pharmaceutical analysis, Study notes of Pharmaceutical Analysis

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CHROMATOGRAHY

By

SAHADEV PARMAR

Classification of chromatography

A. Mode of Separation „ Adsorption (NPC, LSC)– separates molecules based on polarity, least polar eluting first „ Partition - Separates molecules based on combination of solubility parameters, partition coefficients, and polarity, most polar eluting first. „ Ion exchange – Separates molecules on basis of molecular charge „ Size exclusion (GPC) – separation based on molecular size, largest eluting first „ Affinity – based on affinity with ligand B. Basis of Mobile Phase „ Liquid Chromatography – LLC, LSC „ Gas chromatography – GLC, GSC

Two common approaches are used to bring the mobile phase and stationary phase into contact. In column chromatography, the stationary phase is placed in a narrow column through which the mobile phase moves under the influence of gravity or pressure. The stationary phase is either a solid or a thin, liquid film coating on a solid particulate packing material or the column’s walls. In planar chromatography the stationary phase coats a flat glass, metal, or plastic plate and is placed in a developing chamber. A reservoir containing the mobile phase is placed in contact with the stationary phase, and the mobile phase moves by capillary action.

Thin layer chromatography (TLC)

TLC is solid-liquid chromatography. Adsorbent or solid phase (polar ) which does not dissolve in the associated liquid phase or mobile phase (nonpolar) e.g. Silica Gel (SiO 2 ) & Alumina (Al 2 O 3 ) which are polar and eluent is liquid phase (hexane + ethyl acetate). Test substances are spotted onto a TLC plate coated with the adsorbent and allowed to elute up the plate by capillary action. The More polar substances bind strongly to the adsorbent and elute SLOWER while less polar substances bind weakly to the adsorbent and elute FASTER as the stationary phase is polar. The strength of interactions between the adsorbent and eluting components vary approximately in this order:

Salt formation > coordination > H-bonding > dipole-dipole > van der Waals

(More Polar) (Less Polar)

is dried to 110^0 C for 30 mins in oven and this process is called as activation of the adsorbent as water in the

slurry has to be removed otherwise it will block the adsorption (e.g. in silica gel when used as SP, the active part for adsorption are silanols or Si- OH grp which undergoes reversible H- bonding with the analyte become deactivated in presence of water). So the heating of SP activate the adsorbent by evaporation of water molecules. Sometimes silica become highly active and the polar compounds get strongly retained on it, so then we have to deactivate the silica. So water is playing an important role for activation and deactivation of adsorbent in SP.

After the plate has been dried than sample is applied to the plate 2-2.5 cm above from the edge of plate either with the aid of capillary or micropipette or microsyringe as a uniform spot.

A TLC developing chamber or jar contains a filter paper wrapped over its inside wall boundary, a covering lid and solvent mixture or mobile phase. When filter paper is completed wetted with mobile phase (MP move into the filter paper due to capillary action) indicate that chamber equilibrated with the solvent. After the chamber is equilibrated with MP then lid is taken off and the TLC plates are placed vertically in the TLC chamber and again lid is placed. (Applied analyte spots must be above from the level of the MP in the TLC chamber, otherwise the analyte may dissolve in the MP resulting in sample loss) The MP move due to capillary action in SP which is porous in nature and separates the compounds on the basis of polarity. Now the analyte which have stronger affinity for SP i.e. polar retained while nonpolar analyte move along with the nonpolar mobile phase and there retention factor is calculated with respect to distance travelled by the mobile phase. Generally MP for TLC is hexane & ethyl acetate. We start with higher % of nonpolar hexane (say 95%) and lower % of polar ethyl acetate (say 5%). In this concentration range mostly nonpolar analytes goes along with the hexane and occur at the top of the TLC plate (most nonpolar) but polar compounds retained on SP and they are separated by increasing % of ethyl acetate from 5- 6%, 6- 7%, 7- 20 % etc for polar compounds as they get retained on TLC plate and not showing any distance travelled from the origin line. As the % of the polar mobile phase (ethyl acetate) is increased polar compounds starts to move from the origin line or sample application line and attained a particular distance with particular concentration of the solvent. So the distance travelled by the analyte is dependent upon the % of nonpolar and polar MP in the solvent mixture.

