.

Different solid forms of the same chemical compound can exhibit different physical and chemical properties including different solubility and dissolution profiles, which in turn affect the bioavailability and stability of the drug substance. For this study, the high-performance liquid chromatography (HPLC) analysis was carried out for any possible correlation between the solid state properties and the purity/impurity profile of AMT batches with various powder pH. The HPLC system consisted of a 616E pump, a 996 photodiode array (PDA) detector, and a degasser module; data were acquired and processed using a Millennium software Version 2.1 (all from Waters, Milford, MA). The chromatographic separations were performed on Spherisorb (Waters) C-18 columns (250 mm × 4.6 mm, with a particle size of 5 μm). The mobile phase composition, column temperature, and detector wavelength for determination of assay and related substance were determined using the USP 25 method.

ANALYSIS:- HPLC analysis was used to study the profile of the purity/impurities within different samples. The profiles of the purity/impurities of different AMT batches were investigated using standard USP 25 method. USP has introduced several important impurities of AMT: A (6-aminopenicillanic acid), B (L-amoxicillin), C (amoxicillin diketopiperazines), D (penicilloic acids of amoxicillin), E (penilloic acids of amoxicillin), F (3-(4-hydroxyphenyl) pyrazin-2-ol), and I (α-hydroxyphenylglycine). The results of HPLC analysis for AMT samples with different powder pH showed that the samples had acceptable purity/impurity profile (in the USP range), and the total percentage of the impurities in each sample was less than 1%, which was comparable to the standard AMT. The results showed that A, B, C, D, E, and F impurities had no significant variations within different samples (with powder pH from 4.39 to 4.97). However, different samples showed various concentrations of I (α-Hydroxyphenylglycine) impurity. Of interest, as the powder pH increased, the resulting chromatograph showed a noticeable decrease in the impurity I peak intensity is shown by arrows.

8. SEPARATION OF AMINO ACIDS:- Amino acids can be separated by HPLC by using cation–exchange chromatography in which acids can be separated according to their strengths on strong anion exchangers, with the weakest acids

emerging first, either by elution with a strong acid or, since acid dissociation depends on pH, by gradient elution with buffers of decreasing pH. Amino acids, which add protons to form cations in the pH range below their isoelectric points, can be separated on cation–exchangers by gradient elution with buffers of increasing pH; here the more acidic components emerge first and the most the most basic last.

Fig: Amino acid analysis; photometer senses the amino-acid-ninhydrin complex (post column reaction) at 570 nm.

9. SEPARATION OF FUROSEMIDE USP & RELATED COMPOUNDS FROM FUROSEMIDE TABLET FORMULATION:-

Furosemide: Mol.wt 330.75 Furosemide or frusemide is a loop diuretic used in the treatment of congestive heart failure and edema. It is most commonly marketed by Sanofi-Aventis under the brand name Lasix. It has also been used to prevent thoroughbred and standard bred race horses from bleeding through the nose during races. Along with some other diuretics, Furosemide is also included on the World Anti-Doping Agency's

banned drug list due to its alleged use as a masking agent for other drugs.

Method Conditions:- Column : Cogent Bidentate C18™, 4μm, 100Å Catalog No. : 40018-75P Dimensions : 4.6 x 75 mm Mobile phase : 70% Water, 30% THF 1% Acetic Acid Flow rate : 1.0 mL/minute Peaks : 1. Furosemide 2. Related compound Injection Volume: 20 μL Detection : UV 254 nm Temperature : 25°C.

This active pharmaceutical ingredient, Furosemide USP and its related compounds can be a difficult molecule to chromatograph with conventional L1 (C18) columns due to silanol activity.The excellent peak shape of Furosemide and its related compound-A when used with HPLC; baseline resolution is achieved betweenthis specified impurity and Furosemide. The active is easily separated from excipients in this tablet formulation.

10. SEPARATION AND DETECTION OF SULFONAMIDE IN ITS TABLET FORMULATION:-

Sulfonamides are used for the treatment of infections and promotion of growth of livestock and fish. The residue of these veterinary drugs in food is of serious concern, due to the risk to human health (resistance to drugs and allergic or toxic reactions).Method Conditions:- Column : Cogent Bidentate™, 4μm, 100Å Catalog No. : 40018-75P Dimensions : 4.6 x 75 mm Mobile phase: Reverse Phase Gradient : Solvent A: 100% DI water + 0.1% formic acid Solvent B: 100% acetonitrile Time %A %B 0.00 70 30 0.00 – 0.20 70 30 0.20 – 5.00 0 100 5.00 – 10.00 0 100 10.00 – 10.01 70 30 10.01 – 15.00 70 30 Flow rate : 1.0 mL/min. Injection Volume: 10 μL Peak : 1mg of the compound dissolved in 1 ml of substituted sulfonamide (m/z 468) concentration for UV. Analysis : 0.5 mg/mL and for LC/MS: 0.1 mg/mL Detection : A: UV 259 nm B: LC/MS: Atmospheric Pressure Chemical Ionization in positive mode: APCI+, Single Ion Monitoring.

DISCUSSION:- The current sulfonamide detection methods are based on UVabsorption, but there is a need for methods detecting residues belowthe maximum residue limits (MRL). LC/MS is a method of choice. Asimple and quick RP gradient was used to transfer the HPLC/UVmethod to LC/MS for analysis of basic sulfonamides. APCI ionizationmode was more advantageous than ESI.

CONCLUSION The wide applicability, speed, & sensitivity of HPLC have resulted as a powerful tool for the-

Quantitative/qualitative analyses of amino acids, nucleic acids, proteins in physiological samples.

Measuring levels of active drugs, synthetic by-products, and degradation products in pharmaceuticals.

Measuring levels of hazardous compounds such as pesticides and insecticides.

Monitoring environmental samples. Purifying compounds from mixtures.

Man is always worried about the purity of anything next to him. Analysis is a science that gives the solution for. Convenience is great boon of science, the convenience the method, the more it will be popular. HPLC method is the method just heat the sample it will ultimately say its story.