Nowadays precoated silica plates in which silica is absorbed on aluminium plate are available supplied by Merck.

Detection of compounds on TLC plates:

1. Ultraviolet Light — some organic compounds illuminate or fluoresce under short-wave UV light
2. Iodine Vapor — forms brown/ yellow complexes with organic unsaturated compounds and get added to double bonds
3. Fluorescent — compounds fluoresce when placed under UV light. Other can be thin layer adsorbents which contain a fluorescent dye so that when the plate is placed under UV light, the separated compounds show blue, black , green areas against fluorescent background. E.g. zinc silicate

Column Chromatography

In column chromatography three way equilibrium between sample, adsorbent, solvent occurs. A solvent is chosen that gives Rf about 0.35. Elution of compound is detected by using TLC.

A. Column packing can be done as Slurry packing and Dry packing.

A column is prepared by placing a small plug of glass wool in the bottom of the cylindrical glass column, followed by a small layer of sand. The column is then packed with the solid adsorbent phase (silica gel). Slurry of adsorbent in solvent is prepared with the same solvent to be later used in separation. The slurry is carefully and slowly poured into the column after it is partially filled with solvent in order to prevent disturbance of the sand. The solvent is allowed to drain as the silica packs tightly. Once the solvent just barely becomes level with the silica (without drying it!), another small layer of sand is applied carefully without disturbing the silica.

B. Sample application

**1. Dissolved in mobile phase (~ 25% solution for viscous sample)

2. Dry load - Adsorption on silica (1:2 ratio)**
3. Neat - non viscous sample

The compound mixture is dissolved in a minimum amount of solvent (same as in the column) and very carefully added to the top of the column using a Pasteur pipette. After allowing the compound to adsorb into the column, solvent is continually added to the top of the column until each band resolves and is carefully collected.

With colored substances, the bands may be directly observed and collected as they run off the column. However, with colorless compounds, the development can be observed by collecting many small fractions of the eluting solvent and testing each by thin layer chromatography under UV lamp.

C. Adsorbents

Stationary phases used in order of polarity includes Alumina (highest Polarity), Magnesium oxide, Carbon, Silica , Starch, Cellulose (lowest Polarity). The most commonly used adsorbent in column chromatography is silica. It is available in different particle size and shapes. Greater the particle size, lower is the surface area, lower the number of theoretical plates, lower is the efficiency of the column. Lower the particle size, higher is the efficiency of column.

Generally the particle size of silica for column chromatography : 60-200 μm

For Thin Layer chromatography (TLC) pre coated plates : 10 μm

For High Performance Liquid Chromatography (HPLC): 3-10 μm

Stationary phases (adsorbents) may be acidic or basic. Silica is acidic – has preferential ability to retain amines and other basic compounds. Alumina and magnesia are basic – they preferentially retain acidic compounds.

Adsorption – a boundary reaction between dissolved substance and solid substance, weak and reversible interactions (weak bonds) between two phases. The interactions includes Dipole Interactions (Bonding between two atoms of different electronegativity), Hydrogen bridge bonds (Electrostatic bonds between hydrogen of one molecule and strongly electronegative element of the other molecule (F,O, N,S), Pi-Complex (Adduct of electrophilic species with C-C double bond).

D. Activity of Stationary Phase

Adsorbents require activation before use. Activity of silica is due to free silanol groups Si – OH grp. Addition of water results in loss of activity and too much heating results in loss of activity. Exposure to moisture increases physically adsorbed water on surface and decreases interaction between solute and silanol. So silica activated by heating at 110oC for 30 min as at this temp as adsorbed water present on surface is removed and max number of silanols are present. Heating silicas above 200^0 C leads to conversion of silanol groups to siloxane groups (Si-O- Si). This decreases the ability of surface to retain polar materials. Alumina – heat at 200oC for 4h (active), too long heating at high temperature (1000oC) results in total loss of activity. If fresh silica or alumina is too active, deactivation is done by adding water.

Brockman Activity Scale for Alumina

1. Grade I – most active, heating at 350oC for few hrs
2. Grade II – 2-3 % water added
3. Grade III – 5-7 % water added

Three types of silanols –

Free silanols – low concentration, strong binding to basic solutes, gives broad and tailing peaks

Fully hydroxylated – high concentration of geminal and

Associated silanols – most favored (less acidic)

The disadvantage of silica gel includes it start to dissolves at pH 8 due to breakage of siloxane bond. Purity of silica gel is important. Metal contaminants can complex with chelating solutes – lead to tailing peaks or even complete retention

Alumina – Alumina is a network of aluminium and oxygen atoms. Active points are both the Al3+^ centers and the connecting O2-^ atoms. Natural alumina is basic, however it is also available in basic or neutral forms. Chemisorption (Irreversible binding) more probable in alumina mainly when analyte contain COOH grp, so used only for specific purposes.

Elution pattern in normal phase column i.e. stationary phase is polar and MP is

nonpolar

Normal Phase

Alkanes olefins aromatics organic halogen compounds sulfides ethers nitro compounds esters/aldehydes/ketones alcohols/amines sulfones sulfoxides amides carboxylic acids

In NP Chromatography polarity of the mobile phase determines the elution time. Solvents in order of increasing polarity –

Eluotropic Series

Pentane/hexane <pet ether <cyclohexane <xylene <toluene <diethyl ether <chloroform <dichloromethane <THF <acetone <dioxane <acetonitrile <methanol <water

A: Reaction of surface silanol with chlorodimethylsilane

B: Reaction of surface silanol with trifunctional silane

C: Reaction of surface silanol with trifunctional alkoxysilane

Reacting silanol with chlorodimethylalkyl silanes or chloroalkoxy silanes – transform polar stationary phase to non polar stationary phase, chain length varying.

HPLC (HIGH PERFORMANCE LIQUID CHROMATOGRAPHY)

HPLC is a reverse phase liquid chromatography. HPLC is used for thermally labile, high molecular weight, peptides, non volatile compounds. Almost any compound that can be retained by a column can be separated by a column. HPLC separations have been achieved based on differences in polarity, size, shape, charge, specific affinity for a site, stereo, and optical isomerism. Pressure is 1000-6000 psi, for these double reciprocating pumps are used which produce pulse free stream of mobile phase. It can be operated via both isocratic and gradient elution. Particle size is generally 5 μm for analytical columns. Generally a guard column is there before main column which have particle size 30 μm to filter the impurities. Sample is introduced to column by either by auto sampler which uses injection needle, take sample from sample vial placed in the sample rack which is a part of HPLC unit or the sample can be introduced manually via injection in the column.

HPLC system has following component

1. Mobile reservoir
2. Reciprocating pumps to build the pressure (1000 psi – 6000 psi)
3. Auto sampler or injection system
4. Column placed in oven
5. Detector
6. Sample collector

Chromatographic terms:

Column dead time, retention time

Poor

resolution Shoulder

Baseline drift

t 0 = column dead

time = time an unretarded compound needs to pass the column, tR = retention time

Capacity factor (K):

If the substance is e.g. tR = tm, the capacity factor is k' = 0.

not retained by the stationary phase,

Selectivity (α):

Resolution:

……….. For Gaussian peak

R = ¼ × (α-1) × √N × K' / 1+ K' -----------------For non Gaussian peak

Plate number and plate height

The number of theoretical plates is proportional to the column length L. The longer a column, the more theoretical plates.

Van Deemter Curve reflects the relationship between theoretical plate height H and the linear velocity u of the

mobile phase. Since the plate height H is a measure for the band broadening of the injection peak of a substance, the path of the H(u) curve of a separation column is of great interest for determining the chromatographic conditions for practical applications.

Gas chromatography

The compound separated with gas chromatography must be volatile or can be derivatized so that become volatile and it must be thermally stable. Carrier gas either Nitrogen, Helium, hydrogen are used as mobile phase. It can be Gas solid chromatography (GSC) or Gas liquid chromatography (GLC). In GLC mobile phase is a gas and the stationary phase is a liquid coated either on a solid packing material or on the column’s walls.

It is most valuable for the compounds of relatively low polarity or nonpolar. The polar compounds which contain carboxylic acids, alcohols or primary amines as functional grps badly tailed due to interactions of these functional grps with stationary phase. To overcome this problem pyrolysis GC or photolysis is done.

In pyrolysis GC the sample for analysis is heated to very high temp. (800^0 C) for an extremely short time then the thermally destroyed samples are swept into gas chromatograph.

A stationary phase of high boiling point liquid like silicone grease is supported on an inert granular solid like diatomaceous earth or celite. This celite or diatomaceous earth or celite has one problem as it also have free hydroxyl grps which can cause support sample interaction. So these hydroxyl grp must be modified by silanization agent like trichlorosilane. More recently, solid supports made from glass beads or fluorocarbon polymers have been introduced. These supports have the advantage of being more inert than diatomaceous earth. The main criteria for selecting a stationary phase are that it should be chemically inert, thermally stable, of low volatility, and of an appropriate polarity for the solutes being separated. The stationary phase is often high boiling point organic compounds which includes polyethylene glycols , methyl phenyl siloxane, squalene and esters of adipic acid, succinic and pthalic acid. Another important characteristic of a gas chromatographic column is the thickness of the stationary phase. Separation efficiency improves with thinner films. The most common film thickness is 0.25 μm.

Nonvolatile analytes must be chemically converted to a volatile derivative before analysis. For example, amino acids are not sufficiently volatile to analyze directly by gas chromatography. Reacting an amino acid with 1- butanol and acetyl chloride produces an esterfied amino acid. Subsequent treatment with trifluoroacetic acid gives the amino acids volatile N -trifluoroacetyl- n -butyl ester derivative.

The steps involved in GC

1. A vaporized sample is injected onto the chromatographic column. Second, the sample moves through the column through the flow of inert gas.
2. Separation occurs as a result of unique equilibrium established between the solutes and the stationary phase (the GC column).
3. The components are recorded as a sequence of peaks as they leave the column.

N PHARMACEU

Gas chromatography columns

1. Packed Columns are made up of glass, nickel or stainless steel. They are typically 1.5-10 m long and 2- mm in diameter. Contain inert support materials like diatomaceous earth for liquid stationary phase.
2. Capillary (Open Tubular) Columns are typically 10-50 m long and 0.3-0.5 mm in diameter. Capillary or open tubular columns are constructed from fused silica coated with a protective polymer. They are more efficient than packed columns. They are composed of two major parts: tubing and stationary phase. A thin film (0.1-10.0 micro meters) of high molecular weight, thermally stable polymer is coated onto the inner wall of small diameter tubing. I. Support coated open tubular (SCOT) a thin layer of a solid support, such as a diatomaceous earth, coated with a liquid stationary phase is attached to the capillary’s inner wall. II. Porous layer open tubular (PLOT) Capillary GC columns in which the stationary phase is based on an adsorbent or a porous polymer

III. Wall-coated open tubular stationary phase

(WCOT) consist of a capillary tube whose walls are coated with liquid

Oven Temperature

Columns are kept in a heated oven. Column temperature relies on the boiling point of the sample being analyzed, and should be slightly higher than its boiling point. Temperature must be strictly controlled within fractions of degrees.